Ageing and Dementia: Current and Future Concepts 9783709161395, 3709161398

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K. A. Jellinger R. Schmidt M. Windisch (eds.) Ageing and Dementia Current and Future Concepts

Spriuger-Verlag Wieu GmbH

Prof. Dr. Kurt A. Jellinger

Institut fUr KIÎnÎsche Neurobiologic, Kcnyongassc 18/7 A-I070 Wien, Austria

Prof. Dr. Reinhold Schmidt Univcrsitătsklinik

ftir Neurologie, Auenbruggerplatz 22

A-803ti Gra:;:, Austria

Dr. Manfred Windisch JWS Research Forschungslabor GmbH, Rankengasse 28 A-S020 Gra:;:, Austria

This work is subjcct to copyright. AH rights are reserved, whether ilie whole or part of the material is concemed, specifically those of translation, reprinting, re-use of jllustrations, broadcasling, reproduction by photocopying machines ar similar means, and storage in data banks. Producl Liability: The publisher can give no guarantee for aii the information contained in this book. This does also refer to information about drug dosage and application thereof. In every individual case the respective user must check ils accuracy by consulting other pharmaceuticalliterature. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. © 2002 Springer-Verlag Wien Originali y pu blished by Springer- Verlag Wien ~ ew Yar k 2002 Softcover reprint of the hardcover lst edition 2002 Typesetting: Best-Set Typesetter Ltd., Hong Kong Printing: A. Holzhausen's Nfg., A-1140 Wien Printed an acid-free and chlorine-free bleached paper SPIN: 10867608 CIP data applied for

With 64 (partly coloured) Figures

ISBN 978-3-211-83796-2 ISBN 978-3-7091-6139-5 (eBook) DOI 10.1007/978-3-7091-6139-5

Preface Ageing and dementia are among the most important subjects of basic and clinical neurosciences as well as of socioeconomics and health care in the 21 st century. Alzheimer di sease is the leading cause of dementia in elderly people but it s aetiology and pathogenesis are still unresolved, and it s relationship to brain ageing needs further elucidation. Although a variety of risk factors of dementia in advanced age are known, the impact of some of them including small vessel disease are still under discussion. Recent advances in molecular biology and genetics have provided great progress in the understanding of some basic problems of brain ageing and dementia, but more inten sive mutual cooperation and exchange between basic scientists and clinicians are necessary in order to further elucidate the many still open problems. With this idea in mind, the International Symposium on "Ageing and Dementia" was organized by JSW Research in cooperation with the Austrian Alzheimer Society, the Department of Neurology, University of Graz School of Medicine, and the L. Boltzmann Institute of Clinical Neurobiology, Vienna, at Graz on September 28-30, 2001. Within a series of biannual conferences, this workshop offered a carefully selected programme by internationally renowned invited speakers, although, due to the sequelae of September 11, it had to be reorganized within a very short time. It is focused on three key issues: important factors that contribute to the deleterious effects of brain ageing, problems of detection and conversion of mild cognitive impairment, and possible - preventive and therapeutic - methods of altering these effects. This volume presents the updated papers dedicated to vascular risk factors and the impact of white matter le sions, oxidative stress and other pathogenic factors of dementia, mild cognitive impairment and preclinical Alzheimer disease, the current role of biological markers in the early diagnosis of dementia, current and future treatment strategies of Alzheimer disease including receptor modulation, neurotrophic agents, inhibition of amyloid mismetabolismldeposition and neurofibrillary degeneration. In view of the recently suspended Alzheimer vaccination trials, these pharmacological targets are of major current interest. It is hoped that this volume will be helpful and informative to all those who are interested and working in the field of brain ageing and dementia. Finally, we would like to acknowledge the sponsors of this conference and Springer-Verlag Wien NewYork for its excellent publication work. ViennaiGraz, July 2002

K. A. Jellinger, R. Schmidt, M. Windisch

Contents Jellinger, K. A.: Vascular-ischemic dementia: an update. van Dijk, E. J., Prins, N. D., Vermeer, S. E., Koudstaal, P. J ., Breteler, M. M. B.: Frequency of white matter lesions and silent lacunar infarcts . . . . . . . . . . . . . . . Kapeller, P., Schmidt, R., Enzinger, cn., Ropele, S., Fazekas, F.: CT and MRI rating of white matter change s Schmidt, R., Fazekas, F., Enzinger, C., Ropele, S., Kapeller, P., Schmidt, H.: Risk factors and progression of small vessel disease-related cerebral abnormalities Schmidt, H., Fazekas, F., Schmidt, R.: Microangiopathy-related cerebral damage and angiotensinogen gene: from epidemiology to biology . . . . . . . . . . . . . . . . . . Fazekas, F., Ropele, S., Schmidt, R.: Can small-vessel disease-related cerebral abnormalities be used as a surrogate marker for vascular dementia trials? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Perry, G., Nunomura, A., Cash, A. D., Taddeo, M. A., Hirai, K., Aliev, G., Avila, J., Wataya, T., Shimohama, S., Atwood, C. S., Smith, M. A.: Reactive oxygen : its sources and significance in Alzheimer disease Arendt T.: Dysregulation of neuronal differentiation and cell cycle control in Alzheimer's disease Kienzl, E., Jellinger, K., Janetzky, B., Steindl, H., Bergmann, J.: A broader horizon of Alzheimer pathogenesis: ALZAS - an early serum biomarker? . . . . . . . . Smith, G.: Is mild cognitive impairment bridging the gap between normal aging and Alzheimer's disease? Fischer, P., Jungwirth, S., Krampla, W., Weissgram, S., Kirchmeyr, W., Schreiber, W., Huber, K., Rainer, M., Bauer, P., TragI, K. H.: Vienna Transdanube Aging "VITA": study design, recruitment strategies and level of participation Almkvist, 0., Axelman, K., Basun, H., Wahlund, L.-O., Lannfelt, L.: Conversion from preclinical to clinical stage of Alzheimer's disease as shown by decline of cognitive function in carriers of the Swedish APP-mutation . . . . . . . . . . . . . . Kurz, A., Riemenschneider, M., Drzezga, A., Lautenschlager, N.: The role of biological markers in the early and differential diagnosis of Alzheimer's disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Schmitt, F. A., Cragar, D., Ashford, J. W., Reisberg, B., Ferris, S., Mobius, H.J., Stoffler, A.: Measuring cognition in advanced Alzheimer's disease for clinical trials : Windisch, M., Hutter-Paler, B., Schreiner, E.: Current drugs and future hopes in the treatment of Alzheimer's disease Flores-Flores, C., Nissim, A., Shochat, S., Soreq, H.: Development of human antibody fragments directed towards synaptic acetylcholinesterase using a semisynthetic phase display library Giacobini, E.: Long-term stabilizing effect of cholinesterase inhibito rs in the therapy of Alzheimer' disease

1 25 41 47 53 61 69 77 87 97

105 117 127 135 149 165 181

VIII

Contents

Fisher, A., Brandeis, R., Haring, R., Bar-Ner, N., KIiger-Spatz, M., Natan, N., Sonego, H., Marcovitch, I., Pittel, Z.: Impact of muscarinic agonists for successful therapy of Alzheimer's disease Geerts, H., Finkel, L., Carr, R., Spiros, A.: Nicotinic receptor modulation: advan tages for successful Alzheimer's disease therapy Winblad, B., Mobius, H. J., Steffler, A.: Glutamate receptors as a target for Alzheimer's disease - are clinical results supporting the hope ? Kesslak, J. P.: Can estrogen playa significant role in the prevent ion of Alzheimer 's disease? Fahnestock, M., Garzon, D., Holsinger, R. M. D., Michalski, B.: Neurotrophic factors and Alzheimer's disease: are we focu sing on the wrong molecul e? ... .. Humpel, C., Weis, c.: Nerve growth factor and cholinergic CNS neurons studied in organotypic brain slices Ruether, E., Alvarez, X. A., Rainer, M., Moessler, H.: Sustained improvement of cognition and global function in patients with moderately severe Alzheimer's disease: a double-blind, placebo-controlled study with the neurotrophic agent Cerebrolysins Muresanue.Ds.E; Rainer, M. , Moessler, H.: Impro ved global function and activities of daily living in patients with AD:.a placebo-controlled clinical study with the neurotrophic agent Cerebrol ysins Mucke, H. A. M.: Genomics and dementia - new drug targets ahead? Permanne, B., Adessi, C., Fraga, S., Frossard, M.•J., Saborio, G. P., Soto, C.: Are ~- sheet breaker peptides dissolving the therapeutic problem of Alzheimer 's disease? Munch, G., Deuther-Conrad, W., Gasic-Milenkovic, J.: Glycoxidative stress creates a vicious cycle of neurodegeneration in Alzheimer's disease - a target for neuroprotective treatment strategies? Iqbal, K., Alonso, A. del C., EI-Akkad, K , Gong, C.-X., Haque, N., Khatoon, S., Tsujio, I., Grundke-Iqbal, I.: Pharmacological targets to inhibit Alzheimer neurofibrillary degene ration Solomon, B., Frenkel, D.: Generation and brain delivery of anti-aggreg ating antibodi es against l3-amyloid plaques using phage display technology Rockenstein, E., Mallory, M., Mante, M., Alford, M., Windisch, M., Moessler, H., Masliah, E.: Effects of Cerebrol ysin" on amyloid-B depo sition in a tran sgenic model of Alzheimer's disea se Heiser, M., Hutter-Paier, B., Jerkovic, L., Pfragner, R., Windisch, M., BeckerAndre, M. , Dieplinger, H.: Vitamin E binding protein Afamin protects neuronal cells in vitro Jellinger, K. A.: Recent developm ents in the pathology of Parkinson 's disease

189 203 2 17 227 24 1 253

265 277 287 293 303 309 32 1 327 337 347

Vascular-ischemic dementia: an update K. A. Jellinger Institute of Clinical Neurobiology, Vienna, Austria

Both the clinical criteria and morphologic substrates of dementia resulting from cerebrovascular disease and its relation to Alzheimer disease and other age-related brain changes are controversiaL In clinical and autopsy studies in the Western world the prevalence of vascular-ischemic dementia (VID) is around 7-10%, while vascular cognitive impairment without dementia is much more frequent and the risk of poststroke dementia is increased in patients with prestroke cognitive decline. In contrast to previous suggestions that VID was largely the result of large hemispheral infarcts, according to recent studies, it is most commonly associated with widespread small ischemic or vascular lesions (microinfarcts, lacunes) throughout the CNS with predominant subcortical lesions in the basal ganglia and white matter or in strategically important brain regions (thalamus, hippocampus). The lesion pattern of rare "pure" VID , which is related to arteriolosclerotic and hypertensive microangiopathy, differs from that in mixed type dementia (Alzheimer disease and cerebrovascular lesions) that more often shows larger hemispheral infarcts. Another form of VID that is not infrequent in very old subjects is hippocampal sclerosis, a selective damage to the hippocampus that is often accompanied by multiple other cerebrovascular lesions. Both, mild Alzheimer type pathology and small vessel disease-associated subcortical vascular pathology appear to be common and may interact in causing cognitive decline, but the impact of cerebrovascular lesions on cognitive impairment and dementia needs to be further elucidated. Summary.

Introduction

While Alzheimer disease (AD) becomes widely accepted as the most common caus e of dementia in adv anced age (Jorm and Jolley, 1998), the role of cerebrovascular disease (CVD) and ischemic brain lesions in cognitive decline remains controversial and confusing. Similar to recently refined clinical and morphologic criteria for AD and other degenerative dementias (see Jellinger, 1999, 2001d), four clinical diagnostic criteria for vascular-ischemic dementia (VID) supported by the Hachinski Ischemic Score (HIS) in its original (Hachinski et aL, 1975) or modified form (Small , 1985) and by neuroimaging data are currently used (Table 1). Several class I and II studies that compared

K. A. Jellinger et al. (eds.), Ageing and Dementia Current and Future Concepts © Springer-Verlag/Wein 2002

Two others NS

Not limited NS to single narrow category

Yes

+

ADDTC (Chui et al., 1992)

CI

Yes

None

Excluded mechanisms of brain dysfunction

Delirium

Structural neuroimaging

±Focal signs

NS

Yes (probable or possible VaD)

Yes (probable VaD) No (possible VaD)

±

± Focal signs No

Neurologic examination

Yes Delirium Focal signs aphasia psychosis Alzheimer or other brain disease

Yes Delirium

No

H

Yes No

Yes NS

Yes NS

Yes No

AI

Included mechanisms of brain injury

± (Probable VaD : temporal relation for single lesion) No (possible VaD)

Yes (prob able VaD : within 3 mo, abrupt onset or stepwise progression) No (possible VaD)

Yes, by clinical jud gement

No

Causal relationship

AI acute ischemia ; CI chronic ischemia; H hemorrhag e; NS not specified; ± supportive but not necessary for diagnosis; DSM-IV Diagnostic and Statistical Manual of Mental Disord ers, 4th Ed.; NINDS-AJREN Nation al Institute of Neurological Disord ers and Stroke - Association Internationale pour la Rech erche et l'Enseignement en Neurosciences; A D DTC Alzheimer Disease D iagnostic and Trea tment Centers; VaD vascular dementia

NS

NINDSAIREN probable VaD (Roman et al., 1992)

On e other

Yes

Atherosclerosis

DSM-IV (1994)

NS

NS

Memory Cognitive Cerebrovascular impairment impairment disease

Hachinski Ischemic Score (1975)

Criteria

Definition of dementia

Table 1. Comparison of clinical criteria for vascular-ischemic dementia (VID)

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~ 5'

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Vascular-ischemic dementia: an updat e

3

Table 2. Autopsy series showing prevalence of ViD (co mpleme n t to Markesb ery, 1998) Year

Location

Authors

1962 1970 1972 1975 1977 1985 1986 1987 1987 1988 1989 1990 1994 1995 1995 1997 1998

England England US Switzerland Sweden Finland Switzerland Sweden Canada US US Austria Sweden No rway US US (CE R A D ) Canada

1999 2001 2001

Japan Japan Austria

Corsellis Tomlinson et al. Birkett Todorov et at. Sourander et al. Molsa et al. Ulrich et al. Alafuzoff et al. Wade et al. Joachim et al. Boller et al. Jellinger et al. Brun Ince et al. Markesb er y Hulette et al. Bowler et al. (*reexamination) Seno et al. Akat su et al. Jellinger (de me ntias/ probable A D)

No. of cases 167 50 24 776 258 58 54 55 65 150 54 675 175 69 557 1929 122 122 270 810/540

ViD 46 9 14 132 72 11

9 13 6 3 4 106 59 4 13 6 4/5* 42 60 80/7

% 27 18 58 22 28 19 17 23 9 2 7 16 34 5 2 0.03 3/4* 35 22 9.8/1.3

clinical diagnoses and neuropathological findings in referencal cohorts, similar to population-based studies, reported low sensitivity for these criteria (average 50 %, range 20 to 89%), but high er specificity (a verage 87% , range 64 to 98%) (Galasko et aI., 1994; Victoroff et aI., 1995; Moroney et aI., 1997; Gold et aI., 1998). Due to their highly variable specificity and sensitivity (Wetterling et aI., 1996; Moroney et aI., 1997) , inconsistency in th e diagnosis of VID has been recognized (Bowler et aI., 1997; Erkinjuntti, 1999; Chui et aI., 2000; Ikeda et aI., 2001). Since the criteria chosen to diagnose VID will influence estimate s of its incidence and prevalence, as well as its recognition and treatment, new re search criteria, e.g. for subcortical vascular dementia, have been proposed (E rkinjuntt i et aI., 2000), and the need for prospective clinico-pathological correlation studies has been emphasized (Chui et aI., 2000; Knopman et aI., 2001). R ecent clinicopathological validation studies of four sets of clinical criteria for vascular dementia demonstrated that these are not interchangeable. The ADDTC criteria for possible IVD wer e found to be the most sensitive for the detection of IVD, while the DSM-IV criteria and the NINDS-AIREN criteria for possible IVD may be more effective in excluding mixed dementia. Giv en th eir inability to detect the vast majority of cases of IVD , th e ICD-IO criteria and the ADDTC and NINDS-AIREN criteria for probable IVD should be revised (Gold et aI., 2002; G ertz et aI., 2002). Furthermore, morphological substrates of dementia resulting from CVD remain confusing, since it constitutes a multifactorial disorder related to

4

K. A. Jellinger Table 3. Risk factors for VID

Advancing age Hypertension Diabetes mellitus Myocardial infarction Cardiac disease (especially atrial fibrillation) Elevated LDL cholesterol Homocystein Prior strokes

Cerebral atrophy Alcohol abuse Cigarette smoking Low educational attainment Asian background CADASIL Familial cerebral amyloidosis

a wide variety of pathological lesions (Table 2 and 3), the clinical significance of which and its relation to concurrent AD and other age-related changes of the brain, in particular, subcortical white matter lesions (WML) , remains controversial (Vinters et aI., 2000). Neuropathological criteria have not been established for VID. The California ADDTC (Chui et aI., 1992) and the NINDS-AIREN criteria (Roman et aI., 1993) did not suggest specific details for the neuropathological diagnosis of VID, but indicated that histopathological confirmation of the brain was necessary to confirm the presence of multiple infarcts. Complicating the diagnosis of VID are other pathologic entities coexisting with vascular lesions that could lead to cognitive decline. Many of these can be found by postmortem examination; thus, autopsy examination of the brain is critical in the definitive diagnosis of VID. However, it should be emphasized that the presence of vascular lesions found at autopsy does not prove they cause cognitive decline, underscoring that thorough clinico-pathological correlation is essential to establish a definitive diagnosis of VID. Epidemiology of VID

While VID previously was considered the second commonest type of dementia after AD (Fratiglioni et aI., 1991; Hebert and Brayne, 1995; Erkinjuntti, 1999; Meyer et aI., 2001; Dib, 2001), in the Western world it ranges after AD (60-75%), dementia with Lewy bodies (DLB) (10-20%) , other nonAlzheimer dementias (about 10%) at place 3 or 4. In most memory clinicbased series, CVD is considered to be the cause of dementia in no more than 8-10% of affected subjects (Vinters et aI., 2000). Evaluation of 11 pooled European population-based clinical studies of persons 65 years and older revealed an age-standardized prevalence of 6.4% for all causes of dementia, 4.4% for AD, and 1.6% for VaD (Lobo et aI., 2000). VID accounted for 15.8% of all dementia cases. The prevalence ofVID ranged from 0.0% to 0.8% at ages 65 to 69 and from 2% to 8.3% in the 90 and older age group in the different countries. In a Canadian clinical study of 603 demented patients, 12.1 % had VID and 12.6% had mixed AD/VID (Rockwood et aI., 2000). In Asian countries, VID may be more common than AD. Studies from Japan revealed that the prevalence of VID was more than double that of AD (Veda et aI., 1992; Yoshitake et aI., 1995). White et al.

Vascular-ischemic dementia: an update

5

(1996) studied demented Japanese-American men, aged 71-93, living in Hawaii and found that 34% had AD and 30% had VID. The prevalence of VID was similar to that found in Japan, but the rate of AD was higher than in Japan. In China, VID was reported to be 1.5 times that of AD (Li et al., 1991); however, a more recent study found 31% of dementia patients had VID and 61% had AD (Chui et al., 1998). According to recent studies, in Japan, almost half the patients appear to have VID with neuroradiological confirmation as compared to 35% AD and 17% of dementia resulting from other causes (Ikeda et al., 2001). Another Japanese study quoted a population prevalence of 3.8% of all typ es of dementia in the over 65 year old (2.1% AD, 1.0% VID, 0.7% other dementias), but did not include autopsy verification (Yamada et al., 2001). The prevalence of VID increases linearly with age and shows geographic variation, with age-standardized incidence ratios (SIR) ranging from 0.42 to 2.68 (Dubois and Hebert, 2001). Its incidence is estimated 6-12 cases/l,OOO persons over the age of70 years (Hebert and Brayne, 1995). Mean duration of VID is around 5 years and survival is less than for the general population and for AD. Vascular cognitive impairment (VCI) without dementia is the most prevalent form of cognitive impairment among people over the age of 65 years often progressing to VID (Wentzel et al., 2001). Their rates of institutionalization and mortality are significantly higher than those of pepole without cognitive impairment and are similar to that of patients with AD (Rockwood et al., 2000). The prevalence of VID in autopsy series ranges from 0.03% up to 58%, with reasonable values, based on recent diagnostic consensus criteria, between 4 and 10% (Table 2). A review of autopsy studies of patients with dementia from 1962 to 1995 from many different countries revealed an overall mean rate of 17.3% for VID (Markesbery, 1998). However, more recent reports show a prevalence rate of 2-9%. Recent studies may reflect a referral bias heavily weighted with AD subjects from clinics or hospitals where AD predominates. However, this was not the case in the Nun Study in which the authors found only three pure VID cases out of 118 demented subjects at autopsy (Snowdon and Markesbery, 1999). VfD was seen in 9.8% in a 12 years ' consecutive autopsy series (19892000) of demented aged individuals « age 55 years) mainly derived from two large centers (acute and chronic hospitals) in Vienna, but only in 1.3% of patients with the clinical diagnosis of possible or probable AD with a mean age of 81.3 ± 6.9 SD) years (Table 2). Alzheimer type pathology was seen in 82.5 and 92.6%, respectively, with "pure" AD in only 44 and 52%, respectively, and mixed type dementia (AD + VID) in 3.3 and 1.3%, respectively (Jellinger, 2001). For comparison, in recent autopsy series from two Japanese geriatric hospitals of demented subjects with an average age of 82 to 83.5 years, the incidence rates for AD, VID, mixed dementia, and other dementias, were 34, 35, 11, and 20%, respectively in one (Seno et al., 1999), and 46, 22, 6, and 26% (including 18% of Lewy body disease), respectively, in the other (Akatsu et al., 2001). In contrast, among 1,929 autopsy cases of demented subjects collected by 10 medical centers participating in the neuropathology program of the Consortium to Establish a Registry for Alzheimer's

6

K. A. J ellinger

Disease (CERAD), there were only 6 cases ( = 0.03%) in whom autopsy revealed only cerebral infarction without morphological features of AD or other neurodegenerative disorders. In 4 cases, dementia was clinically indistinguishable from AD except for a history of focal neurologic deficits , suggesting that "pure" VID is uncommon (Hulette et aI., 1997). Risk factors of VID

The risk factors for VID are shown in Table 3. Several recent reviews of the subject are available (Gorelick, 1997; Skoog, 1998; Meyer et aI., 2001). Many of the risk factors for VID are the same as for stroke. Age is an extremely important risk factor for VID. Its prevalence doubles every five to ten years after age 65 (Hofman et aI., 1991). Other non-modifiable risk factors include genetic predisposition and Asian background. However, modifiable risk factors for VID include hypertension, diabetes mellitus, atrial fibrillation, elevated cholesterol, alcohol abuse, and cigarette smoking. Independent predictors of poststroke dementia (PSD), observed in up to 28% within three years after stroke, are aging , preexisting cognitive decline related to CVD (around two-thirds) and AD (one-third), severity of the neurological deficit on admission, diabetes mellitus, silent infarcts, and WMLs/leukoaraiosis (Desmond et aI., 2000; Pohjasvaara et aI., 2000; Henon et aI., 2001), while dementia in subcortical ischemic-vascular disease correlates best with hippocampal and cortical atrophy (Fein et aI., 2000). A few examples of VID are transmitted on an inherited basis. Small numbers of patients with familial amyloid angiopathies develop dementia after multiple cerebral hemorrhages or infarcts. Patients with cerebral hemorrhage with amyloidosis have been described in Iceland (HCHWA-I) and the Netherlands (Dutch form) (HCHWA-D) . Both are dominantly inherited disorders and have amyloid deposited in cerebral arteries and arterioles. HCHWA-I causes intracerebral hemorrhages in younger individuals (20-30 years old). Cystatin C, a cysteine protease inhibitor, is present in blood vessel walls in HCHWA-I. A single mutation on the cystatin C gene on chromosome 20 has been demonstrated in this disorder (Abrahamson et aI., 1992). HCHWA-D affects normotensive patients in the age range of 40-60 years. The amyloid fibrils are similar to those found in AD. Tournier-Lasserve et a1. (1993) described an autosomal dominant disorder linked to chromosome 19, termed "cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy" (CADASIL), which is increasingly recognized as a cause of stroke and cognitive alterations in mid-adult life. The major clinical features are migraine headaches, subcortical strokes, psychiatric symptoms, and cognitive decline. Pathologically, there is a widespread arteriopathy in medium and small arteries that contain a granular osmiophilic material (GaM). Identification of GaM in skin , muscle, or nerve by electron microscopy allows a specific diagnosis of the disease. There are multiple subcortical infarcts, microbleeds, and leukoencephalopathy that show a different lesion pattern from sporadic arterio-

Vascular-ischemic dementia: an update

7

sclerotic encephalopathy (Auer et al., 2001; Oberstein et al., 2001), and are inversely correlated with disability and cognitive performance (Bruening et al., 2001) . CADASIL is due to a mutation in the Notch-3 gene, which encodes a transmembrane receptor protein, which is expressed in vascular smooth muscle cells (Joutel et al., 1996). A 210-kDa Notch-3 cleavage product is present at the cytoplasmic membrane of these cells (Joutel, 2000). Several kindreds with a similar condition but with autosomal recessive pattern of inheritance (CARASIL - cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy) and same unique clinical features (association with severe degenerative changes in the lumbar spine and other joints, alopecia) and usual absence of extracellular GOMs have been reported in Japan (see Yamagawa et al., 2002). Many studies have shown that the £4 allele of apolipoprotein E (apoE) is a major risk factor for AD ; however, studies of apoE in VID have yielded variable results. Although several recent studies have described an increased frequency of apofie-l in patients with dementia with stroke (Slooter et al., 1997; Hofman et aI., 1997; Hebert et al., 2000), others from different countries have not confirmed this (Traykov et al., 1999; Alafuzoff et al., 2000; Barba et al., 2000). Again, varying definitions and criteria and the potential overlap with AD in clinical studies make interpretation of these data difficult. Major neuropathologic substrates of VID

The morphologic types of dementia associated with CVD are summarized in Tables 4 and 5. In a simplified manner, the major morphologic lesions associated with VID are as follows: Table 4. Major morphological types of vascular dementia (modified from Garcia and Brown, 1992; Roman et al., 1993) 1. Classical multiinfarct encephalopathy (MIE) multiple large (sub /territorial) infarcts in cortex and white matter/basal ganglia in territories of large cerebral arteries, MCA, MCA + PCA; involving left or both hemispheres 2. Strategic infarct dementia (SID) Small or medium-sized infarcts/ischemic scars in functionally important brain regions: thalamus; hippocampus (PCA), ba sal forebrain (ACA), bilaterally or dominant hemispheres 3. Microangiopathic (small vessel infarct) dementia (SMV A) a) Subcortical arteriosclerotic leukoencephalopathy Binswanger (SAE) Multiple small infarcts in basal ganglia + white matter with preservation of cortex b) Multilacunar state Multiple microinfarcts (scars up to 1.5 ern 0) in basal ganglia, hemisphereal white matter, pontine basis Multiple cortico-subcortical microinfarctions (mixed encephalopathies) c) Granular cortical atrophy Multiple small scar s within border zones ACA MCA in one/both hemisph eres

8

K. A. J ellinger

Table 5. Dementia associated with cerebrovascular disease (Vinters et al., 2000) A. MultifocallDiffuse Disease: 1. Multiple atherosclerotic/watershed infarcts (large artery/borderzone territories) 2. Anti-PL-related ischemia 3. "Granular atrophy" of cortex (multifocal cortical microinfarcts scars) 4. Multiple lacunar infarcts (due to microvascular disease or microatheroma) 5. Binswanger subcortical leukoencephalopathy (BSLE) [? linked to #4] 6. CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) 7. Angiitis (PCNSA, granulomatous angiitis; some cases linked to CAA) 8. Cerebral amyloid angiopathy (CAA) +/- infarcts, hemorrhages (AD variant?) - Familial forms including Dutch, Icelandic, British 9. Miscellaneous angiopathies (FMD, Moyamoya) 10. Cortical laminar necrosis (post-cardiac arrest, hypotension) 11. Extreme dilatation/enlargement of brain parenchymal perivascular spaces B. Focal Disease/Strategically Placed Infarcts: 1. Mesial temporal (including hippocampal) infarcts/ischemia/sclerosis 2. Caudate and thalamic infarcts (especially DM nucleus, bilateral damage) 3. Fronto-cingulate infarcts (ACA territory) 4. Angular gyrus infarct (dominant cerebral hemisphere) Anti-Pl. anti-phospholipid; PCNSA primary angiitis/arteritis of the central nervous system; FMD fibromuscular dysplasia; ACA anterior cerebral artery; DM dorsomedial

1. Classical multiinfarct encephalopathy (MIE) following several thromboembolic insults with multiple infarcts in the cortex and white matter or basal ganglia in the territories or border zone areas of large cerebral arteries, mainly in the territories of the middle cerebral artery (MCA), less frequently of the other major cerebral arteries, involving the left or both hemispheres (Meyer et al., 2001). 2. Microangiopathic (small vessel infarct) dementia (SMVA) with multiple lacunar infarcts, measuring up to 1.5cm3 , located in the basal ganglia, hemispheral white matter, cerebral cortex or hippocampus. They are caused by microvascular disease related to hypertension (lipohyalinosis, hyaline arteriolosclerosis with microaneurysms, etc.), cerebral microembolism of vascular or cardiac origin such as atrial fibrillation, etc. (Garcia and Brown, 1992; Esiri et al., 1997; Vinters et al., 2000). According to their predominant location, the following types are distinguished: Strategic infarct dementia (SID) with small to medium-sized infarcts or ischemic scars in strategically important brain regions (thalamus, especially in the dorsomedial nucleus supplied by the thalamoperforant artery, and the mesial temporal area or hippocampus, both in the supply area of the posterior cerebral artery (PCA), in the basal forebrain (fronto-cingulate infarcts) in the territory of the anterior cerebral artery (ACA), bilaterally or in the dominant hemisphere. - Subcortical arteriosclerotic (leuko)encephalopathy Binswanger (SAE) with multiple small cystic or lacunar infarcts or scars in the basal ganglia and white matter, often associated with diffuse white matter

Vascular-ischemic dementia: an update

9

lesions, with preservation of the cerebral cortex and subcortical Ufibers. They are caused by hypertensive microangiopathy, reduced perfusion and disorders of the blood-brain barrier, CADASIL, and, rarely, by cerebral amyloid angiopathy (Gray et a1., 1985; Vinters et al., 2000). - Multilacunar state with multiple microinfarcts, lacunes or vascular scars in basal ganglia, hemispheral white matter and brainstem basis caused by either occlusion of deep perforating arteries in the basal ganglia, centrum semiovale, or brainstem due to lipohyalinosis or arteriosclerosis (Fisher, 1979; Vinters et al., 2000) or by a leak of blood or fluid into the perivascular space around small abnormal vessels (Wardlaw et al., 2001). - Mixed encephalopathies with multiple cortical and subcortical microinfarcts Granular cortical atrophy with multiple small cortical microinfarcts and scars within the border zones between the ACA and MCA in one or both hemispheres, usually caused by hypoperfusion related to stenosis of the internal carotid arteries or by cerebral embolism. 3. Postischemic encephalopathies - Cortical laminar necrosis: extensive damage to the cerebral cortex due to cardiac or respiratory arrest (hypotension, anesthesia accidents, etc.) - Multiple postischemic lesions in cortex, basal ganglia, and other brain areas. - Hippocampal sclerosis, with selective involvement of the hippocampus ranging from neuronal loss and glial scar formation to frank infarction. White matter lesions

More recent studies have placed emphasis on deep white matter lesions in VID. This has been catalyzed by CT and MRI studies showing alterations in deep white matter. The term "leukoaraiosis" has been used to describe the white matter rarefactions found on CT and MRI scan (Hachinski et al., 1987) . The true meaning of these lesions is controversial, and rarefactions in the white matter can be found in VID, AD, and in aged normal subjects. Merino and Hachinski (2000) summarized data about leukoaraiosis and concluded that it occurred most frequently in association with age , hypertension, lacunar and subcortical strokes, and carotid artery atherosclerosis. Several neuropathological alterations are associated with WML. One explanation are incomplete non-cavitary ischemic zones in deep white matter that are characterized microscopically by myelin, axon, and oligodendroglia loss , mild reactive astrocytosis, sparse macrophage reaction, and hyaline fibrosis of arterioles. Brun (1994) suggested that these result from hypotension and narrowing of arterioles. After chronic cerebral hypoperfusion in the rat, axonal damage and demyelination in the white matter have been observed indicating that the WM is more susceptible to chronic cerebral hypoperfusion than the gray matter (Wakita et al., 2002). A clinical-imagingneuropathological study of periventricular white matter hyperintensities in a

10

K. A. Jellinger

series of autopsied elderly female subjects demonstrated that they were not related to white matter volume, stroke, or dementia (Smith et aI., 2000). The pathogenesis and the cognitive correlates of cerebral WMLs, common in the aged brain, are controversial (De Groot et aI., 1998; Fazekas et aI., 1998; Inzitari et aI., 2000). However, recent community-based neuroimaging and comparative clinico-pathological studies indicate 1. that WML give some evidence for an association with known vascular disease risk factors and cerebral microangiopathy, but 2. that they are not closely related with cognitive decline (De Groot et aI., 1998; Schmidt et aI., 2000; Fernando et aI., 2001). Erkinjuntti (2000) suggested a subclassification of subcortical Vlf) in which lacunar infarcts and ischemic WMLs related to small vessel disease are the major pathologic features. Clinical findings include WMLs on imaging studies, a history of small strokes or multiple transient ischemic attacks, and mild or occasionally absent focal neurologic signs. Positron emission tomography studies demonstrate that subcortical vascular lesions can cause a reduction in cerebral cortical metabolic activity. Sultzer et aI. (1995) showed that periventricular and deep white matter hyperintensities and subcortical lacunar infarcts in patients with Vlf) caused a decline in mean glob al cortical metabolism. The reduction in metabolic rate was lower in patients with lacunar infarcts in the basal ganglia or thalamus. Lower cognitive function tests correlated with the extent of the white matter lesions. Kwan et aI. (1999) found that cortical global metabolic rates were lower in patients with subcortical strokes and cognitive impairment or dementia compared with patients with subcortical stroke and no cognitive impairment. The right frontal cortex showed a lower metabolic rate in all stroke groups. Subtle decline in executive functioning and visual memory, even in non-demented patients. The pattern of cognitive impairment after subcortical lacunes is consistent with models of subcortical-frontal circuites (Kramer et aI., 2002). The entity entitled "subcortical arteriosclerotic encephalopathy, type Binswanger" (Pantonie and Garcia, 1995) is controversial and poorly understood. Importance of small vascular lesions for VID

Infarcts from small vessel occlusion or lacunar infarcts are most often found in cerebral white matter, basal ganglia, thalamus, pons, and cerebellar white matter. Lacunar infarcts are small cavitary lesions «1.5cm in diameter) most often caused by occlusion in small arteries or arterioles and are associated with hypertension or diabetes mellitus. Some may be associated with hemorrhage. Yoshitake et aI. (1995) found multiple lacunar infarcts in 42 % of Japanese patients with dementia. The prevalence of cognitive decline in those with multiple lacunar infarcts is variable and probably dependent on multiple factors including widespread diffuse cerebrovascular disease, cognitive reserve, and the presence of large infarcts. The idea that the diagnosis of multi-infarct dementia (MID) requires a brain tissu e loss exceeding 100ml is a persistent component of neuro-

Vascular-ischemic dementia: an update

11

mythology. In fact, Tomlinson et al. (1970) showed that although all patients with losses of over 100ml of brain tissue were demented, many demented patients had infarcts totalling less than 100 ml. Those totalling more than 20 ml were significantly more frequent in demented subjects than in controls, and marked difference between the two groups was present at 50ml cut-off. In other words, a relative small aggregate volume of infarcts mayor may not contribute to dementia, probably depending on its location, while destruction of a large volume of cortex may be necessarily followed by dementia. In contrast to Tomlinson et al. (1970) who compared the morphologic findings in un selected demented and non-demented patients, Del Ser et al. (1990) studied a group of 40 patients who showed only vascular lesions on histological examination with senile plaque counts below the level necessary for the diagnosis of AD, and compared demented with non-demented ones. They found that the volume of the infarcts was significantly greater in the dementia group, but there was marked overlap between the groups, and the essential volume of infarcted tissue did not reach 100ml in most demented patients. These data were confirmed by more recent studies who found only a nonsignificant trend for lobar infarcts to occupy more cerebral hemisphere volume in VID than in nondemented stroke patients (Esiri et al., 1997; Vinters et aI., 2000). The location of vascular lesions causing VID appears to be more important than the size of the lesion. Multiple brain regions have been implicated in VID, including dominant angular gyrus , anterior or posterior cerebral artery territory, superior middle cerebral artery territory, left anterior corona radiata, bilateral medial thalamus, dominant caudate nucleus, anterior internal capsule, hippocampus, amygdala, and basal forebrain (Markesbery, 1998). Imaging studies demonstrated contradictory results regarding stroke location and VID (Bowler and Hachinski, 1995; Erkinjuntti et al., 1999). More recently, Pohjasvaara et al. (2000) suggested that poststroke dementia is not caused by a single factor but by a combination of factors including volume of infarct, right or left superior middle cerebral artery infarcts, left thalamic-cortical connection infarcts, frequency of left hemispheric infarcts, extent of white matter lesions, medial temporal lobe atrophy, and level of education of the subject. Comparing the neuropathological findings in 18 elderly, nondemented subjects with 19 elderly nondemented subjects who had CVD, many of them with a "stroke", and 24 elderly subjects who had CVD but no other pathology to account for dementia, all of them with no or only very mild Alzheimer type pathology, Esiri et al. (1997) reported the following findings: Microvascular brain damage in the form of severe cribriform or lacunar change and associated white matter damage and microinfarction were correlated with a history of dementia. Severe lacunar state was much more common and microinfarct somewhat more common in the demented group with CVD than in the nondemented group with CVD (p = 0.0006 and p = 0.03, respectively). Other findings of note were that CAA had a greater prevalence in the group with dementia and CVD (p = 0.0028), and the last group lacked evidence of macroscopic infarction more often than the first (p = 0.034). There was a nonsignificant trend for the ratio of infarcted vs noninfarcted tissue in one cere-

12

K. A. Jellinger

Table 6. Macroscopic cerebral infarction in a single cerebral hemisphere in cerebrovascular disease (CVD): prevalence of microscopic vascu lar changes (Esiri et a l., 1997) Type of cerebral lesion

Un demented (UCVD) n ( %)

% cerebral hemisphere infarcted Single cerebral infarct Multiple cerebral infarcts No macroscopic cerebral infarct Microscopic vascular changes: Cribriform changes Severe cribriform change Microinfarction Co ngophilic angiopathy (CAA)

D emented (DCVD) n ( %)

1.93 11 00

1.48 3 8

4 4

12 3 5 4

13# (63) (16) (26) (21)

22 7 15 8

(92) * (71) ** (63)* ** (33)0

* P = 0.03; ** P = O.OOO?; *** P = 0.0031; 0 P = 0.0067 vs nil congophil angiopathy (controls); oop = 0.014, more prevalent than in D CVD ; "P = 0.034, more prevalent than in UCVD

Table 7. Su mmary of types of brain parenchymal injury in vascular ischemic dementia (Vinters et al., 2000) N of Pat.

Category Demented -

1/

-

Cognitively impaired Cog. normal

8 5 1 4 2

Mean age (year) 80.9 81.2 76 76.8 77.5

(5M/3F) (3M/2F) (M) (3M/IF) (1M/IF)

N with cystic infarcts

Lacune

Microinfarcts

Hippocampal injury

1 2 0 1 1

1 5 0 4 2

4 4 0 2 2

4 2 1 3 1

bral hemisphere to be higher in the dementia + CVD group than in the CVD group without dementia (Table 6) . T he circle of Willis athero ma was not more severe in the CVD dementia group than in the undemented stroke or control subjects, while the dementia groups often showe d the arteriolosclerotic and hypertensive form of small vessel disease. In their recent review on neuropathological substrates of VID, Vinters et al. (2000), based on autopsy findings in 20 patients (age range 68-92 years), also emphasized the correlation of this uncommon entity with widespread small ischemic lesions distributed throughout the CNS. They observed cystic infarcts over 1 em in diameter in four, lacunes an d microinfarcts in 12 brains each, an d hippocampal injury in 11 cases. Many brains showed more than one type if ischemic lesion, most of them being associated with severe atherosclerosis and arteriolosclerosis, while CAA was seen in one single brain with severe A D . However, in two cognitively normal controls, simi lar multiple ischemic cerebral lesions were seen (Table 7) . These data can be largely confirmed in a personal series of 55 patients with moderate to severe dementia (Mini-Mental State/MMSE/ < 20/30) and four cognitively impaired (MMSE > 20/30) plus three cognitively normal controls

Vascular-ischemic dementia: an update

13

Table 8. Summary of types of brain injury in vascular-ischemic encephalopathy

Category Dementia Cognitively impaired Cognitively normal Total two one 10 mm)

Rating Scale WML and cut-off for severe lesions

n.s. not specified; * Rotterdam Study (Br eteler et al., 1994); t Lund Stud y (Lindgren et al., 1994); :j: Helsinki Ageing Stud y (Ylikoski et al., 1995); § Cardiovascular Health Study (Bryan et al., 1997; Longstreth et al., 1996, 1998); II Atherosclerosis Risk Tn Communities Stud y (Bryan et al., 1999; Liao et al., 1996); !j[ Austrian Stroke Prevent ion Study (Schmidt et al., 1996, 1997); **Rotterdam Scan study (de Leeuw et al., 2001; Verm eer et al., 2001); tt Preval ence of periventricular (PV) and subcortical (SC) WML, respectively; :j::j: PV and SC WML rated separately on a 3 point scale (cut-off for PV lesions extending into the SC white matter, and for SC WML confluent and early confluent lesions)

10

n.s.

Severe (%)

RS *

Study

Any (%)

WML

Table 2. Prevalence of WML and silent lacunar infarcts in population-based studies with MR imaging

e:..

e-e

(l>

~

£

e

III

10mm) in persons with lacunar infarcts compared to those with non-lacunar infarcts (53 versus 29%) . They also show higher prevalences of moderate to severe periventricular WML (diameter lesions >5mm) in persons with border-zone infarcts compared to those with non borderzone infarcts (Mantyla et al., 1999b). However others find no difference in prevalence or severity of WML on MRI in infarct subtypes (Schmidt et al., 1992). In patients with primary intracerebral hematoma the prevalence of early confluent and confluent WML on MRI is 45% and of silent lacunar infarcts is 51% (Offenbacher et al., 1996). Severe WML and silent lacunar infarcts were more frequent in patients with deep (basal ganglionic and thalamic) primary intracerebral hematoma compared to hemorrhages involving the lobar regions (Inzitari et al., 1990; Offenbacher et al., 1996). In patients with clinical manifest lacunar infarction, the presence of multiple small lacunar infarcts was associated with WML (Boiten et al., 1993). Progression of WML and small lacunar infarcts was associated with symptomatic lacunar stroke compared to territorial stroke at study entry (van Zagten et al., 1996). Incidence in population based studies

Very little data are available on incidence or progression of lesions over time. The Austrian Stroke Prevention Study found progression within 3 years in 17.9% of the 273 participants, of which 9.9% was minor (less than 4 new punctate lesions) and 8.1% was marked (more than 4 punctate lesions or a transition to early confluent or confluent lesions). Age was not associated with lesion progression (Schmidt et al., 1999). The main determinant of progression was the lesion load at baseline. A longitudinal study on gait disturbances and WML found a significant overall increase of WML volume from 3.1 to 4.2ml in a mean follow-up of 4 years. The periventricular WML showed the most marked increase (Whitman et al., 2001). Progression may be underestimated as a result of selective survival of the less severely affected (Briley et al., 2000; Inzitari et al., 1997; Miyao et al., 1992; Tarvonen-Schroder et al., 1995). In the Austrian Stroke Prevention Study this selection effect will be even larger since people with an incident stroke, which was an end point in that study, were excluded. Summary and conclusions

Reported prevalence of white matter lesions and silent lacunar infarcts varies largely among studies. Differences in study design and assessment of lesions may account for the large variation in the frequencies reported. Any WML are seen in almost all persons over the age of 70 years and studied with modern MRI techniques. Severe WML are seen in 10 to 33% and

36

E. J. van Dijk et al.

silent lacunar infarcts in 6 to 20%. Higher age is associated with higher prevalences of any WML, severe WML and silent lacunar infarcts. Women and Afro-Americans tend to have more severe WML and silent lacunar infarcts in some studies. Because of heterogeneity in dementia and stroke populations it is difficult to compare frequencies across populations. WML are more often seen in dementia, especially in vascular dementia but also in Alzheimer dementia and Lewy body dementia. WML and silent lacunar infarcts are associated with clinical manifest lacunar infarcts and deep hemorrhagic stroke. Harmonisation of methodological issues and assessment of lesions is needed for future studies on small vessel disease related cerebral lesions. Also there is a need for more data on incidence and progression of lesions in both population-based studies and patient series. Acknowledgements The Netherlands Organisation for Scientific Research (grant 904-61-096) and the Netherlands Heart Foundation (grant 97.152) supported this study. M. Breteler is a fellow of the Royal Netherlands Academy of Arts and Sciences.

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lations with plasma concentrations of naturally occurring antioxidants. Stroke 27: 2043-2047 Schmidt R , Fazekas F, Hayn M, Schmidt H, Kapeller P, Roob G, et al. (1997) Risk factors for microangiopathy-related cerebral damage in the Austrian stroke prevention study. J Neurol Sci 152: 15-21 Schmidt R, Fazekas F, Kapeller P, Schmidt H , Hartung HP (1999) MRI white matter hyperintensities: three-year follow-up of the Austrian Stroke Prevention Study. Neurology 53: 132-139 Shimada K, Kawamoto A , Matsubayashi K , Ozawa T (1990) Silent cerebrovascular disease in the elderly. Correlation with ambulatory pressure. Hypertension 16: 692699 Tarvonen-Schroder S, Kurki T , Raiha I, Sourander L (1995) Leukoaraiosis and cause of death: a five year follow up. J Neurol Neurosurg Psychiatry 58: 586-589 van Swieten JC, van den Hout JH, van Ketel BA, Hijdra A, Wokke JH, van Gijn J (1991) Periventricular lesions in the white matter on magnetic resonance imaging in the elde rly. A morphometric correlation with arteriolosclerosis and dilated perivascular spaces. Brain 114: 761-774 van Swieten JC, Kappelle LJ , Algra A, van Latum JC, Koudstaal PJ, van Gijn J (1992) Hypodensity of the cerebral white matter in patients with transient ischemic attack or minor stroke: influence on the rate of subsequent stroke. Dutch TIA Trial Study Group. Ann Neurol32: 177-183 van Zagten M, Boiten J, Kessels F , Lodder J (1996) Significant progression of white matter lesions and small deep (lacunar) infarcts in patients with stroke. Arch Neurol 53: 650-655 Vermeer SE , Koudstaal PJ , Oudkerk M, Hofman A, Breteler MM (2002) Prevalence and risk factors of silent brain infarcts in the population-based Rotterdam Scan Study. Stroke 33: 21-25 Wahlund La, Barkhof F, Fazekas F , Bronge L, Augustin M, Sjogren M et al. (2001) A new rating scale for age -related white matter changes applicable to MRI and CT. Stroke 32: 1318-1322 Waldemar G , Christiansen P, Larsson HB , Hogh P, Laursen H, Lassen NA et al. (1994) White matter magnetic resonance hyperintensities in dementia of the Alzheimer type: morphological and regional cerebral blood flow correlates. J Neurol Neurosurg Psychiatry 57: 1458-1465 Whitman GT, Tang T , Lin A , Baloh RW (2001) A prospective study of cerebral white matter abnormalities in older people with gait dysfunction. Neurology 57: 990-994 Wiszniewska M, Devuyst G, Bogousslavsky J, Ghika J, van Melle G (2000) What is the significance of leukoaraiosis in patients with acute ischemic stroke? Arch Neurol57: 967-973 Ylikoski A, Erkinjuntti T, Raininko R, Sarna S, Sulkava R , Tilvis R (1995) White matter hyperintensities on MRI in the neurologically nondiseased elderly. Analysis of cohorts of consecutive subjects aged 55 to 85 years living at home. Stroke 26: 11711177 Authors' address: M. M. B. Breteler, MD, PhD, Department of Epidemiology and Biostatistics, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands, e-mail: [email protected]

CT and MRI rating of white matter changes P. Kapeller l ,2, R. Schmidtl-, Ch. Enzlnger", S. Ropele2, and F. Fazekas'< 1 Deptartment of Neurology, and 2MR I Centre, Karl Franzens University, Graz, Austria

Summary. The impact of white matter changes (WMC) detectable on CT or MRI on various diseases like ischemic stroke and intracerebral hemorrhage and their association with cognitive impairment was and still is under debate. To assess WMC in a qualitative and/or semiquantitative fashion rating scales have been developed. For MRI most widely used scales are the scales of Manolio, Fazekas, Schmidt, and Scheltens. Most recently a new scale extending earlier suggestions has been introduced by Wahlund et al. applicable for both CT and MRI. This article will review strengths and weaknesses of these rating scales and will discuss further requirements and future perspectives.

Introduction The detection of cerebral white matter changes (WMC) on computed tomography (CT) or magnetic resonance imaging (MRI) led to considerable discussion concerning the clinical relevance of these abnormalities. Their possible association with ischemic stroke (Mantyla et aI., 1999), intra cerebral hemorrhage (ICH) (Offenbacher et aI., 1996) or dementia (Fazekas et aI., 1994) put their presence, pattern and extent in the centre of interest of many research groups. One way to further improve our knowledge concerning WMC is to enlarge the number of investigated individuals, i.e. to pool data for increasing statistical power. Collaboration between different sites is needed to reach this goal and has been established. However, common form of evaluating WMC either by CT or MRI is also crucial. This includes both data generation which may be accomplished with different hard and soft ware, e.g. due to different scanners and scanning parameters and data analyses. Visual rating scales for WMC may overcome some of these problems as they are applicable to different settings or image quality and they have few "technical" requirements. However, available scales are highly heterogeneous and few of them have been tested regarding interrater agreement. Moreover, no data are available concerning the correlation between different scales. One of the goals of the "European Task Force on Age Related WMC" has been to provide more insight into the performance of the various scales in use

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(Scheltens et al., 1998) . Most recently Wahlund et al. (2001) introduced a new rating scale for CT and MRI. Furthermore, in a comparison of scales it was attempted to address differences in reliability and distribution of abnormal ratings which may account for either a low sensitivity or ceiling effects to potentially enable a comparison of data obtained with both modalities. Work in this concern is currently in progress. Some widely used scales

The scales of Fazekas and Schmidt (Fazekas et al., 1987; Schmidt et al., 1992), Scheltens et al. (1993) , and Manolio et al, (1994) are frequently used scales which are tested for their inter and intrarater reliability. While the scales of Fazekas, Schmidt and Scheltens are very similar, the scale of Manolio is a somehow different approach to evaluate WMC using a template image and a text description to rate WMC and PVC as one entity on a 0-9 point scale in a single slice. The scales of Fazekas et al. (1987) and Schmidt et al. (1992) separately account for PVC and WMC on a 0-3 point scale. For PVC 0 = no lesion, 1 = caps or pencil thin lining, 2 = smooth halo and 3 = irregular extending into deep white matter. For WMC 0 = no lesion, 1 = punctuate foci, 2 = beginning confluence of foci and 3 = large confluent areas. Further more the scale accounts for the number of WMC quoted 0 = no lesion, 1 = 1-4 lesions, 2 = 5-9 lesions and 3 = more than 9 lesions. In an additional study the authors provide histopathological correlates which underline the importance of a separate assessment of WMC and PVC (Fazekas et al., 1991, 1993). The Scheltens scale (Scheltens et al., 1993) also accounts separately for WMC and PVC but the assessment is even more detailed. This scale additionally accounts for the anatomical distribution of lesions using a 0-6 point scale. For PVC 0 = absent, 1 = 5 mm and CT

CT > MRI

29% 31% 22% 23 %

16% 12% 22% 3%

MRI

= CT

56% 57% 56% 76%

p-value n.s. < 0.02 n.s. < 0.01

MRI magnetic resonance imaging, CT computed tomography, p -value level of significance

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Table 3. Interrater agreement by means of Kappa statistics for the scale of Wahlund et al. (2001) . As expected the agr eement concerning infra tentorial lesions was low for CT Location frontal parieto-occiptal temporal basal ganglia infratentorial MRI magnetic tomography

MRI

CT

0.8560 0.8749 0.6033 0.6535 0.5826

0.7723 0.7155 0.5103 0.4707 0.1845

resonance

imaging,

CT computed

allowed to obtain higher agreement than that of CT scans. Agreement was best for supratentorial lesions, infratentorial changes reached fair to good agreement in case of MRI but virtually no agreement was to obtain for CT (Table 3). Conclusions

The use of different scan parameters and scanners with different hard and software still causes considerable differences in image quality between sites. Therefore the use of absolute quantitative measurements for measuring and comparing WMC severity is currently still difficult and time consuming at least between different centres. Visual rating scales may overcome many of these technical diversities and are in addition easy methods to evaluate the presence, extent and distribution of WMC. However, several limitations should be taken into account when using visual rating scales. The greatest disadvantage is their non-linearity. Moreover, the criteria for rating WMC should be more specifically defined. Otherwise rating scales invite to subjective interpretation which may influence inter- as well as intra-rater reliability (Mantyla et al., 1999). This may especially be difficult for follow up investigations. Another problem may be the low specificity of WMC on the conventional MR image. Hyperintense signal on T2 weighted scans may result from various types of histopathology. Therefore WMC of different aetiology and with different functional consequences can not be adequately discerned and may be analysed together, i.e. as one entity. Centres participating in multicentre comparisons should stick with a few scales fulfilling certain criteria: the scales should provide information on the presence, extent and distribution of WMC and they should be tested in regards to their interrater reliability. In addition it would be preferential to know about their correlation with quantitative measurements and to know their reliability in evaluating the evolution of WMC over time. Information on the latter is currently scarce (Schmidt et al., 1999) or not available at all. Moreover, some general agreements concerning the definition and nomencla-

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ture of WMC are needed and more information on their "normal distribution" would be desirable. Work addressing some of these requirements is currently in progress. References Erkinjuntti T, Ketonen L, Sulkava Ret al. (1987) Do white matter changes on MRI and CT differentiate vascular dementia from Alzheimer's disease? J Neurol Neurosurg Psychiatry 50: 37-42 Fazekas F, Kleinert R, Offenbacher H et al. (1991) The morphologic correlate ofincidental punctate white matter hyperintensities on MR images. AJNR Am J Neuroradiol 12: 915-921 Fazekas F , Chawluk J, Alavi A, Hurtig H, Zimmerman R (1992) MRI signal abnormalities at 1.5T in Alzheimer's dementia and normal aging. AJNR Am J Neuroradiol8: 421-426 Fazekas F, Kleinert R, Offenbacher H et al. (1993) Pathologic correlates of incidental MRI white matter signal hyperintensities. Neurology 43: 1683-1689 Fazekas F, Schmidt R, Fazekas G, Kapeller P (1994) The relevance of white matter changes to vascular dementia. In: Leys D, Scheltens P (eds) Vascular dementia. Current issues in neurodegenerative diseases, vol 6. ICG Publications, Dordrecht, 133-154 Manolio T, Kronmal R, Burke G et al. (1994) Magnetic resonance abnormalities and cardiovascular disease in older adults. The Cardiovascular Health Study. Stroke 25: 318-327 Mantyla R , Aronen H, Salonen 0 et al. (1999) Magnetic resonance imaging white matter hyperintensities and mechanisms of stroke. Stroke 30: 2053-2058 Offenbacher H , Fazekas F, Schmidt R, Koch M, Fazekas G , Kapeller P (1996) MR of cerebral abnormalities concomitant with primary intracerebral hematomas. AJNR Am J Neuroradiol17: 573-578 Scheltens P, Barkhof F, Leys D et al. (1993) A semiquantitative rating scale for the assessment of signal hyperintensities on magnetic resonance imaging. J Neurol Sci 114: 7-12 Scheltens P, Barkhof F, Leys D, Wolters E , Ravid R, Kamphorst W (1995) Histopathologic correlates of white matter changes on MRI in Alzheimer's disease and normal aging. Neurology 45: 883-888 Scheltens P, Erkinjunti T, Leys D et al. (1998) White matter changes on CT and MRI: an overview of visual rating scales. Eur Neurol 39: 80-89 Schmidt R, Fazekas F, Kleinert G et al. (1992) Magnetic resonance imaging signal hyperintensities in the deep and subcortical white matter. A comparative study between stroke patients and normal volunteers. Arch Neural 49: 825-827 Schmidt R, Fazekas F, Kapeller P, Schmidt H, Hartung H (1999) MRI white matter hyperintensities: three-year follow-up of the Austrian Stroke Prevention Study. Neurology 53: 132-139 Van Swieten J, Hijdra A, Koudstaal P, van Gijn J (1990) Grading white matter lesions on CT and MRI: a simple scale. J Neural Neurosurg Psychiatry 53: 1080-1083 Wahlund L, Barkhof F, Fazekas F et al. (2001) A new rating scale for age-related white matter changes applicable to MRI and CT. Stroke 32: 1318-1322 Authors ' address: P. Kapeller, MD, Department of Neurology and MRI Centre, Karl Franzens University Graz, Auenbruggerplatz 22, A-8036 Graz, Austria, e-mail: [email protected]

Risk factors and progression of small vessel disease-related cerebral abnormalities R. Schmidt', F. Fazekas' >, C. Enzinger", S. Ropele-, P. Kapeller' >, and H. Schmidt' D epartment of Neurology, 2MR Centre, and 3 Institute of Medical Biochemistry and Medical Molecular Biology, Karl -Franzens University, Graz, Austria 1

A three year follow-up of 273 participants (mean age 60 years) of the Austrian Stroke Prevention Study provides first information on the rate and clinical predictors of progression of small vessel disease related cerebral abnormalities including white matter changes and lacunes. White matter hyperintensity progression was found in 17.9% of individuals over the 3 year period. New lacunes occurred in 2.2% of subjects. The overall frequency of progression of small vessel disease related brain changes was 19%. Diastolic blood pressure and early conflu ent or confluent white matter hyperintensities at baseline predicted lesion progression. Genetic association studies in the setting of the Austrian Stroke Prevention Study described that polymorphisms in the renin angiotensin system (RAS) increase the susceptibility for progression of cerebral small vessel disease. Homozygosity for the T allele of the M235T polymorphism of the angiotensinogen gene was associated with a 3.19-fold increased risk for lesion progression ind ependently of arterial hypertension. These data suggest that drugs influencing the RAS system may allow to intervene with an unfavorable course of cerebral small vessel disease.

Summary.

Introduction

Small vessel disease related brain lesions including white matter hyperintensities (WMH) and lacunes are commonly observed on MRI scans of elderly individuals (Pantoni and Garcia, 1995). It has been suggested that these abnormalities progress gradually over time with the accumulation of vascular risk factors, and ultimately may result in extensive subcortical arteriosclerotic encephalopathy with concomitant cognitive decline but also with gait disturbances and falls (Awad et al., 1986). Yet, only one systematic study on the actual frequency of progression of such abnormalities has been performed so far (Schmidt et al., 1999). Very little is known about risk factors for and the clinical consequences of lesion progression. Longitudinal data on the

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natural course of cerebral small vessel disease are needed as they will allow to better judge the clinical importance of this type of brain damage. Associations of progression with specific risk factors will probably enable to identify high risk groups as targets for therapeutical intervention. Frequency of progression of small vessel disease related brain abnormalities

To the present the Austrian Stroke Prevention Study is the only investigation that sytematically explored the frequency and risk factors of progression of cerebral small vessel disease. A detailed description of the sampling procedure, selection criteria and the diagnostic work-up is given elsewhere (Schmidt et al., 1999).The follow-up cohort of this study consisted of 273 study participants who underwent MRI scanning at baseline and after 3 years. The baseline and follow-up scans of each study participant were read independently by three experienced investigators blinded to the clinical data of study participants. WMH were specified and graded according to our scheme into absent (grade 0), punctate (grade 1), early confluent (grade 2), and confluent (grade 3) abnormalities (Fazekas et al., 1988). The number of WMH was recorded and categorized into 0, 1-4, 5-9, and >9 lesions. The change in number was again categorized into 0, 1-4, 5-9 and > 9 lesions. Progression of WMH was then graded as absent, minor or marked. A change from baseline by one to four punctate lesions was defined as minor. If there was a difference of more than four lesions, or a transition to early confluent or confluent WMH, the change was considered to be marked. Lacunes were considered to represent lesions isointense to cerebrospinal fluid on T2- and T1-weighted MRI sequences with a maximal diameter of 10mm involving the basal ganglia, the internal capsule, the thalamus, brain stem or the centrum semiovale. Final rating of lesion progression relied on majority judgement of the three assessors. In case of complete disagreement consensus was found in a joint reading session. The kappa-values of interrater agreement for progression of small vessel disease related brain abnormalities ranged from 0.61 to 0.69. Table 1 shows the frequency and extent of progression of lesions at followup as indicated by the three raters and the combined judgement of raters. As can be seen from this table, at the 3-year follow-up examination lesion progression was noted in 19% of subjects, 17.9% of individuals demonstrated progression of WMH, and 2.2% had newly occurring lacunes. Progression of small vessel disease related cerebral abnormalities: conventional risk factors and baseline MRI findings

Individuals with progressing lesions were older, had a higher systolic and diastolic blood pressure, and demonstrated higher grades and numbers of WMH at the baseline MRI. When simultaneously entering these variables and gender into a logistic regression model, diastolic blood pressure and

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49

Table 1. Frequency and degree of progression of small vessel disease-related brain abnormalities in the Austrian Stroke Prevention Study after three years of follow -up Raterl WMH-progression * Minor Marked Total Lacunes progression Overall progression

41 19 60 7 63

(15.0%) (7.0%) (22.0%) (2.6%) (23.1%)

Rater 2 33 (12.1%) 24 (8.8%) 57 (20.9%) 9 (3.3%) 61 (22.3%)

Rater 3 27 21 48 6 52

(9.9%) (7.7%) (17.6%) (2.2%) (19.0%)

Combined 27 22 49 6 52

(9.9%) (8.1%) (17.9%) (2.2%) (19.0%)

* A change from baseline by one to four punctate lesions was defined as minor. A difference of more than four lesions, or a transition to early confluent or confluent WMH was considered to be marked evidence of confluent WMH at baseline remained the only significant and independent predictors of lesion progression. Progression of small vessel disease-related cerebral abnormalities: genetic association studies

A significant contribution of genetic influences in the etiology of WMH has only recently been demonstrated in an US study on World War II veteran twins (Carmelli et al., 1998). This investigation reported a probandwise concordance rate for severe WMH of 61% in monozygotic, and of 38% in dizygotic twins , compared with a prevalence rate of 15% for the entire population. The estimated heritability of WMH volume was 73% . Based on these data small vessel disease of the brain can be conceptualized as a multifactorial disorder with both genetic and environmental factors influencing its presence and severity. Search for genetic susceptibility factors by association studies is promising, because the well defined phenotype of small vessel disease related cerebral abnormalities will probably result in less heterogeneity than can be expected when studying other forms of cerebral ischemic damage. Association studies are most powerful when they examine functional polymorphisms in candidate genes. In the Austrian Stroke Prevention Study we developed a model to identify candidate genes based on possible pathways leading to cerebral small vessel disease and it's morphological consequences (Schmidt et al., 2000). Conventional risk factors indicated by epidemiological studies provide a helpful hint for the selection of good candidate genes. Genes involved in blood pressure regulation are good choices because arterial hypertension is the most consistent risk factor for small vessel disease related brain damage in crosssectional studies (Pantoni and Garcia, 1995). Consequently, genes coding for the renin-angiotensin system (RAS) were our primary targets. A previous study by Markus et al. (1995) showed an association between the liD ACE polymorphism and lacunar stroke. In the Austrian Stroke Prevention Study

R. Schmidt et al.

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Ba seline

Follow up

Fig. 1. Bas eline and 3 year follow-up MRI study in a participant of the Austrian Stroke Prevention Study. Corresponding images at high to supraventricular levels are displayed. At each level marked progression of white matter hyperintensities (arrows) was noted

Table 2. Logistic regression model of risk factors for progression of small vessel disease rel at ed brain abnorm aliti es Variable TT genotype Hypertension Antihypert ensive therapy Age (years) Sex (male) Diabetes Cardiac disease Fibrinogen (mg/dl)

~

SE

Df

P

OR

95% CI

1.16 1.12 - 0.51 0.04 -0.14 -0.37 0.13 -0.002

0.37 0.50 0.55 0.03 0.34 0.72 0.36 0.002

1 1 1 1 1 1 1 1

0.002 0.03 0.35 0.22 0.68 0.60 0.72 0.43

3.19 3.06 0.60 1.04 0.87 0.69 1.14 1.002

1.54; 6.63 1.15; 8.20 0.21; 1.75 0.98; 1.10 0.45; 1.68 0.17; 2.82 0.56; 2.29 0.99; 1.01

we reported that genetic variations at the angiotensinogen gene locus are strongly associated with small vessel disease-related brain lesions (Schmidt et al., 2001). Associations with progression of small vessel disease-related brain abnormalities were noted for the angiotensinogen M235T pol ymorphism. As can be seen from Table 2 homozygosity for the T allele relat ed to a 3.19 fold increased likelihood for lesion progression th an the other genotypes . Th e mechanism(s) responsible for the effect(s ) of the angiotensinogen T235

Progression of cerebral small vessel disease

51

variant on progression of small vessel disease-related cerebral abnormalities remains speculative at this time. It is clear from our data that elevated blood pressure can only partly explain this association because homozygosity for the T allele predicted lesion progression independently of arterial hypertension. One explanation for the relationship is that the T235 variant represents merely a marker in linkage disequilibrium with a close by etiologically important polymorphism. Conceivably, this could be mutations in the promoter region of the angiotensinogen gene which were seen to be in tight linkage disequilibrium with T235 and caused elevated angiotensinogen expression. Mutations in the promoter regions were studied in the Austrian Stroke Prevention Study and the results are reported in a second article of this supplement. Angiotensinogen is the precursor peptide of the vasoactive hormone angiotensin II which has multiple pro-atherogenic effects including induction of smooth muscle cell hypertrophy, stimulation of vascular fibrosis , plasminogen activator-inhibitor-1 stimulation, free radical formation and increased endothelin secretion (Burnier and Brunner, 2000). Most importantly, in the context of our results, there exists an independent RAS in the brain that might contribute to or amplify cerebral small vessel disease, an effect that might not be reflected in the systemic circulation (Brunnemann et al., 1992). Our data suggest that components of the RAS playa role in the etiology of arteriolosclerosis independently of their effects on blood pressure. Consequently, intervention in the RAS could exert beneficial effects on the evolution of small vessel disease related brain damage and its clinical consequences beyond what can be expected from the lowering of blood pressure alone. Acknowledgement The genetic studies of the Austrian Stroke Prevention Study are supported by Austrian Science Fund, project P13180-MED

References Awad lA, Johnson PC, Spetzler RF, Hodak JA (1986) Incidental subcortical lesions identified on magnetic resonance imaging in the elderly. II. Postmortem pathological correlations. Stroke 17: 1090-1097 Brunnemann B, Fuxe K, Ganten D (1992) The brain renin-angiotensin system: localization and general significance. J Cardiovasc Pharmacol 19 [Suppl 6]: S51-S62 Burnier M, Brunner HR (2000) Angiotensin II receptor antagonists. Lancet 355: 637-645 Carmelli D, DeCarli C, Swan G, Jack LM, Reed T, Wolf PA, Miller BL (1998) Evidence for genetic variance in white matter hyperintensity volume in normal elderly male twins. Stroke 29: 1177-1181 Fazekas F , Niederkorn K, Schmidt R, Offenbacher H , Horner S, Bertha G , Lechner H (1988) White matter signal abnormalities in normal individuals: correlation with carotid ultrasonography, cerebral blood flow measurements, and cerebrovascular risk factors. Stroke 19: 1285-1288 Markus HS, Barley J, Lunt R, Bland 1M , Jeffery S, Carter ND, Brown MM (1995) A ngiotensin-converting enzyme gene deletion polymorphism. A new risk factor for lacunar stroke but not carotid atheroma. Stroke 26: 1329-1333

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Pantoni L, Garcia JH (1995) The significance of cerebral white matter abnormalities 100 years after Binswanger's report. A review. Stroke 26: 1293-1301 Schmidt H , Fazekas F, Kostner GM, Schmidt R (2000) Genetic aspects of microangiopathy-related cerebral damage. J Neural Transm [Suppl] 59: 15-21 Schmidt R , Fazekas F, Kapeller P, Schmidt H, Hartung H-P (1999) MRI white matter hyperintensities. Three-year follow-up of the Austrian Stroke Prevention Study. Neurology 53: 132-139 Schmidt R , Schmidt H , Fazekas F, Launer LJ , Niederkorn K, Kapeller P, Lechner A, Kostner GM (2001) Angiotensinogen polymorphism M235T, carotid atherosclerosis, and small vessel disease-related cerebral abnormalities. Hypertension 38: 110-115 Authors' address: R. Schmidt, M.D. , Department of Neurology, Karl-Franzens University, Auenbruggerplatz 22, A-8036 Graz, Austria, e-mail : [email protected]

Microangiopathy-related cerebral damage and angiotensinogen gene: from epidemiology to biology H. Schmidt', F. Fazekas--', and R. Schmidt--' 1

Institute of Medical Biochemistry and Medical Molecular Biology, 2 Department of Neurology, and 3 MR Centre, Karl-Franzens University, Graz, Austria

Microangiopathy-related cerebral damage (MARCD) is a common finding in the elderly. It may lead to cognitive impairment and gait disturbances. Arterial hypertension and age are the best accepted risk factors for MARCD. Genes involved in blood pressure regulation, like genes encoding the proteins of the renin-angiotensin system (RAS) therefore represents good candidate genes for MARCD. Plasma angiotensinogen level is a major determinant of the RAS act ivity. Positive correlation between angiotensinogen gene expression and RAS activity, as well as blood pressure were observed. Common mutations described in the AGT promoter were able to alter AGT expression in cell culture. We described that 4 frequent mutations at the AGT promoter are combined in 5 haplotypes coded as A (-6:g, -20:a, -152:g, -217:g), B (-6:a, -20:c, -152:g, -217:g), C (-6:a, -20:c, - 152:a, -217:g), D (-6:a, -20:a, -152:g, -217:g), and E (-6:a, -20:a, -152:g, -217:a). The B haplotype was significantly associated with MARCD in the cohort of the Austrian Stroke Prevention Study (p = 0.005). The association was independent of hypertension, which pinpointed to a possible role of the local RAS in this relationship. Investigation of the promoter activity of the AGT gene in astrocytes suggests that expression of this gene may be modulated by the haplotype.

Summary.

Definition of the Phenotype

Microangiopathy-related cerebral damage (MARCD) includes early confluent and confluent white matter changes as well as lacunar infarcts (Schmidt H et al., 2000). The definition is based on histopathological findings demonstrating that both of these changes are associated with arteriolosclerosis (Awald et al., 1986; Fazekas et al., 1993; Van Swieten et al., 1991a). MARCD is a common MRI observation in elderly persons and may lead to cognitive impairment and gait disturbances as it progresses (Bots et al., 1993). Identification of individuals prone to the development of MARCD and early control of causal factors could reduce the risk of these common clinical

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problems of the elderly. It is unclear which factors other than advancing age and arterial hypertension predispose individuals to MARCD (Van Swieten, 1991b; Schmidt R et al., 1997) Evidence for genetic factors in MARCD

MARCD is probably a multifactorial disorder with both genetic and environmental factors influencing its presence and severeity. A recent investigation of Carmelli et al. (1998) supports the presence of a substantial genetic background in MARCD. This study on World War II veteran twins has shown a heritability index of 0.73 for white matter hyperintensity (WMH) volume meaning that up to 73 % of the interindividual variation seen in WMH volume can be explained by genetic factors . The probandwise concordance rates for extensive white matter lesions, defined as >0.5% oftotal intracranial volume, were 61% in monozygotic and 38% in dizygotic twins at a prevalence of 15% for the entire study population. This gives a relative risk estimate of 4 for monozygotic and 2.5 for dizygotic twins compared to the risk of the general population. Altough a heritability index in general should be interpreted cautiously and its value strictly applies only to the investigated population, the high estimate for WMH volume stresses the need for further investigations on genetic factors in relation to these brain changes. Angiotensinogen as a candidate gene for MARCD

The consistent association between MARCD and arterial hypertension (Pantoni et al., 1995; Van Swieten et aI., 1991) suggests that genes involved in the regulation of blood pressure may contribute to the strikingly high heritability in this phenotype. The renin-angiotensin system (RAS) is a major regulator of blood pressure. Plasma angiotensinogen (AGT) synthesized by the liver is processed to angiotensin II (AT-II) by the serial action of renin and angiotensin converting enzyme (ACE). The plasma level of AGT is rate limiting in this cascade (Lynch et aI., 1991). Plasma AGT concentration is positively correlated with RAS activity and blood pressure in humans and in animal models (Lynch et al., 1991; Kim et al., 1995; Yang, 1999). Structure of the human AGT gene

The mature human AGT (MW 61.400 D) protein is 452 aminoacides long and has a 14% carbohydrate content. Present data suggest that beside being the extracellular reservoir of angiotensin peptides, AGT exerts no other function (Lynch et aI., 1991). The AGT gene is located on Chr1q. It spans 12kb and contains 5 exons (Fukamizu et al., 1990). The second exon contains 859 nucleotides and encodes the signal sequence and angiotensin I peptide. This exon carries two frequently investigated polymorphisms, namely the T174M

Angiotensinogen and cerebral small vessel disease

55

and the M235T, which are both located downstream of the renin cutting site. Sequence analysis of the 5' -flanking region of the human AGT gene revealed the existence of several putative regulatory elements like AGCE2 (human angiotensinogen core promoter element 2), AGCE1 (human angiotensinogen core promoter element 1), 5'-AGCE2, glucocorticoid responsive elements, estrogen responsive element, heat shock responsive element, cAMP responsive elements (Kim, 1995; Yanai et aI., 1996, 1997a, b) . AGT expression in the liver

The primary site of AGT synthesis is the liver. The basal transcription of the human AGT gene in liver cells is controlled by a suprisingly short region from -32 to +44bp (Yanai et al., 1996). It has been shown that deleting the fragment from -16 to +44 bp reduces promoter activity by 95%, even though the TATA box is located outside of this region. The AGCE1 element (from position - 9 to - 25 relative to the transcriptional start site) between the TATA box and the transcriptional start site was identified as a key regulator of AGT expression. In vitro substituted mutations within AGCE1 had a more profound effect on AGT gene transcription than mutations in the TATA box (Kim et al., 1995; Yanai et al., 1997). The commonly observed, naturally occurring sequence alterations at position -6 and -20, have indeed been shown to alter transcriptional efficiency of the AGT promoter (Jeunemaitre et al., 1992; Yanai et al., 1997; Inoue et al., 1997; Zhao et al., 1999). Angiotensinogen expression in the brain

AGT is expressed in several organs (fat, brain, vascular wall, in small amounts in lung , kidney, ovary, testis, adrenal gland, heart, spinal cord) and cell types (adipocytes, astrocytes, fibroblasts , vascular smooth muscle cells) (Paul et al., 1993). Not only AGT but also other components of the RAS system including renin, ACE and angiotensin receptors are present in these tissues. The major source of AGT in the brain seems to be the astrocyte (Bunnemann et al., 1992; Wright et al., 1992). This is supported by the facts that AGT mRNA appears in the fetal brain as astrocytes develop and that cell lines derived from human and rat astrocytomas are able to produce AGT. Cerebrospinal fluid AGT is also derived from astrocytes as AGT does not cross the blood brain barrier. There is however a large variation in AGT expression among astrocytes according to their location in the brain. Positive association exists between angiotensinogen polymorphisms and MARCD

In our previuos investigation we could observe a positive association between T235 variant and the presence of MARCD in the cohort of Austrian Stroke Prevention Study (Schmidt Ret al., 2001). The T235 allele also enhanced the

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risk for the progression of small vessel disease related brain abnormalities. The association was present in T235 homozygotes but not in heterozygotes. We hypothesized that the association between the T235 allele and MARCD is not causal but the T235 mutation rather represents a marker being in linkage dysequilibrium with a causal mutation in its vincinity. Our focus of interest were variants located in the promoter region of the AGT gene as th ey were shown to alter the expression of angiotensinogen. Using TTGE (temporal temperature gradient electrophoreses) for screening the AGT promoter for mutations, we detected 4 polymorphic sites at positions -6:g/a, -20:a/c, -152:g/a, and - 217:g/a in our cohort (Schmidt H et al., 2001). The 4 polymorphic alleles were present in form of 5 haplotypes which we coded as A (-6:g, -20:a, -152:g, -217:g), B (-6:a, -20:c, -152:g, -217:g), C (-6:a, -20:c, -152:a, -217:g), D (-6:a, -20:a, -152:g, -217:g), and E (-6:a, -20:a, -152:g, -217:a). Among the single nucleotide polymorphic alleles the -20:c in homozygous state was associated with a significantly higher risk for MARCD (OR = 2.5, P = 0.04), while the association between MARCD and -6:a allele was borderline significant (OR: 1.5, P = 0.054). The B haplotype which carries both the -6:a and the -20:c alleles, showed an even stronger effect on MARCD frequency than any of the single nucleotide alleles. The relative risk for B homozygotes was 8 fold increased (p = 0.003), and also B heterozygotes showed a trend to increased MARCD risk (OR:1.8, p = 0.1). There was a significant linear association between B haplotype copy number and MARCD risk suggesting the presence of a gene-dose effect for the haplotype. A similar gene-dose relationship was not seen for the single nucleotide polymorphic alleles. In summary, usinghaplotypes instead of the single nucleotide markers increased the strength of the association as expected if a true causal relationship exists. The association between AGT promoter haplotype and MARCD is independent of hypertension

Interestingly, the association between B haplotype and MARCD was, contrary to our primary hypotheses, not mediated by hypertension. The presence of hypertension rather masked the effect of the haplotype due to the higher baseline risk for MARCD in this group. In the light of the strong correlation between angiotensinogen expression, plasma level and RAS activity this finding suggested that the association between MARCD and AGT promoter haplotype is mediated by an altered activity of the local rather than the systemic RAS. In preeclampsia it has been shown that the M235T variant can modulate the AGT expression in decidual arteries (Morgan et al., 1997). Possible pathological pathways linking the AGT promoter haplotype to MARCD

Conceivably, the association between the AGT B haplotype and MARCD might be mediated by an altered expression of AGT in the brain, which in turn

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may lead to an altered local availability of AGT. Our preliminary results on the haplotype dependent promoter activity of the AGT gene showed that the B haplotype leads to an increased promoter activity in astrocytes (unpublished data). If tissue RAS activity is also regulated by the AGT level as is the systemic RAS, then changes in AGT concentration may result in an altered level of AT-II at this site. AT-II acts on a variety of cell types in the brain (Bunnemann et aI., 1992; Wright et aI., 1992). In the present context its effect on vascular smooth muscle cells is of particular interest. AT-II, a potent regulator of vascular tone can lead to vasoconstriction but also to vasodilation in the cerebral arterioles (Wei et aI., 1978; Whalley et aI., 1988; Haberl et aI., 1990, 1996). AT-II promotes vascular smooth muscle cell hyperplasia and hypertrophy (Taubman et aI., 1989; Weber et aI., 1994; Marrero et aI., 1995; Ushio-Fukai et aI., 1998). It modulates NADHINADPH oxidase and extracellular superoxide dismutase activity in the vessel wall (Rajagopalan et aI., 1996; Fukai et aI., 1999). It was also reported to alter the production of extracellular matrix proteins in the vessels (Kakinuma et aI., 1998; Ford et al., 1999). Therefore, alterations in local AT-II availability might result in imbalance of physiological processes like brain perfusion, autoregulation of cerebral blood flow, the oxidative state of the vessel wall, or blood brain barrier function. Each of these processes might be involved in the development of MARen. In summary, if our results can be replicated in independent studies, then the B haplotype might serve as a genetic marker for the identification of individuals prone to develop microangiopathy-related brain damage and its clinical consequences. The etiological understanding of the association of small vessel disease related cerebral damage with genetic variants in the RAS system independently of arterial hypertension might also point to possible favorable effects of drugs acting on the RAS system beyond what can be expected from lowering blood pressure alone. Acknowledgements The gen etic studies of the Austrian Stroke Prevention Study are supported by the Au strian Science Fund, Project P13180 and Project P15440 and the Austrian National Bank Jubileumsfond Project 7776. The technical assistance of Ing. H. Semmler, I. Polzl, A. Thaci and W. Treu is appreciated.

References Awad lA, Johnson PC, Spetzler RF, Hodak JA (1986) Incidental subcortical lesions identified on magnetic resonance imaging in the elderly. II. Po stmortem histopathological correlations. Stroke 17: 1090-1097 Bots ML , van Swieten JC, Breteler MMB , de Jong PTVM, van Gijn J, Hofman A , Grobbee DE (1993) Cerebral white matter lesions and atherosclerosis in the Rotterdam study. Lancet 341: 1232-1237 Bunnemann B, Fuxe K, Ganten D (1992) The brain renin-angiotensin system: localisation and general significance. J Cardiovasc Pharmacol 19[5uppl 6]: S51S62

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Carmelli D , DeCarli C, Swan G, Jack LM, Reed T, Wolf PA, Miller BL (1998) Evidence for genetic variance in white matter hyperintensity volume in normal elderly male twins . Stroke 29: 1177-1181 Fazekas F, Kleinert R , Offenbacher H , Schmidt R , Kleinert G, Payer F, Radner H , Lechner H (1993) Pathologic correlates of incidental white matter signal hyperintensities. Neurology 43: 1683-1689 Ford CM , Li S, Pickering JG (1999) Angiotensin II stimulates collagen synthesis in human vascular smooth muscle cells. Involvement of the AT(l) receptor, transforming growth factor-beta , and tyrosine phosphorylation. Arterioscler Thromb Vase Bioi 19: 1843-1851 Fukai T, Siegfried MR, Ushio-Fukai M, Oriendling KK , Harrison DO (1999) Modulation of extracellular superoxide dismutase expression by angiotensin II and hyp ertension. Circ Res 85: 23-28 Fukamizu A, Takahashi S, Seo MS, Tada M, Tanimoto K, Uehara S, Murakami K (1990) Structure and expression of the human angiotensin gene. J Biol Chern 265: 7576-7582 Haberl RL, Anneser F, Villringer A , Einhaupl KM (1990) Angiotensin II induces endothelium dependent vasodilation of rat cer ebral arterioles. Am J Physiol 258: H18401846 Haberl RL, Decker-Hermann PJ, Hermann K (1996) Effect of renin on brain arterioles and cerebral blood flow in rabbits. J Cereb Blood Flow Metab 16: 714-719 Inoue I, Nakajima T, Williams CS, Quackenbush J, Puryear R , Powers M, Cheng T, Ludwig EH, Sharma AM, Hata A , Jaunemaitre X, Lalouel JM (1997) A nucleotide Substitution in the promoter of human angiotensinogen is associated with essential hypertension and affects basal transcription in vitro. J Clin Invest 99: 1786-1797 Jeunemaitre X, Soubrier F, Kotelevtsev YV, Lifton RP, Williams CS, Charru A , Hunt SC, Hopkins PN, Williams RR, Lalouel JM (1992) Molecular basis of human hypertension: role of angiotensinogen. Cell 71(1) : 169-180 Kakinuma Y, Hama H, Sugiyama F, Yagami K, Ooto K, Murakami K, Fukamizu A (1998) Impaired blood-brain barrier function in angiotensinogen deficient mice. Nat Med 4: 1078-1080 Kim HS, Kr ege JH, Kluckman KD, Hagaman JR, Hodgin JB, Best CF, Jenette JC, Coffman TM, Maeda N, Smithies 0 (1995) Genetic control of blood pressure and the angiotensinogen locus. Proc Nat! Acad Sci USA 92: 2735-2739 Lynch KR, Peach MJ (1991) Molecular biology of angiotensinogen. Hypertension 17: 263-269 Marrero MB , Schieffer B , Paxton WO, Heerdt L, Berk BC, Delafontaine P, Bernstein KE (1995) Direct stimulation of Jak/STAT pathway by the angiotensin II AT j receptor. Nature 375: 247-250 Morgan T , Craven C, Nelson L, Lalouel JM , Ward K (1997) Angiotensinogen T235 expression is elevated in decidual spiral arteries. J Clin Invest 100: 1406-1415 Pantoni L, Garcia JH (1995) The significance of cerebral white matter abnormalities 100 years after Binswangers report. A review. Stroke 26: 1293-1301 Paul M, Wagner J, Dzau VJ (1993) Gene expression of the renin-angiotensin system in human tissues. J Clin Invest 91: 2058-2064 Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Oriendling KK , Harrison DG (1996) Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. J Clin Invest 97: 1916-1923 Schmidt H , Fazekas F, Kostner GM, Schmidt R (2000) Genetic aspects of microangiopathy-related cerebral damage. J Neural Transm [Suppl] 59: 15-21 Schmidt H, Fazekas F, Kostner GM , van Duijn CM , Schmidt R (2001) Angiotensinogen gene promoter haplotype and microangiopathy-rel at ed cer ebral damage. Results of the Austrian Stroke Prevention Study. Stroke 32: 405-412 Schmidt R, Fazekas F, Hayn M, Schmidt H, Kapeller P, Roob G , Offenbacher H, Schumacher M, Eber B, Weinrauch V, Kostner GM, Esterbauer H (1997) Risk

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factors for microangiopathy-related cerebral damage in the Austrian Stroke Prevention Study. J Neurol Sci 152: 15-21 Schmidt R, Schmidt H, Fazekas F, Launer LJ, Niederkorn K, Kapeller P, Lechner A, Kostner GM (2001) Angiotensinogen polymorphism M235T, carotid atherosclerosis, and small vessel disease-related cerebral abnormalities. Hypertension 38: 110-115 Taubman MB , Berk BC, Izumo S, Tsuda T, Alexander RW, Nadal-Ginard B (1989) Angiotensin II induces c-fos mRNA in aortic smooth muscle. J Bioi Chern 264: 526530 Ushio-Fukai M, Alexander RW, Akers M, Griendling KK (1998) p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. J BioI Chern 273: 15022-15029 Van Swieten JC, Van den Hout JHW, Van Ketel BA, Hijdra A, Wokke JHJ, Van Gijn J (1991a). Periventricular lesions in the white on magnetic resonance imaging in the elderly. A morphometric correlation with arteriolosclerosis and dilated perivascular spaces. Brain 114: 761-774 Van Swieten JC, Geykcs GG, Derix MMA, Peeck BM, Ramos LMP, van Latum JC, van Gijn J (1991b) Hypertension in the elderly is associated with white matter lesions and cognitive decline. Ann Neurol 30: 825-830 Weber H, Taylor SD, Molloy CJ (1994) Angiotensin II induces delayed mitogenesis and cellular proliferation in rat aortic smooth muscle cells. J Clin Invest 93: 788-798 Wei AP, Kontos HA, Patterson JL (1978) Vasoconstrictor effect of angiotensin on pial arteries. Stroke 9: 487-489 Whalley ET, Wahl M (1988) Cerebrovascular reactivity to angiotensin and angiotensinconverting enzyme activity in cerebrospinal fluid. Brain Res 438: 1-7 Wright JW, Harding JW (1992) Regulatory role of brain angiotensinogens in the control of physiological and behavioral responses. Brain Res Rev 17: 227-262 Yanai K, Nibu Y , Murakami K, Fukamizu A (1996) A cis-Acting DNA Element located between TATA box and transcription initiation site is critical in response to regulatory sequences in human angiotensinogen gene. J Bioi Chern 271: 15981-15986 Yanai K, Matsuyama S, Murakami K, Fukamizu A (1997) Differential action of AGCEF2 upon cell type-dependent expression of human angiotensinogen gene. FEBS Lett 412: 285-289 Yanai K, Saito T, Hirota K , Kobayashi H , Murakami K, Fukamizu A (1997) Molecular variation of the human angiotensinogen core promoter element located between the TATA box and transcription initiation site affects its transcriptional activity. J BioI Chern 272(48): 30558-30562 Yang G, Merril DC, Thompson MW, Robillard JE, Sigmund CD (1994) Functional expression of the human angiotensinogen gene in transgenic mice. J Bioi Chern 269: 32497-32502 Zhao YY, Zhou J, Narayanan CS, Cui Y, Kumar A (1999) Role of CIA polymorphism at -20 on the expression of human angiotensinogen gene. Hypertension 33: 108-115 Authors' address: H . Schmidt, M.D ., MSc, Institute for Medical Biochemistry and Medical Molecular Biology, Karl-Franzens University , Harrachgasse 21, A -8010 Graz, Austria, e-mail: [email protected]

Can small-vessel disease-related cerebral abnormalities be used as a surrogate marker for vascular dementia trials? F. Fazekas, S. Ropele, and R. Schmidt Department of Neurology and MR Centre, Karl Franzens U niversity, Graz, Austria

Summary. Clinical scales for measuring the effectiveness of diseasemodifying therapies in patients with vascular dementia are of limited sensitivity. Moreover, they cannot serve to directly probe the potential of a drug in slowing further progression of vascular damage. Assessment of morphologic abormalities that reflect ischemia-related cerebral tissue changes and have a bearing on cognitive function could serve to address both of these aspects and, if sensitive enough, could constitute an ideal surrogate for measuring progression of the disease. For drug licensing agencies a validated surrogate has to meet several requirements: First, the surrogate must predict the future clinical course. Second, the effect of treatment on the disease must be explained by the effect of the treatment on the surrogate; the treatment needs to affect clinical outcome by working through mechanisms related to the surrogate. Third, evidence must exist that treatments of various classes affect the surrogate in the same and predictable manner. Apart from these requirements, regulatory guidelines may also allow the use of even an unvalidated surrogate if it is considered reasonably likely to predict future clinical outcome or disease activity. Various morphologic measures, particularly those using conventional or more sophisticated MRI techniques have already shown a close correlation to neuropsychologic functions. If these associations can also be confirmed in longitudinal studies and following specific treatments, morphologic markers will play a major role in future clinical trials of vascular dementia.

Introduction

Vascular dementia (VD) accounts for probably only a small portion of demented individuals. In pathologic series, the frequency of cases with dementia related presumably to vascular changes remains mostly below 10% to 20% (Jellinger, 2001). However, the number of individuals in whom vascular changes contribute to evolving cognitive deficits may be considerably higher especially when extending considerations by the concept of vascular cognitive impairment (Bowler et aI., 1999; Rockwood et al., 2000). If and to what extent

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the evolution of VD might be prohibited or at least slowed down by appropriate treatment is still unknown. In part this may be a consequence of the difficulties of diagnosing VD per se. Although more recent formulations of research criteria have also included evidence of ischemic damage by computed tomography (CT) or magnetic resonance imaging (MRI) as a prerequisite (Chui et al., 1992; Roman et al., 1993), the variability in defining dementia as related to a vascular cause has remained high (Pohjasvaara et al., 2000). Even the assumptions which type and extent of vascular changes would be sufficient to cause dementia are quite diverse which suggests that the contribution of ischemic lesions to neuropsychologic impairment occurs via different pathophysiologic mechanisms that, consequently also may require different kinds of therapeutic intervention. Another limitation of earlier VD trials may come from the fact that clinical measures may not be sensitive enough to detect progression of VD over the relatively short period of time that drug trials usually can be performed (Desmon et al., 1999). Inhomogeneous distribution of other clinical deficits from cerebrovascular disease may further add to variability among VD patients and make treatment-group comparisons difficult. As a consequence, it would appear tempting to define treatment effects not only by clinical aspects but, given the high sensitivity of MRI, by effects on progression of morphologic changes themselves (Chui, 2000). We, therefore, will examine the type of morphologic changes that may lend themselves to longitudinal monitoring, and the MRI techniques that could be used for data acquisition and analysis. Subsequently, we will also briefly discuss the concepts of surrogacy in clinical trials. Morphological changes appropriate for longitudinal monitoring

When deciding on the types of morphologic abnormalities that might serve best as outcome variables for interventional trials in VD, it appears necessary to distinguish between the different mechanisms causing vascular cognitive impairment. Most unfortunately we have also to accept that coexisting degenerative brain changes which may playa leading role in the development of VD (Henon et al., 2001) cannot be displayed by CT and at least conventional MRI. Accumulating morphologic damage from multiple infarcts and strategic infarct dementia are well recognised entities (Roman et al., 1993). Quite simplistic multi-infarct dementia can be thought of as reflecting the sum of neurologic deficits (including higher cortical functions) accumulated from complete or incomplete territorial infarcts. Strategic infarct dementia is attributed to localised ischemic damage to functionally important cortical and subcortical areas such as the angular gyrus, the thalamus or to other areas relevant for higher cortical functions. Both these types of dementia are usually associated with stroke symptoms as well. Risk-factor patterns, the possibility of recurrence and the possibilities of therapeutic intervention probably do not differ much from those of strokes with primarily sensory or motor deficits.

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Therefore, monitoring of the number, size and location of large infarcts, though relatively easy to perform, does not appear to provide much specific information regarding prevention or amelioration of cognitive deficits in VD drug trials. Dementia associated with haemorrhagic cerebrovascular disease is also a category of its own (Roman et al., 1993). On the one hand, MRI has provided more insight into morphologic findings associated with intracerebral bleeding such as focal hemosiderin deposits reported both in cerebral amyloid angiopathy and hypertensive bleeding (Roob et aI., 2000). On the other hand, cognitive consequences of ICR are likely to follow similar mechanisms as those assumed for multi-infarct and strategic-infarct dementia with the exception of meningeal hemosiderosis. The latter is a rare disorder which typically presents with a triad of cognitive impairment, hypaccusis and spastic paraparesis. Meningeal hemosiderosis develops from repeated bleeding into the CSF spaces which leads to accumulation of hemosiderin at the CNS surfaces and thereby to toxic damage of the adjacent neuronal structures. Small-vessel disease with dementia is yet another cause of VD and constitutes the most promising type of vascular cognitive impairment for morphologic monitoring (Erkinjuntti et al., 2000; Inzitari et al., 2000). The clinical manifestations of small-vessel disease consist usually of a subcortical dementia syndrome which is characterised by memory deficits, abnormal executive functions and psychomotor retardation frequently accompanied by personality and mood alterations. Of course the causes and consequences of smallvessel disease are also quite heterogenous and so diverse aetiologies as hypertensive microangiopathy or cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) may be at the origin of microangiopathy related ischemic tissue damage. For various reasons the morphologic evaluation of microangiopathyrelated tissue changes in association with evolving cognitive impairment appears quite promising (Erkinjuntti et al., 2000) . First, the pattern of lesions indicating small-vessel disease is quite specific. It comprises of lacunar lesions of the basal ganglia, thalami and brain stem and of mostly incomplete ischemic damage to the periventricular and deep white matter. Secondly, these abnormalities are quite frequent and th e few studies that addressed their progression using magnetic resonance imaging (MRI) have shown a sizeable accumulation of damage over time even in asymptomatic elderly individuals (Schmidt et al., 1999; Wahlund et aI., 1996) . It would be expected that the range of progression was even higher in individuals with already more extensive changes at onset (Veldink et al., 1998). Clearly, evaluation of these morphologic abnormalities should be complemented by measurements of associated loss of brain parenchyma, i.e. evolving brain atrophy, in general and at specific locations (Pohjasvaara et al., 2000) . More recent studies appear to indicate that e.g. volume loss of the hippocampus is not only a predictor of worse cognitive performance in Alzheimer's disease but also in VD (Hanyu et al., 2000). A set of T 2-weighted and FLAIR images should suffice to monitor the accumulation of lacunes and white matter damage. Like in MS, semiautomatic

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Table 1. Suggested conv entional sequences for evaluation of microangiopathy-related tissue damage Fast Spin Echo (FSE) Axial plane FOV250mm Reel. FOV 75-100% Acq. matrix 2562 or 256 5 mm slice thickness 0.5 mm inter slice gap 24-28 slices 2NEX Flow compensation Echoes per shot 8-16 TE 100-120ms TR 4,000-6,000ms

Fast FLAIR

X

192

Axial plane FOV 250mm Reel. FOV 75-100% Acq. Matrix 2562 or 256 5 mm slice thickness 0.5 mm inter slice gap 24-28 slices 1NEX Flow compensation Echoes per shot 8-24 TE 100-140ms. TR 6,000-10,000ms TI 2,000-2,400ms

TlW-3DGE (MPRAGE)

X

192

Cor or sag plane FOV256mm Reel. FOV 75-100% Acquisition matrix 2562 1-1.5 mm slice thickness 1NEX Flow compensation FA 15-25 ° TE 4-7ms TR 10-20ms

techniques for lesion outlining can then be used to generate quantitative data (Filippi et al. , 1998). For brain atrophy measurements 3D-data sets appear desirable and, using appropriate coregistration algorithms, have been shown to be very sensitive for even minor changes in brain volume (Fox et al ., 2000). Along these lines the table provides a suggestion of MRI pulse sequences which should suffice to obtain such data set. This sequences are currently also used in LADIS (leukoaraiosis and disability) an ongoing European multicenter study to explore the long-term clinical consequences of ischemic white matter damage of the brain. Additional techniques as magnetisation transfer and diffusion-weighted imaging may allow both for more sensitive and more specific assessments of tissue damage (Fazekas et al., 2000). Magnetisation transfer probes especially the integrity of myelin while diffusion-weighted imaging abnormalities strongly reflect the loss of structural elements. With the advance of MR scanners, these sequences can now be performed at many sites and comparable data acquisition schemes can easily be implemented. Appropriate analysis techniques like the generation of histograms have been developed in parallel that allow to calculate magnetisation transfer or diffusion data for the entire brain or for specifically segmented parts thereof (Cercignani et al. , 2000). Morphologic lesions as a surrogate marker for treatment trials of vascular dementia A surrogate is an outcome other than the clinical outcome that can reliably predict it . For various reasons it is tempting to consider the morphologic abnormalities described above as possible surrogates in treatment trials of YD . Given the sensitivity of MRI, there might be a higher probability to

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detect treatment effects with smaller sample sizes like in multiple sclerosis (Molyneux et al., 2000). Besides demonstrating effects regarding the development or progression of dementia, it could also become possible to directly document the treatment mechanism(s) if a reduction in the progression of vascular changes was shown to go hand in hand with preserved cognitive functioning. Also, morphologic changes are attractive because of their objectivity and, once acquired morphologic data can be repeatedly analysed in various ways. Unfortunately, given the current state of knowledge on the association of vascular changes with cognitive dysfunction, we are still far away from being allowed to consider ischemic tissue changes as a surrogate for vascular cognitive impairment or even YD. So far , there exist no compelling data on an association of progressing tissue changes ascribed to small vessel disease with decreasing cognitive performance. For serving as a validated surrogate, it would also be necessary that the effect of treatment on the clinical disease was explained by the effect of the treatment on the surrogate. Furthermore, evidence would have to exist that treatments of various classes can affect the surrogate in the same and predictable manner.

Conclusions

To advance knowledge all efforts should be undertaken to add the various morphologic measures outlined above to patient evaluation in future treatment trials of dementia in general and specifically in those of YD . Parallel acquisition of clinical and morphologic data will help to show if the concepts of surrogacy, at least in determining future clinical disease, can be fulfilled. Also, morphologic and neuropsychologic measures should be added to large treatment trials targeting the prevention of overall progression of vascular disease e.g. by the use of blood-pressure and lipid lowering drugs or platelet antiaggregants. This may be a first step to look at treatment interactions with progression of morphologic damage. If this was established, one could further advance the concept of at least unvalidated surrogacy, i.e. it can already now be assumed that progressing morphologic changes per se are not beneficial but rather a sign of ongoing brain damage, even if this was not always noted clinically. In these attempts, highly sophisticated techniques should be employed both for image acquisition and for data analysis to extract the maximum information possible.

References Bowle r J, Steenhuis R , Hachinski V (1999) Conceptual background to vascular cognitive impairment. Alzh eimer Dis Assoc Disord 13[Suppl 3]: S30-S37 Cercignani M, Iannucci G, Rocca M, Comi G, Horsfield M, Filippi M (2000) Pathologic damage in MS assessed by diffusion-weighted and magnetization transfer MRI. Neurology 54: 1139-1144

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Chui H (2000) Vascular dementia, a new beginning: shifting focus from clinical phenotype to ischemic brain injury. Neurol Clin 18: 951-978 Chui H, Victoroff J, Margolin D, Jagust W, Shankle R, Katzman R (1992) Criteria for the diagnosis of ischemic vascular dementia proposed by the State of California Alzheimer's Disease Diagnostic and Treatment Centers. Neurology 42: 473-480 Desmond D , Erkinjuntti T, Sano M, Cummings J , Bowler J, Pasquier F, Moroney J, Ferris S, Stem Y, Sachdev P, Hachinski V (1999) The cognitive syndrome of vascular dementia: implications for clinical trials. Alzheimer Dis Assoc Disord 13[Suppl 3]: S21-529 Erkinjuntti T, Inzitari D, Pantoni L, Wallin A , Scheltens P, Rockwood K, Desmond D (2000) Limitations of clinical criteria for the diagnossis of vascular dementia in clinical trials : is focus on subcortical vascular dementia a solution? Ann NY Acad Sci 90:3262-3272 Fazekas F, Ropele S, Bammer R , Kapeller P, Stollberger R , Schmidt R (2000) Novel imaging technologies in the assessment of cerebral ageing and vascular dementia. J Neural Transm [Suppl] 59: 45-52 Filippi M, Horsfield M, Ader H, Barkhof F, Bruzzi P, Evans A, Frank J, Grossman R, McFarland H, Molyneux P, Paty D, Simon J, Tofts P, Wolinsky J, Miller D (1998) Guidelines for using quantitative measures of brain magnetic resonance imaging abnomrmalities in monitoring the treatment of multiple sclerosis. Ann Neurol 43: 499-506 Fox N, Jenkins R , Leary S, Stevenson V, LosseffN, Crum W, Harvey R, Rossor M, Miller D, Thompson A (2000) Progressive cerebral atrophy in MS: a serial study using registered, volumetric MRI. Neurology 54: 807-812 Hanyu H , Asano T, Iwamoto T, Takasaki M, Shindo H , Abe H (2000) Magnetization transfer measurements of the hippocampus in patients with Alzheimer's disease, vascular dementia, and other types of dementia. AJNR Am J Neuroradiol21: 12351242 Henon H, Durieu I, Guerouaou D , Lebert F, Pasquier F , Leys D (2001) Poststroke dementia: incidence and relationship to prestroke cognitive decline. Neurology 57: 1216-1222 Inzitari D, Erkinjuntti T , Wallin A, Del Ser T, Romanelli M, Pantoni L (2000) Subcortical vascular dementia as a specific target for clinical trials. Ann NY Acad Sci 90: 35103521 Jellinger K (2001) Neuropathologic substrates of ischemic vascular dementia. J Neuropathol Exp Neurol60: 658-659 Molyneux P, Miller D, Filippi M, Yousry T , Kappos L, Gasperini C, Ader H, Barkhof F (2000) The use of magnetic resonance imaging in multiple sclerosis treatment trials: power calculations for annual lesion load measurement. J Neuro1247: 34-40 Pohjasvaara T, Mantyla R , Salonen 0 , Aronen H , Ylikoski R, Hietanen M, Kaste M, Erkinjuntti T (2000) How complex interactions of ischemic brain infarcts, white matter lesions, and atrophy relate to poststroke dementia. Arch Neurol 57: 12951300 Pohjasvaara T, Mantyla R, Ylikoski R , Kaste M, Erkinjuntti T (2000) Comparison of different clinical criteria (DSM-III, ADDTC, ICD~10, NINDS-AIREN, DSM-IV) for the diagnosis of vascular dementia. National Institute of Neurological Disorders and Stroke-Associahtion Internationale pour la Recherche et l'Enseignement en Neurosciences. Stroke 31: 2952-2957 Rockwood K, Wentzel C, Hachinski V, Hogan D, MacKnight C, McDowell I (2000) Prevalence and outcomes of vascular cognitive impairment. Vascular Cognitive Impairment Investigators of the Canadian Study of Health and Ageing. Neurology 54: 447-451 Roman G, Tatemichi T, Erkinjuntti T, Cummings J, Masdeu J, Garcia J, Amaducci L, Orgogozo J, Brun A, Hofman A , Moody D , O'Brien M, Yamaguchi T , Grafman J, Drayer B, Bennett D, Fisher M, Ogata J, Kokmen E , et al (1993) Vascular dementia:

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diagnostic criteria for research studies. Report of the NINDS-AIREN International Work Group. Neurology 43: 250-260 Roob G, Lechner A , Schmidt R, Flooh E , Hartung H , Fazekas F (2000) Frequency and location of microbleeds in patients with primary intracerebral hemorrhage. Stroke 31: 2665-2669 Schmidt R, Fazekas F, Kapeller P, Schmidt H, Hartung H (1999) MRI white matter hyperintensities: three-year follow-up of the Austrian Stroke Prevention Study. Neurology 53: 132-139 Veldink J, Scheltens P, Jonker C, LJ L (1998) Progression of cerebral white matter hyperintensities on MRI is related to diastolic blood pressure. Neurology 51: 319-320 Wahlund L, Almkvist 0 , Basun H, Julin P (1996) MRI in successful ageing, a 5-year follow-up study from eighth to ninth decade of life. Magn Res Imag 14: 601-608 Authors' address: Dr. F. Fazekas, Department of Neurology, University of Graz, Auenbruggerplatz 22, A-8036 Graz, Austria

Reactive oxygen: its sources and significance in Alzheimer disease G. Perry', A. Nunomura-, A. D. Cash', M. A. Taddeol, K. HiraP, G. Aliev", J. Avila5, T. Wataya6, S. Shimohama'', C. S. Atwood', and M. A. Smith' 1 Institute of Pathology, and "Department of Anatomy, Case Western Reserve University, Cleveland, OR, U.S.A. 2D epartment of Psychiatry and Neurology, Asahikawa Medical Colleg e, A sahikawa, and 3 Pharmaceutical Research Laboratories I, Pharmaceutical Research Division, Takeda Chemical Industries Ltd. , Osaka, Japan 5 Centro de Biologfa Molecular, Universidad Aut6noma de Madrid, Madrid, Spain 6D epartment of Neurology, Kyoto University, Kyoto, Japan

Summary. Over the past decade, oxidative stress has been established as the earliest cytological feature of Alzheimer disease and an attractive therapeutic target. The major challenges now are establishing the source of the reactive oxygen and what oxidative stress tells us about the etiology of Alzheimer disease. These are complex issues since a variety of enzymatic and nonenzymatic processes are involved in reactive oxygen formation and damage to macromolecules. In this review, we consider disease mechanisms that show the greatest promise for future research. A decade ago , Earl Stadtman and colleagues demonstrated increased reactive carbonyls in Alzheimer disease (AD) , presenting the first compelling evidence for oxidative damage in AD (Smith et a1., 1991). Prior to these studies, most work focused on measuring parameters such as lipofuscin or global enzymes levels or activities that are only weakly related to oxidative stress and, generally, findings were not reproducible between studies. However, with the advent of reactive carbonyl analysis, a specific oxidative change was examined and its increases were confirmed by others (Smith et a1., 1996, 1998). Subsequent cytochemical analysis, of carbonyls as well as a variety of other specific oxidative changes, showed increased oxidative damage involving every category of macromolecules (Smith et a1., 1994, 1996, 1997a; Sayre et a1., 1997; Nunomura et a1., 1999). Interestingly, damage was restricted to the lesions, neurofibrillary tangles and senile plaques, when only crosslinking modifications were examined (Smith et a1., 1994), whereas more labile changes were found throughout the cell body of all vulnerable neurons but absent from neuronal processes and glial cells (Smith et a1., 1996, 1997a; Nunomura et a1., 1999). These findings raised the hypothesis that reactive oxygen formation exceeds defenses in the cytoplasm of, previously con-

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sidered, "unaffected" neurons in AD. Studies to begin to determine the source of these alterations are concentrated on what is the primary site for reactive oxygen in all cells, the mitochondria. Studies of mitochondrial abnormalities in AD have met with much controversy primarily centered on whether there are mtDNA genotypes associated with AD (Corrall-Debrinski et al., 1994; Wallace et al., 1997). Our analysis instead focused on cellular changes in mitochondria restricted to vulnerable neurons. We used quantitative in situ hybridization and immunocytochemistry to show that mitochondrial DNA and protein levels were increased 3-4 fold in AD , whereas direct examination of mitochondrial representation in neurons of biopsy specimens showed little variation in normal mitochondria (Hirai et al., 2001) . Ultrastructural analysis showed that increased DNA and protein levels were present in lysosomes or free in the cytosol. While these data demonstrate a mitochondrial abnormality, it does not link oxidative damage to it because damage is primarily restricted to the endoplasmic reticulum. One hypothesis linking oxidative damage and mitochondrial abnormalities suggests that increased mitochondria targeting to autophagosomes is responsible for release and accumulation of redox-active metals in the cytosol through turnover of mitochondrial metalloproteins (Fig. 1). In light of the aforementioned hypothesis, we analyzed sites of metalcatalyzed peroxidase activity and found that they were restricted to the endoplasmic reticulum and lipofuscin residual bodies. While the latter lacked oxidative damage, the former exactly matches the hypothesis. Importantly, these metals, iron and copper, catalyze the precise types of oxidative damage found in AD, principally those relying on 8-hydroxyguanosine (80HG), carbonyls and tyrosine nitration. Interestingly, cytosolic metal binding sites promoting redox activity are entirely dependent on nucleic acids sensitive to Sl nuclease (single strand specific), i.e. non base paired RNA and DNA. This location is consistent with the extensive damage to RNA in AD because -OR radical's attack is diffusion limited. Whether proteins, e.g. A~ or L , are also involved in the coordination requires further study although our previous studies show that this is likely (Smith et al., 1997b). lysosome lipofusci n

mitochon~

_"'H20 ~J(Fe~ll)D . ~ .. Fe(II O 2-

nucle ic acids . . lipids proteins

Cu(l)

oOH

Cu(II )

O2 ascorbate RNA

Fig. 1. Hypothetical relationship between mitochondrial shuttling to autophagosomes (lysosomes), release of redox active iron and copper to the cytosol and oxidative damage to cellular compartments

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Our hypothesis suggests a complex interplay between mitochondrial turnover, nucleic acids and metals as being responsible for the location of damage. The proposed model suggests leaking of H 20 2 from mitochondria may be important, but since we found no evidence of abnormalities in normalappearing mitochondria in cases of AD, it is unclear whether the release of H 20 2 at physiological levels is sufficient to generate increased reactive oxygen when redox-active metals are increased. Therefore, there may be a nonmitochondrial source of H 2 0 2 such as NADPH oxidase (Shimohama et al., 2000; Pappolla et al., 2001) or xanthine oxidase, the enzyme that metabolizes purines such as those turned over from mitochondria into hypoxanthane and uric acid . We examined xanthine oxidase levels and found they too were increased with the same cytosolic distribution presented by oxidative damage. The possibility that a cytoplasmic oxidase is important may also present itself. A paradox of these models is that they present self-sustaining reactive oxygen formation. The neurons respond to the increased reactive oxygen assault by redirecting more glucose through the pentose phosphate pathway, stimulating kinase activation and inducing heme oxygenase. Due to these responses, substantial damage to biological molecules occurs, and neurons sustaining this damage remain for years. This aspect is perplexing because cellular defenses may not be the only factors defining damage. Phosphorylation state, of r (Perez et al., 2000; Takeda et al., 2000a,b) and neurofilament heavy and medium subunits (Wataya et al., 2002), controls their susceptibility to adduction reactions by reactive carbonyls. This reveals another way of viewing the high level of damage in AD as homeostatically regulated, suggesting the possibility that oxidative damage may even play some regulatory role in protecting neurons from death during stress. The pathological changes of AD, A~ and 't accumulation, also impact oxidative damage in a surprising way since their deposition is marked by a decrease in oxidative damage (Nunomura et al., 2000, 2001). While the mechanism for this reduction is still controversial (Cuajungco et al., 2000), it does make the case that oxidative damage and response are controlled and precede the classic lesions. It also makes the case that the lesions, while likely secondary, cannot be dismissed as inert because whatever they mark is critical to oxidant balance. Therefore, therapeutic efforts to disturb this balance must consider the potential effects, both positive and negative, of lesion formation (Smith et al., 2000; Perry et al., 2000, 2001). Oxidative changes in AD mark a novel balance of neuronal function, one where glucose utilization is rerouted to antioxidant defenses, and stress responses occur potentiating amyloidogenesis (Yan et al., 1995) while antioxidant proteins are upregulated. What is missing from this data, however, is the initiator of the changes. In this regard, analysis of oxidative damage among normal individuals of various ages show that there is an age-dependent increase in oxidative damage and mitochondrial abnormalities after age 40 years suggesting that the compensations of AD may be an accentuation of those of normal aging (Nunomura et al., unpublished observation). This view is consistent with A~ and r deposits in normal aging . AD might be consistent

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with a pleotrophic shift in compensatory reaction yielding new relations between regulator elements while remaining homeostatic. The mitochondrial abnormalities we described above may provide a clue as to the initiation of these phenomena. Mitochondria (Hirai et aI., 2001) and endoplasmic reticulum (Perry and Smith, unpublished observations) are both being shuttled to the autophagosome in AD, a seemingly wasteful process since both organelles are involved in sustaining and renewing the protein and energy needs of the extensive neural network that connects and defines the brain. However, analysis of microtubules, the tracks on which neuritic components move from the cell body, showed that there is a marked reduction of microtubule density in AD, with the presence of neurofibrillary tangles having no effect on the AD-dependent diminution. Further, we also found a significant diminution in microtubules in aging indicating that microtubule alterations are extremely proximal events in disease pathogenesis. We suspect that the changes in mitochondria and endoplasmic reticulum also are related to diminished axonal transport (Fig. 2). We do not know why microtubules are reduced during AD and aging; however, when considering the high energy demands of the brain, 25% of basal metabolism, intriguing clues emerge. Evolutionary selection of our species has worked to promote exceptionally high brain function that requires major energy production. Maintaining the intellectual effectiveness of the post-pubescent adult, a level that defines humanity may require a myriad of compensations to maintain brain function as available resources shrink during aging. During this process, neurons must tread carefully between life and death - ATP depletion or lowered function throughout their long life. Diminished axonal transport (Terry, 1996, 2000; Praprotnik et aI., 1996a,b),

Q

Normal

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Alzheimer

Fig.2. Mitochondrial abnormalities in Alzheimer disease may result from reduced axonal transport with consequent increased autophagocytosis in the cell body

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microtubules (Cash et aI., unpublished observations) and Na/K ATPase (Hattori et aI., 1998) are all measures neurons can and do take during AD to reduce their energy consumption. In aging , the changes result in subtle cognitive loss, a precursor to AD. We suggest that in AD the need for dramatic compensation leaves the system unable to maintain normal function. The energy rationing while near "normal" function when completely adopted leads to loss of function that may spare neurons from death but leave them nonfunctioning. An analogy to these hypotheses can be found in hibernation where neurons have lowered their energy demands and are refractory to injury (Zhou et aI., 2001). That reduced brain metabolism is the earliest change in AD in genetically predisposed individuals (Small et aI., 1996), and like oxidative damage is seen with aging. This finding suggests that metabolic diminution may be the most critical underlying feature, one that requires massive compensatory changes of the scope observed in all individuals suffering from AD. References Corral-Debrinski M, Horton T, Lott MT, Shoffner JM , McKee AC, Beal MF , Graham BH, Wallace DC (1994) Marked changes in mitochondrial DNA deletion levels in Alzheimer brains. Genomics 23: 47]-476 Cuajungco MP, Goldstein LE, Nunomura A , Smith MA, Lim JT, Atwood CS, Huang X, Farrag YW, Perry G , Bush AI (2000) Evidence that the B-amyloid plaques of Alzheimer's disease represent the redox-silencing and entombment of AB by zinc. J BioI Chern 275: 19439-19442 Hattori N, Kitagawa K, Higashida T, Yagyu K, Shimohama S, Wataya T, Perry G, Smith MA, Inagaki C (1998) Cl--ATPase and Na +/K +-ATPase activities in Alzheimer's disease brains. Neurosci Lett 254: 141-144 Hirai K, Aliev G, Nunomura A , Fujioka H, Russell RL, Atwood CS, Johnson AB, Kress Y, Vinters HV, Tabaton M, Shimohama S, Cash AD, Siedlak SL, Harris PLR, Jones PK, Petersen RB, Perry G, Smith MA (2001) Mitochondrial abnormalities in Alzheimer's disease. J Neurosci 21: 3017-3023 Nunomura A, Perry G, Pappolla MA, Wade R, Hirai K, Chiba S, Smith MA (1999) RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer's disease. J Neurosci 19: 1959-1964 Nunomura A, Perry G , Pappolla MA, Friedland RP, Hirai K, Chiba S, Smith MA (2000) Neuronal oxidative stress precedes amyloid-B deposition in Down syndrome. J Neuropathol Exp Neurol59: 1011-1017 Nunomura A , Perry G, Aliev G, Hirai K, Takeda A , Balraj EK, Jones PK , Ghanbari H , Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA (2001) Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol60: 759-767 Pappolla MA, Omar RA, Chyan Y-J, Ghiso J, Hsiao K, Bozner P, Perry G, Smith MA , Cruz-Sanchez F (2001) Induction of NADPH cytochrome P450 reductase by the Alzheimer B-protein. Amyloid as a "foreign body". J Neurochem 78: 121-128 Perez M, Cuadros R, Smith MA, Perry G, Avila J (2000) Phosphorylated , but not native, tau protein assembles following reaction with the lipid peroxidation product, 4hydroxy-2-nonenal. FEBS Lett 486: 270-274 Perry G, Nunomura A, Raina AK , Smith MA (2000) Amyloid-B junkies. Lancet 355: 757 Perry G, Zhu X, Smith MA (2001) Do neurons have a choice in death? Am J Pathoi 158: ]-2

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Praprotnik D , Smith MA, Richey PL, Vinters HV, Perry G (1996a) Plasma membrane fragility in dystrophic neurites in senile plaques of Alzheimer's disease: an index of oxidative stress. Acta Neuropathol 91: 1-5 Praprotnik D , Smith MA, Richey PL, Vinters HV, Perry G (1996b) Filament heterogeneity within the dystrophic neurites of senile plaques suggests blockage of fast axonal transport in Alzheimer's disease. Acta Neuropathol 91: 226-235 Sayre LM, Zelasko DA, Harris PLR, Perry G, Salomon RG, Smith MA (1997) 4Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer's disease. J Neurochem 68: 2092-2097 Shimohama S, Tanino H, Kawakami N, Okamura N, Kodama H, Yamaguchi T, Hayakawa T, Nunomura A , Chiba S, Perry G, Smith MA, Fujimoto S (2000) Activation of NADPH oxidase in Alzheimer's disease brains. Biochem Biophys Res Commun 273: 5-9 Small GW, Komo S, La Rue A , Saxena S, Phelps ME, Mazziotta JC, Saunders AM , Haines JL, Pericak-Vance MA, Roses AD (1996) Early detection of Alzheimer's disease by combining apolipoprotein E and neuroimaging. Ann NY Acad Sci 802: 7078 Smith CD, Carney JM, Starke-Reed PE, Oliver CN, Stadtman ER, Floyd RA, Markesbery WR (1991) Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc Natl Acad Sci USA 88: 1054010543 Smith MA, Taneda S, Richey PL, Miyata S, Yan S-D , Stern D, Sayre LM , Monnier VM , Perry G (1994) Advanced Maillard reaction end products are associated with Alzheimer disease pathology. Proc Natl Acad Sci USA 91: 5710-5714 Smith MA, Perry G, Richey PL, Sayre LM, Anderson VE, Beal MF, Kowall N (1996) Oxidative damage in Alzheimer's. Nature 382: 120-121 Smith MA, Harris PLR, Sayre LM , Beckman JS, Perry G (1997a) Widespread peroxynitrite-mediated damage in Alzheimer's disease. J Neurosci 17: 2653-2657 Smith MA, Harris PLR, Sayre LM , Perry G (1997b) Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci USA 94: 9866-9868 Smith MA, Sayre LM , Anderson VE, Harris PLR, Beal MF, Kowall N, Perry G (1998) Cytochemical demonstration of oxidative damage in Alzheimer disease by immunochemical enhancement of the carbonyl reaction with 2,4-dinitrophenylhydrazine. J Histochem Cytochem 46: 731-735 Smith MA, Jo seph JA, Pe rry G (2000) Arson: tracking the culprit in Alzheimer's disease. Ann NY Acad Sci 924: 35-38 Takeda A , Perry G, Abraham NG, Dwyer BE, Kutty RK, Laitinen IT, Petersen RB , Smith MA (2000a) Overexpression of heme oxygenase in neuronal cells, the possible interaction with tau. J BioI Chern 275: 5395-5399 Takeda A, Smith MA, Avila J, Nunomura A, Siedlak SL, Zhu X, Perry G, Sayre LM (2000b) In Alzheimer's disease, hem e oxygenase is coincident with Alz50 , an epitope of L induced by 4-hydroxy-2-nonenal modification. J Neurochem 75: 12341241 Terry RD (1996) The pathogenesis of Alzheimer disease: an alternative to the amyloid hypothesis. J Neuropathol Exp Neurol 55: 1023-1025 Terry RD (2000) Cell death or synaptic loss in Alzheimer disease. J Neuropathol Exp Neurol59: 1118-1119 Wallace DC, Stugard C, Murdock D, Schurr T, Brown MD (1997) Ancient mtDNA sequences in the human nuclear genome: a potential source of errors in identifying pathogenic mutations. Proc Natl Acad Sci USA 94: 14900-14905 Wataya T, Nunomura A, Smith MA, Siedlak SL, Harris PLR, Shimohama S, Szweda LI , Kaminski MA, Avila J, Price DL, Cleveland DW, Sayre LM , Perry G (2002) High molecular weight neurofilament proteins are physiological substrates of adduction by the lipid peroxidation product hydroxynonenal. J Biol Chern 277: 4644-4648

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Yan SD, Yan SF, Chen X, Fu J, Chen M, Kuppusamy P, Smith MA , Perry G, Godman GC , Nawroth P, Zweier JL, Stern D (1995) Non-enzymatically glycated tau in Alzheimer's disease induces neuronal oxidant stress resulting in cytokine gene expression and release of amyloid B-peptide. Nat Med 1: 693-699 Zhou F, Zhu X, Castellani RJ, Stimmelymayr R , Perry G , Smith MA, Drew KL (2001) Hibernation, a model of neuroprotection. Am J Pathol158: 2145-2151 Authors' address: G. Perry, Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, U.S.A., e-mail : [email protected]

Dysregulation of neuronal differentiation and cell cycle control in Alzheimer's disease T. Arendt Depar tment of Neuroanatomy, Paul Fle chsig Institute for Brain R esearch, University of Leipzig, Federal Republic of Germany

Summary. Degeneration in Alzheimer's disease primarily occurs in those neurons that in the adult brain retain, a high degree of structural plasticity and, is associated with the activation of mitogenic signaling and cell cycle activation. Brain areas affected by neurofibrillary degeneration in Alzheimer's disease are structures involved in the regulation of "higher brain functions " that become increasingly predominant as the evolutionary process of encephalization progresses. The functions these areas subserve require a life-long adaptive reorganization of neuronal connectivity. With the increasing need during evolution to organize brain structures of increasing complexity, these processes of dynamic stabilization and de-stabilization become more and more important but might also provide the basis for an increasing rate of failure. The hypothesis is put forward that it is the labile state of differentiation of a subset of neurons in the adult brain that allows for ongoing morphoregulatory processes after development is completed but at the same time renders these neurons particularly vulnerable. Interferring with neuronal differentiation control might, thus , be a potential strategy to prevent neurodegeneration in Alzheimer's disease and related disorders.

AD pathology follows the pattern of neuroplasticity which is a developmental pattern

Neurofibrillary degeneration in Alzheimer's disease (AD) is not evenly distributed throughout the brain but follows a certain hierarchy (Braak and Braak, 1991). It primarily affects those neuronal systems that playa crucial role in "higher brain functions" and, thus, become increasingly predominant as the evolutionary process of encephalization progresses such as hippocampus , neocortical association areas and cholinergic basal forebrain neurons. At the same time, these areas of the brain take the longest to mature during childhood and adolescence (Braak and Braak, 1996) and display a high degree of structural plasticity throughout life (Arendt et aI., 1998a). It is, therefore, hypothesized that those neurons primarily affected in AD, are neurons that in the normal adult brain retain immature features and are in a "labile state of

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T . Arendt developmental sequence limb ic cortex

young

degree of plasticity in the adult brain plastic

vulnerability aga inst PHF vulnerable

• transentorhina l co rtex • entorhinal co rte x • sub iculum I CA 1

non-primary association cortex • areae 37 , 40 , 46

primar:v sensory assoclatlon cortex • areae 7, 18,22

primary sensory and motor cortex • areae 17 , 41, 4

old

rigid

resistant

Fig. 1. Areas of the brain that take the longest to mature during childhood and adolescence retain a high degree of plasticity throughout life and display the highest degree of vulnerability during aging and in AD (modified after Arendt, 2001)

differentiation" which renders them particularly vulnerable (Arendt, 2001) (Fig. 1). Mitogenic signaling in neurons is abnormally activated in AD

Neurodegeneration in Alzheimer's disease (AD) is associated with the appearance of aberrant neuritic growth profiles that precede formation of paired helical filaments (Arendt et aI., 1986, 1997) making a primary pathogenetic role of aberrancies of growth and proliferation regulating mechanisms in neurons very likely. Numerous neurotrophic and potentially mitogenic compounds are elevated early in the course of the disease and are found highly enriched in plaques (Table 1 and 2). A variety of these factors activate an intracellular cascade of mitogenic signaling mediated through the mitogen activated protein kinase (MAPK) pathway that is also involved in modulation of the expression and posttranslational processing of APP and tau protein (Greenberg et al., 1994; Mills et al., 1997; Sadot et al., 1998). The activation of cell surface receptors of these trophic factors is linked to the downstream MAPK-cascade by p2lras, a small G-protein also activated by nitric oxide (NO) and intermediates generated through oxidative stress (Yun et al., 1998) (Fig. 2). During brain development, p2lras is involved in the regulation of the GO/G 1 transition of the cell cycle and might, thus, be a critical regulator for cellular proliferation, differentiation and apoptosis (Borasio et al., 1996; Ferrari and Greene, 1994). In AD, the p2lras-protein as well as major identified downstream elements of the MAPK-cascade, such as Raf-kinase,

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Table 1. Plaques are enriched in growth promoting factors and/or their receptors -

-

basic fibroblast growth factor (bFGF) hepatocyte growth factor (HGF/SF) platelet-derived growth factor (PDGF) epidermal growth factor receptor (EGF-R) TrkA and TrkB receptors SIOO beta proteoglycans (heparan sulfate keratan sulfate dermatan sulfate chondroitin sulfate) intercellular cell adhesion molecule ICAM-I integrins collagen laminin telencephalin Modified after Arendt (2001)

Table 2. Tangle bearing neurons and dystrophic neurites contain growth associated proteins -

GAP-43 Thy-1 collagen IV laminin integrin receptor VLA6 heparin binding growth associated molecule (HB-GAM) transforming growth factor (TGF)-beta 2 neuronal growth associated protein SCG10 spectrin Nand C terminal APP-fragments Modified after Arendt (2001)

p14-3-3, mitogen activated protein kinase kinase (MAPKK, MEK) and the MAP-kinases ERK1 and EKR2 are elevated at very early stages of the disease (Arendt et al., 1995b; Gartner et al., 1995, 1999) (Fig. 3,4). The strong expression of these kinases associated with neurofibrillary degeneration as well as the subcellular translocation of MEK, ERK1 and ERK2, indicating their activation, suggests that a developmentally immature condition of neurons might be of critical importance in the pathomechanism of AD.

Neuronal differentiation control in AD is critically impaired early in the course of the disease The re-expression of developmentally regulated genes in AD as well as the induction of posttranslational modifications and accumulation of gene prod-

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ucts to an extent which goes beyond those observed during regeneration indicate a process of de-differentiation. Recent evidence for a dysfunction of cell cycle regulation in AD in various cell types including neurons (Arendt et al., 1996, 1998b; McShea et al., 1997; Nagy et al., 1997a,b; Smith and Lippa, 1995; Vincent et al., 1996; Busser et al., 1998) supports the original suggestion of a link between neurodegeneration in AD and cell-cycle related events (Heintz, 1993; Arendt, 1993). Expression of the cyclin-dependent kinases cdk4 and cdk6 that are critical regulators of the activation and orderly progression through the cell cycle as well as their positive and negative regulators, cyclins and inhibitors of the INK4-family, respectively, are elevated in AD (Fig. 5). Observations of an increased expression of the oncogene p21ras and a cell cycle dysregulation in AD, are paralleled by experimental in vitro studies showing that expression of p21ras in primary human or rodent cells results in a permanent G1 arrest. This arrest induced by p2lras is accompanied by accumulation of p16INK4a, and is phenotypically identical to premature cellular senescence (Serrano et a1., 1997) . Induction of dominant-inhibitory p21ras, furthermore, can rescue neuronally differentiated PC12 cells from death caused by NGF withdrawal, implying a relationship between proliferative capacity and cell death. Recent studies have indeed shown that a high degree of structural neuronal plasticity might neurons predispose to neurofibrillary degeneration and cell death in AD (Arendt et al., 1995a, 1998a). Activation of an autocrine loop might exacerbate neurodegeneration

NO might be a key mediator linking cellular activity to gene expression and long-lasting neuronal responses through activating p21ras by redox sensitive modulation (Dawson et al., 1998). In AD, nNOS can be detected in pyramidal neurons in early stages of degeneration. Expression of nNOS in these neurons is highly co-localized with p21ras and p16INK4a(Luth et al., 2000) (Fig. 6). Thus, an autocrine loop may exist within cells, whereby NO activates p21ras that in turn leads to cellular activation and stimulation of NOS expres-

Fig. 2. Cartoon of principal components of the p21ras-dependent signaling pathway involved in the control of cellular proliferation, differentiation and apoptosis Fig. 3. Expression of p21ras quantified by ELISA and detected by immunohistochemistry. P21ras is elevated early in the course of the disease and is highly enriche d in plaques, plaque-associated astrocytes as well as in tangle-bearing neurons (modified after Gartner et a1. , 1995) Fig. 4. Expression of the MAP kinases ERK1/2 quantified by ELISA and detected by immunohistochemistry. ERK1/2 are elevated early in the course of the disease. The subcellular translocation into neuronal nuclei is an indicator of enzyme activation (modified after Arendt et al., 1995b)

cyclin 0

CDK4 cyclin D p16/NK4a p27KJP 1

CDK4

CDK5 CDK1

Fig. 5. Critical regulators of the entry and progression through the cell cycle, i.e. markers of the Gl-phase, S-phase and G2-phase are highly expressed in potentially vuln erable cortical pyramidal neurons in AD

Fig. 6. Double immunofluorescence for nNOS, p21ras and p16 INK4a. The ab errant expression of nNOS in pyramidal neurons is highly co-loc alized with p21ras and p16INK4a. Interneurons expressing nNOS con stitutively, on the contrary, express neither p2lras nor p16INK4a (arrow) (modified after LOth et aI., 2000)

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sion (Lander et al., 1995). The co-expression of NOS and p2lras in neurons vulnerable to neurofibrillary degeneration early in the course of AD clearly provide the basis for a feed back mechanism that might exacerbate the progression of neurodegeneration in a self propagating manner. This self perpetuation of a process likely associated with limited prospects of physiological control and termination might be the critical switch converting two potentially neuroprotective mechanisms such as NO and p2lras dependent signaling into a disease process leading to slowly but continously progressing neuronal death. Interference with mitogenic signaling mechanisms aiming at stabilizing the state of neuronal differentiation and preventing cell cycle re-entry might, thus, be a potential strategy to prevent neurodgeneration or at least to slow down its rate of progression.

Acknowledgements Support by the Bundesministerium fur Bildung, Forschung und Technologie (BMBF), Interdisziplinares Zentrum fur Klinische Forschung (IZKF) at the University of Leipzig (0IKS9504, Project Cl) and the European Commission (QLK6-CT-1999-02112) is gratefully acknowledged.

References Arendt Th (1993) Neuronal dedifferentiation and degeneration in Alzheimer's disease. BioI Chern Hoppe-Seyler 374: 911-912 Arendt Th (2001) Alzheimer's disease as a disorder of mechanisms underlaying structural brain self-organization. Commentary. Neuroscience 102: 723-765 Arendt Th, Zvegintseva HG, Leontovich TA (1986) Dendritic changes in the basal nucleus of Meynert and in the diagonal band nucleus in Alzheimer's disease-a quantitative golgi investigation. Neurosci 19: 1265-1278 Arendt Th, Bruckner MK, Bigl V, Marcova L (1995a) Dendritic reorganization in the basal forebrain under degenerative conditions and its defects in Alzheimer's disease. II. Ageing, Korsakoff's disease, Parkinson's disease, and Alzheimer's disease. J Comp Neurol 351: 189-222 Arendt Th, Holzer M, Grossmann A, Zedlick D, Bruckner MK (1995b) Increased expression and subcellular translocation of the mitogen activated protein kinase kinase and mitogen-activated protein kinase in Alzheimer's disease. Neurosci 68: 5-18 Arendt Th, Redel L, Gartner U, Holzer M (1996) Expression of the cyclin-dependent kinase inhibitor p16 in Alzheimer's disease. Neurorep 7: 3047-3049 Arendt Th, Schindler C, Bruckner MK, Eschrisch K, Bigl V, Zedlick D, Marcova L (1997) Plastic neuronal remodeling is impaired in patients with Alzheimer's disease carrying apolipoprotein e4 Allele. J Neurosci 17: 516-529 Arendt Th, Bruckner MK, Gertz HJ, Marcova L (1998a) Cortical distribution of neurofibrillary tangles in Alzheimer's disease matches the pattern of neurones that retain their capacity of plastic remodelling in the adult brain. Neurosci 83: 9911002 Arendt Th, Holzer M, Gartner U, Bruckner MK (1998b) Aberrancies in signal transduction and cell cycle related events in Alzheimer's disease. J Neural Transm [Suppl] 54: 147-158

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Borasio GD, Ma rkus A , Heumann R, Ghezzi C, Sampietro A, Wittinghofer A, Silani V (1996) Ras p21 protein promotes sur vival and differentiation of human embryonic neural crest-derived cells. Neurosci 73: 1121-1127 Braak H , Braak E (1991) Neuropathological stageing of Alzheimer related changes. Acta Neuropathol 82: 239-259 Braak H, Braak E (1996) Development of Alzheimer-related neurofibrillary changes in the neocortex inversely recapitulates cortical myelogenesis. Acta Neuropathol 92: 197-201 Busser J, Geldmacher DS , Herrup K (1998) Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer's disease brain. J Neurosci 18: 28012807 Dawson TM, Sasaki M, Gonzales-Zulueta M, Dawson VL (1998) Regulation of neuronal nitric oxide synthase and identification of novel nitric oxide signaling pathways. Prog Brain Res 118: 3-11 Ferrari G, Greene LA (1994) Proliferative inhibition by dominant-negative Ras rescues naive and neuronally differentiated PC12 cells from apoptotic death. EMBO J 13: 5922-5928 Gartner U , Holzer M, Heumann R , Arendt Th (1995) Induction of p21ras in Alzheimer pathology. Neuroreport 6: 1313-1316 Gartner U , Holzer M, Arendt Th (1999) Elevated expression of p21ras is an early event in Alzheimer's disease and precedes neurofibrillary degeneration. Neurosci 91: 15 Greenberg SM, Koo EH, Selkoe J, Qiu WQ, Kosik KS (1994) Secreted beta amyloid precursor protein stimulates mitogen activated protein kinase and enhances tau phosphorylation. Proc Nat! Acad Sci USA 91: 7104-7108 Heintz N (1993) cell-death and the cell-cycle - a relationship between transformation and neurodegeneration. Trends Biochem Sci 18: 157-159 Lander HM, Ogiste JS, Pearce SFA, Levi R, Novogrodsky A (1995) Nitric oxidestimulated guanine nucleotide exchange on p21 ras. J Biol Chern 270: 70177020 Luth HJ, Holzer M, Gertz HJ, Arendt Th (2000) Aberrant expression of nNOS in pyramidal neurons in Alzheimer's disease is highly co-localized with p21 ras and p161NK4a. Brain R es 852: 45-55 McShea A, Harris PL , Webster KR, Wahl AF, Smith MA (1997) Abnormal expression of the cell cycle regulators P16 and CDK4 in Alzheimer's disease. Am J Pathol 150: 1933-1939 Mills J , Charest DL, Lam F, Beyreuther K, Ida N, Pelech SL, Reiner PB (1997) Regulation of amyloid precursor protein catabolism involves the mitogen-activated protein kinase signal transduction pathway. J Neurosci 17: 9415-9422 Nagy Z, Esiri MM, Cato AM, Smith AD (1997) Cell cycle markers in the hippocampus in Alzheimer's disease. Acta Neuropathol 94: 6-15 Nagy Z , Esiri MM, Smith AD (1997) Expression of cell division markers in the hippocampus in Alzheimer's disease and other neurodegenerative conditions. Acta Neuropathol 93: 294-300 Sadot E , Jaaro H , Seger R, Ginzburg I (1998) Ras-signaling pathways: positive and negative regulation of tau expression in PC12 cells. J Neurochem 70: 428431 Serrano M, Lin A W, McCurrach ME, Beach D, Lowe SW (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88:593-602 Smith TW, Lippa CF (1995) Ki-67 immunoreactivity in Alzheimer's disease and other neurodegenerative disorders. ] Neuropathol Exp Neurol 54: 297-303 Vincent I, Rosado M, Davies P (1996) Mitogenic mechanisms in Alzheimer's disease? J Cell Bioi 132: 413-425

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Yun HY, Gonzalez-Zulueta M, Dawson VL , Dawson TM (1998) Nitric oxide mediates Nmethyl-D-aspartate receptor-induced activation of p21 ras . Proc Nat! Acad Sci USA

95: 5773-5778 Authors' address: T. Arendt, Paul Flechsig Institute for Brain Research, Department of Neuroanatomy, University of Leipzig, Jahnallee 59, D-04109 Leipzig, Federal Republic of Germany, e-mail: [email protected]

A broader horizon of Alzheimer pathogenesis: ALZAS - an early serum biomarker? E. Kienzl', K. Jetlinger', B. Janetzky", H. SteindP, and J. Bergmann' VIENNA HEALTH BOARD and L. B. Institute of Clinical Neurobiology, Vienna, Austria -Universitatsklinikum Carl Gustav Dresden, University of Technology, Dresden, Federal Republic of Germany 3 Institute of Applied Microbiology (lAM), University of Agriculture, Vienna, Austria 4 ALTEGEN Inc, Hamburg, Federal Republic of Germany 1

Summary. Recently, a novel risk gene protein expressed in elderly patients with the diagnosis of Alzheimer disease (AD) was discovered on chromosome 21 within the APP (amyloid precursor protein) region. This 79 amino acid protein, ALZAS (Alzheimer Associated Protein) contains the ~-amyloid peptide 1-42 fragment, the APP transmembrane signal, and a unique 12 amino acid c-terminal which is not present in any known allele of the APP gene. Reverse transcription-Pf.R revealed that the transcript of ALZAS was expressed in cortical and hippocampal regions of human Alzheimer disease brain as well as in leukocytes derived from AD patients. Most specifically, an endogenous antibody was found in patients with confirmed AD, in patients with depression, and in subjects suggested to have presymptomatic AD, where it was directed against epitopes within the intron encoded amino acid c-terminal sequence.

Introduction

Since Alzheimer's first case report in 1907 until recently, very little was known about the causes of Alzheimer disease (AD), except that it is associated with extensive neuronal loss, extracellular amyloid deposits and intracellular neurofibrillary tangles. Therefore, the theory of AD pathogenesis was based on its pathological hallmarks: plaques, with their core of ~-amyloid peptide (A~), cleaved from amyloid precursor protein (APP), and tangles which consist of hyperphosphorylated microtubule-associated tau protein. Since 1990, it is evident that AD can be caused by autosomal dominant mutations of different genes: the APP gene, located at chromosome 21, and Presenilin 1 (PSl) and Presenilin 2 (PS2), encoded at chromosomes 14 and 1. As familial forms account for only 4-8% of all AD cases , genetic risk factors that significantly increase the likelihood of developing late-onset AD are still to be identified.

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Apolipoprotein (Apo) E , the major serum protein involved in cholesterol metabolism, transport and storage, is polymorphic and encoded by three alleles (ApoEe2, -3, -4), of which the ApoEe3 allele is the most common. The prevalence of AD subjects having either one or two copies of the ApoEe4 allele (45-60%) significantly exceeds that in control populations (Saunders, 2000; Selkoe, 2000). Among the biological markers that may improve the diagnostic accuracy for AD, are genetic mutations that cause or increase the risk of AD. However, at present, no biological markers for sporadic AD are available, which are superior to exact clinical and neuroimaging data. There is no objective ante mortem biochemical assay, that relates to the pathophysiology of AD. Moreover, the search for a test, which is non-invasive, simple, cheap and userfriendly, should therefore be directed at accessible body fluids (Consensus Report, 1998; Jellinger and Rosler, 2000). The aim of the present molecular genetic studies was to identify genes that may contribute to the risk of psychiatric / neurodegenerative disorders in the aged. The method already successfully applied to find alternative genes as putative causative factors of neurodegenerative disorders is "disease gene discovery by positional searching" (DGDPS). Such genes are transcribed in any orientation within the chromosomal locus occupied by another gene. These strategies gave rise to the detection of a new gene, referred to as "Alzheimer associated", not yet described in the literature, and the expression of the related protein. Transcription of the Alzheimer Associated (ALZAS) gene, which is encoded within the APP gene at human chromosome 21 (Fig. 1), is programmed by a variety of regulatory elements implying gene activation by many different factors. Materials and methods

Blood sampling Patients with clinical probable AD and age matched control subjects were recruited fro m several neurology and psychiatry departments. They were classified according their cognitive decline, using strict clinical diagnostic criteria either for major depression according to DSM-IV (American Psychiatric Association, 1994) and/or probable AD according to NINCDS-ADRA criteria (McKann et al., 1984), with Mini-Mental State Examination (MMSE) scores < 24 (Folstein et al., 1975). Blood samples were collected and stored at -20 0

•

ELISA Assay ELISA was performed according th e protocol of lAM: The te st is performed in a standard antibody-trap ELISA platform. A sample of 50 III of diluted serum in buffer was added to the ELISA plate coated with a chemically synthesized epitope of a 20 aa ALZAS peptide fragment. Endogenous anti-ct l2ab in the serum trapped on the epitope is detected with enzyme linked anti-human IgG and measured.

ALZAS - early biomarker for AD?

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Post mortem brain tissue Immunohistochemical and morphological studies of brain specimens of patients fulfilling the clinical criteria for major depression, AD, and nondemented age-matched controls were performed using routine staining and immunohistochemcial methods for ~-amyloid and tau protein.

Immunohistochemistry Sections of frozen postmortem brain tissue were mounted on precoated Superfrost slides and stored at -20°e. For immunocytochemical labelling the standard method of lAM was applied. Several steps, including incubation with the primary antibody (mono-specific anti-ctl2 IgG rabbit antibody) and incubation with the secondary antibody. Rabbit ct12ALZAS antibodies suggested to recognize with high affinity epitopes on ALZAS-antigen were applied with a fluorophore-conjugated secondary antibody. Sections were examined on a Confocal Microscope equipped with Argon LASER.

Results

The capture ELISA was developed to measure levels of the antibody in serum using chemically synthesized epitopes of the ct12 end of ALZAS (Fig. 1). Humans expressing ALZAS have been found to express specific endogenous antibodies directed against epitopes within the amino acid (aa) terminal sequence which is considered "foreign" by the immune system. In patients with mild cognitive impairment (Mel) suggesting presymptomatic dementia/AD, increased levels of the antibody were found as well as in some patients with major depression, suggesting that the antibody may be related to a predisposition for AD. The mean level of the endogenous antibodies in serum varied with the progression of the disease (lower in confirmed AD, as compared to the assumed presymptomatic and suspected AD patients with the diagnosis of major depression (Fig. 2a--c). Immunohistochemical studies on frozen human post mortem AD brain tissue confirmed the localization of ALZAS in neurofibrillary tangles and neuritic plaques in hippocampus and frontal cortex (Fig. 3a, b) . Young human control subjects and patients with Huntington disease were negative.

Discussion

The neuropathological diagnosis of AD based on the presence of a suitable large number of neuritic plaques and neurofibrillary tangles (Mirra et aI., 1993) is no guarantee that all these cases necessarily share the same pathogenesis even if the ~ amyloid (A~) cascade of AD may be valid (Selkoe, 2001). Recent studies have shown a significant correlation between cognitive decline and A~1-42 containing amyloid plaques supporting the important role of A~ in mediating initial pathogenic events in AD dementia (Parvathy et aI., 2001).

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Many studies confirm the AB toxicity in inducing oxidative stress, lipid peroxidation, mitochondrial damage, ion channels, and disorders of calcium homeostasis (Butterfield et al., 2001; Lin et al., 2001). The larger B-amyloid precursor protein (APP) is regarded as single source of the insoluble form of AB. However, it is still uncertain if its neurotoxicity is mediated by intracellular or extracellular events (Rhee et al., 1998; Wilson et al., ).999; Pimplikar, 2002). The immunological answer may explain the variety of inflammatory proteins including complement activation products, acute phase proteins, cytokines, and cell adhesion molecules identified in the vicinity of plaques in AD brain and support the hypothesis that inflammation is implicated in AD (McGeer et al., 2000; Strohmeyer and Rogers, 2001).

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APP, the precursor of A~ , occurs in all cells and tissues of the body, but the clinically important A~ depositions occur predominantly in the brain. APP consists of at least 10 isoforms that are generated by alternative mRNA splicing, principally of exons 7, 8, and 15, of the APP gene on chromosome 21 (Li et al., 1999). It is suggested that a percentage of APP may function as a cell surface receptor on neurons, transducing signals from the extracellular matrix to the interior of the cell (Oiu et al., 1995). In neurons, APP is present in axon terminals (lkin et al., 1996). APP expressed in lymphocytes, monocytes, neutrophils, macrophages, and microglia may correlate with the dual functions of nonadherence and adherence in the immune sys-

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Fig. 3. a ALZAS immunoreactivity in AD (fresh frozen pm brain tissue): frontal cortex, showing neurofibrillary tangle (arrow) b ALZAS immunoreactivity in AD (fresh froze pm brain tissue): hippocampus, multiple plaques (arrow)

tern. Microglia in the CNS is functionally adequate to macrophages and constitute the main effector cells of the immune response in the CNS. APP expression at the cell surface of lymphocytes and monocytes can be increased when the cells are exposed to activation signals such as antibodies to the antigen receptors of Band T cells and secrete amyloidogenic A~ peptides (Bullido et al., 1996). A novel small gene in an intron of the APP gene, found in brain tissue and leukocytes, has a single-nucleotide polymorphism related to AD,

ALZAS - early biomarker for AD?

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and contains the A~ sequence (Fig. 1) suggesting that A~ deposits in AD brain also may derive from ALZAS peptide that may not only be produced locally but also may be derived from the circulation. The mechanisms involved in the delivery of A~ across the blood-brain barrier (BBB) are not clear, but it has been suggested that A~ can enter into the brain via the BBB (Koudinov et al., 1994). Circulating Aj3 may contribute to cerebrovascular amyloid, one of the pathological features of AD (Wisniewski and Wegiel, 1994). ALZAS-peptide may also clarify "neuroinflammation" which so far has not been adequately described for AD. The immune staining of neurofibrillary tangles with the ALZAS antibody gives rise to speculate that ALZAS in some way may be "upstream" of tau in the AD pathology cascade. Moreover, the explanation of late-onset depression as a risk factor and first sign for dementia may be found in the strong immunreactivity against the "ALZAS" - protein and activation of the HPA-axis. Findings from casecontrol studies were confirmed by the EURODEM Group meta-analysis suggesting a history of late-onset depression as risk factor for dementia (Geerlings et al., 2000). Postmortem studies demonstrated that depression can be characterized by specific histopathological changes in both neurons and glial cells (Kokmen et al., 1991). Furthermore, a reduced volume of the hippocampus has been reported in subjects with a history of depression (Evenhuis, 1997). The loss of hippocampal volume is suggested to be correlated with cumulative hippocampal injury and repeated stress during recurrent depressive episodes.

The following working hypothesis is suggested

-

ALZAS protein has been found to be expressed in AD brain tissue Lymphocytes crossing the BBB may transport ALZAS protein Neuronal cells may recognize ALZAS as a pathogenic factor of the EAE-type and both microglia and astrocytes may induce an autoimmune inflammatory reaction. Via "inflammation", elevated cytokine/chemokine expression and activation of kinases (MEK/ERK pathway), ALZAS peptide may induce Tau-phosphorylation ALZAS may compete with APP for insertion sites in intercellular and cell surface membranes, which may disrupt neural and lipid signaling and could induce membrane breakdown observed in AD.

Further studies are neded to further elucidate (1) the role of ALZAS in the pathogenesis of AD and (2) its validity as an early biological marker for both AD and late onset depression.

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References Bullido M, Munoz-Fernandez MA, Recuero M, Fresno M, Valdivieso F (1996) Alzheimer's amyloid precursor pprotein is expressed on the surface of hematopoietic cells upon activation. Biochim Biophys Acta 1313: 54-62 Butterfield DA, Drake J, Pocernich C, Castegna A (2001) Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid B-peptide. TIMM 7: 548-554 Evenhuis HM (1997) The natural history of dementia in ageing people with intellectual disability. J Intellect Disabil Res 41: 92-96 Geerlings MI , Scho evers RA, Beekman AT, Jonker C, Deeg DJ, Schmand B, Ader HJ, Bouter LM , Van Tilburg W (2000) Depression and risk of cognitive decline and Alzheimer's disease. Results of two prospective community-based studies in The Netherlands. Br J Psychiatry 176: 568-75 Ikin AF, Annaert WG, Takei K, De Camilli P, Jahn R , Greengard P, Buxbaum JD (1996) Alzheimer amyloid protein precursor is localizsed in nerve terminal preparations to RabS-containing vesicular organelles distinct from those implicated in the synaptic vesicl e pathway. J BioI Chern 271: 31783-31786 Jellinger KA, Rosler N (2000) Neuropathology and biological markers of deg enerative dementias. Internist 41: 524-537 Kokmen E, Beard CM , Chandra V, Offord KP, Schoenberg BS, Ballard DJ (1991) Clinical risk factors for Alzheimer's diseasee: a population-based case-control study. Neurology 41: 1393-1397 Koudinov A , Matsubara E, Frangione B, Ghiso J (1994) The soluble form of Alzheimer's amyloid beta protein is complexed to high density lipoprotein 3 and very high density lipoprotein in normal human plasma. Biochem Biophys Res Commun 205: 11641171 Li QX, Fuller StJ. Beyreuther K, Masters CL (1999) The amyloid precursor protein of Alzheimer disease in human brain and blood. J Leuk Bio 66: 567-574 Lin H, Bhatia R, Lal R (2001) Amyloid beta protein forms ion channels: implications for Alzheimer's disease pathology. FASEB J 15: 2433-2444 McGeer PI, McGeer EO, Yasojima K (2000) Alzheimer disease and neuroinflammation. J Neural Transm [Suppl] 59: 53-57 Mirra SS, Hart MN, Terry RD (1993) Making the diagnosis of Alzheimer's disease. A primer for practicing pathologists. Arch Pathol Lab Med 117: 132-144 Parvathy S, Davies P, Haroutunian V, Purohit DP, Davis KL , Mohs RC, Park H , Moran TM, Chan JY , Buxbaum JD (2001) Correlation between ABx-40-, ABx-42-, and ABx-43-containing amyloid plaques and cognitive decline. Arch Neurol58: 20252032 Pimplikar SW (2002) ABPP , apoptosis and Alzheimer's disease. J Alzheimer Dis 4: 39-40 Qiu WQ, Ferreira A, Miller C, Koo EH, Selkoe OJ (1995) Cell-surface f3-amyloid precursor protein stimulates neurite outgrowth of hippocampal neurons in an isoformdependent manner. J Neurosci 15: 2157-2167 Rhee SK, Quist AP, Lal R (1998) Amyloid beta protein-(1-42) forms calcium-permeable, Zn2 + -sensitive channel. J BioI Chern 273: 13379-13382 Saunders AM (2000) Apolipoprotein E and Alzheimer disease: an update on genetic and functional analyses. J Neuropathol Exp Neurol 59: 751-758 Selkoe DJ (2000) The genetics and molecular pathology of Alzheimer's disease: roles of amyloid and the presenilins. Neurol Clin 18: 903-922 Selkoe DJ (2001) Clearing the brain's amyloid cobwebs. Neuron 32(2): 177-180 Strohmeyer R, Rogers J (2001) Molecular and cellular mediators of Alzheimer's disease inflammation. J Alzheimer Dis 3: 131-157 The Ronald and Nancy Reagan Research Institute of the Alzheimer's Association and the National Institute on Aging Working Group (1998) Consensus report of the Working Group on "Molecular and biochemical markers of Alzheimer disease". Neurobiol Aging 19: 109-116

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Wilson CA, Doms RW, Lee VM (1999) Intracellular APP processing and A beta production in Alzheimer diseas e. J Neuropathol Exp Neurol58: 787-794 Wisniewski HM and Wegiel J (1994) ~-amyloid formation by myocytes of leptomeningeal vessels. Acta Neuropathol87: 233-241 Authors' address: Prof. K. Jellinger, MD , Institute of Clinical Neurobiology, Kenyongasse 18, A-I070 Vienna, Austria, e-mail: [email protected]

Is mild cognitive impairment bridging the gap between normal aging and Alzheimer's disease? G. Smith Department of Psychiatry and Psychology and Alzheimer's Disease Research Center, Ma yo Clinic, Rochester, MN , U.S .A.

Summary. Mild cognitive impairment (MCI) is one label applied to cognitive dysfunction that is beyond normal aging but of insufficient magnitude to qualify for the diagnosis dementia. Various opinions exist about the proportion of people who fall in this boundary condition, the rate at which they will progress to a full dementia syndrome, and the nature and extent of cognitive impairment at the time of MCI diagnosis. There are at least four important dimensions along which studies of boundary conditions must be compared in order to understand the discrepant findings . The first is the population frame sampled to establish MCI cohorts. The second is the nature of memory complaints. The third and fourth respectively, are the type of memory assessment and number and type of other cognitive domains assessed to identify and follow these groups. The contributions of each of these dimensions to the diagnosis of MCI and it's outcome are discussed.

Introduction

The early detection of Alzheimer's Disease (AD) has been a long sought goal in the neurosciences. This is because by the time the clinical diagnosis is established, significant neuropathological changes in the brain have taken place (Troncosco et al., 1996; Davis et al., 1999; Gilmor et al., 1999). Successful treatment at this stage may be difficult . Ideally, one would like to identify persons who are presymptomatic but at risk by virtue of a positive family history, genotype, or a cognitive and neuroimaging profile. Neurocognitive efforts at early identification of AD have focused on memory, as the consensus is that changes in memory are the earliest identifiable behavioral manifestations of AD. The difficulty is that normal aging also involves a change in memory function (Petersen et al., 1992). Thus overlap in distributions of memory function impede efforts to distinguish normal aging and earliest manifestations of AD. Over the past few decades, a variety of terms and diagnostic approaches have been promulgated to label the overlap or boundary area between normal aging and AD. Many approaches to discriminating amongst these two popu-

K. A. Jellinger et al. (eds.), Ageing and Dementia Current and Future Concepts © Springer-Verlag/Wein 2002

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COGNITIVE CONTINUUM

Normal

•

• Mild cognitive impairment

•

•

•

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•

Fig. 1. Conceptual framework for mild cognitive impairment

lations that create this boundary area have been offered. Diagnostic criteria have included: Benign and Malignant senescent forgetfulness (Kral, 1962) Age Associated Memory Impairment (Crook et a1., 1986), Age Consistent Memory Impairment and Late life Forgetfulness (Blackford and LaRue, 1989), Aging Associated Cognitive Decline (Levy, 1994), Questionable Dementia (Devanand et a1., 1997) and Mild Cognitive Impairment (Flicker et al., 1991). Mild cognitive impairment (MCI) has recently gained some acceptance as an important clinical concept and as a target for potential therapeutic intervention (Bowen et a1. , 1997; D aly et a1., 2000; Petersen et a1., 1999). As suggested in Fig. 1, MCI refers to that overlapping stage of cognitive function that could represent the lowest end of normal cognitive function or the earliest signs of clinically probable AD. In our clinical and research endeavors at the Mayo Clinic Alzheimer's Disease Research Center, we adopted MCI criteria, as listed in Table 1. In subsequent research, we noted that people so defined had a high, (but not absolute) risk of progressing to a full dementia syndrome. Our conversion rate is about 12 % per year over the initial 7 years of follow-up. We have found that persons with mild cognitive impairment as compared to controls and AD patients MCI patients have intermediate degrees of hippocampal atrophy (Jack et a1. , 1999), intermediate changes in regional metabolic pattern (Kantarci et a1. , 2000) and apolipoprotein E allelic frequencies similar to AD patients (Petersen et al., 1995) and that these same variables help predict progression to dementia. Table 1. Mild cognitive impairment criteria (Petersen et al., 1997) -

Memory complaint Normal general cognitive functioning Normal activities of daily living Memory impaired for age Not demented Clinical Dementia R ating Scale 0.5

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Table 2. Comparision of studies of boundary conditions Study

Pet ersen et al. (1999)

Morris et al. (2001)

Ritchie et al. (2001)

T arget sample

Mild Cognitive Impairment

CD R -.S a)DAT b) incipient DAT c) Questionable D em entia

a) "MCl" b) Age As sociat ed Cognitive D ecline

Questionable D em entia

Annual con version rate

12%

a) 12 % c) 4%

a) 6.5%

6%

Sampling Frame

Community Internal Med icine Practice

A dvertiseme nt solicited volunteers

General Practitioner Network

Adverti sement solicited volunteers

Target Sample

Community

Convenience

Population

Convenience

Memory tests

Immediat e and D elayed recall of unstructured verbal materi al

None (only immediate stru ctured rec all in other research)

Immediate and Delayed recall of structured verbal material

Immediate and D elayed recall of structured and unstructured verb al material

Other Cognitive D omains

Attention, Language , Visuospatial

None

Attention, Language, Visuospati al

Attention , Language, Visuospatial, E xecutive Function

b) 7 %

b) 28 %

Albert et al. (2001)

D AT D em entia of th e Alzheimer's Type

Of course, other diagnostic approaches continue to be offered and to evolve. These various diagnostic categories all seem to have the same underlying goal. That is, to identify persons at a significantly elevated risk to progress to the full dementia syndro me. They describe people whose cogni tive functioning is not normal, but who do not meet full criteria for dementia. A variety of studies have now been published regarding the rate at which people falling in thi s boundary condition actually have an underlying AD , the rate at which they will progress to a full dementia syndrome, and the nature and extent of cognitive impairment at th e time of diagnosis. A summary of selected studies in provided in Table 2. A re view of this literature reve als at least four important dimension s along which studies of boundary conditions must be compared in order to understand their discrepant findings : 1) The population sampled 2) The nature of memory complaint, 3) The typ es of memory assessment used 4) The number and type of other cognitive domains assessed. Population sampled

At Mayo, persons diagnosed as Mer initially present as a part of a clinical sample. The Mayo AD Patient Registry seeks to identify any person from our

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Community Internal Medicine practice who has a new complaint regarding memory recorded in their medical record. This complaint regarding memory may arise from the person themselves, their family or significant others, or based on the medical evaluation of their primary care physician. The complaint often arises in the course of the primary physicians review of systems during general medical evaluation, or evaluation of a different condition, or may be the specific reason medical care was sought. However, it is important to recognize that this represents a spontaneous memory complaint from a clinical sample. This is in distinction to MCI "patients" who are identified or solicited as part of a representative sample of older adults, or a normal volunteer sample. There is a clinical concern for Mayo Clinic patients. Other studies that have identified mild cognitive impairment in a clinical context include those by Tierney et al. (1996) and Bowen et al. (1997). A variety of other studies of boundary conditions have identified their cohorts by selecting normal, older adults (e.g. Albert, 2001). These may be controls in clinical trials or longitudinal aging studies, or may be a general population of normal elderly selected to be representative of the entire population. These persons are distinct, however, from those described above in that no one has proactively identified memory concerns in these persons. Populations of mild cognitive impairment are often culled from these normal samples by using a psychometric cut-off based on their memory performance. Thus, by definition, these are normal patients scoring at the lowest end of the memory score distributions. Selection in this fashion necessarily increases the likelihood that these MCI patients arise from the normal population depicted in Fig. 1, rather than cognitively impaired population. Nature of memory complaint Differences in the nature of memory complaints across studies is a corollary of the recruitment method. In clinical samples any memory complaint is generally "spontaneous" . It arise as a concern from some member of the health care process (e.g. patient, family, provider). Such memory concerns especially from family or physicians may have a better correspondance with objectively established cognitive dysfunction. In studies that recruit general or normal samples, memory complaint is typically established by administration of standardized subjective ratings of memory function. Numerous studies have demonstrated that scores on such instruments are more likely to associate with mood or self-efficacy than with actual cognitive dysfunction (Smith et al., 1996; Taylor et al., 1992) . Nature of memory assessment Another key difference across studies of boundary conditions is their approach to memory assessment. The term "memory impairment" has been used to describe a wide variety of cognitive impairments. These may have

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different neurosubstrates. Most studies have focused on episodic memory. However, even within the realm of episodic memory, differences may exist in the extent to which impairments are associated with risk for subsequent decline to dementia. It may be important to distinguish between encoding and retrieval phases of memory tasks. Encoding is typically assessed by aspects of immediate recall while retrieval is assessed by delayed recalL The encoding phase of memory is sensitive to aging effects alone, it may be insensitive relative to delayed recall phases for detecting incipient dementia (Petersen et al., 1992, 1994). For example, a recent study of nine people who died and were observed to have healthy brains, compared to five people who were nondemented at death but were observed to have AD changes in the brain, showed no cognitive differences between the groups. However, all memory test scores in this very small study focused on immediate recall (Goldman et al., 2001). Numersous studies suggest delayed recall measures appear to be most sensitive for early discrimination of dementia (Bondi et al., 1994; lvnik et al., 2000; Tierney et al., 1996) Studies which focus on immediate recall (encoding) versus delayed recall (retrieval) in estalishing the memory impairment criteria for MCI may engender very different samples. Number and type of cognitive domains assessed

The number and type of non-memory cognitive domains assessed in studies of MCI is important since MCI criteria typically stipulate no dementia. Commonly accepted dementia criteria require impairment in at least two cognitive domains. Since MCI patients have memory impairment, rigid statistical applications of dementia criteria exclude from MCI samples any person with scores falling below some cutoff in any non-memory domain. As the number of cognitive domains increases, there is an increasing probability that at least one other cognitive measure will fall below a given cutoff by chance alone. Since all cognitive domains tend to be correlated at least to a moderate degree (-.3 at a minimum), excluding non-demented persons with memory impairment from MCI samples because of a low score in another domain increases the probability that the memory scores for the remaining patients are spuriously low. A recent study provides an example of this problem (Ritchie et al., 2001). In this study 7 non-memory domains were assessed. Seventy five percent of persons with a loosely defined memory impairment (scores < -1 s.d. below age norms) also had at least one other score fall below this cutoff. Of the remaining 25% of their "MCI" patients, only 7% continued to have scores below their cutoff at follow-up. These investigators may have excluded persons with true memory impairments in their strict application of cufoffs. How can a person have two cognitive domains falling below an impairment cutoff and not have dementia? Two fundamentals of neuropsychology are relevant in this regard. First, a low score is not necessarily the same thing as impairment. A certain percentage of the normal population will score below clinical cutoffs on any measure. If the cutoff is liberally set at -1 s.d., as in the example above, 16% of the general population will fall below the cutoff.

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In persons with appropriate histories (e.g. low academic attainment, low IQ) it is entirely possible for a score below this cutoff to be deemed clinically normal for that person. Dementia criteria require a decline from a higher level of cognitive function. The second fundamental issue is that low scores on tests do not necessarily imply "significant impairment in social or occupational functioning" (APA, 1994). There are people who live normal day to day lives that would "fail" the Wisconsin Card Sorting Test. Some of these people are probably neuropsychologists! The neuropsychological battery used in the Mayo ADPR was developed before age appropriate norms were available on most tests of executive function. As a consequence executive function was not well measured in our patients. As we and others have begun measuring executive functioning in MCI patients we have noted that many do have poor scores in this cognitive domain. Yet, the clinician's judgment is that for these patients impairments in memory and executive function do not reach the criteria of substantially interfering with social or occupational functioning .

Conclusion

Boundary concepts remain important to dementia research and clinical practice. One such concept, Mild Cognitive Impairment appears associated with biomarkers of AD even before patients met AD criteria. MCI patients are at elevated risk for progressing to dementia. MCI samples are comprised of patients with incipient dementia and normal older persons with poor cognitive function. The relative proportion of each group in an MCI sample will be influenced by the sampling frame (clinical vs. general or normal) , the nature of the memory complaint (spontaneous vs. elicited), the type of memory assessment used in selecting patients (e.g. immediate vs. delayed recall) and the number, type and interpretation of measures of non-memory domains. These factors need to be considered when comparing outcomes from studies of boundary conditions.

Acknowledgements Supported by National Institute on Aging Grants AG15866 and AG16574.

References Albert MS, Moss MB, Tanzi R, Jones K (2001) Preclinical prediction of AD using neuropsychological Tests. J Int Neuropsychol Soc 7: 639-639 American Psychiatric Association (1994) Diagnostic and statistical manual of mental disorders IV. APA, Washington DC Blackford RC, LaRue A (1989) Criteria for diagnosing age associated memory impairment. Dev Neuropsychol 5: 295-306

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Bondi MW, Monsch AU, Galasko D, Butters N, et al (1994) Preclinical cognitive markers of dementia of the Alzheimer type. Neuropsychology 8: 374-384 Bowen J, Teri L, Kukull W, McCormick W, McCurry S, Larson E (1997) Progression to dementia in patients with isolated memory loss. Lancet 349: 763-765 Crook T, Bartus RT, Ferris SH, Whitehouse P, Cohen GD , Gershon S (1986) Ageassociated memory impairment: proposed diagnostic criteria and measures of clinical change - Report of a National Institute of Mental Health Work Group. Dev Neuropsychol2: 261-276 Daly E, Zaitchck D, Copeland M, Schmahmann J, Gunther J, Albert M (2000) Predicting conversion to Alzheimer's disease using standardized clinical information. Arch Neurol57: 675-680 Davis KL, Mohs RC, Marin D, et al (1999) Cholinergic markers in elderly patients with early signs of Alzheimer disease. JAMA 281: 1401-1406 Devanand DP, Folz M, Gorlyn M, Moeller JR, et al (1997) Questionable dementia: clinical course and predictors of outcome. J Am Geriatr Soc 45: 321-328 Flicker C, Ferris SH , Reisberg B (1991) Mild cognitive impairment in the elderly: pr edictors of dementia. Neurology 41: 1006-1009 Gilmor ML, Erickson JD , Varoqui H, et al (1999) Preservation of nucleus basalis neurons containing choline acetyltransferase and the vesicul ar acetylcholine transporter in the elderly with mild cognitive impairment and early Alzheimer's disease. J Comp Neurol 411: 693-704 Goldman WP, Price JL, Storandt M, Grant EA, McKeel DW, Rubin EH, Morris JC (2001) Absence of cognitive impairment or decline in preclinical Alzheimer's disease. Neurology 56: 381-367 Ivnik RJ, Smith GE, Petersen RC, Boeve BF, Kokmen E, Tangalos EG (2000) Predictive accuracy of four approaches to interpreting neuropsychological test data. Neuropsychology 14: 163-177 Jack CR, Petersen RC, Xu YC, O'Brien PC, Smith GE, Ivnik RJ, Boeve BF, Waring SC, Tangalos EG, Kokmen E (1999) Prediction of AD with MRI-based hippocampal volume in mild cognitive impairment. Neurology 52: 1397-1403 Kantarci K, Jack CR Jr, Xu YC, Campeau NG , O 'Brien PC, Smith GE, Ivnik RJ, Boeve BF, Kokmen E , Tangalos EG, Petersen RC (2000) Regional metabolic patterns in mild cognitive impairment and Alzheimer's disease: a 1H MRS study. Neurology 55: 210-217 Kral VA (1962) Senescent forgetfulness: benign and malignant. Can Med Assoc J 86: 257260 Levy R (1994) Aging-associated cognitive decline. Int Psychogeriatr 6: 63-68 Petersen RC , Smith G, Kokmen E, lvnik RJ, Tangalos EG (1992) Memory function in normal aging . Neurology 42: 396-401 Petersen RC, Smith GE, Ivnik RJ, Kokmen E, Tangalos EG (1994) Memory function in very early Alzheimer's disease. Neurology 44: 867-872 Petersen RC, Smith GE, Ivnik RJ, Kokmen E, Tangalos EG, Tsai M-S, Schaid DJ, Thibodeau SN , Kurland LT (1995) Apolipoprotein E status as a pr edictor of the development of Alzheimer's disease in memory-impaired individuals. JAMA 273: 1274-1278 Petersen RC, Smith GE, Waring S, Ivnik RJ, Tangalos EG, Kokmen E (1999) Mild cognitive impairment: clinical characterization and outcome. Arch Neurol: 303-308 Ritchie K, Artero S, Touchon J (2001) Classification criteria for mild cognitive impairment: a population-based validation study. Neurology 56: 37-42 Smith GE, Petersen RC , Ivnik R , Malec JF, Tangalos EG (1996) Subjective memory complaints, psychological distress, and longitudinal change in objective memory performance. Psychol Aging 11: 272-279 Taylor JL, Miller TP, Tinklenberg JR (1992) Correlates of memory decline: a 4-year longitudinal study of older adults with memory complaints. Psychol Aging 7: 185193

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Tierney MC, Szalai JP, Snow WG, et al (1996) Prediction of probable Alzheimer's disease in memory-impaired patients: a prospective longitudinal study. Neurology 46: 661665 Troncosco JC, Martin LJ, Dal Forno G, Kawas CH (1996) Neuropathology in controls and demented subjects from the Baltimore Longitudinal Study of Aging. Neurobiol Aging 17: 365-371 Authors' address: G. Smith, PhD, Department of Psychiatry and Psychology, Mayo Clinic , 200 1st St SW, Rochester, MN 55905, USA, e-mail: [email protected]

Vienna Transdanube Aging "VITA": study design, recruitment strategies and level of participation P. Fischer' >, S. Jungwirth>, w. Krampla', S. Weissgram-, W. Kirchmeyr-, W. Schreiber-", K. Huber2, M. Rainer", P. Bauer', and K. H. Tragp,3 1 Ludwig Boltzmann Institute for Aging Research, 2D epartment of General Psychiatry, University Hospital for Psychiatry, 3 Danube Hospital, and 4 Institute for Medical Statistics, Vienna, Austria

Summary. The Vienna Transdanube Aging study "VITA " is a prospective, interdisciplinary cohort-study of all 75-years old inhabitants of the 21. and 22. district of Vienna (n = 1,745), which started in May 2000. The study design is described in this paper for the first time. The main scientific question of the study concerns the prediction of incident dementia in the elderly. The main statistical analysis will compare 8 predictors: episodic memory, verbal fluency , subjective memory complaints, depression, APOE-E4, MAO-B activity in thrombocytes, MRT hippocampal atrophy, and MRT atrophy of the substantia innominata. The whole investigation comprises medical and psychosocial interviews, psychological tests, psychiatric and neurological scales, blood characteristic, genetic factors and cranial magnetic resonance imaging. Various variables will be compared with each other concerning sensitivity and specificity of prediction of cognitive decline. The dependent variable of the intended statistical analysis will be the individual's difference between Mini Mental State Examination scores at the two times of investigation. A high level of participation in geriatric epidemiological studies increases the general applicability of results but recruitment procedures must not ignore the individual's right to privacy and integrity. Using a liberal recruitment procedure as recommended by the local ethics commission the level of participation is between 36.7% and 44.3%. Introduction

The VITA (Vienna Transdanube Aging) study is a large prospective longitudinal study on aging and especially mental aging. The study design and recruitment strategy are firstly described in this paper. The study investigates every 75-years old inhabitant of a geographically defined area of Vienna. The main aim of the VITA is the prediction of incident cases of dementia in a community-based age-cohort after 30 months.

K. A. Jellinger et al. (eds.), Ageing and Dementia Current and Future Concepts © Springer-Verlag/Wein 2002

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Since age is the by far most strongest predictor of cognitive decline in the elderly, we try to minimize the variance of age. We investigate the whole cohort of elderly born between May 1st , 1925 and April 30th , 1926, living in the geographical area of Vienna, which lays on the left side of the Hive Danube (Vienna Transdanube). This is a working class area. The first crosssectional investigation is carried out between May I", 2000 and October 31th, 2002 and subjects are invited to participate in the sequence of their birth, which means that older subjects of the cohort are seen earlier. Various variables will be compared with each other concerning sensitivity and specificity of prediction of cognitive decline. The main statistical analysis will compare 8 predictors: episodic memory, verbal fluency, subjective memory complaints, depression, APOE-E4, MAO-B activity in thrombocytes, MRT hippocampal atrophy, and MRT atrophy of the substantia innominata. The whole investigation comprises medical and psychosocial interviews, psychological tests, psychiatric and neurological scales, blood analysis, genetic factors and cranial magnetic resonance imaging. The dependent variable of the intended statistical analysis will be the individual's difference between Mini Mental State Examination (MMSE) scores at the two times of investigation (Folstein et al., 1975). We do not favour the construct of a dichotomous fashion of mental decline in subjects with versus without Alzheimer's "disease", but we favour the construct of a cognitive continuum, i.e. Alzheimer's dementia (AD) as a chronic age-associated disorder. Following this theory every 75-years old inhabitant of Vienna-Transdanube will have his individual rate of cognitive decline (Morris et al., 1999). Because most studies use the MMSE for the description of the decline of individual patients (Han et al., 2000; Mendiondo et al., 2000; Small et al., 2000) we chose this scale as our primary outcome measure.

Material and methods The VITA study, a population-based cohort study, tries to investigate the early clinical manifestations and risk factors of AD. The main scientific question is: how is AD predicted best? Prediction is tried with short screening tests, which are designed to be used by general practitioners. These psychometric variables will be compared with quantitative data from MRT, blood biochemistry, genetic data, psychiatric scales, and extensive neuropsychological testing. Whether a patient suffers from AD or not will be diagnosed in a reinvestigation 30 months after the first investigation. The dependent variable of the intended statistical analysis will be the individual's difference between MMSE scores at the two times of investigation (Folstein et aI., 1975; Han et aI., 2000; Mendiondo et aI., 2000; Small et al., 2000) . The study is being conducted in Vienna-Transdanube, a geographically defined, urban working class area of Vienna, consisting of two districts (21 st and 22 nd districts of Vienna) with 91.064 inhabitants. The study is organized, planned and financed by the Ludwig Boltzmann-Institute for Aging Research with the help of the Danube-Hospital, a large community hospital in care of these two districts. This modern hospital has been opened 1992. The main investigator and organisator of the study comes from the Department of General Psychiatry at the University Hospital Vienna.

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All general practitioners, psychiatrists and neurologists of the 21st , 22nd and the neighbouring districts of Vienna received written information about the VITA study at its beginning. We also popularized the aims and the procedure of the investigations of the VITA study by several articles in local newspapers. A report in the televison news has acquainted broad levels of the population with the VITA study.

Sample selection and contact procedure In Austria every inhabitant has to be registrated in the local registration office, which favours population-based studies. According to the voting evidence (1st March 2000) 1,745 people (693 male, 1,052 female) of our study-region were born between 1st May 1925 and 30 th April 1926, thus having their 75th birth-day in the year of the beginning of the study. Successively, we are inviting all, institutionalized and noninstitutionalized, individuals of this age-cohort to participate in the study: 1. For the initial contact, to each subject in the study population a personal letter was sent explaining the study and the importance of participation, but also stating that participation is voluntary. Individuals received this letter of invitation to participate in the VITA study according to their exact birth day: first invitations were sent to first born individuals of the population. At one time 80 invitations were sent out simultaneously. The invited subjects were asked to contact the research group by phone. 2. Subjects not responding to our contact letter were called (when a telephone-number was available) four times in three weeks between 9 a.m. and 4 p.m . Telephone numbers were only available for 70% of individuals, the rest has so called "secret numbers". A second contact letter was sent to subjects who could not be contacted as described in point 1. and 2. after an interval of at least 2 months. Subjects still not responding were again tried to be contacted by telephone as described in point 2. 3. The contact procedure as described in point 2. was repeated a third and last time for subjects not contacted previously. According to the recommendation of the local ethic commission there was no attempt to visit subjects personally without consent.

Participation and refusal There were three levels of participation. They are listed in order of priority from the point of view of the VfTA study organizers: total investigation (screening-assessment and clinical investigation), or screening-assessment only, or telephone interview only. Subjects unwilling to participate were asked for their reason for refusal and answers were documented. These answers were interpreted by the interviewer and categorized by him as shown in table 1. Participants had to sign informed consent, as approved by the local ethics' commission.

Financial motivation to participate After the complete invitation procedure of the first 240 potential participants, a financial compensation for participation of 300 ATS (comparable to 25 USD) was introduced in order to increase the level of participation. This procedure had been recommended by various international geriatric psychiatrists and psychologists. The financial reward was now mentioned in the first contact letter. All other details of the contact procedure remained identical.

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Total investigation (screening assessment und clinical investigation) The screening-assessment and the clinical investigation took place at different days . The screening-assessment took 60-90 minutes and it was carried out by a clinical psychologist. It comprises the following areas of interest and neuropsychological tests: history of education, history of profession, handedness, Pocket Smell Test (McCaffrey et al., 2000), questionaire on psychosocial activities, memory complaints (Jorm et al., 1997; Geerlings et al., 1999), Intracategorical Delayed Selective R eminding Test (Fischer et al., 1990; Bancher et a\., 1996), verbal fluency (Mattis, 1973), short form of the Mini-Mental-State Examination (orientation, attention, calculation, copy) , Clock Drawing Test (Manos, 1994; Mendez et al., 1992), paper-pencil concentration task (Gatterer, 1990) , Draw a Person-Test (Ziler, 1975; Koppitz, 1968; Ericsson et aI., 1991, 1994), and Informant Questionaire of Cognitive Decline (Jorm et aI., 1989). At the request of the subjects the screening took place in the Danube-Hospital or at their home. Informed consent was obtained from the subjects. At the time of the screening the subjects were informed about the specific procedure of the clinical investigation. The clinical investigation was carried out in the Danube-Hospital and was taking 6 hours (without breaks, and gratis-breakfast, and gratis-dinner). The following investigations were carried out: taking blood (venous puncture); neuropsychological testing (clinical psychologist): test battery of the consortium to establish a registry for Alzheimer's disease (Morris et aI., 1989), Fuld Object Memory Test (Fuld, 1980), Tokentest (Huber et aI., 1983), Trail Making Test (AlB) (Reitan, 1956), Aachener Aphasia Test: Subtests Naming & Language Comprehension (Huber et al., 1983); psychiatric investigation (psychiatrist): drug history, nutrition (Kiyotaro et aI., 1994), physical activities history of neurological and psychiatric illness in the family medical history, Clinical Dementia Rating (Hughes et al., 1982), Short Geriatric Depression Scale (15 Item-Version) (Sheik and Yesavage, 1986), Hamilton Depression Scale (Hamilton, 1960), State / Trait Anxiety Inventory (Spielberger et aI., 1970; Laux et al., 1981), Physical Self-Maintenance Scale and Instrumental Activities of Daily Living (Lawton and Brody, 1969), life-event scale (newly developed), DSM-IV criteria for Alzheimer dementia (Sass et aI., 1996), DSM-IV criteria for major depression, Clinical diagnostic criteria for frontotemporal dementia (Th e Lund and Manchester Groups, 1994); neurological investigation (neurologist): somatic status, neurological status, unified Parkinson's disease rating scale - motoric (Fahn et al., 1987), Hachinski's ischemic scale (Hachinski et al., 1975; Rosen et aI., 1980; Fischer et al., 1991), diagnostic criteria for Alzheimer dementia (McKhann et al., 1984), for vascular dementia (Roman et al., 1993), and for dementia with Lewy bodies (Byrne et aI., 1991; McKeith et al., 1996), and a cranial magnetic resonance imaging with reconstruction of the hippocampal formation. The subjects were informed about their medical results ten days after the clinical investigation had taken place. They got copies of their blood- and magnetic-resonanceimaging results and a structured short report for their general practitioners.

Data handling and analysis All information collected on participants was coded. Identification of individual participants was possible only by their study number. One copy of the data is delivered to the Institute of Medical Statistics of the University of Vienna and fed into a computerized data base using MS-ACCESS. Information was then analysed using the SAS-PC package.

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Results

Subject recruitment, level of participation and reasons for refusal

The first 480 subjects (6 times 80 subjects) selected from the 1745 potential subjects (beginning with the oldest) were invited by maiL They were born between May 1st, 1925 and August 6t h, 1925. After the complete contact procedure, 83 subjects (17.3%,31 males, 52 females) could not be contacted personally. Additionally, 3 subjects (0.6%) had died before the first contact (information by relatives) and to 5 subjects (1 %) the letter could not be delivered. 7 subjects (1.5%) were kept in evidence as they were willing to take part in the VITA study, but they were unable to participate at this particular time. One-hundred-seventy-six subjects agreed to participate in the VITA study. These are 36.7% of the intent-to-contact population. Because we had no process contact in 83 subjects a more optimistic estimation of level of participation would be 44.9% (176 of 392 subjects contacted). Of these 176 subjects 24 (5%, 5 males, 19 females) completed an telephone interview, 7 (1.5%, 1 male, 6 females) completed the screening assesment only and 145 subjects (30.2%, 49 males, 96 females) completed the total investigation (screening assessment and clinical investigation). Of the 397 subjects (82.7%) contacted, a total of 206 subjects (42.9%, 107 males, 198 females) refused any kind of participation in the VITA study. The following objective reasons for refusal of participation are specified by the interviewer: lack of interest (N = 97; 20.2%), somatic illness (N = 54; 11.2%), negativism(N = 25;5 .2%), anxiety (N = 11;2.3%), lackoftime (N = 10;2.1 %), depression (N = 6; 1.3%) and forgetfulness (N = 3; 0.6%) (Table 1). Development of participation fraction following the invitation procedure

Three contact letters, one every four weeks, were sent to subjects who could not be contacted with a previous letter (Table 2). After the first contact letter, 150 (31.2%) of 480 potential participants could not be reached. Of the 330 (68.8%) subjects contacted, 155 (32.3%) subjects agreed to participate in one kind of investigation of the VITA study (telephone interview: N = 20-4.2%; screening assessment only: N = 7-1.5% ; total investigation: N = 128-26.7%). Of the 150 participants not contacted yet , 53 (11.0%) could be reached with the second letter. Seventeen (3.5%) subjects agreed to participate in one kind of investigation of the VITA study (telephone interview: N = 2-0.4% , total investigation: N = 15-3.1 %). The third and last letter resulted in contact with 14 (2.9%) of the 97 remaining subjects still not responding. Four (0.8%) agreed to participate in one kind of investigation of the VITA study (telephone interview: N = 2-0.4%; total investigation: N = 2-0.4%). The remaining 83 (17.3%) potential participants got no further invitation by mail or phone at any stage of the study. Eigthyfour (17.5%) inhabitants had

P. Fischer et al.

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Table 1. VITA-Study -

level of participation

Contact Letter Refusal N_ 206 42,9%

(max. 3 tomes)

II

I I

Reasons f or Refusal

N=480

I

I

I

No Contact

~

N=83 17.3%

N.397

H l

Evidence N =7

-1

Deceased N =3 0,6%

1,5%

Retu rnod 10 sen oo r N=5 0%

82 ,7 %

'1

Participation N . 176 36 ,7%

~

I I Telephon e loten lew

Kind of Participation

I I

II

Somal lc Illness N=54 11 , 2~o

Nega tivis m N=25

II II

5.2% Anxie ty N= I '

Depre ssion N=6 1,3% Fo rgetfu lness N= 3 0,6%

I I I

I I TotD l ln,"e'iti ~ Btio n

A_~~ me n l n nty

(Scr eeni ng A ssessm ont + Clin ical lnvest,gatiOn) N= 145

N=7 1,5%

Lac k of Time N= 10 2 ,1 %

I

2,3 %

Scre en ing

N~4

5%

II II II II

No Inle rest N=97 20 .2%

I

30 ,2"':'

a secret telephone number which was not available to the organizers of the study . Fiftyfour subjects, who could not be contacted wit h three lett er s had such a secret number. That means that 67.5% of subje cts not contacted had a secret te lephone number. The second and third contact lett er increased the participation fraction by 4.3% (N = 21 of 480) . Thereof, 0.8% (N = 4) completed a te lephone in terview an d 3.5% (N = 17) completed the total investigation (screening assessment + clinical investigation) .

Table 2. D evelopment of level of participation Contact letter 1. contact letter N = 480 2. contact letter N = 150 3. contact letter N = 97

No contact

Contact

General participation

Telephone Interview

Screening Assessment only

Total Investigation

N = 150 31.2 % N = 97 20.2% N = 83 17.3%

N = 330 68.8% N = 53 11.0% N = 14 2.9%

N = 155 32.3% N = 17 3.5 % N = 4 0.8%

N = 20 4.2% N = 2 0.4% N=2 0.4%

N = 7 1.5 % N = 0 0% N = 0 0.0%

N = 128 26.7 % N = 15 3.1% N = 2 0.4%

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"VIT A " Vienna-Transdanube-A ging Table 3. Level of participation with and without financi al reward Contact L ett er (ma x. 3 limes) with financial compensation N=240 withou t ttnsn cte! compensstion N=240

I No Con tact: N ~41

17,1%

N- 42 17,5%

c

Co ntact:

N=90 3 7,5 %

4 ,6 c/a

N=13 5,4 %

'a."

.o .t--

Participation

N=ll

-

I N=86 35 ,9%

r--

0

.-

N- 198 82,5%

N- 199 82 ,9%

Telep hone ln tervl e..

.------

I

Screeni ng

-

Assess ment

'"a.

N=3 1,3%

onl ~'

~

N=4 1.7 %

0

Tutu l

Inu~ t( ~utlun

"0

c

.-

...

'----

N=7 2 30,0010

N=73 30 .4 %

'------

Left: data for participants with finan cial compe nsation; right: data for participants without financial compensa tion; all perc entages are referred to th e respecti ve total gro up

L evel of participation with and without financial rewa rd

After th e complete invitation procedure of the first 240 potential participants, a financial compensation for participation of 300 ATS (equivalent to 25 USD) was introduced to increase the participation fraction . This financi al compensation was paid to subjects who took part in both the screening assessment and the clinical investigation. The comparison of the participation fractions for the two groups with (group 1) and without (group 2) financi al compensation is shown in Table 3. There was no increase in level of participation at all. Compared to 30.0% (N = 72) of group 1 (with financial compensation) , 30.4 % (N = 73) of group 2 (without financial compensation) took part in the total investigation. There is no differ ence in participation fraction between group 1 and group 2 (X2 = 0.01). Discussion

Epidemiological research is the basis of health promotion for th e elderly and becomes increasingly important du e to the dramatic increase of longevity. A high level of participation in geriatric epidemiological studies strongly in -

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creases the general applicability of results (Herzog et al., 1983; Vogt et al., 1986; Neaton et al., 1987; Morgan et al., 1997). Recruitment of motivated participants who meet the study inclusion and exclusion criteria is, beside fund raising, the most demanding task in geriatric epidemiology. This issue of recruitment into such programmes has not been communicated frequently. Inclusion of the target population by adequate sampling of in- and outpatients and ensuring high response fractions are central issues to minimize selection bias. Aggressive recruitment strategies have shown to be effective in attaining high percentages of participation (Ives et al., 1992; Norton et al., 1994; RiedelHeller et al., 2000) , but both ethical and legal considerations hinder these procedures (Heun et al., 1997). The recruitment procedure must not ignore the individual's right to privacy and integrity (Strauss et al., 1998). While level of recruitment in population-based studies reach 75% (Riedel-Heller et al., 2000) to 93% (Norton et al., 1994) after personal visits prior to informed consent, recruitment proportions lay between 24% (Heun et al., 1997) and 34 % (Schmidt et al., 1994) with liberal recruitment strategies (e.g ., letter of invitation and/or phone call). Some studies demonstrated the effect of positive attitude from the participants towards research for high levels of participation, especially with longitudinal investigations (Strauss et al., 1998) . This positive attitude could be influenced by public relation strategies like information to general practitioners, local newspapers, priests and senior citizen organizations (Riedel-Heller et aI., 2000). The general level of participation in the VITA study so far was 36.7%: 145 (30.2 %) of 480 contacted subjects took part in the total investigation, 7 (1.5 %) agreed to the screening assessment only, and 24 (5%) were interviewed by phone. A total of 83 (17.3 %) subjects could not be reached by neither mail nor telephone. Only 5 of the letters were returned to sender, so 475 reached the recipients. Telephone numbers were not available for 67.5% of these nonresponding subjects. The frequent secret numbers in Austria burden epidemiological studies like the VITA. The repeated mailing procedure in the VITA study proved to be successful as the participation fraction increased by 4.3% (21 cases). Only three studies with comparable sample selection and recruitment methods were found in the literature (Ives et al., 1992; Heun et al., 1997; Schmidt et aI., 1994). The participation fraction of the VITA study is similar to the participation fractions reported in these studies. It is higher than in the more recent (21.1 %) and lower than reported in the previous study (37%). The study of Ives et al. (1992) used a single letter with financial reward of 30.- USD and telephone interviews of various aggressivity. Telephone numbers were available for every patient. The study of Heun et ai. (1997) used a liberal recruitment strategy, but also sent 3 consecutive letters. The reason for refusal of participation with the highest frequency was "No interest" (20.2 %). Persons responding this way seemed not to be depressed as judged by our experienced telephone interviewer. This percentage of "no interest" is comparable to Heun et al. (1997) after mailing the initial letter. This unconcern had been tried to invalidate with the support of local media.

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Perhaps more emphasis should have been given to the fact that help of elderly subjects for scientific research is urgently needed. Another frequent reason for refusal of participation was "somatic illness" (11.2%). It was stated less frequently (3.0%) as a reason of refusal in another study (Heun et al., 1997), which investigated subjects older than 60 years. People in our investigation stated that they had already undergone a number of medical investigations and did not want to increase this number of contacts to medicine by participating in the VITA study. Somatic comorbidity in the older elderly is obviously hard to investigate with liberal recruitment strategies (Launer et al., 1994). Refusal because of psychiatric morbidity (anxiety, depression, forgetfulness) seemed to be no problem according to the judgement of our telephone interviewer, but this needs to be ascertained by other epidemiological procedures. To our knowledge there is no published gerontoepidemiological study on the effect of paying a financial compensation for participation. We tried to increase the participation fraction by granting a financial compensation (300 ATS /25 USD purchasing power). Unexpectedly there was no increase at all in level of participation. As it is unusal to be paid for a medical investigation that fact could have induced anxiety. It is possible that incorporation of a monetary incentive would increase participation, but not at the level chosen. Recruitment strategies depend on scientific questions. In cross-sectional prevalence studies rather aggressive strategies are necessary in order not to underestimate disease prevalences. In longitudinal studies, i.e. in incidence studies, the motivation for long-term participation is more important. Individuals carrying the disease at the beginning of the study are not selected aggressively, because one would have no interest in recruiting prevalent cases. Because recruitment procedures should not ignore the individual right for privacy and integrity, liberal recruitment strategies have to be developed further. Acknowledgements This work was supported by the Ludwig Boltzmann Institute of Aging Research. We are grateful to the many coworkers in the Danube-Hospital of the City of Vienna (Dr. K. Bauer, Dr. M. Haushofer, Dr. W. Hruby, H. Hombauer, Dr. W. Kristoferitsch, U. Laure, Dr. E. Ogris) for their help in our research. The authors would also like to thank the other members from the VITA-study group [Dr. W. Danielczyk, Dr. G. Gatterer, Dr. K. Jellinger (Vienna), Dr. M. Kalousek, Dr. P. Pietschmann, Dr. P. Riederer (Wurzburg, Germany), Dr. H.-J. Zapotoczky (Graz, Austria)], and scientists from other institutions like Dr. AU. Monsch (Basel, Switzerland), Dr. H. Foerstl and Dr. H. Bickel (Munich, Germany), and Dr. F. Reischies (Berlin, Germany) for their constructive comments. The statistical planning and advice came from the Institute of Medical Statistics of the University of Vienna (A.Auterith, Dr. P. Bauer, T. Lang, S. Scholz).

References Bancher C, Jellinger K, Lassmann H, et al (1996) Correlations between mental state and quantitative neuropathology in the Vienna longitudinal study on dementia. Eur Arch Psychiatry Clin Neurosci 246: 137-146

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Launer LJ, Wind A W, Deeg DJH (1994) Nonresponse pattern and bias in a communitybased cross-sectional study of cognitive functioning among elderly. Am J Epidemiol 139: 803-812 Laux L, Glanzmann P, Schaffner P, Spielberger CD (1981) Das State-TraitAngstinventar. Theoretische Grundlagen und Handanweisung. Beltz Test GmbH, Weinheim Lawton M, Brody E (1969) Assessment of older people: selfmaintaining and instrumental activities of daily living. Gerontologist 9: 179-186 Manos PJ (1994) Ten-point clock test sensitivity for Alzheimer's disease in patients with MMSE scores greater than 23. Int J Geriatr Psychiatry 14: 454-458 Mattis S (1973) Dementia Rating Scale. Psychological Assessment Resources, Inc. , USA Me Caffrey RJ, Duff K, Solomon GS (2000) Olfactory dysfunction discriminates probable Alzheimer's dementia from Major depression: a cross-validation and extension. J Neuropsychiat Clin Neurosci 12: 29-33 Me Keith IG, Galasko D, Kosaka K, et al, for the Consortium on Dementia with Lewy Bodies (1996) Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop. Neurology 47: 1113-1224 McKhann G, Drachman D, Folstein M, et al (1984) Clinical diagnosis of Alzheimer 's disease: report of the NINCDS-ADRDA work group under the auspices of department of health and human services task force on Alzheimer's disease. Neurology 34: 939-944 Mendez MF, Ala T, Underwood KL (1992) Development of scoring criteria for the clock drawing task in Alzheimer's disease. J Am Geriatr Soc 40: 1095-1099 Mendiondo MS, Ashford JW, Kryscio RJ, et al (2000) Modelling mini mental state examination changes in Alzheimer's disease. Stat Med 19: 1607-1616 Morgan A , Harris M, Boyce P, et al (1997) Has social psychiatry met its Waterloo? Methodological and ethical issues in a community study. Austr NZ J Psychiatry 27: 411421 Morris JC, Heyman A, Mohs RC, et al and the CERAD investigators (1989) The consortium to establish a registry for Alzheimer's Disease (CERAD), part 1. Clinical and neuropsycho-logical assessment of Alzheimer's disease. Neurology 39: 1159-1165 Morris MC, Evans DE, Hebert LE, Bienias JL (1999) Methodological issues in the study of cognitive decline. Am J Epidemiol 149: 789-793 Neaton JD , Grimm RH Jr, Cutler JA (1987) Recruitment of participants for the multiple risk factor intervention trial (MRFIT). Contr Clin Trials 8: 41-53 Norton MC, Breitner JCS , Welsh KA, et al (1994) Characteristics of nonresponders in a community survey of the elderly. J Am Geriatr Soc 42: 1252-1256 Reitan RM (1956) Trail M aking Test. Manual for administration, scoring, and interpretation. Indiana University, Indianapolis Riedel-Heller SG, Schork A , Matschinger H, et al (2000) Recruitment procedures and their impact on the prevalence of dementia. Neuroepidemiology 19: 130-140 Roman GC, Tatemichi TK, Erkinjuntti T, et al (1993) Vascular dementia: diagnostic criteria for research studies. R eport of the NINDS-AIREN international workshop. Neurology 43: 250-260 Rosen WG, T erry RD , Fuld PA, et al (1980) Pathological verification of ischemia score in differentiation of dementi as. Ann Neurol 7: 486-488 SaB H , Wittchen HU, Zaudig M (1996) Diagnostisches und statistisches Manual psychischer Storungen DSM-IV. Hogrefe, Gottingen Schmidt R, Freidl W, Fazekas F, et al (1994) The Mattis Dementia Scale : normative data from 1001 healthy volunteers. Neurology 44: 964-966 Sheikh Jl, Yesavage JA (1986) Geriatric depression scale (GDS). Recent evid enc e and development of a shorter version. Clin Gerontol 5: 165-173 Small BJ, Fratiglioni L , Viitanen M, et al (2000) The course of cognitive impairment in preclinical Alzheimer disease. Arch Neurol57: 839-844

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Spielberger CD, Gorsuch RL, Lushene RE (1970) STAI, manual for the the State-TraitAnxiety-Inventory. Consulting Psychologist Press, Palo Alto Strauss Ev, Fratiglioni L, Jorm AF, et al (1998) Attitudes and participation of the elderly in population surveys: data from a longitudinal study on aging and dementia in Stockholm. J Clin Epidemiol51: 181-187 The Lund and Manchester Groups (1994) Clinical and neuropathological criteria for fronto-temporal dementia. J Neurol Neurosurg Psychiatry 57: 416--418 Vogt TM, Ireland CC , Black D , et al (1986) Recruitment of elderly volunteers for a multicentre clinical trial: the SHEP pilot study. Contr Clin Trials 7: 118-133 Ziler H (1975) Der Mann-Zeichen-Test in detailstatistischer Auswertung. Aschendorf, MUnster Authors' address: Prof. P. Fischer, M.D ., Ph.D., Psychiatrische Klinik AKH, Wahringer Gurtel 18-20, A-1090 Wien, Austria, e-mail: [email protected]

Conversion from preclinical to clinical stage of Alzheimer's disease as shown by decline of cognitive function in carriers of the Swedish APP-mutation O. Ahnkvist>', K. Axehnan-, H. Basun', L.-O. Wahlundt, and L. LannfeltDivisions of 1 G eriatric Medi cine and 2E xperimental G er iatrics, Karolinska In stitutet , D ep artment of Clinical Neuroscienc e, Occupational Th erapy, and Elderly Ca re Research (NEUROTEC), Huddinge Uni ver sity Hospital, Stockholm, 3 D epartment of Public H ealth/Geriatrics, Uppsala Universit y H ospital, Uppsala, and 4 D ep artment of Psychology, Stockholm University, Stockholm, Swede n

Summary. The characterisation of the borderline syndrome between normal cognitive function and Alzheimer's disease (AD) , often mentioned as Mild Cognitive Impairment (MCI) has been a goal for recent re search. However, a variety of de finitions of MCI-like syndromes and the uncertainty about the final diagnosis have hampered progress. To overcome these problems, the present study will describe cognitive function in two healthy mutation carriers and two matched non-carriers of the Swedish double mutation family, during the time period when carriers con vert to a symptomatic stage, i.e., true preclinical AD , and finally into the stage when a clinical diagnosis of AD is first possible. The findings question the generality of common MCI concepts and the commonly held beliefs about cognitive features in late preclinical stage of AD .

Introduction Ever since it was found that there is a possibility to treat the symptoms of Alzheimer 's disease (AD) with cholinesterase inhibitors, it has been a challenge to start tr eatment as early as po ssible. However, there is no definitive biological marker of AD, therefore th e diagnosis has to rel y on clinical judgement. Furthermore, clinical standard procedures of diagnosis require a substantial cognitive decline in memory and least on e other cognitive domain as well as clear changes in daily life to fulfil an AD diagnosis (American Psychiatric A ssociation, 1994; McKhann et al., 1984). Thus, this state-ofaffairs manages to recognise AD patients only when the disease process has been going on for a considerable time. This has initiated attempts to identify AD earlier in th e disease course in the preclinical stage. It has been suggested that a solution would be to define a borderline syndrome between normal

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function and dementia. The most popular notion of this syndrome is Mild Cognitive Impairment (MCI; Flicker et al., 1991; Petersen et al., 1999). The borderline syndrome between normal function and dementia has not only been defined as MCI, but a large number of alternative concepts have been suggested to cover this phenomena. The first to appear was the differentiation between benign and malign forgetfulness (Kral, 1962), followed by Age-Associated Memory Impairment (AAMI; Crook et al., 1986), and AgeRelated Cognitive Decline (ARCD; American Psychiatric Association, 1994) and many others. These various definitions vary in terms of cognitive function(s) in focus, size of decline and capacity to fulfil the demands of daily living, which results in a corresponding variation of predictive power to indicate future development of dementia or AD. To overcome the methodological shortage of previous research on MCI, a solution would be to investigate individuals who definitely will develop AD. This challenge is met in the present study, in which we will describe the disease course in terms of cognitive function in a couple individuals, who are carriers of AD-mutations. These individuals have been followed by repeated neuropsychological assessments for several years during which they convert from an asymptomatic condition, through a symptomatic condition, and finally into a condition that could be diagnoses as clinical AD. Interestingly, the symptomatic condition may be viewed as a model of MCI or actually the true description of this syndrome. Material and methods

Subjects Four individuals will be described, two individuals (AD! and ADz) who develop AD due to a double mutation in the APP gene on chromosome 21 (Axelman et al., 1994; Mullan et al., 1992) and two individuals from the same family without the APP-mutation serving as healthy controls (He! and HCz). Two deceased individuals from the Swedish APP family have come to autopsy. These cases show neuropathological findings that are typical for AD (Lannfelt et al., 1994). The study was approved by the Ethical Board at Huddinge University Hospital.

Diagnosis The diagnosis of AD was consistent with the criteria of DSM-IIl-R (American Psychiatric Association, 1987) and Mel was defined according to most commonly used criteria (Petersen et al., 1999) requiring subjective and objective memory impairment but otherwise well functioning.

Procedure All adult individuals of the Swedish Alzheimer family were invited to biannual clinical examination according to a standard protocol for suspected dementia used at the Department of Geriatric Medicine, Huddinge University Hospital (Wahlund et al., 1999). This protocol included medical history, somatic examination, neurological status, psychiatric

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Table 1. Time of clinical evaluation in relation to time of expected clinical onset of AD for two individuals converting to clinical AD and two matched healthy control subjects Time of clinical evaluation Subject AD! ADz C! Cz

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- = denotes years prior to mean for clinical onset in this specific family ; + denotes years after mean for clinical onset status, sampling and routine analyses of blood, urine and cerebrospinal fluid , routine ECG and EEG, magnetic resonance imaging, single photon emission tomography, and neuropsychological assessment. This procedure was repeated three times for each of the four subjects. In order to define a common time scale that is related to the family specific onset of clinical AD, the subject's age was subtracte d by the family specific onset, which was 53 years based on data fro m medical records on demented individuals of this family (Axelman et al., 1994). Thus, the preclinical stage is indicated by negative time values and clinical stage by positive. In Table 1, an overview is presented of time for clinical evaluation for the four subjects.

Neuropsychological assessment The neuropsychological assessment included five sub-tests (Information, Digit Span, Similarities, Block Design, and Digit Symbol) from the Wechsler Adult Intelligence Scale - Revised (Wechsler, 1981), two sub-tests (Vocabulary and Figure Classification) from the Swedish Durernan-Salde battery (Dureman and Salde, 1959) , Boston Naming (Lezak, 1995), FAS verbal fluency (Lezak, 1995), copying and retrieval after 30 minutes of the Rey-Osterrieth Complex Figure (Lezak, 1995), Corsi Block Tapping (Lezak, 1995), Rey Auditory Verbal Learning (Lezak, 1995), free recall (Word FR) and recognition (Word Rec.) of a words (Backman and Forsell, 1994) , Face recognition (Almkvist et al., 1986) , Trailmaking A and B (Lezak, 1995), Simple Reaction time and computerised FingerTapping (Iregren et al., 1996) . The administration and scoring of these 20 tests was performed by the present author, it required approximately 3 hours. The Full Scale Intelligence Quotient (FSIQ) was calculated based on the five WAIS-R sub-tests. Digit Span forward and Corsi Block Tapping were administered according to an up-and-down procedure and the score was calculated as the 50% correct level (Smith et al., 1983). All other scoring was done according to manuals or articles referred to above.

Results

Health status

There was no evidence of poor health in the two non-carriers at any of the three examinations. The two mutation carriers both fulfilled the clinical crite-

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ria of AD at the third examination which was performed at the same time as the expected clinical onset of dementia in this AD family in one individual (t = -0.1; AD!) and some years later in the other individual (t = + 3.1; AD 2) . In contrast, the two mutation carriers showed no symptoms that could indicate AD at the first examination, which was performed 3-4 years prior to the expected clinical onset of AD in this family. However, AD! had some cognitive problem and AD 2 had hypertension. At the second examination (t = -2.2; AD! and t = -0.8; AD 2 ) cognitive decline was apparent in AD! and AD 2 demonstrated some isolated cognitive impairment. At his stage both mutation carriers fulfilled the criteria of Mild Neurocognitive Disorder (MND) according to DSM-IV but not MCI (Petersen et al., 1997), because the episodic memory was slightly but not markedly impaired. Cognitive function

The neuropsychological test performance was evaluated in relation to a reference group of healthy individuals at Huddinge University Hospital by a transformation of raw scores into z-scores. The neuropsychological test results for four individuals - two mutation carriers and two non-carriers - at three evaluations are illustrated in Fig. 1. Case ADJ. Case AD! worked as laboratory technician. The level of education was 9 years compulsory school. At first clinical evaluation (t = -3.6), no subjective symptoms were reported. However, one test result was abnormal (z < -1.64 or alternatively percentile - 1.64) as well as global cognitive function as measured by FSIQ (z = -0.6) and the MMSE score which was at top level (30). Routine EEG was slightly abnormal with episodes of left hemispheric frontotemporal 4-7 Hz activity. Brain imaging with CT and CBFHMPAO were normal. At the second clinical evaluation (t = -2.2), the same pattern appeared in cognitive function as previously noted, although the result on the inductive reasoning test had become still more impaired (z < -2.1). The constructive task (Block Design) showed a marked decline with almost 2SDs from first to second evaluation (z decreased from +0.7 to -1.0), but all other neuropsychological test results were above the critical value of abnormality as exemplified by FSIQ (z = -0.4) and MMSE (score = 29). Symptoms of impaired concentration was reported, but memory problems and other difficulties in daily life were denied. Again, the same type of abnormal EEG activity was observed, although more accentuated compared to the first evaluation. At the third evaluation (t = -0.1), global cognition had deteriorated (FSIQ, z = -1.2; MMSE = 21), as well as the inductive reasoning (z = -2.3), constructive performance (z = -1.8) and TMTB test (z = -1.8). Interestingly, episodic memory performance (RAVL total learning and retention as

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Fig. 2. Depolymerization of microtubules and inhibition of their assembly by AD P-tau. a The assembly of microtubules was determined turbidimetrically at 350 nm using 83 J..lg/ rnl (curve 2) or 333J..lg/ml (curve 1) of a tau isoform. The arrow indicates the addition of AD P-tau (154 ug/ml) . The 't4- and the 't3-promoted microtubules were the most resistant to disruption by AD P-tau. Not shown in this figure, the addition of assembly buffer as a control did not show any disruption of the microtubule assembly. The exact cause of an initial small increase in turbidity by the addition of AD P-tau to microtubules assembled with 83 ug/ml normal tau (curve 2) remains to be understood; it might be due to a small degree of bundling of microtubules before their disassembly. b The microtubule assembly reaction was carried out using 2 mg/ml of tubulin and 83 ug/ml concentration of a tau isoform alone (curve 1) or either mixed with 67J..lg/ml (curve 2) or 133J..lg/ml (curve 3) of AD P-tau. AD P-tau inhibited the microtubule assembly-promoting activity of all six tau isoforms. However, the inhibition was the greatest in the case of r-il, as compared with 't3L , 't3 and 't4. c Negative stain electron micrographs showing the products of microtubule assembly with 't3L a in the absence and b in the presence of AD P-tau. Not shown in this figure similar findings were observed with all other 't isoforms (reproduced with permission from Alonso et al., 200lb)

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the pivotal involvement of abnormal hyperphosphorylation in neurofibrillary degeneration. Protein kinases and protein phosphatases involved in the abnormal hyperphosphorylation of tau

The state of phosphorylation of a phosphoprotein is a function of the balance between the activities of the protein kinases and the protein phosphatases that regulate its phosphorylation. Tau, which is phosphorylated only at serine/ threonine residues, is a substrate for several protein kinases such as glycogen synthase kinase-3, cyclin dependent protein kinase-5, protein kinase A, calcium and calmodulin-dependent protein kinase-II, and stress-activated protein kinases (for review see Iqbal et al., 2000). However, to date, the activities of none of these protein kinases have been reproducibly shown to be upregulated in AD brain. In contrast, the activities of protein phosphatase (PP)-2A and PP-1 are compromised by -20-30% in AD brain (Gong et al., 1993, 1995), and the phosphorylation of tau that suppresses its microtubule binding and assembly activities in adult mammalian brain is regulated by PP2A and not by PP-2B (Gong et al., 2000). PP-2A also regulates the activities

Fig. 3. In vitro polymerization of AD P-tau into tangles of PHF/SF and the effects of dephosphorylation and deglycosylation. AD P-tau, 0.4 mg/ml , a without treatment, b dephosphorylated by alkaline phosphatase (AP) or c deglycosylated by endoglycosidase FIN- glycosidase F, was incubated for 90min and the products of the assembly were examined by negative stain electron microscopy. Dephosphorylation, but not deglycosylation, completely abolished AD P-tau polymerization. Bar represents 50nm. (Insets) PHF at higher magnifications. Arrows label examples of 10-15 nm (straight) and 4nm (arrowhead) filaments. d AD P-tau, O.4mg/ml, was incubated as above to induce assembly and the aggregated protein was separated from the non-aggregated protein by centrifugation at 35°C and 100,000 X g for 15 min. The pellet (P) was resuspended to its original volume and equivalent samples of the original mixture (0), the supernatant (S) and the pellet (1, 2 and 4 X) were analyzed by Western blots by using Tau-l antibody and dephosphorylation of the proteins on the blot with AP. e The amount (Mean ± SD of 4 values) of AD P-tau present in the original and the supernatant fractions was quantitated by scanning the immunoblots. f SDS-PAGE (10% gel) of AD P-tau and blot of a lane from the same gel developed with Tau-l antibody after dephosphorylation. One strip (8 ug of protein/lane) was stained with Coomassie blue (C) and another strip (Zug of protein/lane) was developed with Tau-l antibody after dephosphorylation ofthe proteins on the membrane (B). g For in vitro dephosphorylation and deglycosylation of AD P-tau, aliquots of AD P-tau were treated with (2) or without (1) the addition of AP to dephosphorylate (panels labeled 92e, Tau-l and PHF1) or endoglycosidase FIN- glycosidase F to deglycosylate (panels labeled GNA and PNA) the proteins as described. The immunoblots were developed with 92e (dilution 1/5,000) to detect the total amount of tau, Tau-l (1/50,000) to detect dephosphorylated tau, and PHF1 (1/250) to detect phosphorylated tau. The Increase in Tau-l staining and decrease in PHFI staining show dephosphorylation of AD P-tau. The immunoblots were developed with lectin GNA or PNA to detect glycosylation. Decrease in the staining with the lectins shows deglycosylation of AD P-tau by the glycosidase (reproduced with permission from Alonso et aI., 2001a)

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of several tau kinases in brain. Inhibition of PP-2A activity by okadaic acid in metabolically active rat brain slices results in abnormal hyperphosphorylation of tau at several of the same sites as in AD, not only directly by a decrease in dephosphorylation but also indirectly by promoting the activities of CaM Kinase II (Bennecib et aI., 2001), MAP kinase kinase (MEK1I2), extracellular regulated kinase (ERK 1/2) and P70S6 kinase (Pei et aI., in preparation). PP-2A and PP-l make more than 90% of the serine/threonine protein phosphatase activity in mammalian cells (Oliver and Shenolikar, 1998). The intracellular activities of these enzymes are regulated by endogenous inhibitors. PP-1 activity is regulated mainly by a 18.7kDa heat stable protein called inhibitor-l (I-I) (Cohen et aI., 1988; Cohen, 1989). In addition, a structurally related protein, DARPP-32 (dopamine and cAMP-regulated phosphoprotein of apparent molecular weight 32,000) is expressed predominantly in the brain (Walaas and Greengard, 1992). I-I and DARPP-32 are activated on phosphorylation by protein kinase A and inactivated at basal calcium level by PP-2A. Thus, inhibition of PP-2A activity would keep I-I , DARPP-32 in active form and thereby result in a decrease in PP-l activity. In AD brain a reduction in PP-2A activity might have decreased the PP-l activity by allowing the upregulation of the I-lIDARPP-32 activity. PP-2A is inhibited in the mammalian tissue by two heat-stable proteins: (i) the I1PPZA, a 30kDa cytosolic protein that inhibits PP-2A with a ki of 30nM and (ii) the IlP2A, a 39kDa nuclear protein that inhibits PP-2A at ki of 23nM (Li et aI., 1995, 1996). Both I 1PP2A and IlpzA have been cloned from human kidney (Li et aI., 1996a, b). I1PPZAhas been found to be the same protein as the putative histocompatibility leukocyte antigen class Il-associated protein (PHAP-1). This protein, which has also been described as mapmodulin, pp32 and LANP (Ulitzur et aI., 1997) is 249 amino acids long and has apparent molecular weight of 30 kDa on SDSPAGE. IlpzA, which is the same as TAF-11) or PHAPII, is a nuclear protein that is a homologue of the human SETa protein (von Lindern et aI., 1992). In a preliminary study we have found that the level of I/P2A is ~20% increased in AD brains as compared with age-matched control brains (Gondal et aI., in preparation). Pharmacologic therapeutic targets to inhibit AD through inhibition of neurofibrillary degeneration and outcome measures

The most promising therapeutic approaches to inhibit neurofibrillary degeneration and consequently AD are (1) to inhibit sequestration of normal MAPs by the AD P-tau and (2) to inhibit the abnormal hyperphosphorylation of tau. The latter can be carried out either by inhibiting the activity of a PP-2A inhibitor or by inhibiting the activity of one or more tau kinase activities that are critically involved in converting normal tau into an abnormal state whereby it sequesters normal MAPs. Development of therapeutic targets requires (i) a compelling scientific rationale for a therapeutic target and (ii) the availability of a practical outcome measure(s). Tau is primarily a neuronal protein and its level in CSF is a reliable measure of the rate of neuronal

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degeneration. The level of this protein both as total tau and as tau abnormally hyperphosphorylated at Ser-396/404 are markedly elevated in AD cases (Hu et aI., 2002). Thus, levels of CSF total tau and abnormally hyperphosphorylated tau offer excellent outcome measures to test efficacy of therapeutic agents towards total neurodegeneration and neurofibrillary degeneration, respectively. These outcome measures can be used to test drugs that inhibit neurofibrillary degeneration either by inhibiting the sequestration of normal MAPs by the AD P-tau or by inhibiting the abnormal hyperphosphorylation of tau.

Acknowledgements We are grateful to J. Biegelson and S. Warren for secretarial assistance. Studies in our laboratories were supported in part by the New York State Office of Mental Retardation and Developmental D isabilities and NIH grants AG05892, AG08076 and NS18105 , Alzheimer's Association (Chicago, IL) grant IIRG-00-2002 and a grant from the Institute for the Study of Aging (ISOA), New York.

References Alafuzoff I, Iqbal K , Friden H , Adolfson R, Winblad B (1987) Histopathological criteria for progressive dementia disorders: clinical-pathological correlation and classification by multivariate data analysis. Acta Neuropathol (Berlin) 74: 209-225 Alonso A del C, Zaidi T, Grundke-Iqbal I, Iqbal K (1994) Role of abnormally phosphorylated tau in the breakdown of microtubules in Alzheimer disease. Proc Nat! Acad Sci USA 91: 5562-5566 Alonso A del C, Grundke-Iqbal I, Iqbal K (1996) Al zheimer's disease hyperphosphorylated tau sequesters normal tau into tangles of filam ents and disassembles microtubules. Nat Med 2: 783-787 Alonso A del C, Grundke-Iqbal I, Barra HS , Iqbal K (1997) Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: sequestration of MAP1 and MAP2 and the disassembly of microtubules by the abnormal tau. Proc Nat! Acad Sci USA 94: 298-303 Alonso A del C, Zaidi T , Novak M , Grundke-Iqbal I, Iqbal K (2001a) Hyperphosphorylation induces self - assembly of tau into tangles of paired helical filaments/ straight filaments. Proc Natl Acad Sci USA 98: 6923-6928 Alonso A del C , Zaidi T, Novak HS, Barra HS , Grundke-Iqbal I, Iqbal K (200lb) Interaction of tau isoforms with Alzheimer's disease abnormally hyperphosphorylated tau and in vitro phosphorylation into the disease-like protein. J Biol Chem 276:37967-37973 Arrigada PA, Growdon JH, Hedley-White ET, Hyman BT (1992) Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease. Neurology 42: 631-639 Bancher C, Brunner C, Lassmann H , Budka H, Jellinger K, Wiehe G, Seitelberger F , Grundke-Iqbal I, Iqbal K, Wisniewski HM (1989) Accumulation of abnormally phosphorylated tau precedes the formation of neurofibrillary tangles in Alzheimer's disease. Brain Res 477: 90-99 Bennecib M, Gong C-X, Grundke-Iqbal I, Iqbal K (2001) Inhibition of PP-2A upregulates CaMKII in rat forebrain and induces hyperphosphorylation of tau at Ser 262/356. FEBS Lett 490: 15-22

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Cohen P (1989) The structure and regulation of protein phosphatases. Ann Rev Biochem 58: 453-508 Cohen P, Alemany S, Hemmings BA, Resink TJ , Stralfors P, Lim Tung HY (1988) Protein Phosphatase-1 and Protein Phosphatase-2A from rabbit skeletal muscle. Meth Enzymol 159: 390-408 Dickson DW, Farlo J , Davies P , Crystal H, Fuld P, Yen SH (1988) Alzheimer's disease: a double-labeling immunohistochemical study of senile plaques. Am J Pathol132: 86101 Dickson DW, Crystal HA, Mattiace LA, Masur DM, Blau AD , Davies P , Yen S-H, Aronson M (1991) Identification of normal and pathological aging in pro spectively studied non-demented elderly humans. Neurobiol Aging 13: 179-189 Finch C, Tanzi RE (1997) Genetics of aging. Science 278: 407-411 Goedert M , Spillantini MG, Jakes R, Rutherford D, Crowther RA (1989) Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease. Neuron 3: 519-526 Gong C-X, Singh TJ , Grundke-Iqbal I, Iqbal K (1993) Phosphoprotein phosphatase activities in Alzheimer disease brain. J Neurochem 61: 921-927 Gong C-X, Shaikh S, Wang J-Z, Zaidi T, Grundke-Iqbal I, Iqbal K (1995) Phosphatase activity towards abnormally phosphorylated r: decrease in Alzheimer disease brain. J Neurochem 65: 732-738 Gong C-X, Lidsky T, Wegiel J , Zuck L, Grundke-Iqbal I, Iqbal K (2000) Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain. J BioI Chern 275: 5535-5544 Grundke-Iqbal I, Iqbal K, Quinlan M, Tung Y-C, Zaidi MS , Wisniewski HM (1986a) Microtubule-associated protein tau: a component of Alzheimer paired helical filaments. J BioI Chern 261: 6084-6089 Grundke-Iqbal I, Iqbal K , Tung Y-C, Quinlan M, Wisniewski HM, Binder LI (1986b) Abnormal phosphorylation of the microtubule associated protein tau in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci USA 83: 4913-4917 Hu YY, He SS, Wang X , Duan QH, Grundke-Iqbal I, Iqbal K , Wang JZ (2002) Levels of nonphosphorylated and phosphorylated tau in CSF of Alzheimer disease patients: an ultrasensitive bienzyme-substrate-recycle ELISa. Am J Pathol160: 1269-1278 Hutton M , et al (1998) Association of missense and 5 '-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393: 702-705 Iqbal K, Grundke-Iqbal I (2000) Metabolic hypothesis, mechanism and therapeutic targets of Alzheimer neurofibrillary degeneration. Neurosci News 3: 14-20 Iqbal K , Grundke-Iqbal I, Zaidi T, Merz PA, Wen GY, Shaikh SS, Wisniewski HM, Alafuzoff I, Winblad B (1986) Defective brain microtubule assembly in Alzheimer's disease. Lancet ii: 421-426 Iqbal K, Grundke-Iqbal I, Smith AJ, George L, Tung YC, Zaidi T (1989) Identification and localization of a tau peptide to paired helical filaments of Alzheimer disease. Proc Nat! Acad Sci USA 86: 5646-5650 Iqbal K, Zaidi T , Bancher C, Grundke-Iqbal I (1994) Alzheimer paired helical filaments: restoration of the biological activity by dephosphorylation. FEBS Lett 349: 104-108 Iqbal K, Alonso A del C, Gondal JA, Gong C-X, Haque N, Khatoon S, Sengupta A, Wang J-Z, Grundke-Iqbal I (2000) Mechanism of neurofibrillary degeneration and pharmacologic therapeutic approach. J Neural Transm 59: 213-222 Katzman R, Terry RD , DeTeresa R, Brown R, Davies P, Fuld P, Renling X, Peck A (1988) Clinical, pathological and neurochemical changes in dementia: a subgroup with preserved mental status and numerous neocortical plaques. Ann Neurol23: 138144 Khatoon S, Grundke-Iqbal I, Iqbal K (1992) Brain levels of microtubule-associated protein tau are elevated in Alzheimer's disease: a radioimmuno-slot-blot assay for nanograms of the protein. J Neurochem 59: 750-753

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Khatoon S, Grundke-Iqbal I, Iqbal K (1994) Le vels of normal and abnormally phosphorylated tau in different cellular and regional compartments of Al zheimer disease and control brains. FEBS Lett 351: 80-84 Kopke E , Tung Y-C, Shaikh S, Alonso A del C, Iqbal K, Grundke-Iqbal I (1993) Microtubule associated protein tau: abnormal phosphorylation of non-paired helical filament pool in Alzheimer disease. J Bioi Chern 268: 24374-24384 Lee VM-Y, Balin BJ, Otvos lr L, Trojanowski JQ (1991) A68: a major subu nit of paired helical filaments and derivatized forms of normal tau. Science 251: 675-678 Li M, Guo H , Damuni Z (1995) Purification and characterization of two potent heat-stable protein inhibitors of protein phosphatase 2A from bovine kidney. Biochemistry 34: 1988-1996 Li M, Makkinje A , Damuni Z (1996a) Molecular identification of It PZA , a novel potent heat-stable inhibitor protein of protein phosphatase 2A. Biochemistry 35: 6998-7002 Li M , Makkinje A, Damuni Z (1996b) The Myeloid Leukemia-associated protein SET is a potent inhibitor of Protein Phosphatase 2A. J Bioi Chern 271: 11059-11062 Mah VH, Eskin T A , Kazee AM, Lapham L, Higgins GA (1992) In situ hybridization of calcium/calmodulin dependent protein kinase II and tau mRNAs; species differences and relative preservation in Alzheimer's disease. Brain Res Mol Brain Res 12: 85-94 Morsch R , Simon W , Col eman PD (1999) Neurons may live for decades with neurofibrillary tangles. J Neuropathol Exp Neurol58: 188-197 Oliver Cl, Shenolikar S (1998) Ph ysiologic importance of protein phosphatase inhibitors. Frontiers Biosci 3: 961-972 Poorkaj P, Bird TD, Wijsman E , Nemens E, Garruto RM, Anderson L, Andreadis A , Wiederholt WC, Raskind M , Schellenberg GD (1998) Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann Neurol 43: 815-825 Spillantini MG, Murrell JR, Goedert M , Farlow MR, Klug A , Ghetti B (1998) Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc Nat! Acad Sci USA 95: 7737-7741 Ulitzur N , Rancano C, Pfeffer SR (1997) Biochemical characterization of mapmodulin, a protein that binds microtubule-associated proteins. J Bioi Chern 272: 30577-30582 von Lindern M, van Baal S, Wi egant J, Raap A , Hagemeijer A, Grosveld G (1992) can, a putative oncogene associate d with Myeloid Leukemogenesis, ma y be activated by fusion of its 3' half to different genes: characterization of the set gene. Mol Cell Bioi 12: 3346-3355 Wang l -Z, Gong C-X, Zaidi T , Grundke-Iqbal I, Iqbal K (1995) Dephosphorylation of Alzheimer paired helical filaments by protein phosphatase-2A and -2B . J Bioi Chern 270: 4854-4860 Wang J-Z, Grundke-Iqbal I, Iqbal K (1996) Restoration of biological activity of Alzheimer abnormally phosphorylated by dephosphorylation with protein phosphatase-2A, -2B and -1. Mol Brain Res 38: 200-208 Walaas SI, Greengard P (1991) Protein phosphorylation and neuronal function. Pharmacol Rev 43: 299-349 Weingarten MD , Lockwood AH , Hwo SY , Kirschner MW (1975) A protein factor essential for microtubule assembly. Proc Nat! Acad Sci USA 72: 1858-1 862 Authors' address: K. Iqbal, Ph.D ., Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314-6399 , USA, e-mail: [email protected]

Generation and brain delivery of anti-aggregating antibodies against (i-amyloid plaques using phage display technology B. Solomon and D. Frenkel D ep artment of Molecul ar Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Isr ael

Summary. Antibodies towards the N-terminal region of the ~-amyloid peptide bind to ~-amyloid fibrils, leading to their disaggregation. We generated anti-aggregating ~-amyloid antibodies using filamentous phages displaying th e only four amino acids EFRH found to be the main regulatory site for ~­ amyloid formation. In order to overcome the low permeability of the blood brain barrier for targeting 'anti-aggregating' mAbs to the ~A plaques in the brain, we applied antibody engineering methods to minimize the size of the mAbs while maintaining their biological activity. We found that single-chain antibodies displayed on the surface of the phage are capable of entering the central nervous system (CNS). The feasibility of these novel strategies for the production and targeting of anti-aggregating antibodies against ~-amyloid plaques to disease affected regions in the CNS may have clinical potential for treatment of Alzheimer's disease. Introduction

Site-directed monoclonal antibodies towards the N-terminal region of the ~-amyloid peptide (A~P) bind to preformed ~-amyloid fibrils, leading to their disaggregation and inhibition of their neurotoxic effect (Solomon et al., 1997). Moreover, these antibodies prevent ~-amyloid (A~) formation in vitro (Solomon et aI., 1996) and in vivo (Schenk, 1999). We developed a novel immunization procedure for the production of anti-aggregating ~-amyloid antibodies, using filamentous phages displaying the only four amino acids EFRH found to be the main regulatory site for ~-amyloid formation (Frenkel et aI., 1998). Moreover, we propose the genetically engineered filamentous bacteriophage as an efficient and non-toxic, viral delivery vector to the eNS. Filamentous bacteriophages offer an obvious advantage over other vectors; being bacteriophages they lack the ability to infect mammalian cells as neuron cells unless designed to do so.

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Methods

Preparation of antigen The antigen, consisting of a filamentous phage displaying EFRH epitope on protein surface VIII, was selected from phage peptide library (F88) by anti-Bvamyloid antibody (Frenkel et aI., 2000a).

Immunization procedure The guinea pigs and/or rabbits were immunized intraperitoneally (ip) several times with various doses of phage-EFRH in PBS per injection at 14-day intervals. The animals were challenged with the phage antigens without any adjuvant. Seven days after each injection the animals were bled and their sera tested by ELISA.

Construction of scFv on the phage display The scFv anti-Bvamyloid was prepared from mAb 508, as previously described by Frenkel et al. (2000b). mRNA extraction, first strand cDNA synthesis, PCR amplification of variable heavy (V H) and variable light (V L) genes, and assembly of scFv cassettes were done according to protocols essentially as described (Pharmacia Biotech RPAS manual).

Disaggregation of f3-amyloid fibril by ScFv obtained The effect of ScFv on disruption of the A~ fibril was measured using the ThT reagent (Levine, 1993). The effect of ScFv in preventing A~ mediated neurotoxicity toward cultured cells was measured using rat phenochromocytoma PC12. Viability of the cells exposed to A~ with or without antibody was measured using the MIT assays (Sladowski et aI., 1993).

Detection of phage displayed scFv in mice brain sections The engineered single chain Fv (scFv) antibody fragment 508F fused to filamentous minor coat peptide was used to investigate the ability of the filamentous phage to carry scFv directly to the CNS. The phages carrying scFv conjugated with biotinylated A~P (1-16) were intranasally administered to the mice. Following intranasal administration of one dose of the immunocomplex displayed on the phage, the mice were sacrificed at intervals of 1 and 28 days and their brains taken for further investigation of the presence of phage and immunocomplex. The washed cover slips were then reacted with Streptavidin coupled with CyTM 3 (1 : 50 dilution) for 1 hr at room temperature. The fluorescence was measured with a fluorescence microscope at a final magnification of X10.

Results

The high immunogenicity of filamentous phages enables the raising of antibodies against self-peptides or antigens. Immunization of guinea pigs and/or

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rabbits with EFRH-phage as antigen, in which the ~-amyloid peptide sequence is similar to that in humans, resulted in the production of high affinity (IgG) antibodies in a short period of time (few weeks) (Frenkel et aI., 2000a). Highly specific antibodies (IgG) against the A~P (1 :5000) were received after the fourth injection of EFRH-phage. Similar results were obtained after immunization with 1011 phage per intranasal injection without the adjuvant with an additional administration of antigen. The antibodies resulting from EFRH phage immunization are similar regarding their immunological and anti-aggregating properties to monoclonal antibodies previously studied and to the antibodies raised by direct injection with fibrillar ~-amyloid (Frenkel et aI., 2000a). Ability of phage carrying ScFv to enter/remove AfJP fragment from the brain

ScFv-508F fusion to filamentous minor coat peptide gpIII was used to investigate the ability of scFv to be carried by filamentous phage display system to the CNS. Delivery of the displayed immune complex formed between ScFv and biotinylated ~-amyloid fragment on the filamentous phage was followed in both regions via the olfactory tract in the hippocampus. The fluorescence of immunocomplex was detected in the brains of all the treated animals one day after administration of a single dose. There was no evidence of the biotinylated peptide nor of the ScFv when they were administrated without the phage (Fig. 1). Discussion

Administration of filamentous phages in animals induced a strong immunological response to the phage proteins in all animals tested (Bastein et aI., 1997). In this study, we use a self-anti-aggregating epitope displaced on the recombinant phage as a vaccine, and this technology may be used for the treatment of diseases acquired by abnormal conformational accumulationpeptide diseases, such as Alzheimer's disease. The blood brain barrier imposes obstacles to drug therapies of brain diseases. Therefore, finding a vehicle with the ability to target specific brain regions suffering neuron loss, such as Alzheimer's, may be the key point for treatment of brain amyloidogenic diseases. Due to its linear structure, the filamentous phage has a high permeability to different kinds of membranes (McCafferty, 1990) and, following the olfactory tract, may directly reach the affected sites . Delivery of the displayed immune complex formed between scFv and biotinylated ~-amyloid fragment on the filamentous phage was followed in both the olfactory bulb and in the hippocampus region. The filamentous phage maintains the biological activity of the potential therapeutic molecules (as in the case of the immunocomplex of scFv 508 F with its antigen related to Alzheimer's disease) and efficiently penetrates biological membranes.

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A

c

8

D

Fig. 1. Immunofluorescence detection of filamentous phage displaying ScFv-508F. The ScFv was coupled to biotinylated 1-16 A~P and detected in brain sections following intranasal administration. The presence of 1-16 A~P fragment immunocomplexed with ScFv 508F filamentous phage display in mice olfactory bulb sections (A) hippocampus sections (B) was visualized with Streptavidin coupled with cyrM 3 compared to administration of ~-amyloid alone (C,D)

These preliminary data suggest that filamentous phages could carry therapeutic molecules to the CNS while maintaining their biological properties without any toxic side effects. The feasibility of these novel strategies for the production and targeting of anti-aggregating antibodies against ~-amyloid plaques to disease affected regions in the CNS may have clinical potential for treatment of AD. References Bastcin N , Trudel M, Simard C (1997) Protective immune responses induced by the immunization of mice with recombinant bacteriophage displaying an epitope of the human respiratory syncytical virus . Virology 234: 118-122

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Frenkel D , Balass M, Solomon B (1998) N-Terminal EFRH sequence of Alzheimer's ~-amyloid peptide represents the epitope of its anti-aggregation antibodies. J Neuroimmunol88: 85-90 Frenkel D , Katz 0 , Solomon B (2000a) Immunization against Alzheimer's ~-amyloid plaques via EFRH-phage administration. Proc Natl Acad Sci USA 97(21): 1145511459 Frenkel D, Solomon B, Benhar I (2000b) Modulation of Alzheimer's ~-amyloid neurotoxicity by site-directed single-chain antibody. J NeuroimmunoI106(1-2): 23-31 Levine H III (1993) Thioflavine T interaction with synthetic Alzheimer's disease ~­ amyloid peptides: detection of amyloid aggregation in solution. Protein Sci 2: 404410 McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348: 552-554 Schenk D , Barbour R , Dunn W, Gordon G , Grajeda H ,m Guido T , Hu K, Huang J , Johnson-Wood K, Khan K et al (1999) Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400(6740): 173-177 Sladowski D , Steer SJ, Clothier RH, Balls M (1993) An improved MTT assay. J Immunol Methods 157(1-2): 203-207 Solomon B, Koppel R , Hanan E, Katzav T (1996) Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer ~-amyloid peptide. Proc Natl Acad Sci USA 93: 452-455 Solomon B, Koppel R, Frenkel D, Hanan-Aharon E (1997) Disaggregation of Alzheimer f3-amyloid by site-directed mAb. Proc Natl Acad Sci USA 94: 4109-4112 Authors' address: B. Solomon, Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Tel-Aviv 69978, Israel, e-mail: [email protected]

Effects of Cerebrolysint'" on amyloid-B deposition in a transgenic model of Alzheimer's disease E. Rockenstein', M. Malloryl, M. Mantel, M. Alford', M. Windisch3, H. Moessler', and E. Masliah1,2 1 D ep artment of Neurosciences and 2Departme nt of Pathology, University of Californi a San Diego, School of Medicine, La Joll a, Ca, USA 3Institute of E xperimental Ph armacology, JSW-RESEARCH, Graz, and 4 EBEWE Pharmaceuticals, Research Di vision , Unterach, Austria

Summary. We investigated the potential mechanisms through which Cerebrolysin'Y, a neuroprotective noothropic agent, might affect Alzheimer's disease pathology. Transgenic (tg) mice expressing mutant human (h) amyloid precursor protein 751 (A PP751) cDNA under the Thy-1 promoter (mThy1hAPP751) wer e treated for four weeks with this compound and analyzed by confocal microscopy to asses its effects on amyloid plaque formation and neurodegeneration. In this model, amyloid plaques in the brain ar e found much earlier (beginning at 3 months) than in other tg models. Quantitative computer-aided analysis with anti-amyloid-B protein (A~) antibodies, revealed that Cerebrolysin significantly reduced the amyloid burden in the frontal cortex of 5-month-old mice. Furthermore, Cerebrolysin treatment reduced the levels of A~l-42' This was accompanied by amelioration of the synaptic alterations in the frontal cortex of mThy1 -hAPP751 tg mice . In conclusion, the present study supports the possibility that Cerebrolysin might have neuroprotective effects by decreasing the production of A~l-42 and reducing amyloid deposition.

Introduction The neurodegenerative process in Alzheimer's disease (AD) is characterized by synaptic injury (Terry et al., 1994; Masliah et al., 1997) and neuronal loss (Terry et al., 1981), accompanied by amyloid deposition (Selkoe, 1989), astrogliosis (Beach et al., 1989) and microglial cell proliferation (R ogers et al., 1988; Masliah et al., 1991a). Although the precise mechanisms leading to neurodegeneration in AD are not completely clear, most recent studies have focused on the role of amyloid ~ protein (A~) precursor (APP) and it's products in AD pathogenesis (Masters et al., 1985; Selkoe, 1989). Various studies have implicated APP in AD pathogenesis because: 1) APP mutations

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are associated with familial AD (Goate et al., 1991; Clark and Goate, 1993), 2) APP degradation products are found in AD brains (Sisodia et al., 1990), and 3) overexpression of mutated APP in transgenic (tg) mice results in ADlike pathology (Games et al., 1995). In AD, APP abnormally accumulates in dystrophic neurites of plaques and in synaptic terminals (Cole et al., 1989; Martin et al., 1989; Arai et al., 1991; Cras et al., 1991; Masliah et al., 1992b) and the balance between APP isoforms is altered with a relative shift toward an increase in APP7701751 vs. APP695 (Rockenstein et al., 1995). In view of the pivotal involvement of APP in AD pathogenesis, recent studies have focused on developing treatments aimed at reducing Af3 deposition in the brain. While some studies have tested the efficacy of antiinflammatory agents (Lim et al., 2000), others have investigated the effects of inhibitors of the y-secretase pathway (Moore et al., 2000) and, more recently, the effects of an Af3 vaccine (Schenk et al., 1999). However, it is unclear whether reduction of Af3 load in these models is also associated with preservation of the synaptic and neuronal structure. In this regard, it is possible that therapeutic agents with a neurotrophic activity might have this combined effect. Cerebrolysin" (Cere) (a mixture of peptides and amino acids obtained from porcine brain tissue) is a nootrophic agent that improves memory in patients with mild to moderate cognitive impairment (Ruther et al., 1994a, b). This compound has also shown a neurotrophic activity in vitro (Mallory et al., 1999) and in animal models of neurodegeneration (Francis-Turner and Valouskova, 1996; Masliah et al., 1999; Veinbergs et al., 2000). Then, for the present study, we tested the effects of Cere on plaque formation and neurodegeneration in APP tg mice expressing mutated human (h)APP751 under the control of the murine (m)Thy-1 promoter (mThy1-hAPP751). Based on our results, we conclude that Cere is capable of reducing plaque formation and preventing synaptic damage in this tg model of AD.

Materials and methods Generation ofmThyl-hAPP751 tg mice, treatment regimen and tissue processing The tg mice generated express mutated hAPP751 under the control of the mThy-1 promoter (mThy1-hAPP751) and, for this study, the highest expresser (line 41) tg mice were used (Rocken stein et al., 2001). These tg mice are unique in that, compared to other tg models, amyloid plaques are found in the brain at a much earlier age (beginning at 3 months) (Masliah and Rockenstein, 2000). Genomic DNA was extracted from tail biopsies and analyzed by PCR amplification, as described previously (Rockenstein et al., 1995). Transgenic lines were maintained by crossing heterozygous tg mice with nontransgenic (nontg) C57BL/6 X DBA/2 F1 breeders. All mice were heterozygous with respect to the transgene and the nontg littermates served as controls. Twenty-four 4month-old mice were utilized for the present study (n = 12 mThyl-hAPP751 tg; n = 12 nontg). For each group, 6 mice received daily intraperitoneal (IP) injections of saline and the other 6 received daily IP injections of Cere (Batch# 802772, 5 mllkg) for 4 weeks. In accordance with NIH guidelines for the humane treatment of animals, mice were anesthetized with chloral hydrate and flush-perfused transcardially with 0.9 % saline.

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Brains were removed and divided sagitally. One hemibrain was post-fixed in phosphatebuffered 4% paraformaldehyde (pH 7.4) at 4°C for 48hr and sectioned at 40-!-!m with a Vibratome 2000 (Leica), while the other hemibrain was snap frozen and stored at -70°C for protein analysis.

Determination of hAPP expression Levels of hAPP immunoreactivity were det ermined in brain homogenates by Western blot and in vibratome sections by immunocytochemistry, as previously described (Mucke et aI., 1994). For Western blot analysis, Ifi-ug per lane of cytosolic and particulate fractions, assayed by the Lowry method, were loaded into 10% SDS-PAGE gels and blotted onto nitrocellulose paper. Blots were incubated with the mouse monoclonal antiAPP antibody (Clone 22Cll, 1 :20,000, Chemicon International, Temecula, CA), followed by 1251 protein A. Blots were exposed to PhosphorImager scre ens (Molecular Dynamics) and analyzed with ImageQuant software. For immunocytochemical analysis of hAPP expression, 40-~lm vibratome sections were incubated with anti-hAPP (Clone 22Cl1 , 1: 1000), as previously described (Mucke et aI., 1994). After an overnight incubation at 4°C, binding of primary antibody was detected with the ABC Elite kit (Vector Laboratories, Burlingame, CA) using diaminobenzidine tetrahydrochloride (DAB) with 0.001 % H 20 2 for development. Sections were analyzed, as previously described (Mucke et aI., 1994), with the Quantimet 570C.

Neuropathological analysis and detection of Af3 deposits Vibratome sections were incubated overnight at 4°C with the mouse monoclonal antibody 4G8 (1: 600, Senetek), which specifically recognizes AI). Two methods were used to detect primary antibody binding: 1) Vector ABC Elite kit and DAB/H 20 2 , and 2) FlTCconjugated anti-mouse IgG (Vector). Sections reacted with DAB/H20 2 were examined with a 2.5 x objective of the Olympus Vanox light microscope. The percent area of the hippocampus covered by 4G8-immunoreactive material ("plaque load") was assessed, as previously described (Mucke et aI., 2000), with the Quantimet 570C. The FITC-Iabeled sections were imaged with the laser scanning confocal microscope (LSCM) (BioRad 1024; BioRad Laboratories, Hercules, CA), as described previously (Mucke et aI., 2000). Digitized images were analyzed with the NIH Image 1.43 program in order to determine the number of plaques per unit area and the plaque size. Three immunolabeled sections were analyzed per mouse and the average of individual measurements was used to calculate group means. Additional analysis of the neuropathological alterations in the mThy1hAPP751 tg mice was performed in sections immunolabeled with the antibody against glial fibrillary acidic protein (GFAP, Chemicon), a marker of astroglial cells (Mucke et aI., 2000).

Determination of Af3 levels by ELISA Brain samples of the mouse cortex were homogenized in an ice-cold buffer [5M guanidine-HCI and phosphate-buffered saline (PBS, pH 8.0)] with 1X protease inhibitor cocktail (Calbiochem, San Diego, CA). Homogenates were then mixed for 3-4hrs at room temperature and centrifuged at 16,000 X g for 20 minutes at 4°C. The resulting supernatants were diluted tenfold in Dulbecco's PBS (pH 7.4), containing 5% bovine serum albumin and 0.03 % Tween 20. Quantification of the levels of Al)l_40 and A1)1_42was

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performed with a commercially available sandwich-type ELISA (Bioso urce International, Camarillo, CA).

D etermination of the density of presynaptic terminals Vibratome sections were incubated overnight with the monoclonal antibod y against synaptophysin (Lfl ug/ml, Chemicon), follow ed by FITC-conjugat ed anti-mouse IgG (Vector) and imaging with the LSCM (MRC1024) (Mucke et al., 2000). Once the linear range of fluor escenc e int en sity was determined in sections from nontg mice , this setting was used to collect all images analyzed in the same experime nt (Buttini et al., 1999). For each mouse, 12 confocal imag es (4 per section) from the frontal cortex were obtained. Digitized images were transferred to a Macintosh computer and analyzed with the NIH Image 1.43 software. Th e area occupied by synaptophysin-immunore active presynaptic terminals was qu antified and expressed as a perc ent of the total area imaged, as previously described (Mucke et al., 2000). This method of quantifying synaptophysin-immunor eacti ve presynaptic terminals has been exte nsively used to assess neurodegen er ative alte rations in human brains (Masliah et aI., 1991b, 1992a; Knowless et al., 1998) and in various expe rimental mod els (T oggas et al., 1994; Games et al., 1995; Buttini et al., 1999). Other studies ha ve validated this method by comparison s with quantitative immunobl ots (A lford et al., 1994; Mucke et al., 1994), quantificat ion of syna ptic proteins by ELISA (Brow n et al., 1998; Buttini et al., 1999), and a modified ste reological "disector" approach (E verall et al., 1999; H sia et al., 1999). To ensure objective assess me nt and reli ability of results, brain sections from mice to be compar ed in any given expe riment were blind cod ed and processed in par allel. Once all results wer e ob tain ed , codes were broken an d dat a ana lysis was perfo rmed.

Statistical analysis Analyses were carried out with the StatView 5.0 program (SA S Institute Inc. , Cary, NC) . Di ffer enc es among mean s were assessed by one-way ANOVA with post-hoc Dunnett's. Comparisons betw een 2 groups wer e done with the two-tailed unpaired Student's t-test. Correlation studies wer e carried out by simple regr ession analysis and the null hypothesis was rej ected at th e 0.05 level.

Results

Compared to saline-treated nontg control mice (Fig. lA) , 5-month-old salinetreated mThyl-hAPP75l tg mice showed the formation of amyloid plaques in the frontal cortex (Fig. lB), accompanied by astrogliosis (not shown). After four weeks of Cere tr eatment, mThyl-hAPP75l tg mice showed a significant reduction in the percent area of the front al cortex (Fig . lC, D) and hippocampus (Fig. lD) covered by Ap-immunoreactive plaques. Furthermore, computer-aided quantification showed a decrease both in th e size of the remaining plaques (Fig . lE) and the astr oglial reaction (not shown). Consistent with these findings, analysis of A~ 1-40 and A~1-42 levels by ELISA showed that, in the frontal cortex, Cere significantly reduced the levels of Af31-42' but not Af31-40 (Fig. 2A). In addition, Western blot analysis showed th at Cere had a moderate effe ct on the levels of APP immunoreactivity (Fig. 2B), suggesting

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that the effects of this compound on A~1-42 might be related to regulation of APP expression. To determine if the amyloid-reducing effects of Cere were accompanied by amelioration of the neurodegenerative alterations associated with A~ production, LSCM analysis was performed. This study showed that, compared to saline-treated nontg mice (Fig . 3A), saline-treated mThyl-hAPP751 tg mice displayed a significant loss of synaptophysin immunoreactivity in the frontal cortex (Fig . 3B, E). In contrast, Cere-treated mThyl-hAPP751 tg mice showed levels of synaptophysin immunoreactivity comparable to nontg control mice (Fig. 3C, E) . Discussion

Studies in patients with mild to moderate AD, have shown that Cere improves cognitive performance and that these effects are maintained even 6 months

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after termination of therapy (Ruther et al., 2000). The present study showed that, in mThy1-hAPP751 tg mice, after 4 weeks of treatment Cere reduced plaque formation and neurodegeneration associated with A~ 1-42 production. The mechanisms responsible for these in vivo anti-amyloidogenic and neuroprotective effects of Cere are not completely clear. However, recent studies have shown that this compound can exert a neurotrophic-like activity by: 1) promoting synaptic formation in 6-week-old (Windholz et al., 2000) and aged (Reinprecht et al., 1999) rats, 2) protecting against excitotoxicity (Veinbergs et al., 2000) , and 3) ameliorating cognitive deficits in apolipoprotein E (apoE)- deficient mice (Masliah et al., 1999) . Furthermore, in a manner similar to nerve growth factor (NGF) (Rossner et al., 1998), Cere promotes neuritic outgrowth and cholinergic fiber regeneration (FrancisTurner and Valouskova, 1996; Satou et al., 2000). Interestingly, neurotrophic factors such as NGF are capable of modulating A~ production by regulating APP expression (Rossner et al., 1998). Consistent with this possibility, the present study showed that Cere modulates APP production and a previous in vitro study in a human neuronal cell line (NTIN) showed that Cere promotes synaptic formation by regulating APP expression (Mallory et aL, 1999). Furthermore, other reports have shown that APP is synaptotro phic (Mucke et al., 1994) , and that while secreted a-APP might promote synapse formation, production of A~ I -42 re sults in synapse loss (Mucke et al., 2000). Taken together, these studies suggest that Cere might reduce amyloid formation and neurodegeneration by regulating APP expression. In conclusion, Cere might

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have beneficial effects in AD patients both by decreasing amyloid production and promoting synaptic repair. Acknowledgements This work was supported by NIH grants AGI0869 and AG5131 and by a grant from EBEWE Pharmaceuticals.

References Alford MF, Masliah E , Hansen LA, Terry RD (1994) A simple dot-immunobinding assay for the quantification of synaptophysin-like immunoreactivity in human brain. J Histochem Cytochem 42: 283-287 Arai H , Lee V-Y, Messinger ML , Greenberg BD, Lowery DE, Trojanowski JQ (1991) Expression patterns of f)-amyloid precursor protein (f)-APP) in neural and nonneural tissues from Alzheimer's disease and control subjects. Ann Neurol 30: 686-693 Beach TG, Walker R, McGeer EG (1989) Patterns of gliosis in Alzheimer's disease and aging cerebrum. Glia 2: 420-436 Brown DF, Risser RC, Bigio EH, Tripp P, Stiegler A, Welch E, Eagan KP, Hladik CL, White CL (1998) Neocortical synapse density and Braak stage in the Lewy body

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variant of Alzheimer disease: a comparison with classic Alzheimer disease and normal aging. J Neuropathol Exp Neurol 57: 955-960 Buttini M, Orth M, Bellosta S, Akeefe H, Pitas RE, Wyss-Coray T , Mucke L, Mahley RW (1999) Expression of human apolipoprotein E3 or E4 in the brains of Apoe - j mice : isoform-specific effects on neurodegeneration. J Neurosci 19: 4867-4880 Clark RF, Goate AM (1993) Molecular genetics of Alzheimer's disease. Arch Neurol 50: 1164-1172 Cole GM, Masliah E, Huynh TV, DeTeresa R, Terry RD, Okudea C, Saitoh T (1989) An antiserum against amyloid I)-protein precursor detects a unique peptide in Alzheimer brain. Neurosci Lett 100: 340-346 Cras P, Kawai M, Lowery D, Gonzalez-DeWhitt P, Greenberg B, Perry G (1991) Senile plaque neurites in Alzheimer disease accumulate amyloid precursor protein. Proc Natl Acad Sci USA 88: 7552-7556 Everall IP, Heaton RK, Marcotte TD, Ellis RJ, McCutchan JA, Atkinson JH, Grant I, Mallory M, Masliah E , the HNRC Group (1999) Cortical synaptic density is reduced in mild to moderate human immunodeficiency virus neurocognitive disorder. Brain Pathol 9: 209-217 Francis-Turner L, Valouskova V (1996) Nerve growth factor and nootropic drug Cerebrolysin but not fibroblast growth factor can reduce spatial memory impairment elicited by fimbria-fornix transection: short-term study. Neurosci Lett 202: 1-4 Games D, Adams D, Alessandrini R, Barbour R , Berthelette P, Blackwell C, Carr T, Clemes J , Donaldson T, Gillespie F, Guido T, Hagopian S, Johnson-Wood K, Khan K, Lee M, Leibowitz P, Lieberburg I, Little S, Masliah E, McConlogue L, MontoyaZavala M, Mucke L, Paganini L, Penniman E , Power M, Schenk D, Seubert P, Snyder B, Soriano F, Tan H , Vitale J, Wadsworth S, Wolozin B, Zhao J (1995) Alzheimertype neuropathology in transgenic mice overexpressing V717F l3-amyloid precursor protein. Nature 373: 523-527 Goate A, Chartier-Harlin M-C, Mullan M, Brown J , Crawford F, Fidani L, Guiffra L, Haynes A, Irving N, James L, Mant R , Newton P, Rooke K, Roques P, Talbot C, Williamson R, Rossor M, Owen M, Hardy J (1991) Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature 349:704 Hsia A Y, Masliah E , McConlogue L, Yu G-Q, Tatsuno G, Hu K, Kholodenko D , Malenka RC, Nicoll RA, Mucke L (1999) Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc Nat! Acad Sci USA 96: 32283233 Knowless RB, Gomez-Isla T, Hyman BT (1998) Abeta associated neuropil changes: correlation with neuronal loss and dementia. J Neuropathol Exp Neurol 57: 11221130 Lim GP, Yang F , Chu T, Chen P, Beech W, Teter B, Tran T, Ubeda 0 , Ashe KH, Frautschy SA, Cole GM (2000) Ibuprofen suppresses plaque pathology and inflammation in a mouse model of Alzheimer's disease. J Neurosci 20: 5709-5714 Mallory M, Honer W, Hsu L, Johnson R , Masliah E (1999) In vitro synaptotrophic effects of Cerebrolysin in NT2N cells. Acta Neuropathol 97: 437-446 Martin LJ, Cork LC, Koo EH, Sisodia SS, Weidemann A , Beyreuther K, Masters C, Price DL (1989) Localization of amyloid precursor protein (APP) in brains of young and aged monkeys. Soc Neurosci Abstr 15: 23 Masliah E , Rockenstein E (2000) Genetically altered transgenic models of Alzheimer's disease. J Neural Transm [Suppl] 59: 175-183 Masliah E , Mallory M, Hansen L, Alford M, Albright T, Terry R, Shapiro P, Sundsmo M, Saitoh T (1991a) Immunoreactivity of CD45, a protein phosphotyrosine phosphatase, in Alzheimer disease. Acta Neuropathol 83: 12-20 Masliah E, Terry RD, Alford M, DeTeresa RM, Hansen LA (1991b) Cortical and subcortical patterns of synaptophysin-like immunoreactivity in Alzheimer disease. Am J Pathoi 138:235-246

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Masliah E, Ellisman M, Carragher B, Mallory M, Young S, Hansen L, DeTeresa R, Terry RD (1992a) Three-dimensional analysis of the relationship between synaptic pathology and neuropil threads in Alzheimer disease. J Neuropathol Exp Neurol 51: 404414 Masliah E, Mallory M, Ge N, Saitoh T (1992b) Amyloid precursor protein is localized in growing neurites of neonatal rat brain. Brain Res 593: 323-328 Masliah E , Mallory M, Alford M, DeTeresa R , Iwai A, Saitoh T (1997) Mole cular mechanisms of synaptic disconnection in Alzheimer's disease. In: Hyman BT, Duyckaerts C, Christen Y (eds) Connections, cognition and Alzheimer's disease, Springer, Berlin Heidelberg New York Tokyo, pp 121-140 Masliah E, Armasolo F, Veinbergs I, Mallory M, Samuel W (1999) Cerebrolysin ameliorates performance deficits and neuronal damage in apolipoprotein E-deficient mice. Pharmacol Biochem Bev 62: 239-245 Masters CL, Simms G , Weidemann A , Multhaup G, McDonald BL, Beyreuther K (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Nat! Acad Sci USA 82: 4245-4249 Moore CL, Diehl TS, Selkoe DJ, Wolfe MS (2000) Toward the characterization and identification of gamma-secretases using transition-state analogue inhibitors. Ann NY Acad Sci USA 920: 197-205 Mucke L, Masliah E, Johnson WB, Ruppe MD, Rockenstein EM, Forss-Petter S, Pietropaolo M, Mallory M, Abraham CR (1994) Synaptotrophic effects of human amyloid Bprotein precursors in the cortex of transgenic mice . Brain Res 666: 151167 Mucke L, Masliah E, Yu G-Q, Mallory M, Rockenstein EM, Tatsuno G, Hu K, Kholodenko D, Johnson-Wood K, McConlogue L (2000) High-level neuronal expression of Abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. J Neurosci 20: 4050-4058 Reinprecht I, Gschanes A , Windisch M, Fachbach G (1999) Two peptidergic drugs increase the synaptophysin immunoreactivity in brains of 24-month-old rats. Hi stoch em J 31: 395-401 Rockenstein EM, McConlogue L, Tan H, Power M, Masliah E, Mucke L (1995) Levels and alternative splicing of amyloid Bprotein precursor (APP) tr an scripts in brains of APP transgenic mice and humans with Al zheimer's disease. J Biol Chem 270: 2825728267 Rockenstein E , Mallory M, Mante M, Alford M, Masliah E (2001) Early formation of mature AB deposits in a mutant APP transgenic model. J Neurosci Res 66: 573-582 Rogers J, Luber-Narod J, Styren SD, Civin WH (1988) Expression of immune systemassociated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer's disease. Neurobiol Aging 9: 339-349 Rossner S, Ueberham U, Schliebs R, Perez-Polo JR, Bigl V (1998) The regulation of amyloid precursor protein metabolism by cholinergic mech anisms and neurotrophin receptor signaling. Prog Neurobiol 56: 541-569 Ruther E, Ritter R, Apecechea M, Freytag S, Windisch M (1994a) Efficacy of the peptidergic nootropic drug cerebrolysin in patients with senile dementia of the Alzheimer's type (SDAT). Pharmacopsychiatry 27: 32-40 Ruther E , Ritter R , Apecechea M, Freitag S, Windisch M (1994b) Efficacy of Cerebrolysin in Alzheimer's disease. In: Jellinger KA, Ladurner G, Windisch M (eds) New trends in the diagnosis and therapy of Alzheimer's disease. Springer, Wien New York, pp 131-141 Ruther E, Ritter R , Apecechea M, Freytag S, Gmeinbauer R, Windisch M (2000) Sustained improvements in patients with dementia of Alzheimer's type (DAT) 6 months after termination of Cerebrolysin therapy. J Neural Transm 107: 815-829 Satou T, Itoh T, Tarnai Y, Ohde H, Anderson AJ, Hashimoto S (2000) Neurotrophic effects of FPF-1070 (Cerebrolysin) on cultured neurons from chicken embryo dorsal root ganglia, ciliary ganglia, and sympathetic trunks. J Neural Transm 107: 1253-1262

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Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H , Guido T , Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R , Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, Walker S, Wogulis M, Yednock T, Games D, Seubert P (1999) Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400: 173-177 Selkoe DJ (1989) Amyloid ~ protein precursor and the pathogenesis of Alzheimer's disease. Cell 58: 611-612 Sisodia SS, Koo EH, Beyreuther K, Unterbeck A, Price DL (1990) Evidence that Bamyloid protein in Alzheimer's disease is not derived by normal processing. Science 248:492-494 Terry RD, Peck A , D eTeresa R, Schechter R, Horoupian DS (1981) Some morphometric aspects of the brain in senile dementia of the Alzheimer type. Ann Neurol10: 184192 Terry RD, Hansen L, Masliah E (1994) Structural basis of the cognitive alterations in Alzheimer disease. In: Terry RD , Katzman R (eds) Alzheimer disease. Raven Press, New York, pp 179-196 Toggas SM, Masliah E, Rockenstein EM, Mucke L (1994) Central nervous system damage produced by expression of the HIV-1 coat protein gp120 in transgenic mice. Nature 367: 188-193 Veinbergs I, Mante M, Mallory M, Masliah E (2000) Neurotrophic effects of Cerebrolysin in animal models of excitotoxicity. J Neural Transm 59: 273-280 Windholz E, Gschanes A , Windisch M, Fachbach G (2000) Two peptidergic drugs increase the synaptophysin immunoreactivity in brains of 6-week-old rats . Histochem J 32: 79-84 Authors ' address: Dr. E. Masliah, Department of Neurosciences, University of California San Diego, La Jolla, CA 92093-0624, U.S.A., e-mail: [email protected]

Vitamin E binding protein Afamin protects neuronal cells in vitro M. Heiser--', B. Hutter-Paier-, L. Jerkovlc-, R. Pfragner-, M. Windisch!,

M. Becker-Andre", and H. Dieplinger>' 1 JSW Research GmbH, Graz, 2Vitateq Biotechnology GmbH, Innsbruck, 3 Institute for Pathophysiology, Karl-Franzens-University Graz, and 4Institute of Medical Biology and Human Genetics, University of Innsbruck, Austria

Afamin, an 87kDa human plasma glycoprotein with specific binding properties for vitamin E (a-tocopherol) was recently characterized (Jerkovic, 1997; Vogele, 1999). In the present study the in vitro effects on neuronal cells of native human Afamin, of Afamin pre-loaded with vitamin E (Afamin +), and of vitamin E were investigated. Isolated cortical chicken neurons were maintained either under apoptosis-inducing low serum conditions or exposed to oxidative stress by the addition of H 2 0 2 or beta-amyloid peptide2s_3s• Afamin and vitamin E synergistically enhance the survival of cortical neurons under apoptotic conditions. Furthermore, Afamin alone protects cortical neurons from cell death in both experimental settings. Therefore, the plasma glycoprotein Afamin apparently displays a neuroprotective activity not only by virtue of binding and transporting vitamin E but also on its own.

Summary.

Introduction

Alzheimer's disease (AD) is the most common form of dementia in the elderly and affects about 15 million people worldwide (Honig and Mayeux, 2001). Its cause is still unknown and there is neither a cure nor an universally effective treatment (Tyas et aI., 2001). However, there is a growing body of evidence suggesting that oxidative stress plays a key role in the pathophysiology of neurodegenerative disorders including AD (Mattson, 2000a). Indeed, in AD brains increased lipid peroxidation, oxidative damage to proteins and nucleic acids, protein nitration, accumulation of advanced glycation end products, elevation of iron and a reduction of Mn-superoxide dismutase have been reported (Brera et aI., 2000). These effects have been documented in association with neurofibrillary tangles, neuritic plaques, impaired mitochondrial function and pertubation of calcium homeostasis (Mattson, 2000b). An involvement of free radicals accounts for the two most striking features of AD , the multitude of abnormalities affecting essentially every system and the strict

K. A. Jellinger et al. (eds.), Ageing and Dementia Current and Future Concepts © Springer-Verlag/Wein 2002

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age dependence (Smith and Perry, 2000). Reactive oxygen species (ROS), such as superoxide anions, hydroxyl radicals, and hydrogen peroxide, are produced as a result of normal and aberrant cellular reactions. The role of ROS in the pathophysiology of AD suggests that the reduction of oxidative stress by pharmacological interventions may slow or halt the rate of neurodegeneration (Massoud et al., 2000). Therefore, therapies based on administration of antioxidant and ROS-scavenging substances have been widely proposed for treatment of neurological diseases (Contestabile, 2001). As several clinical trials have shown, vitamin E (a-tocopherol), which functions as an antioxidant, has become attractive as therapeutic agent in the treatment of AD (Sano et al., 1997). Recent studies suggest that this lipophilic free radical scavenger delays the progression of AD by converting free radicals into less damaging compounds, hence avoiding their deleterious effects (Thal, 2000). In plasma, vitamin E is transported by several lipoproteins such as HDL, LDL and VLDL (Herrera and Barbas, 2001), yet no specific transport protein in extravascular fluids like cerebrospinal fluid (CSF) has been reported. Since this vitamin is also found in CSF (Jimenez-Jimenez et al., 1997) and is known to hardly pass the blood-brain barrier (Behl and Moosmann, 2000), specific transport mechanisms are still hypothesized. Jerkovic (1997) discovered a new 87kDa human plasma glycoprotein with a micromolar affinity to vitamin E . This protein was subsequently identified as Afamin, a member of the albumin gene family (Lichenstein et al., 1994). Since Afamin is found in serum and in CSF at concentrations about 60mg/l and 0.2mg/1 respectively, it was hypothesized to serve as a carrier for vitamin E . The following study was performed to investigate the effects of native human Afamin, Afamin pre-loaded with vitamin E, and vitamin E alone on neuronal cells under various cell culture conditions. Material and methods

Purification of Afamin Afamin was purified from lipoprotein depleted human plasma by a combination of various chromatographic methods. Purity of the protein was assessed by reducing SDS-PAGE and silver staining (Jerkovic, 1997).

Tissue culture The cell culture experiments were performed with cortical neurons as described by Wronski et al. (2000) and Hutter-Paier et al. (1998).

Substances Afamin, Afamin pre-loaded with vitamin E , in the following referred to as Afamin +, and vitamin E were tested. For control purpose a buffer containing Tween 80 was used.

Neuroprotective properties of Afamin

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Afamin, vitamin E and buffer-control were diluted with medium in glass tubes. Afamin + was generated by incubating Afamin with various concentrations of vitamin E (Cttocopherol, Sigma Aldrich) for 1 hour at room temperature followed by 10 minutes on ice. All substances were added directly to the cultures from the first day onwards.

In vitro lesions In the 2% low serum assay neurons were lesioned due to a partial withdrawal of FCS (Reinprecht et al., 1998) . For lesion with the beta-amyloid peptide fragment 25-35 (A~25-35) or with hydrogen peroxide (H202), cells were pre-cultured in a medium supplemented with 5% FCS. At the 8th day in vitro (DIV) a 24 hours-lesion was carried out by adding either A~25-35 (Sigma, 20 ~lM) ,pre-agregated for 72 hours, or H 20 2 (100 ~M) . Phosphate buffered saline (PBS) was used as a vehicle control.

MTT viability assay At th e end of each experiment viability of the remaining neurons was assessed with the colorimetric MIT reduction test according to Gutmann et al. (2002). MIT-values (n > 6), are given as mean and s.e.m, Statistical discriminations were made by two-way analysis of variance (ANOVA) followed by the Duncans post hoc test. Differences were considered significant at p :::; 0.05.

Results

Effects of Afamin pre-loaded with different concentrations of vitamin E on the viability of isolated cortical neurons from chicken brain are shown in Fig. 1. Afamin (60 ug/ml) was pre-loaded with various concentrations of vitamin E (0-5200IlM) and subsequently added to the cultures from the first day onwards. MTT assay results, assessed at the 7th DIV, show clear dose-dependent effects of this pre-loaded Afamin (Afamin+) on neuronal viability. Afamin+ and vitamin E curves take a parallel course. However, the neuroprotective effects of Afamin + are more pronounced than those of vitamin E alone. Maximal effects on neuronal viability can be observed with Afamin preloaded with 52-520 11M vitamin E. Yet, significant effects of Afamin + arise already at a concentration of vitamin E as low as 0.052 11M. At a concentration of 60 ug/ml , Afamin alone is as protective as 5.2 11M vitamin E alone. Effects of Afamin and Afamin + on cortical neurons maintained under low serum cell stress conditions are shown in Fig. 2. Degenerative effects on the neurons can be diminished in the presence of Afamin and Afamin +. At concentrations of 30-60 ug/ml, Afamin enhances neuronal viability in a dose-dependent manner (p < 0.05). Afamin + and vitamin E significantly protect neurons from cell death over a broad dose range. However, at concentrations above 151lg/ml Afamin pre-loaded with 130llM vitamin E , effects of Afamin + are significantly higher than those observed in the presence of vitamin E alone. Effects of Afamin and vitamin E on the viability of isolated cortical neurons submitted to oxidative stress by the addition of H 2 0 2 or beta-amyloid peptideg.j, are

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shown in Fig. 3. In both lesion models Afamin and Afamin + are able to counteract neurodegenerative effects. After HzO z lesion (Fig . 3A), 30 and 60 ug/ml Afamin significantly protect cells from degeneration. The activities of vitamin E as well as of Afamin + are comparable to those of Afamin alone. In the A1325-35 lesion model (Fig. 3B) the effects of Afamin alone are more pronounced than those of Afamin + and vitamin E. Afamin acts already at concentrations as low as 0.9 ug/ml, strongly increasing towards higher concentrations. Afamin + and vitamin E curves demonstrate similar courses while their protective effects can be observed only at higher concentrations. Discussion

The use of vitamin E for treatment of AD has increasingly become subject of interest (Tabet et al., 2000). Treatment with vitamin E was shown to slow down the progression of AD (Sano et al., 1997) and might therefore offer a therapeutically relevant solution. Vitamin E acts as an antioxidant, scavenging toxic free radicals and preventing the propagation of radical damage (Christen, 2000). Because of its hydrophobicity vitamin E requires special transport mechanisms. Although several intracellular a-tocopherol binding

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proteins are known (Dutta-Roy, 1999; Zimmer et al., 2000), vitamin E has no specific plasma transport protein. The majority of plasma Vitamin E is transported via the lipoprotein system (Traber and Sies, 1996). The mechanism of transport into extravascular fluids like eSF, follicular and seminal fluid is still unknown. In 1997 the 87 kDa human plasma glycoprotein Afamin was shown to have a micromolar affinity to a-tocopherol (Jerkovic, 1997). Afamin is the fourth member of the human albumin gene family (Lichenstein et al., 1994) next to serum albumin, a-fetoprotein, and vitamin-D-binding protein. Afamin was detected in eSF, follicular and seminal fluid and the amounts correlate well with the concentrations of a-tocopherol (unpublished observations), suggesting vitamin E-transporting properties of Afamin. In the present study, the biological activity of Afamin was investigated in several in vitro cell culture experiments using isolated cortical chicken neurons. In almost all experiments Afamin is most effective at concentrations of 30 and 60llg/ml, which is comparable to values found in human plasma (Jerkovic, 1997). Data obtained in our study suggest a dual role for Afamin: (1) Afamin and vitamin E act synergistically on enhancing the survival of isolated cortical neurons; (2) Afamin itself protects neurons from cell death. The effects of Afamin pre-loaded with vitamin E could be explained by

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Fig. 3. Neuroprotective effects of Afamin and Afamin+ on H 20z (A) and (3-amyloid peptide (B) induced lesion. Cells were lesioned at the 8th DIV with either HzO z (100j.tM) or A(3Z5-35 (20 u.M) for 24 hours and subsequently cell viability was assessed with the MIT assay. Values, scaled to the mean of buffer treated control cultures (=100) are given as mean z s.e.m. from 2 independent experiments (n = 6). X-axis = different concentrations of Afarnin pre-loaded with several concentrations of vitamin E. Y-axis = Neuronal viability in percent

the sustained release of bound vitamin E to the culture medium. Since vitamin E does not readily pass the blood brain barrier (Aigner et aI., 1997), Afamin could serve as a transporter for vitamin E across this barrier. This could offer possibilities for applying a combination of the two substances for pharmaceutical treatments of Alzheimer's or other neurodegenerative diseases.

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The present study is the proof of concept that Afamin counteracts in vitro certain neurodegenerative stress factors including beta-amyloid peptide. These beneficial effects have been shown in the apoptosis-inducing low serum assay as well as in two oxidative stress models (HzOz and beta-amyloid peptide25_3s) . The molecular basis for one of these effects might be the capability of Afamin to bind and transport vitamin E . Since it is known, that neurons maintained under low serum conditions undergo apoptosis (Atabay et al., 1996), it is possible that Afamin acts against this kind of cell death like a growth factor. Furthermore, the effects of Afamin on cortical neurons submitted to oxidative stress by HzO z also suggest a function as a radical scavenger. However, in the HzOz-Iesion model and in the Ap-model, besides Afamin also Afamin + and vitamin E display protective effects. Vitamin E has been shown to prevent cells from oxidative stress-mediated death (Ricart and Fiszman, 2001; Behl, 1999). While lesion with HzO z mimics pathophysiological situations comparable to those of acute neurological diseases like stroke, exposure to beta-amyloid peptides induces a more chronic impairment since these peptides are found in senile plaques of AD brains (Selkoe, 2001). As shown, vitamin E is also able to protect against beta-amyloid-induced cell death (Huang et al., 2000). The data obtained in this study correspond well with these earlier findings, describing a role of beta-amyloid in the oxidative stress hypothesis (Varadarajan et al., 2000). Afamin is particularly effective in the beta-amyloid lesion model. Furthermore, Afamin enhances neuronal viability up to twofold of the control, showing better neuroprotective effects than vitamin E. Therefore, Afamin could act as a free radical scavenger or interact with beta-amyloid peptide fibrils, thus directly inhibiting their toxic effects. So far we can only speculate about the underlying mechanisms of the in vitro neuroprotective action of the human plasma glycoprotein Afamin. Nevertheless, the obtained data of this study lead to the conclusion that Afamin alone is able to protect cortical neurons from neuronal cell death and that Afamin and vitamin E synergistically enhance the survival of isolated cortical neurons. Therefore, Afamin displays a neuroprotective profile not only by virtue of its role in vitamin E transport but also on its own. The precise mechanism clarifying the neuroprotective efficacy of Afamin requires further investigations.

References Aign er A , Wolf S, Gassen HG (1997) Transport und Entgiftung: Ansatze und Perspektiven fur die Erforschung der Blut-Hirn-Schranke. Angew Chern 109: 2542 Atabay C, Cagnoli CM, Kharlamov E, lkonomovic MD, Manev H (1996) Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: protection with antioxidants and glutamate receptor antagonists. J Neurosci Res 43: 465-475 Behl C (1999) Vitamin E and other antioxidants in neuroprotection. Int J Vitam Nutr Res 69(3): 213-219

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Behl C, Moosmann B (2000) Estrogen and other antioxidants in neuroprotection: implications for Alzheimer's disease. In: Poli G, Cadenas E, Packer L (eds) Free radicals in brain pathophysiology, 1st edn. Marcel Dekker, New York Basel, p 467 Brera B, Serrano A, Ceballos ML (2000) B-amyloid peptides are cytotoxic to astrocytes in culture: a role for oxidative stress. Neurobiol Dis 7: 395-405 Christen Y (2000) Oxidative stress and Alzheimer's disease. Am J Clin Nutr 71(2): 621629 Contestabile A (2001) Antioxidant strategies for neurodegenerative diseases. Exp Opin Ther Patents 11(4) : 573-585 Dutta-Roy AK (1999) Molecular mechanism of cellular uptake and intracellular translocation of a-tocopherol: role of tocopherol-binding proteins. Food Chem Toxicol 37(9-10): 967-971 Gutmann B, Hutter-Paier B, Skofitsch G , Windisch M, Gmeinbauer R (2002) In vitro models of brain ischemia: the peptidergic drug Cerebrolysin" protects cultured chick cortical neurons from cell death. Neurotox Res 4: 59-65 Herrera E , Barbas C (2001) Vitamin E : action, metabolism and perspectives. J Physiol Biochem 57(2) : 43-56 Honig LS, Mayeux R (2001) Natural history of Alzheimer's disease. Aging Clin Exp Res 13: 171-182 Huang HM, Ou HC, Hsieh SJ (2000) Antioxidants prevent amyloid peptide-induced apoptosis and alteration of calcium homeostasis in cultured cortical neurons. Life Sci 66(19): 1879-1892 Hutter-Paier B, Steiner E , Windisch M (1998) Cerebrolysin" protects isolated cortical neurons from neurodegeneration after brief histotoxic hypoxia. J Neural Transm 53: 351-361 Jerkovic L (1997) Biochemical characterization of a vitamin E-binding protein from human plasma. Thesis, University of Innsbruck Jimenez-Jimenez FJ, Bustos F, Molina JA, Benito-Leon J, Tallon-Barranco, Gasalla T, Arenas J , Enriquez-de-Salamanca R (1997) Cerebrospinal fluid levels of atocopherol (vitamin E) in Alzheimer's disease. J Neural Transm 104(6-7): 703710 Lichenstein HS, Lyons DE, Wurfel MM, Johnson DA, McGinley MD, Leidli JC, Trollinger DB, Mayer JP, Wright SD, Zukowski MM (1994) Afamin is a new member of the albumin, a-fetoprotein, and vitamin D-binding protein gene family. J Biol Chem 269(27): 18149-18154 Massoud F, Schittini M, Sano M (2000) Vitamin E and other antioxidant treatments for the neurobehavioral aspects of Alzheimer's disease and other neurodegenerative diseases. In: Poli G , Cadenas E, Packer L (eds) Free radicals in brain pathophysiology, 1st edn. Marcel Dekker, New York Basel, p 501 Mattson MP (2000a) Free radical-mediated disruption of cellular ion homeostasis, mitochondrial dysfunction, and neuronal degeneration in sporadic and inherited Alzheimer's disease. In: Poli G, Cadenas E, Packer L (eds) Free radicals in brain pathophysiology, 1st edn. Marcel Dekker, New York Basel, p 323 Mattson MP (2000b) Emerging neuroprotective strategies for Alzheimer's disease: dietary restriction, telomerase activation, and stem cell therapy. Exp Gerontol 35: 489-502 Reinprecht K, Hutter-Paier B, Crailsheim K, Windisch M (1998) Influence of BDNF and FCS on viability and programmed cell death (PCD) of developing cortical chicken neurons in vitro. J Neural Transm 53: 373-384 Ricart KC, Fiszman ML (2001) Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing medi a. Neurochem Res 26(7): 801-808 Sano M, Ernesto C, Thomas RG, Klauber MR, Schafer K, Grundman M, Woodbury P, Growdon J, Cotman CW, Pfeiffer E, Schneider LS, ThaI LJ (1997) A controlled trial

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of Selegiline, a-tocopherol, or both as treatment for Alzheimer's disease. N Engl J Med 336: 1216-1222 Selkoe DJ (2001) Alzheimer's disease: genes, proteins and therapy. Physiol Rev 81(2): 741-766 Smith MA, Perry G (2000) Molecular and cellular aspects of oxidative damage in Alzheimer's disease. In: Poli G, Cadenas E, Packer L (eds) Free radicals in brain pathophysiology, 1st edn. Marcel Dekker, New York Basel, p 313 Tabet N, Birks J , Grimley EJ (2000) Vit amin E for Alzheimer's disease. Cochrane Database Syst Rev 4: CD002854 ThaI U (2000) Trials to slow progression and prevent disease onset. J Neural Transm 59: 243-249 Traber MG , Sies H (1996) Vitamin E in humans: demand and delivery. Annu Rev Nutr 16: 321-347 Tyas SL, Manfreda J, Strain LA, Montgomery PR (2001) Risk factors for Alzheimer's disease: a population-based, longitudinal study in Manitoba, Canada. Int J Epidemiol 30: 590-597 Varadarajan S, Yatin S, Aksenova M, Butterfield DA (2000) Alzheimer's amylo id 13peptide-associated free radical oxidative stress and neurotoxicity. J Struct BioI 130: 184-208 Vogele (1999) Biochemical characterization of the vitamin E-binding properties of Afamin. Thesis, University of Innsbruck Wronski R, Kronawetter S, Hutter-Paier B, Crailsheim K, Windisch M (2000) A brain derived peptide preparation reduces the translation dependent loss of a cytoskeletal protein in primary cultured chick en neurons. J Neural Transm 59: 263-272 Zimmer S, Stocker A, Sarbolouki MN, Spycher SE, Sassoon J, Azzi A (2000) A novel human tocopherol-associated protein: cloning, expression and characterization. J BioI Chern 275(33): 25672-25680 Authors' address: Prof. Dr. H. Dieplinger, Institute of Medical Biology and Human Genetics, Schopfstrasse 41, A -6020 Innsbruck, Austria, e-mail: hans [email protected]

Recent developments in the pathology of Parkinson's disease* K. A. Jellinger Institute of Clinical Neurobiology, Vienna, Austria

Summary. Parkinson's disease (PD) is morphologically characterized by progressive loss of neurons in the substantia nigra pars compacta (SNpc) and other subcortical nuclei associated with intracytoplasmic Lewy bodies and dystrophic (Lewy) neurites mainly in subcortical nuclei and hippocampus und, less frequently in cerebral cortex. SN cell loss is significantly related to striatal dopamine (DA) deficiency as well as to both the duration and clinical severity of disease, The two major clinical subtypes of PD show different morphologic lesion patterns: the akinetic-rigid form has more severe cell loss in the ventrolateral part of SN with negative correlation to DA loss in the posterior putamen, and motor symptoms related to overacitivty of the GABAergic "indirect" motor loop, which causes inhibition of the glutamatergic thalamocortical pathway and reduced cortical activation. The tremor-dominant type shows more severe cell loss in the medial SNpc and retrorubal field A 8, which project to the matrix of th e dorsolateral striatum and ventromedial thalamus, thus causing hyperactivity of thalamomotor and cerebellar projections. These and experimental data suggesting different pathophysiological mechanisms for the major clinical subtypes of PD may have important therapeutic implications. Lewy bodies, the morphologic markers of PD, are composed of hyperphosphorylated neurofilament proteins, lipids, redox-active iron, ubiquitin, and a-synuclein, showing a continuous accumulation in the periphery and of ubiquitin in the central core. a-synuclein, is usually unfolded in a-helical form. By gene mutation, environmental stress or other factors it can be transformed to f3-folding which is sensible to self-aggregation in filamentous fibrils and formation of insoluble intracellular inclusions that may lead to functional disturbances and, finally , to death of involved neurons. While experimental and tissue culture studies suggest that apoptosis, a genetically determined form of programmed cell death, represents the most common pathway in neurodegeneration, DNA fragmentation, overexpression of proapoptotic proteins and activated caspase-3, the effector enzyme of the terminal apopoptic cascade, have only extremely rarely been detected in SN of PD brains. This is in accordance with the rapid course of apoptotis and the

* Dedicated to my friend , Prof. Dr. DI Peter Riederer, on the occasion of his 60th anniversary.

K. A. Jellinger et al. (eds.), Ageing and Dementia Current and Future Concepts © Springer-Verlag/Wein 2002

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extremely slow progression of the neurodegenerative process in PD. The biological role of Lewy bodies and other intracellular inclusions, the mechanisms of the intracellular aggregation of insoluble protein deposits, and their implication for cellular dysfunction resulting in neurodegeneration and cell demise are still unresolved. Further elucidation of the basic molecular mechanisms of cytoskeletallesions will provide better insight into the pathogenesis of neurodegeneration in PD and related disorders. Introduction

Parkinson's disease (PD) or brainstem type Lewy body disease (McKeith et aI., 1996) is a progressive neurodegenerative disorder of advanced age clinically featured by motor symptoms such as bradykinesia, rigidity, tremor and postural imbalance. Subtle cognitive dysfunctions and/or depression are often present early in the disease, whereas in later stages dementia is not infrequent. There is progressive degeneration of the dopaminergic nigrostriatal system and other (sub)cortical neuronal networks associated with widespread occurrence of Lewy bodies (LB) and dystrophic (Lewy) neurites (LN) causing striatal dopamine deficiency and multiple other biochemical deficits and resulting in the variable clinical picture of this heterogeneous disorder. Accepted clinical criteria for the diagnosis ofPD (Gelb et al., 1999) provide a high sensitivity for detecting "parkinsonism" but show a specificity of only 75% for identifying PD and to differentiate it from other LB disorders, e.g. dementia with Lewy bodies (DLB) (Litvan et aI., 1998). For the diagnosis of definite PD histopathologic confirmation is required. Several clinico-pathologic studies have shown that LB disease including PD represent 60-83 %, whereas other degenerative disorders masquerading PD, e.g., multisystem atrophy (MSA), progressive supranuclear palsy (PSP), etc., account for 15-25% ; other entities, referred to as secondary parkinsonism are seen in 5-10% (see Jellinger, 1998, 2001a). Awareness of the high misdiagnosis rate, refinements in the clinical diagnostic criteria and approaches to early diagnosis of PD have led to an improvement in the specifity of PD diagnosis to 85% (Hughes et aI., 2001, 2002; Jellinger, 200la). While recent data on the lesion pattern of the multisystem degeneration in PD have provided better insights into the course and pathophysiology of its clinical subtypes (Graham and Sagar, 1999; Jellinger, 1999), the etiology and pathogenesis of PD remain unclear. Lesion pattern and regional vulnerability

Histopathology of PD is featured by the presence of LBs and LNs in association with variable neuron loss in the midbrain and other subcortical nuclei. In addition to degeneration of the dopamine-containing neurons in the substantia nigra pars compacta (SNpc), other cell groups, including the locus ceruleus (LC) and the basal nucleus of Meynert (NBM), are affected (Jellinger, 1991, 1998, 2001a ; Braak and Braak, 2000). According to the contents of calbindin (CAB) in the midbrain, dopaminergic neurons of SNpc have been divided

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Fig. 1. Neuronal loss in catecholaminergic nuclei of midbrain (intermediate level) in Parkinson's disease. a Distribution of catecholaminergic nuclei; b Distribution of melanized, TH-IR neurons (%) in normal controls; c Average loss of TH-IR neurons in 5 PD cases (duration of illness 7-32 years); d Percent average loss ofTH-IR neurons in PD (no specific clinical type); e Percent loss of TH-IR neurons in akinetic-rigid PD; f percent loss of caspase-3 positive pigmented neurons in PD ; g Percent loss of TH-IR neurons in tremor-dominant subtype of PD; h Percent loss of calbindin-IR neurons in PD. AS dopaminergic cell group AS; CG central gray substance; CB cerebral peduncle, DBC decussation of brachium conjunctivum; M medial group, Mv medioventral group; N nigrosome; RN red nucleus, SNpd substantia nigra pars dorsalis; SNpl substantia nigra pars lateralis, PHD parabrachial pigmented nucleus

into 2 major types showing different vulnerability (Damier et al., 1999): sparsely distributed neurons in a CAB-rich matrix component and densely packed cells in 5 CAB-poor "nigrosomes". There is severe depletion of melanized neurons (45-66%) and tyrosine hydroxylase (TH) immunoreactive (IR) neurons (60-85%, mean 75%) in the A9 group of SNpc (Ma et al., 1995; Halliday et al., 1996), particularly in the ventrolateral tier (area a, 91-97%) followed by the medioventral, dorsal, and lateral areas (Fig. 1b). Cell loss in the "nigrosomes" ranges from 76 to 98%, being most severe in an initially involved area in the caudal and mediolateral part of SNpc (98%) compared to 84% in the adjacent matrix. From here, cell depletion spreads to other nigrosomes, pocket II being more affected (94% cell loss) than the medial matrix of the same level (77%), and finally to the matrix along a caudo-rostral, latero-medial, and ventro-dorsal direction of progression (Damier et al., 1999). The above temporo-spatial order is related to the somatotopic pattern of dopaminergic terminal loss in striatum that is more severe in the dorsal and

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caudal putamen than in the caudate nucleus (Kish et aI., 1988). The degree of SNpc cell loss that shows significant correlations to the decrease of both dopamine and its main metabolite homovanillic acid (HVA) in striatum (Fig. 2), is also closely related to the duration and severity of motor dysfunction (Paulus and Jellinger, 1992; Ma et aI., 1997). There is much less involvement of the A10 group (VTA, nucI. parabrachialis and nucl. parabrachialis pigmentosus) projecting to the cortical and limbic areas (mesocortico-limbic dopaminergic system). These nuclei suffer 40-50% cell loss, while both the periretrorubral A8 region containing only few TH-IR, but CAB rich neurons and the central periventricular gray either show no definite degeneration (McRitchie et aI., 1997) or 20 to 32 % cell loss in A-8 (Fig . Lc, d). Cell depletion in these nuclei shows no correlation to duration of disease (Ma et aI., 1997). With an inverse pattern to the contents of CAB and other calcium-binding proteins, SNpc lesions in human PD are similar to those produced by the neurotoxine I-Methyl-4-phenyl-l,2,3,6tetrahydropyridine (MPTP), the most widely used model of PD (Varastet et aI., 1994). They differ from age-related lesions in the dorsal tier that is involved only in late stages of PD (Fearnley and Lees, 1994; Halliday et aI., 1996) , whereas recent studies showed a 35-41 % reduction in total numbers of pigmented SN cells with severe loss of dopamine transporter (DAT)-IR neurons in the aged (Ma et aI., 1999a). The estimated decrease in the total number of pigmented neurons and neuronal density in SNpc is 9.8 and 7.4% , respec-

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tively, per decade, with about 4.4% decrease per decade in neuronal volume (Ma et al., 1999b) . While neuromelanin shows a continuous accumulation in SNpc with aging , in PD brain it is severely depleted by almost 50% of agematched controls (Zecca et aI., 2001). There is a similar distribution of both reduced intensity of DAT messenger (m)RNA in the remaining SNpc neurons (Counihan et al., 1998) and decreased n-synuclein mRNA expression in SN and cortex of PD brain (Neystat et aI., 1999), with loss of vesicular monoamine transporter VMAT2 (a dopaminergic neuronal marker) in striatum, orbitofrontal cortex, and amygdala already in early stages of PD but not in SN (Ciliax et aI., 1999). Most mesotelencephalic dopamine neurons in A9 and Ala cell groups express high levels of DAT, whereas a smaller subpopulation of mesencephalic and all hypothalamic dopamine cell groups express little or no DAT (Miller et aI., 1999). PD shows an increase of 8hydroxyguanidine (8-HOG) , a common product of nucleic acid oxidation (Zhang et aI., 1999), with similar cell loss but less production of 8-HOG in SNpc in MSA and DLB brains. In addition, SNpc neurons in PD brain show an overexpression of calpain II , a calcium-dependent protease, which is not compensated for by its endogenous inhibitory protein calpastatin (MouattPrigent et aI., 2000). Whereas fibroblastic growth factor-I (FGF) IR and its binding activity are retained in SNpc neurons in PD (Walker et aI., 1998), brain-derived neurotrophic factor (BDNF) expression (Mogi et aI., 1999; Howells et aI., 2000) and the number of trkB mRNA (a high-affinity BDNFreceptor) containing neurons in SNpc and VTA are reduced in PD without decrease of trkB mRNA in the remaining neurons (Benisty et aI., 1998).There is a significant decrease of neurons expressing activated caspase-3, the central effector enzyme of terminale stages of apoptosis (Hartmann et aI., 2000). Reduced expression of the ND 1 subunit of mitochondrial complex I mRNA by 25% and of aldolase c mRNA in melanized SN neurons without relationship to disease duration and L-dopa treatment, with no such changes in the pontine and LC neurones, indicate that dopaminergic SN neurons are more dependent on mitochondrial energy metabolism and oxidative phosphorylation than other brainstem populations (Kingsbury, 2001). These data suggest a selective vulnerability of neurons rich in neuromelanin and caspase-3, with high expression of DAT mRNA (Uhl, 1998), unrelated to their intrinsic capacity of dopamine synthesis (Kingsbury et aI., 1999), low content of calcium binding proteins (protective role by preventing Ca 2 + -influx into cells) (Damier et aI., 1999), and weaker support by both neurotrophins and BDNF and glycolytic enzymes which may be a trigger for the neurodegeneration (Parain et aI., 1999; Howells et aI., 2000). This is supported by preservation of the STN containing calcineurin and paralbumin (calcium-binding protein)-IR neurons (Hardman et aI., 1997) and of non-dopaminergic, GABAergic neurons in SN pars reticulata (SNpr) that are involved only in terminal stages of PD with loss of paralbumin-IR (Halliday et aI., 1996). However, th ere is selective involvement of the parabrachial nucleus of area 10 with neurons rich in TH and GABA but poor in neuromelanin (McRitchie et aI., 1997). Severe decrease of caspase-3 positive pigmented neurons in SNpc in PD (-76%) compared to controls suggests that distribution of this apoptosis-

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Table 1. Biochemical alterations in substantia nigra in Parkinson's disease indicating oxidative stress Decreased

Increased Iron Ferritin? Lactoferrin receptor expression Ratio of oxidized to reduced glutathione (GSSG/GSH) Polyunsaturated fatty acids Mitochondrial monoaminoxidase B Lipofuscin Ubiquitin Cu/Zn-superoxide dismutase Cytotoxic cytokines (TNF-a, IL-l,IL-6) Inflammatory transcription factor NFKB Heme oxygenase-l Nitric oxide Thiobarbitur ic acid 8-hydroxy-2-deoxy guanosine

Ferritin ? GSH (GSSG unchanged); GSH/GSSG ratio) GSH-peroxidase activity Catalase activity Mitochondrial complex ! Calcium binding protein (calbindin 28) Transferrin receptor Vitamins E and C Copper

related enzyme may contribute to their regional vulnerability (Hartmann et al., 2000). The regional increase of 8-HOG corresponding to the pattern of neurodegeneration in PD suggests increased oxidative damage to neuronal cytoplasm (Zhang et al., 1999), supporting the hypothesis that oxidative stress represents a major pathogenic factor in PD (Jenner, 1998; Riederer et al., 2001; McNaught et al., 2001; Shapira, 2001; Mouradian, 2002) (see Table 1). In conclusion, the majority of midbrain dopaminergic neurons that are severely affected in PD are 1. located in the densely populated ventral tier of the SNpc, 2. contain neuromelanin, are rich in DAT and poor in glycolytic enzymes and calbindin, and 3. arborize densely in the striatum and sparsely in the extrastriatal structures. Conversely, most dopaminergic neurons that resist degeneration in PD are 1. located in the scantily populated dorsal tier of the SNpc, 2. contain calbindin and glycolytic enzymes but are poor in DAT, and 3. arborize profusely in the extrastriatal components of the basal ganglia and sparsely in the striatum. Symptom-specific lesion pattern in PD

Dopaminergic fibers, originating from different cell groups in the ventral mesencephalon, i.e., the SNpc (A9 cell group), the VTA (AlO) and the retrorubral area (A8), strongly innervate the striatum and the cortex of the frontal lobe, including the prefrontal cortex, motor and premotor areas (Berger et al., 1991). Therefore, progressive degeneration of the dopaminergic system in PD affects the functioning of both the basal ganglia and cerebral

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cortex, in particular the frontal cortex (Fig. 3). The major clinical subtypes of PD show specific morphologic lesion patterns of pathophysiological relevance (Jellinger, 1999, 2001a). a) In the akinetic-rigid type , the ventrolateral part of SNpc which projects to the dorsal putamen degenerates more severely than the medial part projecting to caudate nucleus and anterior putamen (Fig. 1e) , with a negative correlation between SNpc neuron loss, severity of akinesia-rigidity, and dopamine loss in posterior putamen (Bernheimer et aI., 1973). Damage to the nigrostriatal system caused by SN cell loss produces dopaminergic

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denervation of the striatum whose efflux systems via pallidum, SNpr and thalamus to the cortex and the parallel striato-nigro-pallido-cortical loop to premotor cortex remain intact in early stages of PD. There is a ventromedial gradient loss of TH-IR and DAT-IR fibers and endings (both frequently co-localized in synaptic vesicles and plasma membranes) progressing from dorsal to ventral putamen (Morrish et aI., 1996), with predominant involvement of the metenkephalin-substance P rich , acetylcholin esterase poor striosomes of putamen projecting to the severely involved ventrolateral SNpc. Preservation of the CAB-positive, somatostatin rich matrix showing increased somatostatin mRNA expression (Eve et aI., 1997) and projecting to GABA neurons of SNpr and motor thalamus, as well as of periventricular islands of caudate and nucleus accumbens suggests that the DAT-richest endings are most sensitive towards degeneration (Miller et aI., 1997). Caudate nucleus biopsies in PD patients, in addition to reduced TH immunostaining, revealed differences in substance P and met-enkephalin, both being either normal or variably reduced; low met-enkephalin immunostaining was correlated with the severity of motor clinical symptoms (De Ceballos and Lopez-Lozano, 1999). It has been estimated that the onset of clinical motor symptoms in PD occurs with loss of about 50% of SNC neurons, reduction of striatal DA uptake by 57-80% , and DAT loss of 56% (Bernheimer et aI., 1973; Morrish et aI., 1998; Guttman et aI., 1998). It is preceded by a preclinical phase correlating with "incidental Lewy body disease" (Fearnley and Lees, 1994), the duration and progression of which are still under discussion (Ito et aI., 1999). Dopaminergic denervation of the striatum causes severe loss of dendrites on type I medium spiny neurons, the principal goal of dopaminergic input from SN (Neill et aI., 1988) which, together with ultrastructural findings in PD (Lach et aI., 1992) and progressive loss of TH- and DAT-IR nigrostriatal fibers, suggest transsynaptic degeneration as a possible substrate for the severity of motor deficits and decreased efficacy of dopaminomimetic treatment in late stages of PD (Ito et aI., 1996). Whereas in PD, despite progressive dopaminergic denervation, the striatal matrix and the striatal efferences remain intact, in MSA and PSP, loss of calcineurin-, CAB-, methionin- and substance P-IR neurons in dorsolateral putamen, ventrolateral pallidum and lateral SNpc induce deafferentation of striatal efflux nuclei (Ito et aI., 1996; Hardman et aI., 1997) and, thus, caus e lesion of both striatal afferent and efferent projection systems ("motor loop"). In the akinetic-rigid form of MSA, more severe atrophy in the lateral SNpc causes loss of CAB-IR matrix cells in caudal putamen with transsynaptic degeneration of striatonigral efferences (Hardman and Halliday, 1999a). The effect of reduced dopaminergic input to the putamen but not the caudate causes increased activity of the GABA-ergic inhibitory "indirect" striatal efferent loop via SNpr and GPi lesioned in PD, leading to increased GABA output to the ventrolateral thalamus projecting to the cortex - thalamocortical motor loop - (Fig. 3). This leads via reduced cortical activation by glutamate to an akinetic-rigid syndrome that can be explained by overactivity of STN and GPi (Albin, 1995; Halliday et aI., 1996; Hardman et al., 1999b).

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Additional if not alternative mechanisms may underlie STN hyperactivity. Given the reciprocal innervation on the SNpc and the STN, the dopaminergic deficit may influence the STN activity directly, while the increased excitatory drive to the nigral neurons originating from the hyperactive STN might sustain the progression of the degenerative process (Blandini, 2001). The increased striatal GABAergic activity tends to disappear in the course of the disease and is reduced by L-dopa treatment. Alterations in signaling systems linking dopaminergic and glutamatergic receptors within the GABAergic efferent neurons may induce NMDA receptor changes resulting in long-term changes in glutamatergic synaptic efficacy which leads to alterations in the cortical input to the striatum and modifies striatal GABAergic output, favouring motor complications. Since the striatal matrix and its efferences remain intact, restitution of the dopaminergic transmission by L-dopa substitution maintains the function of the "motor loop", but progressive degeneration with transgression to non-dopaminergic systems in later stages causes loss and reduced stimulation of postsynaptic D2 and muscarinic cholinergic receptors in the striatum by dopamine with unchanged antagonist binding. This uncoupling of receptor systems is considered a major cause of drug resistance of motor symptoms and adverse L-dopa effects (dyskinesia, fluctuation) (Bezard, 2001). b) The tremor dominant type of PD shows less severe total neuronal SNpc loss (mean 52.8 vs. 69%) and less severe depletion in the lateral than in the medial SNpc (Paulus and Jellinger, 1992), while there is damage to the retrorubral A8 field (Fig. 19) that is usually preserved in akinetic-rigid PD (McRitchie et al., 1997; Damier et al., 1999). The A8 area, which (in contrast to A9 and AlO) contains only few TH-IR and DAT-IR but mainly calretininIR neurons (Mouatt-Pringent et al., 1994), is largely independent of striatal influences, but does project to the matrix of the dorsolateral striatum and ventromedial thalamus (Gerfen, 1992; Parent et al., 1999). Both the A8 and AlO areas directly influence the striatal efflux via SNpr to thalamus and from there to the prefrontal cortex (Groenewegen, 1997) (Fig. 3). Tremor synchronous electric activity has been reported in STN, GPi, and ventral intermedial thalamus (VIM) (Obeso et al., 1997), and PET studies suggest increased functional activity of ventral thalamic projections to cortical motor regions (Antonini et al., 1998; Zirh et al., 1998). Recent MRI studies showed locally increased gray matter densities in the VIM of the thalamus contralateral to parkinsonian resting tremor (Kassubek et al., 2002). While autopsy cases of essential tremor, in general, show no pathological changes except for mild SN cell loss (Lee et al., 1999), biochemical analysis of MPTP-lesioned monkeys with tremor showed significant decrease of dopamine in both caudate nucleus and putamen suggesting that nigrostriatal degeneration contributes to rest tremor (Eberling et al., 2000). On the other hand, in both essential and orthostatic tremor, bilateral overactivity of cerebellar connections has been reported (Boecker and Brooks, 1998; Deuschl et al., 2000). These data, differences in biopterin content of CSF between akinetic-rigid and tremor dominant PD, and the relation of dopa uptake between caudate and putamen (Otsuka et al., 1996) suggest different pathophysiologic mechanisms of the

356

K. A. Jellinger

two major clinical subtypes based on differential morphology that have therapeutic implications, in particular for surgical treatment. Since PD is a multisystem disorder, many other extranigral systems are involved (JeHinger, 1998, 2001a; Braak and Braak, 2000): the noradrenergic LC, dorsal vagal nucleus and adrenergic medullary nuclei, serotonergic dorsal raphe nuclei, NBM and other cholinergic brainstem nuclei, e.g. WestphalEdinger nucleus (controlling pupillomotor functions and REM sleep function) , reticular brainstem nuclei controlling somatomotor and autonomic systems (Braak et al., 2000; Benarroch et al., 2000) posterolateral hypothalamus and parts of the limbic system including amygdaloid nucleus, hippocampal formation, anterior cingulate gyrus , limbic thalamic nucleus with prefrontal projection and other CNS regions, e.g. the centre medianparafascicular thalamic sysem (Henderson et al., 2000). On the other hand, histaminic innervation of SN in PD is increased (Anichtchik et al., 2000). Most of the lesions are not random but region-specific, affecting not all neurons containing a specific transmitter or harboring LBs and may explain the complex patters of morphologic, functional, biochemical, and clinical deficits of the disorder including mental decline in PD (JeHinger, 1999,2001). Lewy bodies and neuronal dysfunction

In both PD and DLB, LBs are occurring in two different types: the classical brainstem and cortical type. Classical LBs are intraneuronal cytoplasmic spherical inclusions ranging from 8 to 30 ~ in diameter with a hyaline eosinophilic core and a narrow pale-stained halo. They are composed of radially arranged 7 to 20nm intermediate filaments associated with a granular electron-dense coating material and vesicular structures; the core contains densely packed filaments and dense granular material. Cortical LBs are eosinophilic, rounded, angular or reniform structures without an obvious halo. Ultrastructurally, they are composed of felt-like arranged intermediate filaments and granular material (Forno, 1998). Both LBs and pale bodies - their precursors (Dale et al., 1992) - are diagnostic hallmarks for both PD and DLB, but are not specific for these disorders; they have been described in a variety of conditions as a secondary pathology, e.g. MSA, LB dysphagia, corticobasal degeneration (CBD), motor neuron disease, Hallervorden-Spatz disease (HSD), ataxia-telangiectasia, sporadic and familiar AD (Arai et al., 2001), Down syndrome, Meige syndrome, subacute sclerosing panencephalitis, and normal aging. Given the fundamental nature of a-synuclein con tainting lesions in these disorders, these conditions have been summarized as synucleinopathies (Galvin et al., 2001). LBs are associated with coarse dystrophic neuritic changes - Lewy neurites (LN), also decorated by ubiquitin and u-synuclein as inclusions in axonal processes of neurons. They are most frequent in the central and accessory cortical nuclei of the amygdala, in hippocampus, mainly the CA 23 subfields, the periamygdaloid cortex (PAC), in many brainstem nuclei, in the intermediolateral columns of the spinal cord, olfactory bulb, in sympathic and parasympathic neurons, enteric, cardiac, and pelvic neurons, and in adre-

Recent developments in the pathology of PD

357

nal medulla indicating involvement of multiple neuronal systems (Jellinger, 1991,1999; Braak and Braak, 2000). Absence ofTH-IR suggests that many of these neuritic processes are not derived from dopaminergic neurons. The precise biochemical composition of LBs and LNs is still unknown, but immunohistochemical studies have shown that major components are phosphorylated neurofilament proteins present in both core and periphery, ubiquitin, a heat shock protein targeting proteins for breakdown, enzymes associated with ubiquitin mediated proteolysis and (de)phosphorylation, cytosolic and microtubule-associated proteins except for tau-protein, a-Bcrystallin probably mediating the aggregation of microfilaments, synaptophysin, chromogranin A (suggesting that vesicular structures in LBs may represent degenerating nerve endings), and the presynaptic nerve terminal protein a-synuclein (Spillantini et al., 1998; Baba et al., 1998; Gai et al., 2000; Duda et al., 2000; Galvin et al., 2000, 2001), and synphylin I, an asynuclein interacting protein that promotes LB formation (Wakabayashi et al., 2000). Further components are amyloid ~ peptide (A~), amyloid precursor protein (APP), actin-like protein, ubiquitin, and ubiquitin-pathway associated enzymes (VCH-L1, proteases, ligases, and kinases), a-microglobulin, immunoglobulin S, a-B crystallin (in about 10% of cortical LBs) probably mediating the aggregation of microfilaments, Cu/Zn superoxide dismutase, cytosolic and microtubule-associated proteins except for tau protein, e.g. MAP-2 and MAP-5, calbindin, tubulin, TH, G7 and G9 proteins (Lowe, 1997; G alvin, 2001; McNaught, 2001). In addition, LB contain lipids and redox-active iron (Gai et al., 2000; Castellani et al., 2000). a-synuclein is at present one of the best markers to differentiate LBs and LNs from u-synuclein-negative neurofibrillary tangles (NFT) or Pick bodies (Dickson et al., 1999; Duda et al., 2000). Staining for a-synuclein is replacing staining for ubiquitin as the preferred method for detecting LBs and LNs. Altered u-synuclein is incorporated into LBs, their precursors ("pale bodies"), and dystrophic LNs before ubiquitination; it is aggregated and fibrillated in vitro, morphologically resembling LB-like fibrils (Hashimoto and Masliah, 1999). Colocalization of a-synuclein, synphilin, and parkin within subsets of LBs suggests that parkin may play a role in the posttranslational procession and ubiquitinatin of a-synuclein (Shimura et al., 2001). This may explain why IPD mutations lack LBs. It is associated with torsin, a novel protein in which a mutation causes dominant, early onset torsion dystonia, which may serve as a chaperon for misfolded proteins that require refolding or degradation (Sharma et al., 2001). LBs and related cytoplasmic inclusions express cdk5, a proline-directed protein kinase involved in cell cycle regulation that is likely to catalyse the in vivo phosphorylation of neurofilament proteins. The aberrant accumulation or ectopic expression of cdk-5 and mitogen-activated protein kinase (MAPK), normally not found in neurons and glia, may lead to the formation of pathologic cytoskeletal inclusions (Nakamura et al., 1998). Recent studies on the development of LBs in brainstem suggest an initial intraneuronal appearance of fine dust-like particles related to neuromelanin or lipofuscin, with homogeneous deposition of a-synuclein and ubiquitin with transition into a continuous accumulation of a-synuclein in the periphery and of ubiquitin in the central core of the LB (Wakabayashi et al., 1998; Gay et al., 2000; Gomez-

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358

Tortosa et al., 2000; Braak et al., 2001). More recent studies using triplelabeling for n-synuclein, ubiquitin and thiazin red (TR) confirmed the heterogeneity of nigral and cortical LBs, but showed different localization of these epitopes (Sakamoto et aI., 2002): Immunolabeling either with anti u-synuclein or ubiquitin antibody was diffuse without halo structure in cortical LBs, 80% being TR-positive. In addition to this diffuse staining, over 60% of brainstem LBs exhibited peripheral accumulation of both u-synuclein and ubiquitin, probably representing later stages of LB evolution. Only 25% of nigral LBs were TR positive, but in both types of LBs the TR signal was always concentrated in the center, as was the silver-stained material, suggesting that fibrillary components in the central portion of LBs undergo some conformational changes of their constituents. The later frequent extraneuronal deposition of LBs may be related to disappearance of the involved neurons (Forno, 1998). The mechanism of u-synuclein aggregation in vivo has not yet been elucidated, while in vitro it is modulated by various factors. While a-synuclein usually is unfolded or has an a-helical form, gene mutations, environmental stress or interaction with chaperon molecules can induce a transformation to I)-folding (Duda et al., 2000). In this form, it is sensible to self aggregation in filamentous, amyloid-like fibrils and formation of insoluble intracellular inclusions (Fig. 4). In cultured cells, aggregation of u-synuclein takes place under certain conditions, such as high temperature or low pH (Hashimoto and

PLD2

..

decreased inhibition by A30P mutant?

acidic phospholipids

ERK

decreased activity

..

cell death?

(incl. PA)

disturbed neurotransmitter release, cell growth, synaptic plasticity A35Tbinds more strongly thanWT

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•

increased aggregation

via: BAD ? PKC ?

increased inhibition by overexpre ssion of WT and mutants (A30P and A53T)

decrea sed binding of A30P mutant

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self-aggregation

~

increased by: reduced vesicular transport? reduced degradation? overexpression

haploinsufficiency? .

increased by: mutations truncation metal ions (Fe 3+) AI3-peptide ApoE4

~~~~:sed availability of u- synuclein?

Fig. 4. Model of o-synuclein aggregate formation caused by several factors inducing conversion to a ~ -pleated sheet conformation. In this conformation, a-synuclein is more susceptible to self-aggregation into filamentous protein fibrils forming intracellular inclusions

359

Recent developments in the pathology of PD

Masliah, 1999). Both wild type and mutant types of a-synuclein may undergo self-aggregation and form insoluble fibrillar aggregates with antiparallel ~­ sheet structure upon incubation at physiologic temperature, which was accelerated for both hitherto known PD-linked point mutations (Narhi et al., 1999). Since aggregation has been shown to be a nucleation-dependent process followed by fibril-formation, a-synuclein nucleation may be the ratelimiting step for the formation of LB a-synuclein fibril (Farrer et al., 1999). Since a-synuclein binds to phopholipids associated with a certain conformational change of structure (Davidson et al., 1998), the altered composition of membrane lipids in neurodegeneration may be a prerequisite for the aggregation of a-synuclein (Gai et aI., 2000). Sequestration of redox-active iron and aberrant accumulation of ferric iron causing formation of hydroxyl radicals via the Fenton reaction in PD (Riederer et al., 1999; Linert and Jellinger, 2001) suggest that the iron-catalyzed oxidation reaction also plays a significant role in a-synuclein aggregation in vivo. Decreased a-synuclein mRNA in both rat SN following administration of 6-hydroxydopamine (6-0HDA) (Kholodilov et al., 1999) and in SN and cortex of PD brain in the presence of preserved DAT and YMAT2 suggest that its decrease is an early change in the process leading to neuron degeneration preceding changes in TH and dopamine markers (Neystat et al., 1999). Since immunoreactivity for the heme protein cytochrome c which is located in the intramembrane of mitochondria and is released upon apoptotic stimuli into cytoplasm was detected in LB with defects of cytochrome c oxidase in SNpc in PD (Itoh et al., 1997), aggregation of a-synuc1ein may also be closely related to mitochondrial dysfunction (Hashimoto and Masliah, 1999) (Fig. 5). a-Synuclein has been shown to

OH·

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T

f - - - - Anti-aggregatio n mol ecu les

(J3-syn, APLP2....)

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Fig. 5. Possible mechanisms of neuronal cell death in Lewy body synucleinopathies

disease/

360

K. A. Jellinger

produce neuronal death due to promotion of mitochondrial deficit and oxidative stress (Saha et al., 2000; Hsu et al., 2000). Its cross-linking by advanced glycation products (AGE) thought to playa role in the pathogenesis of neurodegeneration has been observed in advanced PD and incidental LB disease. Since AGEs are both markers of transitional metal-induced as and inducers of protein crosslinking andfree radical formation, AGE promoted formation of LBs may reflect early disease specific changes rather than late epiphenomena (Munch et al., 2000). It is unknown whether LB are cytotoxic or harmless side products or markers of cell damage, while the involvement of the ubiquitin proteolytic system suggests that they may be the structural manifestation of a cytoprotective response designed to eliminate damaged cellular elements, rendering the mutated or damaged protein less toxic than its soluble form suggests that they may be the structural manifestation of a cytoprotective response designed to eleiminate damaged cellular components and to delay the onset of neuronal degeneration (Goldberg and Lansbury, 2000; Chung et al., 2001). The deposits of insoluble proteinaceous fibrils may contribute to dysfunction or death of the involved cells (Duda et al., 2000). On the other hand, LBs being the sequelae of frustranous proteolytic degradation of abnormal cytoskeletal elements may represent - similar to other cellular inclusions, e.g. neurofibrillary tangles in AD, Pick bodies, etc, - end products or reactions to hitherto unknown neuronal degeneration processes that are associated with disturbances of axonal protein transport and finally lead to cell death (Fig. 6). SNpc cell degeneration is preceded by loss of neurofilament (NF) proteins, neuronal TH-immunoreactivity, TH, DAT and NF mRNA, TH- and DAT-proteins, and cytochrome oxidase c indicating functional neuronal damage (Itoh et al., 1997; Jellinger, 1999; Kingsbury et al., 1999). It is accompanied by distribution of melanin with uptake into macrophages (Forno, 1996), astroglial reaction, and proliferation of MHC class II positive microglia, the latter by releasing cytokins, CD 23, nitric oxide, and other substances mediating inflammatory reactions that may be both inducing factors or sequeale of neuronal death (Calignasan et aI., 1998; Riederer et al., 1999; Hirsch, 2000). Programmed cell death and nenrodegeneration

While the causes of neuronal death in PD and related neurodegenerative disorders are still enigmatic, several mechanisms are currently discussed: programmed vs. passive cell death (apoptosis vs. necrosis), and autophagy (Anglade et aI., 1997; Olanow and Tatton, 1999), but the specific pattern of cell loss and the paucity of necrotic changes in SN and other brainstem nuclei in PD have led to the suggestion that programmed cell death (PCD) may be a major mechanism (Tatton and Olanow, 1999; Tatton, 2000). Recent studies demonstrating DNA fragmentation (Mochizki et aI., 1997; Tompkins et aI., 1997; Tatton, 2000) and an upregulation of proapoptotic and cell deathregulating proteins and enzymes in PD (Marshall et aI., 1997; Hartmann and

Recent developments in the pathology of PD

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a -Synuclein

accumulation of toxic substrates

(e.g. Pael-R, ? a-synuclein p22)

I cytoprotection I Fig. 6. Proposed pathogenic cascade leading to neuronal death in familial and sporadic Parkinson disease

Hirsch, 2001) and in an MPTP mouse model (Duan et al., 1999) raise the question whether apoptotis, a specific gene directed form of PCD, may represent a dominant pathway in the selective degeneration of specific neuronal populations. Apoptosis, a morphologically and biochemically well characterized form of PCD to remove unnecessary or damaged cells in various situations, is characterized by nuclear fragmentation, condensation of nuclear chromatin with DNA fragmentation/laddering, breaking up of the cell into membrane bound ultrastructurally well preserved fragments (apoptotic bodies), with preservation of intracellular organelles (mitochondria), nuclear and cellular membranes, and lack of inflammatory reaction (Wyllie et al., 1980; Majno and loris, 1999) . It is carried out by an intrinsic suicidal machinery of the cell and can be triggered by en vironmental stimuli including iradiation leading to DNA damange, oxidative stress, toxins, viruses, withdrawal of neurotrophic suppport. Necrosis is a passive pathological event killing of the cell - arising from spontaneous insults, e.g. stroke or trauma (Levin et al., 1999; Clarke, 1999; Yuan and Yankner, 2000; Reed, 2000).The principal molecular players of th e apoptotic program include: a number of apopoptosis-inducing or death receptors (e.g. Apo-l/Fas) , Apaf-1 (apoptotic protease-activating factor 1) and other apoptosis-initiating factors (AIFS), small proteins released from damaged mitochondria into the cytoplasm in early stags of cell death - proteins/proteases of the caspase/calpain family -

362

K. A. Jellinger Dea t h inducing triggers (toxins, oxid o radical s, AB, cal cium, etc.)

Death s ignals

Lack of growt h fac tor

Death r eceptor (Fas, T ' FRi, etc)

St r ess

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Activ ated --. Cas pase X ~ caspase-3 "-~ Caspase ?

Transcriptional regulat/on of proof antiapoptotic genes

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~[

i

APOPTOSIS c-j un, fax. fas l. , bas

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Fig. 7. Markers for apoptosis in tissue and their localization in the apoptotic cascade: at least four distinct signaling pathways, such as apoptotic triggers, death receptors, lack of neurotrophic factors, and stress, induce complex caspase activation. All these different events may have the activation of caspase-3 in common, finally leading to DNA fragmentation and apoptotic cell death

the Bc1-2 and p53 oncogen families-, mitogen activated protein kinase pathway regulated by neurotrophins (Fig. 7). Once activated, many but not all of them induce proteolysis of specific cellular components and consequently amplify the death signal cascade (Reed, 2000; Stadelmann and Lassmann, 2000; Wang, 2000). Central players of PCD are upstream instigator caspases (8, 9, 10), activating other caspases, namend effector capsases (3, 6 and 7), important regulators of postmitotic neuronal homeostasis (Reed, 2000; Roth et al., 2000). Such downstream caspases and their proteolytic products are recognized markers of apoptosis, their activation representing an irreversible step in the cell death cascade and cells expressing these enzymes are prone to death. The Bcl-2 family includes a highly homologous group of mitochondrial proteins which act either to enhance (Bax, Bid, Bad, Bak, Bcl-xs) or prevent (Bcl-2, Bel-xL) apoptosis by forming homo- or heterotypic dimers which may effect the formation of a permeability transition pore in the mitochondrial membranes and the release of cytochrome c into the cytoplasm (Reed, 2000) .

Recent developments in the pathology of PD

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This leads to formation of a cytoplasmic complex including Apaf-1 and caspase-9, which results in caspase-9 activation and cleaving which, subsequently, activates downstream caspase-3. Similar death-signaling pathways may be activated in neurodegenerarative diseases by proteins belonging to abnormal subcellular structures, such as j3-amyloid, tau protein, u-synuclein , huntingtin, etc. (Wolozin and Behl, 2000). Histochemical methods to detect free 3'ends generated by endonuclease cleavage of genuine DNA during PCD in by in situe terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end leabeling (TUNEL/ISEL) method or in situ end translation with detection by dioxigninantidigoxin have facilitated the study of apoptosis in human disease. However, these and related methods cannot always distinguish between the two modes of cell death (see Stadelmann and Lassmann, 2000). Therefore, these methods have to be supplemented by ultrastructural evidence (different in postmortem material) and by studies on the balance of apoptosis-related proteins (ARP), because increased expression of both death-promoting and -inhibiting proteins regulators, genes, and stress proteins may precede or accompany neuronal PCD (Reed, 2000; Stadelmann and Lassmann, 2000). Studies in experimental models of PD have strongly suggested a role for apoptosis in the related human pathology, since systemic administration of MPTP produces DNA fragmentation with induction of caspase-3 activity (Hartmann et al., 2000; Tatton, 2000), while inhibition of the downstream cellular substrate of caspase-3, poly(ADP-ribose)polymerase (PARP), an enzyme involved in DNA repair, protects against MPTP-induced neurotoxicity (Tatton, 2000). MPTP administration in mice increases nigrostriatal activity of both c-Jun and c-Jun NH2-terminal kinase (JNK), members of the stress-induced protein kinase (SAPK) pathway, which is attenuated by a JNKspecific inhibitor also reducing dopaminergic cell loss in SN (Saporito et al., 2000). MPTP is a mitochondrial enzyme which elicits its action first via monoamine oxidase B-catalysed conversion to its metabolite, MPP+, which is selectively taken up into dopaminergic SN neurons by the DAT. It kills these cells by specific inhibition of mitochondrial complex I which has also been implicated in the pathogenesis of SN cell death in human PD (see Tatton and Olanow, 1999; Tatton, 2000; Reichmann and Janetzky, 2000). Loss of complex I activity may result in decreased ATP production and increased formation of mitochondrially generated reactive oxygen species (ROS) both of which contribute to neuronal cell death via decreased protein pumping and reduced voltage differential across the inner mitochondrial membrane that would elicit opening of the mitochondrial permeability transition pore and subsequent initiation of apoptosis (Cassarino et al., 1999; Tatton and Olanow, 1999; Tatton, 2000). Similar inhibition of complex I activity and subsequent mitochondrial impairment is seen with decreased levels of the antioxidant compound glutathione (GSH) that, in PD, is suggested to precede both complex I and dopamine loss (Jenner, 1999; Tatton and Olanow, 1999; Jha et al., 2000), and has been shown to induce apoptosis in culture. Conversely, apoptosis can be prohibited by overproduction of Bcl-2 due to increase in GSH levels (He et al., 2000).

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In another animal model of PD, intracerebral injection of 6-0HDA causes both apoptotic and necrotic cell death of dopaminergic SN neurons (Choi et al., 1999; Duan et al., 1999), but in dopaminergic cell cultures, 6-0HDA mediates apoptosis via activation of caspases. While not all dopaminergic neurotoxins, e.g. MPP+, appear to induce apoptosis (Choi et al., 1999), partizipation of prostate apoptosis response-4 (Par-4) related to Fe 2+ induced mitochondrial function in experimental PD models has been observed (Oh et al., 1998). The toxin-induced apoptosis can be prevented by expression of Bcl-2 and Bax, suggesting that Bcl-2 related proteins may show a specific interaction with a distinct partner protein or cell-death pathway determining its role as a positive or negative modulator of cell death (Jha et al., 2000; Jellinger, 2000; Hartmann and Hirsch, 2001). DNA fragmentation and definite histologic features of apoptosis in nigral neurons in PD, DLB and related disorders are extremely rare, ranging from a up to 12% (means 1-2 %) of the total dopaminergic SN cell populations, compared to 0-1 % in controls; they are not visible in all PD brains examined in different studies (see Table 2). While very few SN neurons display a reduction in cell size and clumping of nuclear chromatin resembling apoptotis (Graeber et al., 1999), and show mild to moderate expression of proapoptotic proteins, they only occasionally express activated caspase-3 involved in the executive phase of neuronal cell death (Hartmann et al., 2000; Mogi et al., 2000; Tatton, 2000), suggesting a proapoptotic environment and caspase-3 activation probably preceeding SN cell death. Other authors reported increased levels of the antiapoptotic factors Bcl-2 and Bcl-X in basal ganglia and SN of PD brain (Marshall et al., 1997), while others found no differences in the immunoreactiviy of Bcl-2 and Bcl-X and in the expression of Bcl-2mRNA in SN neurons (Jellinger, 2000a; Nicotera, 2000). Some post mortem data suggest that Bax is expressed in SNpc neurons (Vila et al., 2001), although the intensity of Bax-IR was identical in LB and non-LB bearing neurones, suggesting that Bax does not contribute to LB formation (Tortosa, 1997). Reduced Fas and Fas-L in SN neurons with and without LBs but increased Fax and Fas-L in reactive astroglia indicate that the Fas/Fas-L pathway does not play an essential role in regulating SN cell loss (Ferrer, 2000), while SNpc neurons show a significant decrease in FAS-associated proteins with a death domain (FADD) immunohistochemistry that correlates with their known selective vulnerability suggesting that this pathway contributes to TNFmediated apoptosis (Hartmann et al., 2002). A positive correlation between the degree of neuronal loss and the percentage of caspase-3 positive pigmented SN cells with a 76% decrease in PD suggests that this central effector enzyme of apoptosis may contribute to their regional vulnerability (Hartmann, 2001). Increased numbers of SN neurons expressing activated caspase-8, a proximal effector protein of the tumor necrosis factor (TNF) receptor family death pathway, suggest its activation in early stages of cell demise (Hashimoto, 1999). Ultrastructurally activated caspase-3 staining was observed in dopaminergic cells displaying the morphological features of increased protein synthesis, but not in cells with the signs of apoptosis (Hartmann, 2000). Elevated caspase activities and tumor necrosis factor re-

Recent developments in th e pathology of PD

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Table 2. Incidence of DNA fr agmentati on in substantia nigra in various neu rodegenerative disorders Author , yea r Dragunow et al. (1995) Mochizuki et al. (1997) Anglade et al. (1997) Tompkins et al. (1997)

Kosel et al. (1997) Banati et al. (1998) Ol anow et al. (1999) Kingsbury et al. (1998)

Wullner et al. (1999)

Jellinger (2000)

Tatton (2000) Broe et al. (2001)

DX

N

Time (h ) pm

PD juvPD Late PD Co PD PD DLB AD/PD AD Co PD PD PD Co PD MSA DLB PSP Co PD Co PD Co PD DLB PSP CBD Co PD Co DLB Co

3 4 7 6 3 5 7 4 5 3 22 3 3 3 16 4 1 1 14 3 4 3 4

? 3-12.6 1-5 1-3 8.3 ± 2.3 1.7-31 2.5- 24.3 5.5- 20 11-16 3-8 ? 7-30 ? ? 5-3 8.5-35 16 25.5 5.5-48 20- 38 4-42 20-38 4-42 4-12 18-24 12-24 14-24 16-24 2.75-7 8-11 15 ± 13 18 ± 14

5

2 3 3 4 8 4 11 11

Method

TV TV TV TV EM

TV TV TU

TV TU

TV TV TV+ YO

TV TV TV TV TV TV TV TU (prol)

TV TU

TV TV TV ISEL YO

TV TV

% Neurons

0 0 0-4.2 (m 1.2) 0 3.7 6.9 ± 2.2 11.46 ± 1.3 7.8 ± 2.45 1.7 ± 0.65 0.93 ± 0.47 "few" 1/22 brains 0 1.5 0.1 0-12.8 (m = 2.0) 0-19.4 (m = 9.0) 9.3 0 0-10.5 (m = 1.0) 0 0 2.0 ± 1.2 1.3 ± 1.1 0/1080 0/1010 0/1080 0/]010 0 9.0 ± 5.0 0.4 ± 0.1 0 0

A D Alzheimer's disease , iuv iuvenile, Co Controls , PD Parkinson 's dise ase , D LB D ementia with Lewy bodies, M SA multiple system at rophy, PS P progress ive supranuclear palsy, T V T UNEL, CBD corticobasal degeneration, YO YOYO 1, pm post mortem

ceptor R 1 (p53) in SN and increased caspase-3 and Bax Iry accompany nuclear GAPDH (glyceraldehyde-3-phosphate dehydrogenase) translocation and neuronal apoptosis (Tatton, 2000; Vila et al., 2001). Activated forms of both caspase-8 and -9, upstream form known to cleave and activate caspas e3, have been demonstrated in SN neurones in PD (Anderson, 2001), which also show translocation ofDANN repair enzymes (PARP and DANN-PKCS) from the cytoplasm to the nucleus (Love, 2001), but no relationship between MAPK expression and in situ lab eling of nuclear DANN fragmentation or caspase-3 activation has been observed (Ferrer et aI., 2001).

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On the other hand, frequent DNA fragmentation and expression of ARPs have been observed in reactive astrocytes and microglia, indicating their increased turnover in neurodegeneration (Banati et al., 1998; Wullner et al., 1999; Jellinger, 2000). This is confirmed by findings in human subjects with parkinsonism following exposure to MPTP and survival for 3 to 16 years, were signs of active, ongoing neuron loss and clustering of microglia around nigra1 neurons have been observed (Langston et al., 1999) . Lewy bodies in the brainstem are usually negative for TUNEL, ARPs, and activated caspase-3 (Tompkins and Hill, 1997; Jellinger, 2000) as are Pick bodies, the morphologic markers for Pick's disease (Gleckman et al., 1999), and a-synuclein containing glial cytoplasmic inclusions in oligodendroglia in MSA (Probst-Cousin et al., 1998; Jellinger and Stadelmann, 2000). LBs are stained with anti-ERK-2 antibodies, while cytoplasmic granules expressing these kinases are seen in association with a-synuclein deposits in few cortical neurons (Ferrer et al., 2001). These data suggest that nuclear DANN damage, contribution of Bel family members, and activation of the caspases cascade may be involved in the degeneration of dopaminergic neurons in PD but rarely indicate neuronal apoptosis. Specifically, abnormalities of complex I of the mitochondrial respiratory chain (Orth and Schapira, 2001; Silva et al., 2001) , and mitochondrial inhibition of cytochrome c release by reinforcing anti-apoptotic or blocking pro-apototic factors, e.g. by caspase inhibition, which may block the apoptotic programme but will not reverse the underlying cellular dysfunction, are to be considered. In Alzheimer disease (AD) brain an increased risk of neuronal death related to ~-amyloid deposits and neurofibrillary tangles was observed (Lassmann et al., 1995) , but only 0.02 to 0.05% of hippocampal neurons showed apoptotic features and expression of activated caspase-3, which documents the extremely rare occurrence of apoptotic cell death in AD (Stadelmann et al., 1999). The controversial findings in PD and other neurodegenerative diseases, with frequent inability to identify apoptotic cell death in the involved brain regions, are not surprising, since they represent chronic progressive disorders, with cell death occurring over periods of 5-20 years and more. In this case, apoptotic events may be rare at any given time and apoptotic bodies may be rapidly phagozytosed by neighbouring glial cells and therefore difficult to detect. Therefore, the demonstration of rare neurons undergoing apoptosis in some of these disorders, e.g . AD, appears realistic, given the short duration required for completion of apoptosis and the protracted course of neurodegeneration with long preclinical phases and duration of illness (Perry et al., 1998). However, at any time, a small proportion of neurons may undergo morphological changes, such as DNA fragmentation and condensation within the nucleus, hyperchromatic changes in cytoplasm an d dislocation of the nucleus indicating a rapid, transient phase of degeneration. These morphologic changes that contrast with the classical features of necrosis make normal cell function unlikely and may be followed by disappearance of cellular cytoplasm with remaining abnormal nuclei. Such cell withering has been called apoklesis (Graeber et al., 1999). On the other hand, apoptosis, although

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initiated, may not necessarily progress to its completion in neurodegenerative processes. Given that execution of apoptosis requires amplification of caspase-mediated apoptotic signal (Figs. 5 and 7), recent data suggest that in PD and other neurodegenerative disorders, effective apoptotic signal propagation to downstream caspase effects is lacking. This novel ph enomen of avoidance termed abortive apoptosis or abortosis, may represent an exit from the caspase-induced apoptotic program, that ultimalety leads to neuronal survival (Smith et al., 2001). Such factors are found in cells with abundant intracellular filaments (Majno and Joris, 1995) or insoluble intracytoplasmic protein filaments, like NFTs, LBs , Pick bodies, etc. , suggesting that such filaments may contribute to dysfunction or increased vulnerability of the involved cell (Wolozin and Behl, 2000; Duda et al., 2000). However, demonstration of negative DNA fragmentation in SN neurons with LBs (Tompkins and Hill, 1997; Jellinger, 2000), in neurons with Pick bodies (Gleckman et al., 1999) and recent calculations that tangle-bearing neurons in Parkinson-dementia complex and AD may survive for an average of 2.5 years or even up to 20 years (Schwab et al., 1999; Morsch et al., 1999), indicate that these inclusions may not predispose a cell to undergo PCD. This is in agreement with recent data after chronic inhibition of protein phosphatases 1 and 2 causing dephosphorylation of tau protein and neuronal apoptosis, that show different distribution, suggesting that these cytoskeletal changes have no obvious sequelae for the viability of the involved neurons (Arendt et al., 1998). Thus, the biological significance of these inclusions, related to mismetabolism of cytoskeletal proteins and often representing morphologic hallmarks of a specific neurodegeneative disorder, and also the role thay play in neurodegeneration are still undetermined. Light and ultrastructural features of autophagic degeneration, i.e. mild condensation of nuclear chromatin, moderate vacuolation of endoplasmic reticulum , and lysosome-like vacuoles but normal mitochondria which were observed in melanized SN neurons in PD brain (Anglade et aI., 1997) and in hippocampal neurons in AD brain (Stadelmann et al., 1999) suggest alternative mechanisms of cell death not necessarily via apoptosis. They rather reflect the combined action of deficient DNA repair and acceleerated DNA damage within susceptible cell populations (Jellinger and Stadelmann, 2000). Conclusions

Although many in vitro and in vivo data favor apoptosis in PD and other neurodegenerative disorders as a major pathway of PCD, the majority of human brain tissue studies have yielded controversial results and there is increasing evidence for .. alternative mechanisms of neuronal demise in neurodegeneration. Experimental and human studies suggest that cells with DNA fragmentation are injured cells, although not necessarily undergoing apoptosis or necrosis, and that activation of some caspases does not have a significant role in the widespread neuronal death that occurs in PD and other neurodegenerative disorders, although it may contribute to the loss of specifi-

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cally vulnerable neurons. The death cascades may be counteracted by other cellular mechanisms wich limit the activations of the caspase and other deathsignal cascades, suppress oxydoradicals, and stabilize calcium homeostasis and mitochondrial function (Mattson, 2000). This suggests that there may be compensatory mechanisms in neurons that respond to various insults in neurodegeneration (Reed, 2000; Mattson, 2000). Thus, neuronal death in neurodegeneration may represent a form of demise that is neither classical necrosis nor apoptosis (Graeber et al., 1999; Jellinger and Stadelmann, 2000; Smith et al., 2000; Jellinger, 2001b), but may be finally triggered during the terminal period of the patient's life (Lassmann et al., 1995; Kingsbury et al., 1998). Despite considerable progress in the clarification of the molecular mechanisms of neurodegeneration, the intracellular cascade leading to neuronal demise in PD and other chronic progressive disorders remains to be elucidated. Understanding of their molecular pathobiology and causative mechanisms may lead to a better understanding of the pathogenesis of PD and to development of protective strategies and novel approaches for the treatment of this disorder.

Acknowledgements The author thanks Mrs . V. Rappelsberger and Mrs . H. Breitschopf for excellent technical assistance; Mr. E. Mitter-Ferstl, PhD, for computer and secretarial assistance. Part of the work was supported by grants of the Austrian Parkinson Society, the Society for Promotion of Research in Experimental Neurology, Vienna, Austria, and Consultatio GmbH, Vienna, Austria.

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