418 Hydrolysis of amyloid precursor protein (APP) by a brain acid protease

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sAPPI3 products generated by eukaryotic and yeast molecular expression systems, and synthetic peptide fragments of the sAPPs, the activity of sAPPs was localized to amino acids 591-612. Heparinases greatly reduced the actions of sAPPas indicating a role for the heparin-binding domain at the Cterminus of sAPPccs in receptor activation. Mutations in [3APP previously shown to increase I~-secretase cleavage may promote neuronal degeneration by reducing levels of neuroprotective sAPPots.

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response. High level of IL-6 in the CNS may induce an acute phase response, stimulating the expression of APP. We found that APP 695 is the only isoform expressed in relatively pure neuronal culture, while all three isoforms 770,751, and 695 are detected in astrocytes. IL-6 was able to stimulate APP 695 expression in neurons. These results show that IL-6 is produced by astrocytes and is able to induce APP; they also suggest that in the CNS are present the mediators of an acute phase response that may be involved in the pathogenesis of neurodegenerative disorders. (Supported by CNR 1995 grant to G.S.).

415 Role of Calcium in [3 25-35-Induced Neurotoxicity in Cerebellar Granule Cells. A. Scorziello*, O. Meucci, T. Florin, S. Thellung, §M. Salmona, ~G. Forloni and G. Schettini Dept. of Clin. and Exp. Ontology Univ. of Genoa, IST, Unit of Neuroscience CBA, Largo R. Benzi I0, 16132 Genova; §Inst. of Pharmacol. Res. M. Negri, Via Eritrea 62, 20157 Milano, ITALY. We studied the neurotoxic effects of 1325-35 amyloid fragment (13 25-35) on cerebellar granule cells, at different stages of maturation in vitro, in presence of both low and high extracellular KCI ([KCI]o; KCI 5 and 25 mM), and this effects have been correlated with the alterations in calcium homeostasis in basal and glutamate-stimulated conditions. 13 25-35 (25 p.M) was added directly to the culture medium, and its effects on neuronal survival monitored cache 24 hr by means of phase-contrast microscopic analysis and by counting nuclei of intact cells, until the death occurred. Our results show that, the treatment for 3 days with the peptide greatly reduced the survival of I day in vitro (DIV) cultures kept in 5 mM KCI, while sligthly modified the survival of 25 mM KCl-cultared cerebellar granule cells. We also studied the effect of glutamate on survival of undifferentiated cerebellar granules. We report no neurotoxic effect of glutamate on 3 DIV-treated cultures, while in 13 25-35-pretreated cells, a significant glutamate toxicity was observed. Six DIV cells treated with 1325-35 in presence of 25 mM [KCI]o showed a late but significant signs of neurotoxicity after 5 days of exposure to the peptide, and died within 8 days of treatment. Differentiated cerebellar granule cells were also sensitive to glutamate-related neurotoxicity, and this effect was enhanced by 13 25-35 pretreatment. To study the molecular mechanisms underlying the neurotoxic effects of 1325-35, changes in calcium homeostasis, following glutamate stimulation, were evaluated in control and 13 25-35 treated single cell, by using fura 2 and video imaging analysis. 1325-35 did not affect basal [Ca2+]i, while modified glutamate-induced [Ca2+]i increase, causing a sustained plateau phase that persisted even after the removal of the agonist. These results show that 1~25-35 induces neurotoxicity in cerebeUar granule cells and that this effect is related to modifications in the control of calcium homeostasis (Supported by MURST 40% 1995 to G.S.).

417 Amyloid B-Protein F o r m s Cation-Selective Channels Across Excised M e m b r a n e Patches F r o m G n R H Neurons M. KAWAHARA* t, y. KURODAt, N. ARISPE2, and E. ROJAS2,3 IDepartment of Molecular & Cellular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183, Japan, 2Laboratory of Cell Biology and Genetics, NIDDK, NIH, Bethesda, MD 20892, U.S.A., 3Department of Physiology and Biophysics, Faculty of Medicine, University of Chile, Santiago, Chile. Alzheimer's disease 40-residue amyloid B-protein (ABP [1-40]) forms cationselective channels across planar acidic phospholipid bilayer membranes (Arispe, et al., Proc.Natl.Acad.Sci., 90, 10573-10577 (1993)). To determine whether direct incorporation of AI3P [1-40] molecules from the solution also occurs in neuronal membranes, we exposed excised membrane patches from hypothalamic GnRH neurons (GT1-7 cells) to AI3P [1-40] molecules dissolved in saline mimicking the composition of the internal (or external) medium. We now report that although ABP [1-40] molecules can interact with the excised membrane patch from either side, peptide incorporation leading to channel formation occurred within 3-40 minutes and was more efficacious from the inner aspect of the excised patch. As expected, the reversal potential of the ABP [1-40] specific channel currents in a CsCI solution system followed the Cs + gradient. The ABP [-40] channel exhibits spontaneous conductance changes over a wide range (50-500 pS). Zn 2+ (50500 I.tM), reported to bind to ABP [1-40] with high affinity, effectively blocked the channel. This blockade was reversed by the Zn2÷ chelator, o-phenanthroline in the millimolar range (see Arispe, etal:, Proc.Natl.Acad.Sci., (1996, in press)). These properties of the ABP [ 1-40] formed across a neuronal membrane patch are similar to those observed in the bilayer. Furthermore, deposition of ABP [1-40] on GT1-7 cell membranes could be identified by the immunohistochemical method. Neurotoxicity of ABP [ 1-40] to GT 1-7 cells were observed by WS T- 1 assay (a modified MTT assay). Taken together these data support the "Al~P-channel hypothesis" to explain the neurotoxic effects of amyloid g-proteins and suggest a preventive role for Zn 2÷, known to accumulate in hippocampal neurons, in the etiology of Alzheimer's disease. (Supported by Japan Health Science Foundation, FONDECYT 1950774, and Fundaci6n Andes, Chile).

416 Relationship Between IL-6 Production From Type-I Cortical Astrocytes And Induction of Neuronal APP mRNA Expression. G. Schettini*, T. Florin, §M. Grimaldi, A. Scorziello. Dept. of Clin. and Exp. Oncology Univ. of Genoa, IST, Unit of Neuroscience CBA, Largo R. Benzi 10, 16132 Genova, §Dept. of Neuroscience, Section of Pharmacology, Univ. of Naples ITALY. The aim of this study has been to characterize the regulation of IL-6 release and production from cortical type I astrocytes, to assess its role in the regulation of the expression of APP in neurons. We found that IL-6 release and production are under the control of PKA/PKC phosphorylating enzymes. Forskolin, (Bu)2cAMP, 8-Br-cAMP, stimulate in a dose-dependent manner IL-6 production by cortical astrocytes, and the activation of receptors coupled to the adenylate cyclase enzyme, such as VIP, also induces a dose-related release of IL-6 in the culture medium. These effects were selectively antagonized by KT 5720 but not by calphostin C, suggesting that the effects occur through the activation of a cAMP-PKA system. Conversely, the treatment with somatostatin, known to inhibit AC enzyme, resulted in a dose-dependent decrease of IL-6 release, which was mediated by the activation of a PTX-sensitive G-protein. The activation of PKC with PMA also leads to an increase in IL-6 production that was bloked by PKC antagonists or desensitization. Such observations suggest that the activation of receptor systems coupled with AC or PLC enzymes may regulate in physiological conditions the production and release of IL-6. The concomitant interaction with inflammatory stimuli may trigger an exaggerated IL-6 secretory

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Hydrolysis of Amyloid Precursor Protein (APP) by a Brain Acid Protease M. N. Malik*, G. Y. Wen, and H. M. Wisniewski New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA An acid protease which was discovered during the characterization of a high molecular weight (HMW) protease from the brain (JBC, 266, 6594-6599, 1991) was purified from the calf brain. The purification procedure involves ammonium sulfate fraetionation of the cytnsol followed by chromatography on DEAE-Sephaeel. The unbound material from this column was adjusted to 2 mM CaCI2 and MnCI 2 and applied to a ConASepharose 4B column, The column was washed with equilibration buffer followed by elution with 0.6 M NaCI, 0.2 M ,~-methyl-D-mannoside, 0.1 M NaPO o pH 6.5. The eluted fractions were concentrated and applied to a pepstatin-sepharose 4B column and washed with 6 M urea followed by elution with 0.6 M NaCI, sodium bicarbonate buffer pH 8.2. The eluted fractions were concentrated on an Amicon ultrafiltration cell fitted with a YM10 membrane. The molecular weight of the protease was estimated to be 48,000 kDa. Human anti-cathepsln D immunoreacted with the protease band, thus suggesting similarities with cathepsin D. Purified amyloid precursor protein (APP) from human platelets reacted with the protease. As a result of this hydrolysis, peptides of various sizes ranging from 100 to 15 kDa were released. Further characterization of these peptides is in progress. These observations suggest that cathepsin D is involved in the

FIFTH INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE catabolism of APP. Its possible role in generating amyloid beta (A4a) peptide in Alzheimer disease is being investigated. Supported in part by funds from the NYS Office of Mental Retardation and Developmental Disabilities, a grant from the National Institute on Aging, NIH, PO1 AGO 4220 and the fund for the Center for Trace Element Studies and Environmental Neurotoxicology.

of Alzheimer's disease, individual astrocytes or neurones increase their content of ferritin, and then start to degenerate. As their cell membranes rapture, ferritin leaks extracellularly, releasing metal ions which in turn precipitate 13-amyloid. Phagocytic microglia may contribute to the deposition of amyloid, thereby entombing the dying cells within plaques. 1. Connor, J.R. et al. (1992) J. Neurosci. Res., 31: 75. 2. Mantyh, P.W. et al. (1993) J. Neurochem., 61:1171. 3. Kuroda, Y. and Kawahara, M. (1994) Tohoku J. Exp. Med., 174: 263. 4. Bush, A.I. et al. (1995) Science, 268: 1921.

419 Granules in glial cells of patients with Alzheimer's disease are immunopositive for C-terminal sequences of I~-amyloid protein H. Aldyam at', C. Schwab2, H. Kondo t, H. Mori t, F. Kametani t, K. Ikeda t, and Patrick L. McGecr 2 tTokyo Institute of Psychiatry, 2-1-8, Kamikitazawa, Setagaya-ku, Tokyo, 156, Japan, and 2Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, B.C., V6T 1W5, Canada Granular structures that are recognized by antibodies specific for the Cterminal but not the N-terminal sequences of the [~-amyloidprotein(A[~) fragments are present in a subset of microglia and astrocytes in Alzheimer brain tissue. The A[~-eontaining microglia outnumber the Al3-containing astrocytes. Anti-A[~ antibodies employed in this study are as follows (antigen peptide sequence, or the specificity of the antibody, if determined); Ai81(N-terminal Aspt); R17(8-17); 6F/3D(8-17); 2F(17-28); E50(17-31); f~mid(25-35); [~ter40(Cterminal Vale); [~ter42(C-terminal Ala42/Thr43). The immunohistochemical profile indicates that the A~ in these granules is truncated between the residues 17 and 28. A[3 in the vast majority of the granules terminates at the residue 42 or 43. Such A[~-containing glia occur in brain areas with the heavy A[I deposits. They are also found in some aged control subjects but only in areas with the parenchymal A[3 deposits. Microglia within senile plaques frequently contain AI3 granules. At present, whether the intraglial A[~ fragments accumulate as a result of phagocytosis of extracenuinr A~ or are formed intracefiularly by glial cells from amyloid precursor protcin(APP) remains unclear. The absence of A~ fragments that end at Val4°in most glial granules may reflect the relative paucity of A~ fragments ending at Val4°compared with Ala42in the extracellular AI3 deposits. This also infers that these glial A[~ granules are derived from A~ once deposited in brain parenchyma. Recent in vitro studies have shown that some cell types such as dog microglia endocytose Ai3 pcptides or senile plaques amyloid [~ fibrils added to the culture medium. It appears that we have found evidence that this may occur in vivo in Alzheimer brain tissue. The heavy accumulation of N-terminally located epitopes of At3 around these glial cells suggests that the N-terminal fragment of A[3 is cleaved out intracellularly with the C-terminal fragment being more resistant to degradation in glial cells.

421 THE MECHANISM OF AMYLOID 13-PEPTIDE (1-42) TOXICITY IN PC12 CELLS. Fagarasan, M.~'and Eflhimiopoulos, S. Department of Psychiatry, Mount Sinai School of Medicine, New York, USA Severallines of evidence support a causal role of amyloid 13peptide 142 (AI3) in the development of Alzheimer's Disease. However, contradictory data have been reported regarding the direct in vitro and in vivo neurotoxicity of A[3. We found that AI3 induced an increase in flee radical formation during its incubation, for 0 to 2 hours, in water. This effect was dose dependent and was correlated with the appearance of toxicity in PC12 cells. We also determined that A[3 induced arachidonic acid release in a dose- and time-dependent manner. Antioxidants such as N-acetylcysteine, nordihydrognairetic acid, mannit,ol as well as the chelating agent deferoxamine protected PC12 cells against A[3-induced toxicity. Furthermore, we found that antioxidants inin'bited Al3-induced arachidonic acid release and that inlfibitors of cyclooxygenase and lipooxygenase protected PC 12 cells against AI3-induced toxicity. These data suggest that AI3-induced toxicity is mediated through flee radicals generated during its incubation and through activation of the arachidonic acid cascade. We also found that protein kinase C activators (phorbol ester and 1-oleyl2-acetyl-su-glycerol), the museafinic receptor agonist, carbachol and the acethylcholinesterase inh'bitor, tacrine attenuated Al3-induced toxicity in a dose-dependent manner. These data suggest that A[3 stimulates signal transduction pathways and Al3-induced toxicity may be reversed by activation of the phospholipase C/protein kinase C pathway.

420 Plaques Often Contain Ferritin-richAstrocytes or Neurones: Implications for 13-AmyloidDeposition S.R. Robinson I*, D.F. Nuone I and J. Kril2 IVTHRC, University of Queensland, St. Lucia, QLD, Australia, 4072. 2Department of Pathology, University of Sydney, NSW, Australia, 2006. Alzheimer's disease is charaeterised by a high frequency of [3-amyloid plaques in the inferior temporal and entorhinal regions of the cerebral cortex. These plaques are reported to be encircled by microglia, which contain ferritin, the principal storage protein for iron, zinc and aluminium (1). Since low ionic concentrations of these metals precipitate 13-amyloidin vitro (2-4), ferritin may be implicated in amylold deposition in vivo. In the present study, double-label immunocytochemistry and confocal microscopy were used to undertake a detailed examination of the relationship between ferritin-rich ceils and amyloid plaques. The entorhinal cortices from 7 normal, non-demented donors (aged 58-84 years), and 4 individuals who had died with advanced Alzheimer's disease (aged 66-76 years) were fixed in formalin, then cut coronally at 48p.m. Sections were incubated with antisera directed against L-chain ferritin (1:25000; ICN), [3-amyloid (1:100; DAKO) or GFAP (1:2000; DAKO), then processed with streptavidin-HRP-DAB or with streptavidin-FITC. Examination of our double-labelled sections enabled us to confirm that classical plaques (but not diffuse plaques) are normally surrounded by clusters of ferritin-rieh microglia. In addition, we observed that many plaques from both groups of donors have ferritin-rich cells at their centre. Some of these cells are microglia, hut others have larger somata than microglia and their radiating dendrites are generally blebbed and degenerate in appearance. Small ferritin deposits are often seen near their somata and proximal dendrites. Examination of tissue immunolabelled for GFAP suggests that some of these cells are astroeytes, while others more closely resemble neurones. We speculate that during the course

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422 fl-amyloid mediated vasoactivity and vascular endothelial damage M. Mullan*, G. Thomas, C. McLendon, T. Sutton and T. Thomas. Roskamp Laboratories, Institute for Research in Psychiatry, University of South Florida, Tampa, Florida, 33613, USA. Deposits of B-amyloid are apparent in aging and Alzheimer's disease, but the role of the peptide in neurodegeneration is unclear. The free radical theory of aging may also account for Alzheimer type degeneration and consequently links between free radical generation and B-amyloid have been sought. We report that B-amyloid interacts with endothelial cells causing superoxide radical excess and dose dependent alterations in endothelial function with endothelial damage. Treatment of blood vessel having intact endothelium with 13-amyloidproduces characteristic features of endothelial dysfunction such as decreased availability of endothelium derived relaxing factor (EDRF) which is thought to be nitric oxide or a nitrosyl compound. In vitro, these effects can be observed as increased vasoactivity, enhancement of vasoconstriction and inhibition of relaxation responses. These effects can be prevented by treatment with the free radical scavenging enzyme superoxide dismutase. Thus 13-amyloid-endothelial interaction produces a functional excess of superoxide radicals, which can scavenge EDRF. The interaction of nitric oxide with superoxide can also produce potent oxidizing agents, which in turn can cause lipid peroxidation and other degenerative changes. Electron