山口 淳

Based on the neurotrophic properties of astrocytes in response to ischemia, the current work focuses on the mechanism for cultured astrocytes to adapt to a hypoxic environment. Intracellular glucose levels in primary cultured rat astrocytes exposed to hypoxia fell by 30% within 24 h, in parallel with a decrease in glycogen stores. Glycolytic metabolism was crucial for cell survival during hypoxia, as 2-deoxyglucose resulted in rapid ATP depletion and cell death. The mechanism for maintaining glucose levels under these conditions appeared to be mobilization of glycogen stores, rather than increased extracellular uptake of glucose, as gluconolactone (an inhibitor of beta 1-4 amyloglucosidase) induced a rapid fall in cellular ATP in cultures subjected to hypoxia, whereas cytochalasin B was without affect. Addition of cycloheximide diminished the viability of astrocytes in hypoxia, suggesting an obligatory role of de-novo gene expression to respond to hypoxia. Consistently, the results of differential display suggested the induction of glycolytic enzymes, including aldolase 4 (EC 4.1.2.13), hexokinase II (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), and triosephosphate isomerase (EC 5.3.1.1) in the hypoxic culture. Marked induction of these glycolytic enzymes in hypoxic astrocytes was confirmed by Northern blot analysis. These data provide a theoretical basis to understand the ability of astrocytes to tolerate ischemic condition. (C) 1999 Published by Elsevier Science B.V. All rights reserved.

Mutations in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes account for the majority of cases of early-onset familial Alzheimer's disease (AD). Alternative splicing forms of the PS-1 and PS-2 gene products have previously been reported in fibroblast and brain tissue from both familial and sporadic AD patients, as well as from normal tissues and cell lines. We demonstrate here unusual alternative splicing of the PS-2 gene that leads to the generation of mRNA lacking exon 5 in human brain tissue. This product was more frequently detected in brain tissue from sporadic AD patients (70.0%; 21 of 30) than from normal age-matched controls (17.6%; three of 17). In cultured neuroblastoma cells, this splice variant was generated in hypoxia but not under other forms of cellular stress. Hypoxia-mediated induction of this splice variant was blocked by pretreatment of neuroblastoma cells with the protein synthesis inhibitor cycloheximide or antioxidants such as N-acetylcysteine and diphenyl iodonium, suggesting that hypoxia-mediated oxidant stress might, at least in part, underlie the alternative splicing of PS-2 mRNA through de novo protein synthesis. Furthermore, the stable transfectants of this splice variant produced the N-terminal part of PS-2 protein (15 kDa) and were more susceptible to cellular stresses than control transfectants. These results suggest the possibility that altered presenilin gene products in stress conditions may also participate in the pathogenesis of AD.