Inflammatory events in the Central Nervous System (CNS) contribute to the pathology underlying diseases such as Multiple Sclerosis (MS), Alzheimer's Disease (AD) and HIV-1 Associated Dementia (HAD), and activated macrophages/microglia are central to this response. Immunological activation of these cells leads to the production of cytokines that ultimately impact on glial and neuronal function. Macrophages/microglia also respond to cytokines upon engagement of cell surface receptors and activation of complex intracellular signaling pathways. Dysregulation of macrophage/microglial cytokine production and responsiveness is a critical determinant of the neuroimmunologic cascades that typify diseases such as MS, AD and HAD. IFN- and TNF-a are potent activators of macrophages and microglia, and are aberrantly expressed in the CNS in the diseases mentioned above. IFN-y uses Signal Transducer and Activator of Transcription-la (STAT-la) for signal transduction, while the Tumor Necrosis Factor Receptor 1 (TNFRl)-associated death domain (TRADD) protein is critical for TNF-a signaling. We have discovered a novel and unanticipated form of crosstalk between the IFN? and TNF-a signaling pathways that is operational in macrophages/microglia. Specifically, we have demonstrated that IFN-? induces the formation of a nuclear-localized TRADD-STAT-la complex. In this configuration, we propose that TRADD is involved in the dephosphorylation of STAT-la since IFN-?-induced STAT-la phosphorylation, DNA binding activity, nuclear retention and transcriptional activity are enhanced in cells with reduced TRADD levels. We hypothesize that TRADD has a functional role in regulating the response of macrophages/microglia to IFN-?, impacting on cellular activation and subsequent neuroinflammatory responses. The function of TRADD on IFN-?-induced gene expression in macrophages/microglia will be studied, focusing on genes critical for immunological responses (Aim 1). Two strategies are proposed: depletion of TRADD as well as TRADD reconstitution and/or overexpression.
In Aim 2, we will test the hypothesis that TRADD is involved in the STAT-la dephosphorylation process, perhaps by interacting with STAT-la phosphatases to regulate their localization and/or activity. A critical issue is how signaling from the IFN-? receptor induces nuclear localization of TRADD, and subsequent TRADD-STAT-la complex formation. The experiments in Aim 3 will address these questions. Lastly, we will examine the ability of other cytokines that use the STAT pathway to induce TRADD-STAT complex formation, and the repertoire of cell types in which TRADD-STAT interactions occur (Aim 4). Our studies will provide a comprehensive assessment of TRADD function in cytokine signaling, thereby advancing our understanding of this novel signaling pathway in the context of macrophage/microglial activation and neuroinflammation. ? ? ?
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