In our part of the Program Project Grant, we have been investigating the cellular pathology that accompanies GFAP accumulation. We have found that GFAP accumulation leads to several metabolic consequences: 1) activation of an MLK - JNK and p38 pathway, 2) inhibition of proteasome activity, 3) accumulation of small heat shock proteins (shsps), 4) abnormal coiling and bundling of filaments, and 5) induction of autophagy. Each of these responses can occur during the accumulation of either wild type or the R239C mutant of GFAP, but all are exaggerated in cells expressing the mutant protein. Our first major hypothesis, addressed in Specific Aim 1, is that the activation of stress pathways and the inhibition of proteasome activity promotes further increases in GFAP synergistically and makes the astrocytes more susceptible to further stressful stimuli, while the activation of autophagy decreases GFAP accumulation. In addition, we propose that the increases in shsps help to unbundle filaments and promote cell survival. We will investigate important points of the cellular """"""""stress"""""""" pathway to define: 1a) intermediates between MLK activation and JNK and p38 activation, and 1 b. intermediates between the activation of p38 and autophagy, 1.2) We will test if abrogating a shsp response makes astrocytes more susceptible to apoptosis. 1.3) We will work with the Messing lab to assay """"""""stress"""""""" responses in primary cultures of astrocytes from GFAP mutant mice. 1.4) We will expand our studies to include two other common mutations, R239H and R416W. Our second major hypothesis, addressed in Specific Aim 2, is that the effects of GFAP accumulation, through the induction of stress pathways, alter critical astrocyte functions that are deleterious to other cell types in the brain. We will pursue several possible mechanisms that have been suggested by preliminary data. 2.1) We will measure glutamate transport, glutamate currents, and glutamate metabolizing enzymes in astrocytes overexpressing wild type or mutant GFAPs and determine if changes are regulated by specific stress pathways. 2.2) We will determine astrocytic Ca2+ responses to purinergic agonists in cultures, slices, and in vivo. 2.3) We will examine astrocytic modulation of synaptic activity in hippocampal slices (P12-P18). 2.4) We will determine if hippocampal slices from transgenic mice are more prone to develop seizure activity during exposure to 4- AP. 2.5) We will examine coupling between astrocytes and connexin43 levels in cultures and in slices of mouse brain.
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