434 Effects of peptide fragments of β amyloid precursor protein on parallel fiber-purkinje cell synaptic transmission in rat cerebellum

S108

FIFTH INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE

431 Zinc Promotes All Aggregation But Attenuates All Neurotoxieity K.S. Fuson, L.N. Boggs and P.C. May* CNS Research, Eli Lilly and Co., Indianapolis, IN 46285 USA Numerous in vitro studies suggest that AB is directly neurotoxic to cultured rat and human neurons by a mechanism enhanced by peptide aggregation. Recent studies suggest that metal ions, particularly Zn, can modulate AB aggregation in vitro. These observations have led some investigators to suggest that altered zinc metabolism in AD may contribute to AB pathology. In this study, we have explored Zn effects on AB aggregation by the Thioflavin T (ThT) fluorescence assay as well as followed the outcome of these Zn manipulations upon the neurotoxic properties of multiple lots of AB peptide. Addition of 20 -1001aM ZnCI2 to 1001aM AB(1-40) resulted in a prominent increase in ThT fluorescence; Zn concentrations below 2 I.tM had no apparent effect on AB aggregation. To assess Zn effects on AB neurotoxieity, primary rat hippocampal cultures were co-treated with 25 p.M AB1-40 (Bachem Lot# ZM605) and increasing concentrations of Zn. Freshly prepared aliquots of this lot of AB showed prominent neurotoxicity as measured by LDH release after 3-4 days of treatment. Co-addition of Zn caused a dose-dependent attenuation of AB neurotoxicity. Consistent with the ThT fluorescent aggregation studies, a prominent Zn effect was only observed at nominal Zn concentrations of 20-100 laM. As Zn afforded no protection against other neurotoxic challenges, e.g., kainate or H202, these data suggest some specific interaction of Zn with the AB peptide. Interestingly, another lot of AB (# ZM269) which requires "aging" for activation, was still non-toxic when freshly applied to hippocampal cultures in the presence of Zn concentrations previously shown by ThT fluorescence studies to enhance peptide aggregation. Together these data confirm that I~M concentrations of Zn can promote AB aggregation, but suggest that aggregation per se is not sufficient for neurotoxicity.

432 A Possible Role For the NF-KB/Rel Family of Proteins in Mediating AI~Induced Neurotoxidty and Glial Activation K.R. Bales 1, G-.M. Yan2, E. Hamilton-Byrd t, X. Wu 1, and S.M. Pault,2, 3 tLilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN 46285 USA and 2Departments of Pharmacology and Toxicology, 3psychiatry, Indiana University School of Medicine, Indianapolis, IN 46202 USA The amyloid 13peptide (A[3) is deposited in both diffuse and neuritic plaques which are characteristic of Alzheimer's disease (AD). Prominent neurodegeneration occurs around these plaques leading to the hypothesis that All plays a causative role in disease pathogenesis. In fact, the A~ peptide is a well known neurotoxin to most cultured neurons; however neither the cellular nor molecular mechanisms underlying AI3-induced neurotoxicity are known. We have recently identified the nuclear transcription factor NF-v.B as playing a critical role in the vulnerability of cultured cerebellar granule neurons to apoptose in response to bilirubin exposure. We surmised that activation of NF~B following exposure of rat fetal cortical neurons to AI3 may play a similar role. Surprisingly, the normally high basal level of activated NF-~B present in cultured rat fetal cortical neurons, decreases in a time- and dose-dependent fashion following exposure to toxic concentrations of AI3. No corresponding decrease in NF-r,B message or protein (p65) occurs, suggesting that regulation of the level of activated NF-x.B rather than the larger pool of inactivated protein is responsible for the reduced levels of activated NF-rB observed by gel shift analysis. Additionally, we found that upregulation of IkB-a, one of the proteins responsible for retaining inactivated NF-KB in the cytoplasm, may be responsible for the observed decrease in activated NF-~B in cultured cortical neurons following AI3 exposure. Our data suggests that AI~ neurotoxicity may be mediated by increases in IkB-~ gene expression which results in a decrease in activated NF-KB. We have also found that exposure of primary astroglial cultures to AI3 activates NF-~B. We postulate that Al3-induced activation of NF-KB in astroglia may contribute to cytokine release and the inflammatory response which occurs in AD.

433 Neurotoxicity of a carboxyl-terminal fragment of the AIzheimer's amyloid precursor protein. Seong-Hun Kim* and Yoo-Hun Suh Department of Pharmacology, College of Medicine and Department of Molecular Biology, Neuroscience Research Institute, Seoul National University

We have previously shown that a recombinant earboxyl-terminal 105 amino acid ffagment(CT105) of the amyloid precursor protein induced strong nonselective inward current in Xenopus oocytes. Here we investigated the toxic effect of CT105 peptide on the cultured mammalian cells. The CT105 peptide induced a significant LDH release from cultured rat cortical neurons and PC12 cells in a concentration(from 10 raM) and time(from 48 I1) dependent manner. The toxic effect of CT105 was more potent than any fragments of amyloid beta protein(AI3). However the CT105 did not affect the viability of U251, human glioblastoma cells. In contrast to CTI05, A[3 increased LDH release only slightly even at 50raM but significantly inhibited MTT reduction at submicromolar concentration. Among the various neuroprotective drugs tested, only cholesterol which alters the membrane fluidity could attenuate the cytotoxicity of CT105 significantly. The CTI05 peptide formed multiple self-aggregates upon solubilization which might be related to its toxicity. Pretreatmant with CTI05 did not significantly alter the susceptibility of eelis against hydrogen peroxide and glutamate. These results imply that CT peptide can directly attack the cell membrane probably by making pores or nonselective ion channels while A[3 impairs intraeellular metabolic pathway first. Thus it is thought that both CT and AI3 which are formed during the processing of APP may participate in the neuronal degeneration in Alzheimer's disease by different mechanisms.

434 Effects of Peptide fragments of [3 amylold precursor protein on parallel fiber-Purkinje cell synaptie transmission in rat cerebellum. Yoo-Hun Suh I and Nick A. Hartell2 1 Department of Pharmacology, Seoul National Unlversit3,,College of Medicine, Seoul 110-799, Korea 2 Laboratory for synaptic function, Frontier Res. Program, RIKEN, l-Iirosawa, Japan The effects of various protein fragments of bAPP were examined on the parallel fiber-Purldnje cell synapse in the rat cerebellum. Of all 4 proteins examined, I3APP, CTl0~, [32~-3~ and 13146, only application of CTl05 consistently induced large, transient inward currents that were associated with calcium influx. All four proteins, however, induced depression of PFPurkinje eeU synaptie transmission at the site of application through a combination of pre- and postsynaptie effects. Recovery was not observed with either CTt05 or 1325.3~and in the former case, the depression spread to distant sites and was consistently followed by cell death within 30-40 minutes of application. These data indicate that C-terminal fragments of the 13APP can both block synaptie transmission and have neurotoxic effects ands'as such may be considered an alternative candidate for some of the neurotoxie effees of Alzheimer's disease.

435 Amyloid/3 Protein Disrupts Bradykinin-Mediated Ins(l,4,5)P 3 Hsueh-Meei Huang *and Hsiu-Mei Chiang Department of Medical Research, Taichung Veterans General Hospital Talchung, Taiwan, R.O.C. A/3 accumulation and alteration of signal transduction systems are the two major changes accompany Alzheimer's disease. However, interactions of A/3 and signal transduction systems underlie the pathophysiological meaning in AD is not clear. Neurotoxidty effect of A/3 on PC12 cells was evaluated by morphological observations, tetrazolium salt uptake (MTT) and lactate dehydrognnase (LDH) release. In the absence of serum, treatment of PC 12 cells with A/3 (0.1-20 tt M) induced morphological change of the cells in a concentration dependent manner. Although A/3 pretreatment did not induce cell morphological change in the presence of serum, the release of LDH in the medium was increased by 72 % at 5/t M A/3 treatment. Under the similar conditions, the viable cell number was reduced 20 % at 5 g M , and ~.~,'-her t, reduced to 55% at 10 /~ M. Extraeellular addition of A/3 to the medium