Little is known of the molecular mechanisms that trigger oligodendrocyte death and demyelination in many acute central nervous system insults. Since oligodendrocytes express functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type glutamate receptors, we examined the possibility that oligodendrocyte death can be mediated by glutamate receptor overactivation. Oligodendrocytes in primary cultures from mouse forebrain were selectively killed by low concentrations of AMPA, kainate or glutamate, or by deprivation of oxygen and glucose. This toxicity could be blocked by the AMPA/kainate receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). In vivo, differentiated oligodendrocytes in subcortical white matter expressed AMPA receptors and were selectively injured by microstereotaxic injection of AMPA but not NMDA. These data suggest that oligodendrocytes share with neurons a high vulnerability to AMPA/kainate receptor-mediated death, a mechanism that may contribute to white matter injury in CNS disease.

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Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

Cytosolic calcium ([Ca 2ϩ ] i ) is an important mediator of neuronal signal transduction, participating in diverse biochemical reactions that elicit changes in synaptic efficacy, metabolic rate, and gene transcription. Excessive [Ca 2ϩ ] i also has been implicated as a cause of acute neuronal injury, although measurement of [Ca 2ϩ (Rothman and Olney, 1986;Choi, 1992). Studies using primary neuronal culture systems have shown that cultured neurons are selectively vulnerable to brief applications of glutamate (Rothman, 1985; or to selective agonists of the NMDA class of glutamate receptors (Rothman, 1985;Choi et al., 1988;Hartley and Choi, 1989). In contrast, prolonged activation of the AM PA or kainate receptors is required to produce neurotoxicity (Koh et al., 1990).Schanne and colleagues (1979) originally described the dependence of extracellular calcium in hepatocyte toxicity, which provided a potential mechanism for neuronal toxicity with the subsequent discovery that glutamate receptor activation produced neuronal calcium influx (Connor et al., 1987(Connor et al., , 1988Murphy et al., 1987). Additional studies by other investigators led to the hypothesis that calcium entry was responsible for excitotoxic neuronal injury (Choi, 1987) (for review, see Dubinsky, 1993a). A correlation between intracellular calcium levels and glutamate toxicity was suggested by the dependence of the latter on extracellular calcium Rothman et al., 1987 influx during nonlethal AMPA treatment (Marcoux et al., 1988;Eimerl and Schramm, 1994; Lu et al., 1996). The study by Hartley and coworkers (1993) suggested that a direct relationship existed between calcium accumulation and subsequent death in neurons exposed to NMDA. Although previous studies using fluorescent intracellular calcium indicators have noted that lethal excitotoxic insults may be followed by prolonged or delayed [Ca 2ϩ ] i elevation (Ogura et al., 1988;de Erausquin et al., 1990;Randall and Thayer, 1992;Dubinsky, 1993b; Tymianski et al., 1993a;Limbrick et al., 1995), such indicators have failed to demonstrate a consistent difference between intracellular free calcium concentration ([Ca 2ϩ ] i ) in cells during lethal and nonlethal challenges (de Erausquin et al., 1990;Michaels and Rothman, 1990; Tymianski et al., 1993a).One potential interpretation of these results is that there is not a direct relationship between the magnitude of acute [Ca 2ϩ ] i elevation and the extent of subsequent neuronal death. Another possibility is that current methods of [Ca 2ϩ ] i measurement fail to distinguish toxic levels of [Ca 2ϩ ] i . In the present study we examine the hypothesis that conventional, high-affinity calcium indicators (e.g., fura-2) are unable to measure accurately the [Ca 2ϩ ] i in the micromolar range and therefore fail to detect differences in [Ca 2ϩ ] i between lethal and nonlethal excitotoxic exposures. We Received April 18, 1997; revised June 2, 1997; accepted June 12, 1997. This work was supported by National Institutes o...

Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity similar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.