NEUROBIOLOGY OF AGING, VOLUME 1 l, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE BRAIN AMYLOIDOSIS

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change. CAO are numerous in the frontal cortex of Picks's disease, substantia nigra of Parkinson's disease and the motor cortex of amyotrophic lateral sclerosis. In this study, brain tissue from Alzheimer's disease patients as well as from other neurological cases and age matched controls was immunostained with antibodies to AP, C3d and C4d. In all cases examined, CAO were stained positively for AP. CA0 were not frequent, however, in cerebral cortical areas which were heavily affected in Alzheimer's disease. These included the hippocampus, middle temporal and angular cortices. Senile plaques and diffuse amyloid deposits are particularly rich in these areas and stain positively for AP as well as for C3d and C4d. Although the functions of AP are not yet understood, we speculate that AP may play some role in immunological reactions associated with complement activation. (Supported by the Alzheimer Society of B.C. and the American Health Assistance Foundation.)

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EFFECTS OF A NOVEL CARBOHYDRATE, 4-DEOXY-L-THREO-PENTOSE ON NEURAL CELL PROTEOGLYCAN SYNTHESIS AND FUNCTION. *K. Dow, R. Riopelle, M. Bols, E.R. Ison, W.A. Szarek, A. Lyon and R. Kisilevsky. Departments of Pediatrics, Medicine, Chemistry and Pathology, Queen's University and Kingston General Hospital, Kingston, Ontario, Canada K7L 3N6.

Hcparan sulphate proteoglycans (HSPGs) have been implicated in neurite outgrowth. In Alzheimer's disease abnormal neurites are prominent, and HSPGs are a constituent of amyloid plaques, while sulphated glycosaminoglycans (GAGs) decorate neurofibrillary tangles. To further pursue the function of HSPGs in neurite growth and their potential role in angiopathic and plaque amyloid deposition, a novel carbohydrate~ 4-deoxy-~- threo-pentose (4-deoxyxylose) was prepared. This compound ~s the potential to interfere with GAG synthesis. 4-deoxyxylose produced; I) decreased sulphation of proteoglycans by dissociated spinal cord neurons; 2) a profound undersulphation of the GAGs produced by astrocytes; and 3) a dose dependent inhibition of neurite outgrowth by sensory neurons. Some specificity was observed in that primary mouse hepatocytes were unaffected by concentrations as high as I0 mM.

Undersulphation of proteoglycans may be a mechanism which influences neurite outgrowth. The ability to produce undersulphated proteoglycans may provide a useful probe to explore the function of proteoglycans on neurite outgrowth and on beta-amyloid gene expression by neural elements.

Supported by grants from the MRC, MA-10477, MT-3153. MT- 7757, from the NIH, AA 07632-02, from NSERC and the Upjohn Company.

224

MORRBOLOGIE~EL C8~RA~A~IZATICN OF A NOVEL ANTIBOET£ AGAINST AM~DID E~g~NCING FACTC~. *Y. R~bi~ 11e, K. Alizad~-Ehiavi, Z. Ali-]C~n, R. Ne~a, A. ~ e t t e and D. Gauvreau. Dept. of Neurqpatholc~y, McGill University, and INRS-Sante, N~i~l, O~bec, CANA~

Amyloid ~t~ncing fa~r (A~), isola~d fr~ amyloidotic mouse ~ and Alzhelmer (AD) brain =z~pla~, has beem purified to homogeneity, lOuo~ of each A~ • ~ration hlduced accelerated splenic amylo!_ae~_is in mice in 72 to 120 hrs. Based c~ structural criteria and w~tern blotting, both mouse and Alzheimar AEF were i~mtlfied as ubiqultin. ~e AEF polyclo~l antibody was tast~ on ~p~eer~cative sectior~ of 3 ~D braiT~ i n the early and late stagas of evolution, 7 brain~ of Levy eo~y ~ with limbic predcmdzmnce, and 3 nmrmal control ~clmamm. ~I~ ABC ,,,=-=r=,d ~ (mP) ~ of sternber~r was used. It ~s __ee~A~ed with Mab 6.423 et al, 1988), of high ~$=_c__iflcity a~u~t fetal tau ar~ Paired Hali~l fil~r~s (t~F), and Alz 50 (Wol~in et al, 1986). ~ AEF antibody ~ __m~!~ra__ te to ~t~ ~--~=~¢ivity in the edvemtitia, ~a, and erzk~halial cells of l~--~Ln~eal and ¢=re~al vassels infiltrated sen i le colloid, c~as and margins of Mrd. la p~_~,w~u~a

~m.,mcrasct~ ~ ~ ¢~]atzq[~ic r ~ ' t t a s and m~ofi~illary ~m~ias (m~) of AD braln~.

Normal ~ols revealed no =i~r~ of AEF ~moreactlvity.

cortical ~ eodi~ reacted =~=~ly with the AEF antibody, i--.~|rmd tEm~catlr#~ ~ Msb 6.423 and AIz 50. ~ Body

~Iv~ceactivity ~s si~ifice, tly atmr~ed ~ pre-tr~tm~t with a]J<z~i~B ~6~atase. Mab 6.423 and Alz 50 heavily a~rated neuronal t~F'a and ~ c neuriVa=, ~=~iv~y. ~ ~ , ~ ~t ~ =~i-A~ (~i~itln)

b ~ to ~ = y l a ~ ~ i ~ of hi~ ~IZi~ty for cortical NFr's, ~ Bodice, ar~ ~ c neuritea as wall as vascular and plaqua ~loid. AEF (ubiquitin) my be involved in the physiological prou~sing of altered ~loid

p~L~. ~ ~ ~C ~ ~0172 ~d Canadian Parkln=~ R~dati~.

225

INDUCTION OF AMYLOID DEPOSITION BY HIGH MOLECULAR WEIGHT PROTEINS OF THE AMYLOID ENHANCING FACTOR EXTRACTED FROM BRAIN OF PATIENTS WITH ALZHEIMER'S DISEASE. *C61ine Modssette and Francina Gervais. McGill Centre for the Study of Host Resistance and McGill Centre for Studies in Aging, The Montr6al General Hospital Research Institute, 1650 Cedar Avenue, Montr6al, Qu6bec, H3G 1A4

In order to determine the role of the amyloid enhancing factor (AEF) in the deposition of amyloid fibrils in the brain, we have extracted and initiated the purification of this factor. Since its first description in the murine model of secondary amyloidosis, AEF has been reported in amyloidotic tissues from patients with secondary amyloidosis, such as Familial Mediterranean Fever, Familial Polyneuropathy and recently from patients with brain amyloidosis. This factor could play a role in the degradation of the amyloid B-protein precursor (ABPP) in 8 amyloid fibrils.

AEF was extracted from brain samples of patients with Alzheimer's disease by solubilization in 4M glycerol-10mM Tris buffer solution and by ultracentrifugation. Following dialysis against phosphate-buffered saline (PBS), AEF which precipitated was recovered by centrifugation. The biological activity of AEF preparation was monitored by its ability to induce the amyloid A fibril deposition in C57BL/6J mice. When given concomitantly or one day after an inflammatory agent such as silver nitrate, AEF caused appearance of amyloid A fibril deposits within 2 to 5 days. Presence of amyloid deposits in spleen was determined histologically following Congo red staining.

AEF extract did not demonstrate reactions following immunostaining with the anti-serum amyloid P component (SAP) and the anti-B-protein sere. The biological activity of AEF cannot thus be attributed to presence of SAP or B-protein in this extract. Amino acid analysis of the AEF extract showed to contain minute amount of glucosamine demonstrating that AEF is not a glycosaminoglycan nor proteoglycan. AEF extract having a biological activity was electrophoresed in 15% polyacrylamide gel in presence of SDS. After sapa(ating the preparative gel in segments comprising the major protein bands, proteins were electroeluted and recovered in PBS. When proteins of each segment were tested for AEF activity in mice, only the segment containing high molecular weight proteins (>90 kDa) gave positive results. These proteins are now being punfied and tested to identify which protein(s) has (have) AEF activity.

We thank the Brain Tissue Bank, Douglas Hospital Research Centre, Verdun (Quebec) for providing us brain samples from AD patients

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IDENTIFICATION OF THE SECRETED FORM OF THE 751 AMINO ACID 13-AMYLOID PRECURSOR PROTEIN IN PLASMA. R. Barbour, S. Sherard, M. Schlossmacher, M. Podlisny, D. Selkoe and *D. Schenk. Athena Neuroscience, Inc., S. San Francisco, CA 94080 and Department of Neurology, Harvard Medical School and Center for Neurological Disease, Brigham and Women's Hospital, Boston, MA 02115.

I~-AP, a small 4.2 Kd protein, derived from the ~-amyloid precursor protein (I~-APP) is found deposited in senile plaque tissues and cerebral vessel walls of patients with Alzheimer's disease. The source of I3-AP is unknown; however, the possibility exists that it might be circulating in blood. This hypothesis has been addressed by investigating whether I~-APP is found in plasma. Three monoclonal antibodies were made to three distinct regions of 13-APP. The first was to the putative extracellular region (amino acids 444-531); the second was to the Kunitz domain; and the third was to the C-terminal region of 13-APP (amino acids 676-695). These antibodies were used as an affinity matrix to pudfy I~-APP from human control plasma. Western blots were used to evaluate the proteins that were immunoaffinity punfied. Using polyc, lonal antisera to I~-APP to identify the purified proteins, a 125 Kd band that co-migrates with purified ~-APP751, was identified. This protein can be purified from plasma by both the monoclonal antibody to amino acids 444-531, and the monoclonal