The initial aim of the project is to examine the heat-stress response in the central nervous system (CNS) of Lewis rats during the course of experimental autoimmune encephalomyelitis (EAE), a disease involving inflammation, demyelination and extensive reactive gliosis. The heat-stress response will be evaluated by examination of changes in the levels, synthesis and localization of heat-stress protein (HSP) and mRNA resulting from EAE. The degree of disaggregation of polysomes (a characteristic occurrence during heat-stress) and the association of HSPs with the cytoskeleton and/or nucleus during EAE will also be examined. These experiments are proposed to explore a possible association between heat-stress and previous observations that a temporary suppression of transcription and translation of glial fibrillary acidic protein (GFAP) and neurofilament (NF-L) occurs during the acute stages of EAE. Following these in vivo studies, the individual cellular response to heat-stress will be examined by studying primary cell cultures using similar experimental approaches as above. The use of primary cultures should help to tease apart some of the confounding elements present in whole CNS tissue. In addition, the effects of cytokines on the heat-stress response will be examined. It is hoped that these experiments may provide an important link between inflammatory conditions and heat-stress and glial responses. Finally, the heat-stress response of isolated glial precursor cells will be explored. HSPs may play an important role in the organization, structural stability and anchoring of the cytoskeleton, and thus these studies may provide interesting insights into the function of HSPs during development of the CNS. The appearance and inducibility of HSPs will be correlated with the expression of various markers of differentiation, as well as under conditions that stimulate proliferation and lineage selection of these undifferentiated cells. The methodology for these studies includes Northern and Western blotting, in situ hybridization, immunocytochemistry, in vitro protein synthesis, cell-free translation, nuclear-runoff experiments, cell culture, subcellular fractionation and polysome size analysis.
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