Chromatin structure and gene expression at the HNF-L gene locus in Alzheimer's disease (AD) affected temporal neocortex

NEUROBIOLOGY OF AGING, VOLUME It. b)9o ABSTRACTS OF SECOND INTF~RNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE GENETIC ME(THANISMS

322

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brain (B.F.], frontal cortex (]:.G.), hippoeampus (E) and c:~r~bellum (Cb) of post-mortem brain obtained from control patients and those with clinically and histopathologically confirmed A[zheimer's Disease (A.D.). A method of quantitating different mRNAs in a single experiment (PCR Multiplexing) was used to investigate the levels of expression of several genes including Cu/Zn superoxide dis~.utase (SOD), microtubu]e associated protein (Tau), alphal-antichvmotrypsin (alACT) and amyloid precursor protein (APP). In all brain areas examined, APP 695 is the predominant mRNA species while in Cb there is a [larked reduction in the level of APP 751. The APP 695:751:770 ratio is highest in Cb and lowest in F.C. and B.F. while in all areas examined a general trend of APP 695>751>77(] is found. Very little evidence was found to substantiate published reports of an ~berration in expression of the APP gene at the level of transcription in A.D. in these brain regions. A small but measureable increase in SO[) mRNA is observed in F.C., H. and CB brain from A.D. patients; no such incretses are found for (*]ACT mRNA in this disease. While evidence is presented for alternative splicing of the Tau gene, the relative levels of Tau I:Tau II show regional and patient variability. No evidence is found tc implicate increased levels of Tau mRNAs in A.D. These results suggest that, despite pub] [shed claims o} altered gene expression in many neurodegenerative disorders for whlch few, if any etiological mechanisms are known, we need t ~ examine the potential heterogeneity of such diseases by sharing tissues and analyzing them by different methodolegies before a consensus regarding altered brain expression can be reached.

DS and Alzheimer dementia (An) in extende~ f~milzes. The clinical studies include assessment of mental and physical handicap, t h y r o i d functioning, etc. Of 122 probands e n r o l l e d to date (average age: 39.4 years), 8 cases (average age: 55 years) of probable AD were found at initial assessment, one of whom died during the first year of study and neuropathological examination has confirmed the clinical d i a g n o s i s of AD. There were a furthez 1 8 c a s e s of s u s p e c t e d AD (average age: 42 years). About 1/3 of all DS probands were hypothyroid. No abnormalities of zinc were detected in the cohort. Previous reports have indicated a higher than expected frequency of DS in f a m i l i e s with AD. Our h y p o t h e s i s is that in f a m i l i e s with both DS and AD, affected individuals have the same #21 chromosome and that a chromosome rearrangement on an AD-bearing #21 leads to n o n - d i s j u n c t i o n in a subset of these families. Thirtyeight f a m i l i e s are e n r o l l e d in the genetic studies (including cytogenetics, DNA marker studies, and dermatoglyphics) aimed at testing this hypothesis. Of the these, at least 8 f a m i l i e s have a history of p o s s i b l e or p r o b a b l e AD -- often in the p a r e n t of the DS individual. The genetic studies have been performed on 154 members of the e x t e n d e d families. The p a r e n t a l origin of the #21s using both c h r o m o s o m e v a r i a n t s and DNA markers, the s e g r e g a t i o n of such c h r o m o s o m e v a r i a n t s as double and t r i p l e NORs, the extent of r e c o m b i n a t i o n throughout chromosomes 21, and the dermatoglyphic findings in family members will be presented.

288

286 TWIN STUDY OF SENILE DEMENTIA. *A.L.M. 8ergem, E. Kringlen. Department of Psychiatry, University of Oslo, Norway. The Norwegian twin registry has been checked against outpatients and patients in nursing homes and hospitals suffering from all kinds of senile dementia. 140 twin pairs have been found, most of them 75 years and older. In half of the cases both twins are alive. Each twin is classified into the following subgroups of dementia:

Alzheimer's

disease, multi-infarct dementia, alcoholic dementia, other degenerative dementiaS different from Alzheimer's disease and mixed forms. One will compare clinical findings with cerebral computer tomography and brain autopsy, when it is possible. Hopefully this study will throw light on genetic and environmental etiological factors.

Traditional twin analyses of concordance and

AGE OF ONSET OF ALZHEIMER'S DISEASE. M. Mullah, A. Goate, J. Hardy, P. Roques, M. Rossor. Dementia Research Group, Departments of Neurology and Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, London W2 IPG, England. The determinants of the age of onset of familial A[zheimer's disease are not well understood, in a series of pedigrees with clinically diagnosed and histologically confirmed familial Aizheimer's disease ascertained for genetic [iE~kage studies (Hardy et al., this meeting), an analysis ot variance of age of onset was conducted by family membership, by gender and paternal/ maternal affection status, in the early onset pedigrees (in which the weight of evidence suggests linkage to chromosome 21), 85% of the variance was determined by family membership, furthermore, practically none of the variance could be attributed to paternal/maternal inheritance. By contrast, in late onset pedigrees only 55% of the total variance could be explained by family membership. Again, no effect of gender, nor of paternal/ maternal inheritance could be detected. 45% ~I tim total variance in age of onset in this group remains unexplained. Given that previous work has reported these findings in an epidemiologically based sample (Van Duijn & Hsfman: submitted), we can draw the following conclusions:- (a) age nf onset is determined almost completely by the effects of the disease locus in the early onset families. (b) in late onset pedigrees the unknown aetioiogy does not allow conclusion as to the source of variation of age of onset but gender specific and paternal/ maternal inheritance are unimportant.

discordance rates are used. Co-morbidity will be studied, and DNA-

289 testing will be done. Affected first degree relatives will also be included in the study. By July 1990 data on approximately 70 twin pairs will have been collected.

287 DOWN SYNDROME AND ALZHEIMER DEMENTIA: CLINICAL EVALUATION AND GENETIC ASSOCIATIONS. *J.J.A. Holden, M. Chalifoux, M. Wing, R. Smithe, F, Dalziel, C. C l a i r m a n , D. Greet, B.N. White, D. S t a n i s t r e e t , I. Swift, J. F o t h e r i n g h a m , R. M a c l a c h l a n , J. Burley, J. Berg, M. Korossy, D. Robertson, B.D. McCreary. O n g w a n a d a C y t o g e n e t i c s and DNA R e s e a r c h L a b o r a t o r y and Dept, Psychiatry, Queen's University, Kingston, Ontario, K7L 3N6, CANADA We have established a program for b o t h the l o n g i t u d i n a l c l i n i c a l e v a l u a t i o n of p e r s o n s w i t h D o w n syndrome (DS) and studies of the genetic associations of

CHROI~TIN S ~ AND ~ ~ S S I O N AT THE HNF-L LOCUS IN ALZHEIM~'S DISEASE (An) / ~ l ~ NEO0~. *W.J, Lukiw and O.R.C.l~px~hl~, Center for _R~___rc~ in N e u r o d ~ a t ive Disease, M e d i c a l Sciences Building, University of Toronto, Toronto ~ IA8 CANADA. Micrococcal nuclel~e (NN; EC 3 . 1 . 3 1 . I ) di~m~cion was employed to e~amine DNA a~8ociated with nucl___~_ l ~ l a t i o r ~ isolated from AD a f f e c t ~ superior t ~ a l lobe n ~ t i c a l nuclei. 46.1% o f the immolate 5' ul~t,r-e~ DNA ~qt.~ertce o f the single c~oy burton n e u r o f i l ~ l i ~ t chmin (HNF-L) 9 ~ was found to be smeociatad with a m ~ ' w : w " ~ c l ~ fraction in control temporal neooortic~6. This fraction wm~ reduced to 7.4~ in age match~ AIY-affect~ __ru~ct__tex. No d i f f e r ~ in aoce~sibility to the n u c l ~ prob~ ~ found b ~ Anaffected and o ~ t r o l temporal grey a ~ t t e r nuclei for the human p~ion PrP ~ or for the HNF-L ~ in nuclei imolat~d from the primary visual cortex or t ~ cer~llum. An AvaI restriction e ~ d o n u c l m Bite, loc~t~ 124 ~ p~irs upstream from the TATAA box in the HNF-L DNA~ w ~ wm~ also found to be occluded in AD affected t ~ a l lobe nuclei.

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NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE GENETIC MECHANISMS

From t2nis and previous clara we conclude tJnat within ~ A£)affEw~t.e(l n u ~ l ~ , fc~L.~,(yJ ~ in neuronal c h r ~ t i n conf~ti~ (x~cur. I n c r ~ in t/no packing clet~sity of Onrornatin may r~3W~-.e neuron ~ i f i c HNF-L 9(w~et.ra~scripticw~ ~ d alter t)he a~)ility of neur(w~s to 9w~r~-at~ ~ f f i c i e n t levels of r~urofi lambent pro'ceins to maintain normal r~ocx)rtical furw~ti~. Sw~p~x)r1~ecl by tJ~e Ontario Mental FW~alt~ F(xJndati(~1, Naticyr~l ~ i ~ ~ d Engineerir~j Re~=earc-~l O~uncil ~ d M~=~ical Research Council of Canada.

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290 CHARACTERIZATION OF TWO BLOCKS OF CIS-ACTING REGULATORY ELEMENTS MODULATING THE EXPRESSION OF THE ~ENE ENCODING THE ALZHEIMER'S AMYLOID PRECURSOR PROTEINS. D . K . Lahiri and N.K.Robakis. Mount Sinai Medical Center, New York, N.Y. 10029 The promoter region of the Alzheimer's Amyloid Precursor Protein (AAPP) contains several regulatory elements including five copies of the GGGCGCsequence l ocatedbetween positions 107 and - 1 8 8 , consensus sequences recognized by the transcription factors SpI and AP-I, a rich GC region characteristic of housekeepinggenes and a heat shock element (LaFauci et ai.1989). I t has been proposed that an increase in the transcriptional a c t i v i t y of this gene might result in the formation of the amyloid B-protein which aggregates to form the amyloid depositions observed in Alzheimerrs disease and Down's syndrome. To determine the promoter sequence requirements for the expression of the gene encoding the AAPP, chimeric plasmids containing different parts of the promoter region of the ~u~PP gene linked to the bacterial chloramphenicol acetyl transferase (CAT) gene were constructed. Sequencesderived from the 5'flanking region were then tested for their effects on B-amyloid promoter a c t i v i t y by transfection and transient CAT expression in PC12 and HeLa cells. In PC 12 cells there is a significant increase in basal promoter a c t i v i t y after deletion of certain sequences from the 5'-flanking region. The maximum increase in a c t i v i t y was observed when the promoter sequences were reduced to 0.7 kb from 1.2 kb. Similar increase, though less pronounced is observed in HeLa cells. However, further deletion of 150 base pairs (bps) resulted a sharp decrease in promoter a c t i v i t y in both cell lines indicating that this region contains cisacting elements which stimulate the expression of AAPP gene. Interestingly,in both cell lines there was a gradual and significant increase in promoter a c t i v i t y upon further deletion with a construct containing only 250 bps of flanking sequence displaying the strongest promoter a c t i v i t y . This construct contains three of the five GC boxes present in the amyloid promoter. Further deletion of 125 bps abolished all a c t i v i t y . These results indicate that there are two blocks of sequence which modulate the expression of AAPPgene promoter. One block extending from 550 to 600 bps acts as a positive regulator because i t s deletion results in a dramatic decrease in promoter a c t i v i t y in both cell lines. A second block of sequences extending from 250 to 500 bps acts like a negative regulator as their removal results in increase in promoter a c t i v i t y . Our observations suggest that by imposing conformational constraints~ these sequences might control the binding of trans-acting factor(?) on the AAPP gene promoter and thus modulate its expression. The identification of such factors is in progress. 291 PROCESSING OF PRECURSORS OF AMYLOID A4 PROTEIN IN ALZHEIMER'S DISEASE *Andreas Weidemann. Hans Mechler, Colin L. Masters l and Konrad Beyreuther Center for Molecular Biology, University of Heidelberg (ZMBH), Irn Neuenheimer Feld 282, D-6900 Heidelberg 1, F. R. Germany lDepartment of Pathology, University of Melbourne, ParkviIle, Victoria 3052, Australia Cloning and cDNA sequencing have indicated that the A4 amyloid protein is encoded as part of a larger protein. Several different mRNAs arise from the gene by alternative splicing. The three primary translation products are transmembrane proteins of 695, 751 and 770 amino acids (PreA4695, PreA475 1 and PreA4770). Based on recently published data we used polyclonal and monoclonal antibodies to study the biogenesis of PreA4 proteins expressed in different cell lines. Specific antisera able to distinguish various parts of the amyloid protein were obtained by cloning and expression of PreA4 domains as fusionproteins in E.coli. The maturation process of the PreA4 proteins includes tyrosin sulfation, O- and N-glycosylation. The O-glycosylated

forms show a higher molecular weight as well as a higher negative charge compared to the N-glycosylated forms and are also found as phosphorylated proteins. The cells express the polypeptides at the cell surface, but secrete also C-terminal truncated proteins into the medium. Deduced from the di ffe re nt molecular w e i g h ts ~)f transmembrane and secretory forms (17-18 kd) the sile ~f cleavage was proposed to be located in the region of the Nterminus of A4. Although the precise site is still unknown, preliminary results indicate that the primary cleavage event does not generate the N-terminus of A4. It seems to occur within the A4 sequence N-terminal to the transmembrane domain. This finding suggests that an intact A4 molecule is not generated by cellular events involved in the normal proteolysis that leads Io secretion of PreA4 proteins. 292

THE COMMON ORIGIN OF FAD IN THE CALABRIA REGION. *A.C. Bruni(1), M.P. Montesi(1), G.Gei(1), C. Ermio(1), I. Rainero(2), J.F. Foncin(3). (1) SmidSud Lamezia Terme, Italy. (2) Clinica Neurologica, Torino, Italy. (3) Laboratoire EPHE Salpetriere, Paris, France. We have been studying Familial Alzheimer's Disease in two families: family N (pathologically confirmed, Foncin et al. 1973, 1985; sea also Feldman et al. 1963) and family TO (Bergamini et al. 1989). The largest is family N which has been instrumental for the mapping of the FAD gena to chromosome 21 (St. George-Hyslop et al. 1987). It currently lists 6,000 subjects in 13 generations, with 65 affected and 13 obligated carriers with unknown phenotype. Family TO now lists 1,200 subjects in 8 generations with 24 affected persons. Both families have been studied using the same "blanket genealogical method" relying primarily on municipal, parish and State Hospital archives. Clinical presentation of AD is rather uniform; early onset (40+12 in Family N, 44+11 in Family TO, after a mean course of 8 years. Sex ratio (1:1) and segregation ratio (higher than 0.5, probably due to ascertainment bias) are not significantly different in the two families. Recently we discovered in the same region another family with clinicaUy diagnosed FAD. Family RR now lists 120 subjects with 7 affected persons over 4 generations. Remarkably, their clinical picture is also characterized by early onset and terminal myoclonus and grand mal seizures. Mean age at death in this family in 49.5_+13 years. The strong symptomatologic similarity we found in these three families could be explained by allelic identify by descent, as suggested by their close geographic origin. In fact many other (apparently) small clusters with early onset FAD are found in our region. We think that a founder effect could be postulated, due to the relative isolation and stability kept by the Calabrian population until the early 20th century. These conditions are similar to those described by Bird et el. (1988) for the Volga Germans. We hope to be able to prove this hypothesis in the future through the identification of a common (carrier) ancestor, as we did in the past for apparently distinct branches of family N. 293 QUANTIFICATION OF GROWTH-ASSOCIATED PROTEIN 1B mR N A IN N O R MA L AGING AND ALZHEIMER'S K.Rogers, A. Wadhams, D. McLymond, P. D. Coleman. of N e u r o b i o l o g y and A n a t o m y University of Rochester, N.Y. 14642 USA

43 AND ILDISEASE. Department Rochester,

G r o w t h - a s s o c i a t e d protein 43(GAP-43) appears to be a marker of development, growth, and regeneration of neuronal processes and therefore may also be a marker of neuronal plasticity. In Alzheimer's disease(AD) it is proposed that neuronal plasticity is d e c r e a s e d or absent. Therefore, GAP-43 m R N A has been qua nt i fi e d in normal aging and A l z he i me r' s disease in superior frontal gyrus(area8/9). Initially, the total amount of m RN A in each sample was determined by hybridization to a r a d i o a c t i v e l y labelled synthetic oligo-dT probe. Levels of Gap-43 mRNA were determined by hybridization of a ra di oa c t i ve l y labelled rat GAP43 e D N A to serial dilutions of each mR N A sample. F o llo w in g autoradiography, bands were d e n s i t o m e t r i c a l l y scanned and p eak areas integrated. Peak areas were n o r m a l i z e d for the m RN A content of each sample using the oligo-dT hybridization values. Consequently, we have found that GAP-43 levels of 89-97 yearold normal individuals are only 25% of the levels of the normal 50 year-old group. However, there is no significant difference in