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

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activation. Because PN-2/APP is a potent protease inhibitor and possesses growth factor activity, these results indicate that PN-2/APP may participate in the complex events that lead to wound repair. In certain disease states, alterations in platelet release and processing and clearance of PN-2/APP and its derived fragments could lead to pathological cerebrovascular and peripheral perivascular accumulation of these proteins.

ANALYSIS OF PROTEASES IN CEREBROSPINAL FLUID SAMPLES FROM ALZHEIMER'S DISEASE AND CONTROL PATIENTS. *J.M. Pastemack and S.G. Younkin. Division of Neuropathology, Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, 44106 USA.

The 39-42 amino acid polypeptide (B amyloid protein, i3AP) that deposits as amyloid in Alzheimer's disease (AD) is derived from a larger protein referred to as the 8 amyloid protein precursor (BAPP). We are analyzing AD and control cerebrospinal fluid (CSF) for proteases that may participate in cleaving the BAP from this precursor. In each of the full-length forms of the fiAPP, the BAP is located close to the carboxyl- terminus of the precursor where it extends from the extracellular region into the putative membrane-spanning domain. Data from our laboratory indicate (i) that full-length forms of the BAPP in human brain are normally cleaved either within the BAP or on its earboxyl-terminal side to produce large soluble derivatives containing all or part of the BAP peptide and (ii) that there are changes in the relative levels of BAPP soluble derivatives in AD CSF (see abstract by Palmert el al.) suggesting that proteolytic processing of the BAPP may be altered in AD. Cleavage on the amino side of the BAP could play a central role in amyloid deposition either by releasing the 8AP from SAP-bearing soluble derivatives or by cleaving full-length membrane-associated forms to produce small i3AP- bearing membrane-associated fragments from which amyloid could be generated. Soluble extracellular CNS proteases with this specificity would presumably have access to the relevant site in the 8APP, and these proteases would be expected to enter the CSF. Using the technique of substrate gel analysis (also called enzymography), we have identified seven proteases between 45 and 155 kDa as well as several larger proteases in both AD and control CSF. All seven proteases have pH optima between 7.4 and 8.5 and require calcium for maximum activity. Serum proteases assessed in parallel with the CSF proteases have a different profile, indicating that the proteases that we have observed in CSF are not the simple result of serum contamination. We are currently comparing the proteases in AD and control CSF and testing their ability to cleave purified forms of the 8APP.

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STUDIES ON A BRAIN PROTEASE THAT CLEAVES AT THE N-TERHINUS OF K-PROTEIN-RELATED SYNTHETIC PEPTIDES. ~bC.R. Abraham, ,W. Van Nostrand, ,E. Koo, 4J. Meschla, 4S. Sisodla. OBoston University Medical School, Boston, MA 02118, ,University of California, Irvlne, CA 92717, ,Johns Hopkins University School of Medicine, Baltimore, MD 21205.

The finding of the serlne protease inhibitor ~i antlchymotrypsin (ACT) in the amylold deposits of Alzhelmer's disease (AD), Down's syndrome, Hereditary Cerebral Hemorrhage with Amyloldosis of Dutch origin and normal aging, as well as the presence of increased levels of soluble ACT in AD brain when compared to controls, prompted us to search for brain serine proteases which may be involved in the generation of the 4kd B- protein from the much larger B-protein precursor (B-PP).

Synthetic peptides were made according to the sequence flaking the N-terminus of the B-proteln, and the proteolytic activity of various fractlons tested on radlolabeled peptldes which were then separated on High Pressure Liquid Chromatography (HPLC) and subjected to autoradlography. Following several steps of purification, we obtained a fraction able to cleave the synthetic peptlde between methionlne and aspartate, aspartate being the N-terminus of the B-proteln. The activity is dependent on calcium, and inhibited by ACT and also by the secreted form of the B-PP containing the inhibitory domain (also called protease nexln 2, or PN2). When excess enzyme is used, the PN2 is degraded to lower molecular weight forms which can be detected on Western blots. We are now in the process of further purifying the protease using inhibitor and substrate columns, and trying to determine the cleavage sites generated by our protease in the B-PP.

In sammary, our protease may be involved in the abnormal degradation of the B-PP, leading to amylold deposition, which in turn can induce the neuritic response, tangle formation and

neuronal death. (Supported by ADRDA grant number IIRG-89-125 to C.R.A.)

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TRP~SFE~TION OF B-AMYLOID INTERFERES WITH THE DIFFERENTIATION OF THE CHOLINERGIC CELL LINE NG108. *A. Dagcnais I, N.R. Cashman 2, H.D. Durham 2, D. Gauvreau I , J. Nalbantoglu ~ . tINRS-Sant6, Pointc-Claire, Quebec. 2Montreal Neurological Institute, Montreal, Quebec.

The B-amyloid protein is found in neuritic plaques of Alzheimer's disease (AD) and Down Syndrome (DS) cases. This protein is derived from a high molecular weight precursor encoded by differentially-spliced transcripts expressed in the brain and a wide variety of tissues. Faulty processing of the B-amyloid precursor protein (B-APP) may lead to the neuropathology observed in AD and DS.

We are studying the consequences of the expression of such "aberrant" forms of the B-APP on the cholinergic cell line NG108 which can be differentiated in the presence of dibutyryl cAMP (dbcAMP) to extend neurites in culture. We have transfected NG108 with a plasmid construct coding for amino acids 591 to 695 of the B-APP (the B-amyloid/C-terminal domain) under the control of the neurofilament NF-L promoter, a promoter which is induced upon differentiation. The stably transfected cells were similar to NGI08 in terms of morphology and growth characteristics. However, in the presence of dibutyryl cAMP, these transfectants started aggregating and piling up (within 2 days) and remained aggregated, extending very few neurites during the 8-day differentiation protocol. (A control transfectant containing integrated copies of the plasmid pSV2Neo behaved similarly to NG108). This morphological change did not lead to cell mortality since throughout the 8 days, all examined cell lines had the same viability as judged by trypan blue exclusion as well as [3H] thymidine incorporation. Furthermore, the aggregation was reversible upon withdrawal of the dibutyryl cAMP.

We have also obtained stable transfectants where constitutive expression of the B-amyloid/C-terminal domain is directed by the SV40 late promoter. Preliminary results indicate that these transfectants also show changes in cell morphology upon differentiation with dibutyryl cAMP. (Supported by MRC Canada (MA-10407)).

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PURIFICATION OF CLIPSIN, AN ¢tI-ANTICHYMOTRYPSIN-BINDING PROTEASE WHICH CLEAVES THE ~-PROTEIN PRECURSOR. *Robert B. Nelson and Huntington Potter. Department of Neurobiology, Harvard Medical School, Boston, MA 02115.

Three independent lines of evidence suggest that a chymotrypsin-like protease participates in the process of Ill-protein deposition into Alzheimer's amyloid filaments: I) cleavage after a methionine residue in the 13-protein precursor generates the amino terminus of 13-protein; 2) ct l-antichymotrypsin (ACT) is an integral component of amyloid filaments; and 3) the 13-protein precursor includes a protease inhibitor domain having high affinity for chymotrypsin. The discovery of clipsin, a chymotrypsin-like protease in rat brain which binds to ACT in an SDS-stable manner and preferentially degrades memhi'ane-associated amyloid precursor protein, has recently been reported (J. Biol. Chem. 262, March 5 issue). We report here the purification of clipsin to homogeneity. Detergent-extracted membranes from 5- to 6-day-old rat forebrains were treated with 1.0 M MgC12 to solubilize clipsin activity. Clipsin was then precipitated by dialysis against a low-ionic strength buffer, eluted at high salt from a heparin-agarose column, and finally purified using a custom-made subswate column (Ala-Pro-Phe-Sepharose 4B). The eluant from the latter column contained 17% of the starting activity and on silver-stained gels revealed a single 25 kDa doublet. Both bands of the doublet had protease activity as measured by enymography and potentially represent post- Wanshttionally distinct forms of clipsin. Purified clipsin also retained its ability to bind to ACT in an SDS-stable manner. We are currently working to obtain internal protein sequence information from tryptie digests of clipsin to determine if clipsin is a novel enzyme. These partial sequences will subsequently be used to raise domain-specific antibodies to clipsin and to construct degenerate oligonucleotide probes so that clipsin may be cloned from rat and human brain.

210 BASEMENT MEMBRANE HEPARAN SULPHATE PROTEOGLYCAN IS PART OF

ISOLATED ALZHEIMER'S AMYLOID PLAQUES. S. Narindrasorasak, I.