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

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Alzheimer's disease (AD) (3 cases), Down's syndrome (DS) (2 cases) cerebral

hemorrhage with angiopathy (CHWA) (1 case) and normal aging (3 cases).

Our results indicate that:

(i) - arnyloid deposits (amyloid plaques and amyloidaceous vessels) are detected

in all cases by both anfisera.

(ii) - neurofibrillary tangles observed in the brains of AD and DS patients (and a

few in normal aging) were not detected by anti BPA4.

(iii) - An important diffuse amyloid material infiltrated the neuropil only in AD

and DS, particulary in layers II to V where neurofibrillary tangles appear.

The correlation between the presence of diffuse amyloid deposits,

neurofibrillary degeneration and dementia suggest that the amyloid infiltrate

observed in the neuropil of AD and DS patients may exert a direct toxic effect on

the vulnerable neuronal perikarya.

This work was supported by grants from the Caisse Regionale d'Assurance

Maladie Nord-Picardie.

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THE IMMUNOCYTOCHEMICAL LOCALIZATION OF AMYLOID /~-

PROTEIN PRECURSOR IN THE MOUSE BRAIN . *T.

Kawarabayashi, M. Shoji, Y. Harigaya, H. Yamaguchi, S.

Hirai. Department of Neurology, Gunma University

School of Medicine, 3-39-15 Showa-machi, Maebashi,

Gunma 371, Japan.

We investigated the histological localization of

the predicted amyloid B-protein precursor (APP) in

the mouse brain using antisera to the carboxyl- and

amino-terminal regions of APP. Both antisera recog-

nized the same 106-122 kDa proteins of the Triton X

extract of mouse brain by immunoblot analysis. In the

immunocytochemical study, reactivity for these an-

tisera was located on the almost all types of

neurons. These immunoreactive neurons were found in

almost all area examined. Some glial cells and ves-

sels were also immunoreactive. The immunoreactivity

had a granular appearance and was seen in cytosol, ie,

near nuclear membrane , near plasma membrane , in

proximal dendrites and in axons. The immunoreactive

localization by C-terminal antibody was almost similar

to that by N-terminal antibody. These results indi-

cate that the full length of APP exists in the almost

all neurons and some glial cells of the adult mouse

brain. The study of APP in peripheral nerve, and in

the old mouse brain are now in progress.

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C-TERMINAL TRUNCATION OF APP IN CULTURED MAMMALIAN CELLS OCCURS WITHIN B/A4. *S.S. Slsodla, J.F. Meschla, E.H. Koo, D.L. Price. Neuropathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205-2181 USA.

B-amylold protein (B/A4), derived from a larger amyloid precursor protein (APP), is the principal component of senile plaques In cases of Alzheimer's disease. APP, an integral membrane glycoprotein, Is secreted as a C-terminal truncated molecule. The site of cleavage within APP during maturation is presently unknown. The present study employs molecular biological approaches to examine APP sequences that are both necessary and suff ic ient for this processing event to occur in cultured mammalian cells. Our studies provide compelling evidence that APP is cleaved only when associated with membranes and that the cleavage site resides within 11 amino acids upstream of the transmembrane domain. Because this site resides

within the BIA4 reglon, these studies indicate that an intact amyloldogenlc B/A4 fragment is not generated during normal APP catabolism. Results of our ef for ts directed toward biochemical ident i f icat ion of the precise cleavage site wil l be presented.

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ALTERATIONS IN PC12 CELLS OVEREXPRESSING THE A4-C TERMINAL REGION OF THE AMYLOID PRECURSOR PROTEIN. *B. Tate-Ostroff, R. E. Majocha, M. Ventosa-Michelman, W.-G. Chou, S. Zain and C.A. Marotta. Harvard Medical School, Massachusetts General Hospital and McLean Hospital, Boston, MA and University of Rochester Medical School, Rochester, NY.

PC12 cells were transfected with cDNA corresponding to the A4-C-terminal region of the APP. Transfected cells showed an immunologically detectable overabundance of the A4 region in the cell membrane at the light and electron microscope level. In addition, Western blots of cell membrane preparations showed a low molecular weight band immunologically detectable by a monclonal antibody to the A4 region. Conditioned media from transfected cells, but not from untransfected cells, stimulated neurite growth by control cells. Serum free media (SFM) was collected from cultures of transfected cells as well as from untransfected PC12 cells. SFM was concentrated and separated by gel electrophoresis and subsequently stained by Coomasie blue. Analysis revealed dissimilar banding patterns from the SFM of transfected cells when compared to the SFM of untransfected cells. We conclude that amyloid accumulation in PC12 cells causes alterations in the cell membrane and secretion products. A4-amyloid overexpression, and/or the overaccumulation of the peptide within the membrane, may be related to the expression of new unidentified genes in transfected PC12 cells. Supported by AG02126, the American Health Assistance Foundation and the Metropolitan Life Foundation.

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INCREASED EXPRESSION OF ALZHEIMER'S DISEASE B-AMYLOID IN SENESCENT CULTURED FIBROBLASTS

*Nazneen N. DewilY, Mark J. Adler1., J. E. Seogmillert, Earl Shelton$ and Crystal Coronel1.. 1" University of California, San Diego, La Jolla, CA 92093. i: Syntex Research, Pale Alto, CA.

The pathological hallmark of Alzheimer's disease (AD) is the accumulation of neurofibrillary tangles and neuritic plaqueS. The latter contains a 4-5 Id3a amyloid 8-protein fragment which is also found in the blood vessels of the brain of patients. Since amyloid deposition in the brain increases with age even in normal people, we sought to establish whether the disease state bears a direct relationship with normal aging processes. AS a model for biological aging, the process of in-vitro cellular senescence was used. mRNA levels of 8-arnyloid associated with Alzhoimer's disease were compared in human fibroblasts in culture at early passage and when the same fibroblasts were grown to senescence after more than 52 population doublings. A dramatic increase in mRNA levels was observed in senescent fibroblasts compared to early passage cells. Hybridization of mRNA from senescent and early proliferating fibroblests with oligonuclootide probes specific for the three alternatively spliced transcripts of the gone gave similar results, indicating an increase during senescence of all three forms. A similar, though more modest increase in message levels was also observed in early passage fibroblasts made quiescent by serum deprivation; with repletion of serum, however, the expression returned to previous low levels. ELISAs wore performed on cell- extracts from senescent, early proliferating and quiescent fibroblasts, and quiescent fibroblasts repleted with serum for over 48 hours, using polyclonal antibodies to a synthetic poptide of the 8-amyloid precursor. The rasults confirmed that the differences in rnRNA expression were reflected at the protein level. Regulated expression of APP may be an important determinant of growth and metabolic reSponses to serum and growth factors under phys!ological as well as pathological conditions.