324 ABSTRACTS OF

NEUROBIOLOGY OF AGING. VOLUME I1, t990 SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE

GENETIC MECHANISMS

GAP-43 mRNA levels in any age group between normal and AD. Additionally, there is no difference in mRNA content between normal and AD tissues.

Interteukin-ll](IL-113) is an immunomodula tor produced by microglia, astrocytes, and neurons. It is also an astroglial mitogen which induces the expression of the B-amyloid precursor gene. Thus, we have compared the mRNA levels of IL-113 in AD to the l eve ls in normal age-matched cont ro l s us ing the blot hybridization methods previous ly described. An average increase of 65% in IL-113 mRNA was seen in young AD SFG as compared to the age-matched controls. Conversely, an average decrease in IL-113 mRNA of 14% below age-matched controls was obtained for SFG for old AD.

294

REACTION OF rm~NA EXPRESSION FOR STEROID HO~KINE ~ R S AND CALCIU%{ BINDING PROTEINS IN ALZHEI~R BRAIN. *M. Sutherland, L. Wong, c.Mizzen, P.Handley, C.Bergeron, L.Young and D.R.McLachlan Depts. of Physiology, Medicine and Pathology, University of Toronto, Toronto, Ontario Canada

Previous investigations using quantitative Northern analyses in our laboratory have shown that mF4qA expression for the 68kD neurofilament protein (NF-L) is reduced to 14% of control in Alzheimer brain. This decrease correlated strongly with increased heterochromatization of the 5'-prcmoter region of the NF-L gene. To dtermine whether the expressions of other important neuronal genes are similarly affected, we examined specific neuron popu- lations of Alzheimer and Huntington (control) brains using in situ hybridization. Hip~l, neocortical (temporal) and cerebellar paraffin sections were hybridized under high stringen- cy conditions with 3H-antisense ~NA probes for thyroid hormone receptor (c-erbB) , vitamin D hozmone receptor, calbindin and par- valbu-nmn. After autoradiography, cellular grain count was deter- mined using a LECO Image Analyzer.

Hippocampal CA1 cells of Alzheimer brains expressed a statis- tically reliable 35% reduction in m~NAs coding for thyroid hor- mone receptor, vitamin D receptor, calbindin and parvalb~mdn when cc~pared to control, mRNA pool sizes for calbindin and parvalbu- rain but not for steroid hormone receptors, were reduced by 30% in Alzheimer hippocampal CA2 cells. Neocortical expressions of thy- roid hormone receptor and calbindin and cerebellar expression of thyroid hormone receptor were elevated by 28% in Alzheimer but not control brains. There were no differences in the cerebellar expressions of calbindin, parvalb~nin and vitamin D receptor. Since grain counts were corrected for cell size, reduced m~qA expression in Alzheimer brain could not be due to neuronal shrinkage. The results indicate, that unlike NF-L, m~qA pool size for steroid hormone receptors and calci~n binding proteins were only moderately reduced. In a recent experiment using irm~/nofrac- tionation to separate histone Hl°-enriched mono-and di-nuclesome fractions prepared from micrococcal nuclease (~)-digested nuclei frc~ control and Alzheimer temporal neocortex, we discovered that the Alzheimer fractions contained i. 75 times more NF-L hybridi- zable signal per unit of DNA as con~pared to control. In contrast, the same fractions contained an Alzheimer/control signal of only 0.99 for calbindin. Taken together, these experiments suggest that different mechanisms are responsible for the reduction of mRNA expressions in Alzheimer brain.

295

CONSTRUCTION OF A PHYSICAL MAP OF CHROMOSOME 21q IN RELATION TO ALZHEIMER'S DISEASE. *W. Van Hul, G. Van Camp, H. Backhovens, P. Stinissen, A. wehnert, 3. Korenberg, A. vandenberghe, C. Van Broeckhoven. University of Antwerp (UIA), Dept. of Biochemistry, B-2610 Antwerpen, Belglttm and Cedars-Sinai Medical Center, Los ~ngeles, CA 90048, USA.

Genetic linkage analysis studies indicated that a locus predisposing to AlzheIzer's diseases (AD) is present on the proximal long arm of chromosome 21 close to the marker loci D21SI3, D21S16 and D21S1/S11. However, the exact localization of the AD locus relative to these marker locl is still unclear. A major problem hindering the genetic analysis of AD is the lack of sufficient data regarding genetic and physical map poeltlons of markers located in this chromosomal area. Partial genetic mapping data showed that D22S13 is located proxlmal of D21Sl/S11 with the

exact position of D21816 unknown. We reported tight linkage of the marker loci D21S13 and D21S16 in an extended reference pedigree and confirmed close vicinity of both marker loci by pulse field gel electrophoresis. Furthermore, recent data from somatic cell hybrids indicated that D21S16 is the closest marker towards the centromere.

We aimed at constructing a complete pulse fleld map of the region between the centromere and the locus D21SI/SII using several additional marker loci mapped to proximal 21q. At present, the map consists of three major parts. A first part of 2.5 Mb around the D21S23 locus showing the positions of D21S16, D21S46 and D21S48. A second part of 2.0 Mb including the marker loci D21S4, D21Sl10 and D21S52. A third part of 2.8 Mb around the loci D21SI and D21S11 including the locus D21S95. The results obtained are compatible with published genetical and physical mapping data. The gaps in this primary map will be bridged using megabase mapping techniques plus new chromosome 22 markers selected for the Alzheimer's disease region=

296

USE OF A CHROMOSOME 21 JUMP CLONE IN LINKAGE ANALYSIS OF FAMILIAL ALZHEIMER'S DISEASE (FAD). A.P. Walker, R.J. Bartlett, MA. Pedcak-Vance, L.H. Yamaoka, W.-Y. Hung, J.L. Bebout, F. Mechler, S.L. Secore, P.C. Gaskell, F. S, Collins* and A.D. Roses. Duke University Medical Center, Durham NC 27710, USA and "Howard Hughes Medical Institute & Departments of Internal Medicine and Human Genetics, University of MiChigan, Ann Arbor, MI 48109, USA.

A 100kb jumping library was screened with pGSE9 (D21S16) and a rapid PCR method was used to identify non- repetative landing sites, One of 5 positive clones had a unique landing site (AW8-1J, D21S194) which detected a BamH I polymorphism, and localized to 21ql 1.1- 21ql 1.2. No crossovers were seen between the jump clone and D21S16/D21S13 loci, and therefore AW8-1J could be combined with these loci into a single haptotype in order to make linkage analysis of our AlzhelrnsCs disease families more informative. No disequilibrium was seen between AWS-1J and the S16/$13 sites (n=78), The clone was used in linkage analysis of 217 members (64 affecteds) from 28 FAD famities (mean onset = 65.6 years), comprising 25 late onset Alzheimer's disease (LOAD; mean > 60) and 3 early onset Alzheimer's disease (EOAD; mean < 60) families. Linkage analysis of the haplotype AW8-fJ/pGSEg/ pGSM21 in all families excluded linkage of FAD up to approximately 8cM on either side. When only LOAD families were analysed, linkage was excluded to ¢1 lcM. The EOAD families gave a peak Iod score of z = 0.98 at ~ = 0.15. When a femaie : male map ratio of 5.65 was employed, exclusion of linkage of FAD was reduced to .%4cM over all families, and Io +YcM in the LOAD families. The EOAD families gave a peak Iod score of z = 1.22 at ~ = 0.05.

When the analysis was repeated employing the disease status information on only AD affected individuals, linkage of FAD was excluded up to 6cM on either side, both when all families and when only LOAD families were considered. The EOAD families gave a peak Iod score of z = 0.56 at ~ = 0,15. Use of the female : male map ratio of 5.65 in the analysis Of these only AD affected individuals reduced the exclusion of FAD to + 2oM both in all families, and in only the LOAD families.The EOAD families gave a peak led score of z = 0.84 at 8 = 0.05. Updated 2-point iod scores and also multipoint analysis will be presented.

These results while supporting the possibility of linkage of EOAD on chromosome 21, do not support linkage of LOAD to this locus, thereby further suggesting the possibility of genetic heterogeneity in this neurogenetic disorder. The probe has been distributed for testing in EOAD families in other laboratories.

297

PREDOMINANCE OF A-4 OVER ALZ-50 POSITIVE PLAQUES IN N O N - D E M E N T E D ELDERLY *P.V. Arriagada and B.T. Hyman. Massachusetts General H o ~ , Harv'ard Medical Scllocl, Boeto~ MA 02114.

one of ~ e ~ ~ of ~ d ~ a s o ~ ~ p ~ s e : r c e s e 0~nytofd plaques are frequently associated with dystrol0tlic neurites that are AIz-50 immunoreactlve: Some senile plarlnaS, as ~ by ~ h l a t o l o g i c techniques, are also f r ~ noted in the brains of nondemented indlvk~als over the age of 80. We studied the topographic, morphologic and immunof,JetochemP.~charactemt~ of ~ that occur in the ~ ~ in 13 brains of ~ ~ ~ agerange ~ : ~ . ~ mlo'on frozen sections of ~ , ~ ¢xxte, x; ~ afld.soco.ical a~as 20, 21, 22, 41 and 42 were st~lsd w ~ thiofle~n-S (Tlllo-S), ; ~ and AIz-50 a.d ~ amy~d-A4 - L y. ~ ~ ~ e W ~ o two cateOeles: plequ~ v~h Alz.~ ~ nlu~m, ~b~,:Itt1~w recog~za~ onfy with amylofd stains (A4 or ~-S). In ~Is series of ~

2 cases (69 and 75 years) had no ~ ~ by any of me employed t~niquse.Ten out of 13 cu~ had ~ numbers of plaquse o~lambumd ~ in ~ ar~ ~ a.'~d ~n tl~ am~/~dele.in 7 of these cases plaques were detected by Thins and A-4. Two had only A-4 positive

NEUROBIOLOGY OF AGING, VOLUME l l, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE ANIMAL MODELS

325

plaques without Thio-S positive staining and one had Thio-S positive plaques (A-4 unavailable). Only one case (76 years) showed AIz-50 positive plaques and those were located predominantely in the hippocarnpai formation,amygdala and entorhinai cortex. In this case there were also Thio-S and A-4 amyloid positiv0, Alz- 50 negative plaques in the neocortex, amygdaia, entorhinai cortex, hippocampus and subiculum. The remaining 12 cases showed rare or no AIz-50 positive plaques.

All 13 cases contained Thio-S and AIz-50 positive neurofibrillary tangles (NFT) in layer II of enterhinai cortex, as well as in hippocampus and rarely in association cortex. In the two instances in which there were no senile plaques, NFT were found only in layer II of entorhinai cortex.

Our results suggest that (1) plaques associated with AIz-5O positive dystrophic neurites are rare in nondemented elderly, while A-4 amyloid plaques are more frequent. (2) Entorhinal cortex tangles can be seen without concurrent A-4 or Thio-S plaques. (Supported by the Brookdale Foundation, the Educational Commisison for Foreign Medical Graduates, and NIH AG08487). We thank P. Davies, New York, and C. Masters, Melbourne, for the gifts of AIz-50 and anti-A4, respectively.

ANIMAL MODELS

298

MODEL SYSTEMS IN AI 7HEIMER'S DISEASE. *D.L. Price', V.E. Koliatsos', L.J. Martin', L. C. Walker', R. E. Clatterbuck', E. H. Koo', S.S. Sisodia', W. C. Mobley 2 and L. C. Cork ~, Neuropathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205-2181 USA and University of California at San Francisco, San Francisco, California.

This review focuses on two models relevant to AIzheimer's disease (AD): aged monkeys; and monkeys with fimbria-fornix lesions that cause degenerative changes in basal forebrain cholinergic neurons. Older macaques develop a variety of behavioral and brain abnormalities, some of which closely resemble those occurring in older humans, including individuals with AD. Behavioral tests, chosen to assess the integrity of different neural circuits, demonstrate age-related impairments that appear in the late teens. Different animals of the same age show different patterns of behavioral deficits. Brain abnormalities appear at the end of the second decade and include: abnormal fibers/neurites; the presence of B/A4 al-antichymotrypsin in plaques and blood vessels; and abnormal patterns of cytoskeletal epitopes in perikarya. Our findings suggest that: abnormalities of fibers/neurites and deposits of B/A4 increase with age; APP immunoreactivity, normally present in perikarya, proximal dendrites, axons, and nerve terminals, appear in neurites; some neurites are surrounded by deposits of B/A4; and abnormal perikaryal immunoreactivity is a late event. The demonstration of APP immunoreactivity in neurites and the presence of B/A4 in proximity to neurites are consistent with the concept that neurite- derived APP may be one source of B/A4 deposited in brain parenchyma. Unilateral transection of the fornix causes a series of degenerative changes in neurons of the medial septum. Monkeys were allowed to survive for two weeks postoperatively, during which time half of the subjects received intraventricular nerve growth factor (NGF) and the other animals received vehicle alone. The latter animals showed a reduction in the number of choline acetyltransferase-immunoreactive cell bodies within the ipsilateral medial septum. Treatment with NGF almost completely prevented reductions in the number and size of cholinergic neurons. The in vivo effectiveness of mouse MGF on basal forebrain cholinergic neurons suggests that mouse or human recombinant NGF may be useful in ameliorating the age-associated acetylcholine- dependent, memory impairments that occur in nonhuman primates.

299 MOLECULAR BIOLOGY AND TRANSGENETICS OF PRION DISEASES.

*S. B. Prusiner. University of California, San Francisco, California, 94143 USA.

Considerable progress has been made deciphering the role of an abnormal isoform of the prion protein (PrP) in scrapie of ani- mals and Gerstmann-Straussler syndrome (GSS) of humans. Trans- genic mice (Tg 81) that carry and express a Syrian hamster (Ha) PrP gene developed scrapie 75 days after inoculation with Ha

prions; non-Tg mice failed to show symptoms after >500 days (Cell 59:847-857, 1989). Brains of these infected Tg 81 mice featured protease-resistant Ha PrP Sc, amyloid plaques characteristic for Ha scrapie, and 109 ID50 units of Ha-specific prions upon bio- assay. Studies on Syrian, Armenian and Chinese hamsters suggest that the domain of the PrP molecule between codons i00 and 120 controls both the length of the incubation time and the deposition of PrP in amyloid plaques. Ataxic GSS in families shows genetic linkage to a mutation in the PrP gene leading to the substitution of Leu for Pro at codon 102 (Nature 338:342-345, 1989). Discovery of a point mutation in the PrP gene from humans with GSS established that GSS is unique among human diseases - it is both genetic and infectious. These results have revised thinking about sporadic Creutzfeldt-Jakob disease (CJD) sug- gesting it may arise from a somatic mutation. Pulse-chase radio- labeling experiments of scrapie-infected cultures of mouse neuro- blastoma cells indicate that protease-resistant PrP Sc is synthe- sized with th -15 h from a protease-sensitive precursor, consis- tent with the conclusion that PrP C and PrP Sc differ due to a post-translational event. While PrP C is bound to the external cell surface by a glycosyl phosphatidylinositol anchor, PrP Sc was observed to accumulate in the cytoplasm of cultured cells near the Golgi apparatus. Immunoaffinity chromatography with ronoclo- hal antibodies to PrP 27-30 demonstrated copurification o11 PrP Sc and scrapie infectivity. These results combined with tho~;e from many other studies especially molecular biological investlgations assert that PrP Sc is a component of the transmissible particle, and the PrP amino acid sequence controls the neuropathology and species specificity of prion infectivity. The precise mechanism of PrP Sc formation remains to be established. Attempts to demon- strate a scrapie-specific nucleic acid within highly purified preparations of prions have been unrewarding to date (Bio:hem d. 266:1-14, 1990). Whether transmissible prions are compos~d only o-~PrP Sc molecules or they also contain a second component such as small polynucleotide remains uncertain.

3OO

CJD-LIKE AGENTS AND HUMAN DEMENTIAS OF UNKNOWN ETIOLOGY. L. Manuelidis* and E.E. Manuelidis Yale Medical School, New Haven, CT 06510 USA

Creutzfeldt-Sakob Disease (CJD) and scrapie of rodents provide useful general models for the study of human dementias of unknown etiology. These models are clearly relevant for: 1) widespread neuronal degeneration, 2) plaque formation in the CNS and 3) genetic modulation of disease. As in many human dementias, inflammatory infiltrates are not obvious in these infectious models. Plaques reflect neuronal dysfunction and breakdown over a long period of time, regardless of initiating events. Plaques in both CJD/scrapie and human Alzheimer's disease (AD) share several proteins, such as tau. Common pathogenetic pathways may be utilized during plaque formation in both CJD and AD. In the case of CJD/scrapie, a specific membrane glycoprotein (Gp34 or prion protein) remains This protein may be a CJD or scrapie receptor, but is unlikely to be the virus itself. Although the etiology of AD is unknown, a subset of AD cases

could be caused by a CJD-like virus. In this case the virus may target the ~-amyloid membrane protein, possibly as a consequence of host genetics, viral mutation or other factors.

The identification of virus-specific molecules in CJD and scrapie is of paramount importance for clarifying etiological relationships between AD and CJD. Up to the present time, these issues have only been explored on a limited basis in cumbersome transmission studies. However, our recent characterizations and purifications of infectivity in CJD set the stage for isolating virus-specific nucleic acid molecules. In this context, we have developed novel amplification strategies for recovering low pg levels of RNA. Our working hypothesis is that retroviral-like complexes are involved in both CJD and AD. Recent molecular studies in CJD are consistent with this hypothesis.

301

THE PRION PROTEIN GENE, RATHER THAN A LINKED LOCUS, APPEARS TO CONTROL SCRAPIE INCUBATION PERIOD IN MICE. *G.A. Carlson, C.Ebeling, M.Torchia, M.Scott, D.Westaway, S.B.Prusiner. McLaughlin Research Institute, Great Falls, Montana 59401 ; Dept. of Neurology, University of California, San Francisco, California 94143 USA.