This proposal focuses on the hypothesis that cytokines produced systemically and by infiltrating immune cells or resident brain cells, contribute to CNS injury during HIV-infection. To test this hypothesis, a well defined transgenic approach was employed in which the expression of the cytokines IL-6 and IL-3 was targeted to astrocytes using glial fibrillary acidic protein (GFAP)-fusion gene constructs. This has provided us with unique and powerful models to study the neuropathogenic consequences of the constitutive production of cytokines from astrocytes in the intact CNS. Initial characterization of GFAP-IL6 and GFAP-IL3 transgenic mice has unveiled wide-ranging molecular, cellular and functional alterations of the CNS-many of which share similarities to those seen in HIV encephalopathy. Significantly, these studies directly implicate cytokines in having a causal role in the genesis of HIV encephalopathy and other neurodegenerative diseases. Here we propose to develop transgenic mice with expression of the cytokine TNF-alpha targeted to the CNS. Detailed neuropathological assessment in this new model as well as in existing GFAP-cytokine mice will employ an established battery of tests to examine CNS alterations at the molecular and cellular levels, including RNase protection assays, in situ hybridization, northern blot hybridization, protein immunoblot assay, conventional light and laser confocal microscopy of immunolabeled brain sections and electron microscopy. Functional CNS alterations in the GFAP- cytokine mice will be determined at the behavioral and electrophysiological and levels and where possible be linked to specific molecular and cellular alterations. The identification of primary pathogenetic and functional milestones associated with the cerebral expression of the various cytokines will be determined by: i) detailed developmental studies and comparative analysis of the different GFAP- cytokine models, and ii) analyzing the CNS alterations resulting from the grafting of cytokine producing transgenic astrocytes in the normal mouse brain. The neurological impact of additional pathogenetic factors will be assessed: i) in cross-breeding experiments to develop biogenic mice expressing combinations of cytokines (i.e. IL-6+IL-3), and ii) by back- cross breeding GFAP-cytokine mice with SCID mice to develop immunodeficient GFAP-cytokine transgenic animals. These studies will develop models that recapitulate the multi-factorial pathogenetic and immunodeficient environments thought to underlie HIV encephalopathy. Finally, the well characterized GFAP-transgenic mice will be used to identify and assess in vivo the efficacy of drugs targeted at harmful individual cytokine-CNS interactions. This study provides a unique and powerful approach to elucidate the molecular and cellular basis for the CNS pathobiology of cytokines in vivo and can be expected to advance our understanding of HIV-associated neurological disease, help identify critical targets for therapeutic interventions and facilitate the preclinical evaluation of therapeutic strategies.
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