This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Oligodendrocytes are critical regulators of nervous system function and play a central role in many acute neurological disorders, including stroke. Glutamate excitotoxicity contributes to ischemic oligodendrocyte injury; however, it is not clear what the intracellular mechanisms are leading to cell death. This proposal examines a hypothesis that over-activation of glutamate receptors and hypoxia/ischemia reduces oligodendrocyte survival, leading to apoptosis, which is mediated in part by ceramide and prevented by sphingosine-1-phosphate. We use primary cultures of oligodendrocytes and a mouse model of stroke to study the role of sphingolipids in neural cells. We assess cellular activity using fluorescence measurements of cell survival, western blotting, immunoprecipitation and high performance lipid chromatography coupled with mass spectrometry. In cultured oligodendrocytes, we found that stimulation of glutamate receptor induced biphasic increase in endogenous ceramide levels via two different mechanisms. The data suggest that an early increase in C18-ceramide level, which is generated by de novo synthesis and it is not accompanied with sphingomyelin hydrolysis could play a role in protecting oligodendrocytes from glutamate-induced damage. Thus, in isolated mitochondria, we found that C18-ceramide is a powerful inhibitor of mitochondrial permeability transition pore which is a pivotal part of apoptotic machinery. This is the first demonstration that endogenous ceramide could block the pore. The late increase in ceramide levels was accompanied by a substantial sphingomyelin hydrolysis. The data provide further support for our hypothesis and suggest that the late increase in ceramide level could manifest a pro-apoptotic role of ceramide in oligodendrocyte injury. It also raises the question as to what ceramide-generating enzymes are activated by ischemic insult. In the animal model of stroke, brief cerebral ischemia increased ceramide levels in brain without apparent sphingomyelin hydrolysis. These data are in line with our findings in cultured oligodendrocytes and suggest that the early activation of de novo ceramide synthesis occurs in cerebral ischemic brain injury.
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