253 Assembly of soluble β-amyloid peptides into Alzheimer disease amyloid fibrils in rat brain

FIFTH INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE disturbances in the cellular mechanisms available to preserve the genomic integrity. An inherited or environmentally adquired failure in these mechanisms might induce an important depletion of genetic information that could be essential for normal cell replacement processes, leading to degeneration, cell death and accelerated senescence. Supported by EuroEspes Foundation.

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genotype. So far, we cannot demonstrate a n y causative point mutations or relevant polymorphisms in the catalytic domain of the PLC-81 gene in these patients with sporadic AD. In conclusion, although PLC-81 is thought to be playing some important roles in the pathogenesis of AD, these documented pathological and enzymatic changes m a y be derived from other causative factors as well as changed activities of other intracellular messengers. F u r t h e r genomic analysis of other concerning regions of the PLC-81 gene from larger cases is required. Supported in part by grants from the Kato Memorial Trust for Nambyo Research and the Suzuken Memorial Foundation.

250 Alzheimer Disease National Cell Repository P.M. Conneally, D. Fugman, ME. Hodes, J. Tischfield Department of Medical and Molecular Genetics, Indiana University Medical Center, Indianapolis, Indiana 46202. The AD National Cell Repository began in 1989, as a collaboration between Indiana and Duke universities, It was established to provide the research community with large numbers of well characterized, informative families with multiple individuals affected with Alzheimer Disease, The main goal of the Repository is to facilitate research aimed at expanding our understanding of the etiology, pathogenesis, diagnosis, treatment, prevention, and ultimately, potential cure for this disease. The Repository collects and maintains information and biological specimens on well-characterized families with AD in order to provide a resource for use in research projects and to encourage and foster the development of new avenues of AD research. Specifically, the Repository seeks to identify, recruit, gather and maintain information from families with histories of AD. This includes pedigree information; medical records concerning the evaluation, diagnosis, and treatment of patients; documentation of the cognitive, behavioral and social consequences of AD; epidemiological and demographic data, and neuropathological diagnosis information. The data bank has records on 650 families comprising 38,000 individuals. The cell bank has established over 1,600 cell lines. All lines are tested for the presence of mycoplasm contamination. Meticulous care is used to avoid sample mix-up. A bar code system is used in conjunction with the data base to ensure careful tracking of all blood, transformed cells, and DNA samples. A DNA "fingerprint" is generated for all blood samples on arrival which can later be compared with a questionable sample. In conjunction with the collection of these data, the Repository seeks to collect, maintain and distribute DNA and permanent cell lines from these families and to make these samples and corresponding information available for research to qualified investigators throughout the world. The use of common data and sample sets by various researchers employing different approaches is likely to facilitate comparison of data and the emergence of a common understanding of possible interpretations. The inherent control provided by common sample sets is particularly important in research aimed at understanding a disease with significant genetic and phenotypic heterogeneity. To this end, we have already provided over 3,200 samples to nearly 40 researchers throughout the world. For further information, or to request a copy of the Cell Line & DNA Catalog, visit our world wide web site at: http://medgenAupui.edu/~medgen/research/alzheimer/. To request samples, contact the first author at 1-800-526-2839 (in USA) or by emai] at [email protected]. (Supported by NIP, Grant #P30 AG 10133)

251 Genetic Analysis of a Catalytic Domain of P h o s p h o l i p a s e C_~I in P a t i e n t s w i t h Alzheimer's Disease J. Kawamata*, S. Shimohama, H. M a t s u s h i m a , T. Imura, and J. K i m u r a Department of Neurology, Faculty of Medicine, Kyoto University, 54 Shogoin-kawaharaeho Sakyo-ku, Kyoto 606, J a p a n Phosphoinositide-speeifie phospholipase C (PLC) is a key molecule in signal transduction between extraeellular and intraeellular compartments. It catalyzes the phosphoinositides to generate diaeylglyeerol and inositol 1,4,5-triphosphate which serve as intraeellular messengers for protein kinase C activation and intraeellular Ca 2+ mobilization. Previously, we demonstrated that the PLC isozymo, PLC-81, is abnormally accumulated in neurofibrillary tangles, the neurites surrounding senile plaque cores, and the neuropil threads in autopsied brains with Alzheimer's disease(AD). We also found that PLC-81 activity is reduced in the eytosolie fraction prepared from platelets taken from AD patients especially who had apo £ 3/3 genotype compared with age-matched controls. To clarify the pathogenic roles of PLC-81 in AD, we have examined: genomie sequence of the catalytic domain of the PLC-81 gene in 10 patients with early-onset sporadic AD and 10 patients with late-onset sporadic AD which cases h a d significant lower PLC81 activity of platelet compared with controls and carried apo e 3/3

Cellular & Animal Models H

252 Immunohistochemical evidence for AIM0 and A1542 in rat soleus musde in chloroquine-induced myopathy

K. Tsuzukil)*, R. Fukatsu2), Y. Takamam3), T. Yoshida2), Y. Hayashi2), N. Sasaki2), H. Yamaguchi4), N. Fujiil) and N. Takahata2) DDept. of Microbiology and 2)Dept. of Neuropsychiatry, Sapporo Medical University, School of Medicine, South I, West 17, Chuo-ku, Sapporo 060, Japan 3)Dept. ofNeuropsychiatry, Sapporo City General Hospital, Sapporo 062, Japan 4)Collegeof Medical Care and Technology, Gunma University, Gunma 371, Japan Alzhdmer's disease (AD) affected brain is characterized by the deposition of amyloid 13protein (A[~). A[3 is generated from amyloid precursor protein (APP) by the proteolytic cleavages that vary slightlyat the carboxy-terminus, yielding species of 40, and 42(43) amino acids. Chloroquine, a potent lysosomotropicagent, induces myopathy in rats similarto rimmed vacuole (KV) myopathy in humans. We demonstrated immunohistochemicai evidence that A[~, APP N=, C=terminalregions and cathepsin D accumulate in RVs in chloroquine-induced myopathy in rats. Moreover, A[3 immunoreactivity colocalizes with apolipoprotein E, SP-40,40, oh-antichymotrypsin,and uhiquitin. There is striking similaritybetween amyloid lesions in AD and RVs in this experimental model. The purpose of this study, is to ducidate whether A[I is deav~ fium Ai:P, or APP fragments containing AI3 accumulate in the RVs. Three antibodies were used. Two of these antibodies are end-spec'dicfor the C- terminus of A[5:BC40 and BC42 reacts only with AIM0 and AIM2, respectively. The third, A1390/12, a monoclonal antibody, reacts with the N-terminus of AI$, reacting both with APP containing A[3 and cleaved A[3, AIM0 and A[542. Immunohistochemicalstudies were carried out with denervated soleus muscle 14 days after the initial injection of chloroquine, 50 mg/kg of body weight. Transverse paraffin sections were stained according to the standard streptavidin-biotin peroxidase technique. Muscle fibers suffering from chloroquine-induced myopathy contained both cleaved AIM0 and AIM2. AIM2 appeared to predominate over AIM0. In conclusion, our results provide further evidence that chioroquine-induced myopathy in rats is a useful model for understanding APP processing into A[3 in terms of the pathogenesisof AD.

253 Assembly of Soluble l~-Amyloid Peptides into Alzheimer Disease Amyloid Fibrils in Rat Brain R.-W. Shin° *, K. Ogino2), T.C. Saido3), T. Kitamoto °, I.Q. Trojanowski 4)and J. Tateishi s~ Department ° of Neurological Science, Tohoku University Medical School, Sendai 980, Cellular Technology Institute2), Otsuka Pharmaceutical Co., Tokushima 771-01, Departmen#>of Molecular Biology, Tokyo Metropolitan Institute of Medcial Science, Tokyo, Department 4~of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283, USA, and Departmen:) of Neuropathology, Neurological Institute, Kyushu University School of Medicine, Fukuoka 812-82, Japan Deposition of 13-amyloidpeptides (A[~) as amyloid fibrils in senile plaques is one of the pathologic hallmarks of Alzheimer disease(AD) brain. To probe the biological properties of A~ in vivo; we injected synthetic AI3~.~0 into the

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FIFTH INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE

hippocampus and neocortex of adult Sprague-Dawley rats (n= 25). Fifty lag of the peptides freshly dissolved into 5 lal of 0.15M Tris pH 8.8 was used for injection. Following different postinjection survival times (1 day to 7 weeks), the brains were examined by Congo red stain and immunohistochemistryusing several anti-Al~ antibodies. Soluble AI~o formed aggregates in the brains of all rats examined. Congo red staining of the A [~aggregates showed birefringence under polarized microscopy, suggesting the formation of amyloid fibrils. The AI~aggregates were analyzed for N-terminal modification by using antibodies which specifically recognize N-terminal epitopes of AI3, anti-Al~ and anti-A[3 m~E). Anti-A[~ recognizes standard AI3 bearing the first N-terminal residue and anti-AI3m~ recognizesmodified A[3in which the first and secondN-terminal residues are deleted and the third glutamate is converted to pyroglutamate. The AI3aggregates were positively immunostainedwith both antibodies, similar phenomenon as that found for A[3 deposits in AD (T.C. Saido et al., Neuron 14 : 457, 1995). The model system described here will help dissect how A[3 deposits effect the pathobiological processes in AD brain.

254 Age-related changes in blood-braln barrier (BBB) on senescence accelerated mouse (SAM) M. Ucno*1, M. ShinnouI, H. Sakamoto1, I. AEguchl2, M. Hosokawa3 and A.W. Vorbrodt4 1Second Dept. of Pathology, Kagawa Medical School, Kagawa 761-07, 2Dept. of Neurology, Faculty of Medicine, 3Dept. of Senescence Biology, Chest Dis. Res. Inst. Kyoto University, Kyoto 606, JAPAN. 4Dept. of Pathological Neurobiology, NYS Institute for Basic Res. in Dev. Disabil., Staten Island, New York, USA First, we examined age-related changes in brain transfer of blood-borne horseradish peroxidase (HRP) in SAMP8, which showed age-related deficits in leaming and memory. The intracerebral HRP transferred from blood stream was reacted with tetramethyl benzidine (TMB). Positive areas for HRP-TMB reaction products in parenchymaof medial CA1 regions and medial dentate gyrus of the hippocampus,were observed both in 3- and 13-month-oldSAMP8. The area of HRP-TMB reaction products in dark fields in sections through rostral and caudal portions of the hippocampuswas morphometrieallyevaluated. Mean percentage of the positive area for the reaction products to the area of interest in rostral portions of the hippocampuswas significantly higher in 13-month-old SAMP8 than in 3month-old one. On the contrary, age-related changes in mean percentage of the positive area for the reaction products to the area of interest, were not observed in the cortices and caudal portions of the hippocampus. In addition, we observed HRP-TMB reaction products in parenchymaof central parts of the olfactory bulb of, especially 13-month-old, SAMP8. Next, we evaluated age-related changes in BBB to endogenous albumin on the olfactory bulb of SAMP8. The tissue samples were immersion-fixedand embedded at low temperature in Lowicryl K4M. Ultrathin sections were exposed to antimouse albumin antiserumfollowed by protein A-gold. Morphometricalanalysis of the electron micrographs in the olfactory bulb revealed that some percentage of capillaries showed leakage of endogenous albumin and the increase of this leakage, manifested by higher density of the labelling of the subendothelial space, in 11-13month-old SAMP8 was statistically significant as comparedto control group. In addition, some larger than capillary vessel, presumablyvenule, located in the internal granular layer showed leakage of albumin in 11-13-month-old SAMP8. These results suggest that barrier function in the hippocampusand the olfactory bulb is weaker than that in other BBB regions and moreover changes with aging.

In order to elucidate the correlation between brain atrophy and behavioral/learning disturbances in SAMPI0, we performed the open field test, passive avoidance task, and Sidman avoidance task on male SAMPI0 mice and accelerated senescence-resistant SAMRI mice as controls, aged 2, 4, 8, and 12 months (n=7 each). Subsequently the brain of each mouse was removed, brain atrophy was graded (grade 0-3) with emphasis on the Fb/OB regions, and the whole brain weight was measured. Results The grade of Fb/OB atrophy was advanced significantly with age and had a negative correlation with the whole brain weight in SAMP10. In the open field test, the initial latency on the starting square increased significantly with age in SAMPI0. In the passive avoidance task, SAMPI0 showed the same level of memory retention as SAMRI at the retention trial 24 hours later. In the last session of Sidman avoidance task, the whole brain weight correlated negatively with the number of foot-shocks received and positively with the mean duration of avoidance by continuous responses in SAMP10. In addition the grade of Fb/OB atrophy had a positive correlation with the percentage of immediate responses to the foot-shocks. Conclusion Our data suggest that the brain atrophy is responsible for the procedural learning disturbance in SAMPI0.

256 Effect of acidic fibroblast growth factor on basal f o r e b r a i n c h o l i n e r g i c n e u r o n s in s e n e s c e n c e a c c e l e r a t e d mice I. T o o y a m a 1., Y. Aimi 1, K. Sasaki 2, Y. O o m u r a 3, A.-J. Li 4, H. K i m u r a 1 1Institute o f Molecular Neurobiology, Shiga University of Medical Science, Seta, Otsu, 525: 2Division of Bio-Information Engineering, Faculty o f Engineering, T o y a m a University, T o y a m a ; 3Institute o f Bio-Active Science, Nippon Zoki Pharmaceutical Co. Ltd., Hyogo; 4Department o f Physiology, Faculty o f Medicine, K y u s h u University, Fukuoka, Japan W e examined the effects o f chronic administration of acidic fibroblast growth factor (aFGF) on m e m o r y and choline acetyltransferase (CHAT) immunoreactivity in the forebrain o f senescence-accelerated mice (SAM; 1:'8 strain). Subcutaneous injection o f a F G F (aFGF group) or saline vehicle (saline group) once a week into SAM was begun at 3 weeks after birth and continued for 9 months. In the passive avoidance test, the retained latency was significantly longer in the a F G F group than in the saline group. In the Morris test, the m e a n latency to climb on a platform w a s significantly shorter in the a F G F group than in the saline group. The n u m b e r of ChAT-positive neurons in the forebrain s e p t u m w a s greater in the aFGF group than in the saline g r o u p , and was at the level of that in the control mouse strain (R1). The intensity of ChAT staining in the a F G F group appeared slightly weaker than in R1 but significantly stronger than in the saline group. The results indicate that the effect o f a F G F on m e m o r y function in S A M m a y be related to the preservation o f function in septal cholinergic cells.

255 Correlation between Brain Atrophy and Behavioral/Learning Disturbances in Senescence-Accelerated Mouse SAMP10 N. Sedu .1), K. Ohnishi1), H. Yagi1), I. Akiguchil), J. Kimurat), A. Ohta2), K. Higuchi3), and M. Hosokawa3) ~)Department of Neurology, Faculty of Medicine; Z)Department of Psychology, Faculty of Letters; 3)Department of Senescence Biology, Chest Disease Research Institute; Kyoto University. 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan. SAMPI0, an accelerated senescence-prone strain of the SenescenceAccelerated Mouse (SAM), shows spontaneous brain atrophy especially in the forebrain (Fb) and olfactory bulb (OB) regions. Moreover age-related impairment in several learning/memory tasks has been reported in SAMPI0. Materials and Methods

257 Synaptic changes in rat induced by intraventricular infusion of ieupeptln. S.Takauchi, K.Ohara, T. Nakajima, M. Kokai, Y. Morita, K.Miyoshi* Department of Neuropsychiatr3;Hyogn College of Medicine,l-i Mukogawa-cho, Nishinomiya,663 Japan * Department of Psychia~; Kyoto University School of Medicine, Kyoto, Japan We have previously reported that widespread neuritic degeneration and subsequent cytoskeletal changes accompanied with accumulation of abnormal filaments in the neuronal cell body are induced by intraventficular infusion of a protease inhibitor, leupeptin. We show here synaptic changes induced by