1,3-Dinitrobenzene (DNB) induces a selective, focal edematous lesion in the brainstem astrocytes of rats reminiscent of lesions induced by vitamin B, deficiency and chemicals that produce central nervous system (CNS) energy deprivation syndromes. Preliminary data collected in our laboratories provide compelling evidence that selective regional astrocyte vulnerability to DNB is mediated by opening of the mitochondrial permeability transition pore (mt-PTP) with subsequent formation of reactive oxygen species (ROS). It is well established in the literature that the mt-PTP is stabilized in the closed conformation by Bc1-2 and BC1-XL and maintained in the open state by Bax. The central hypothesis of this proposal is that regional expression of Bc1-2 family molecules regulates the differential susceptibility of astrocytes to chemicals that induce oxidative stress. Addressing the following specific questions will test the hypothesis: 1) Does DNB induce translocation of Bax to astrocytic mitochondria? 2) Does modulation of expression of mt-PTP agonist (Bax) or antagonist (Bc1-2/Bcl-XL) proteins alter the toxicity of DNB in astrocytes? 3) Does alteration of the expression of Bax, Bc1-2 or BC1 XL proteins modulate astrocytic sensitivity in vivo? 4) Does DNB induce transient regional inhibition of SDH in neurons and astrocytes in vivo and in vitro, and is the inhibition of SDH linked to induction of the mt-PTP? 5) Does opening of the mt-PTP increase cellular calcium loads thereby altering the ability of cortical and brainstem astrocytes to spatially buffer physiologic ions and maintain cell volume and viability? The Specific Aims will address the functional consequences of altered expression of selected Bc1-2 family proteins and their translocation to the mitochondrial compartment. Enriched primary cortical and brainstem astrocyte cultures will be used as a well characterized in vitro model of DNB-induced encephalopathy. These studies utilize emerging techniques in real-time confocal and high-resolution laser scanning confocal microscopy developed in the laboratory of the PI and collaborators. The recent development of nano-optochemical sensing technology in our laboratories permits a degree of spatial resolution and quantitative measurement of ionic transients hitherto unavailable. Data obtained from the proposed studies will provide a greater understanding of molecular and pathophysiologic mechanisms underlying the selective vulnerability of neuron and astrocyte populations to neurotoxicants and neurodegenerative change.
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