PROJECT SUMMARY The prevalence and incidence of neurological complications in people with HIV (PWH) remains steady for those aging on antiretroviral therapy (ART). Eliminating the tight association between chronic inflammation and HIV reservoirs in the periphery and CNS remain major therapeutic challenges. Elevated osteopontin/secreted phosphoprotein-1 (OPN/SPP1) in PWH having moderate to severe neurological complications was first reported in 2008. Since this time, the importance of high OPN/SPP1 RNA expression in human brain and more widely in neurodegenerative microglia has emerged. However, much remains to uncover about OPN/SPP1's fundamental basic molecular mechanism (s) of action in the brain in HIV infection. In this regard, our investigations over the past several years using in vitro and in vivo experimental models have begun to close the gap. A fundamental concept has emerged from our collective findings: OPN/SPP1 function is required in the CNS for brain recovery and return to homeostasis after infection. For the first time, able to knockdown OPN expression in vivo, we obtained a clearer view that its functional specificity is context- dependent. OPN/SPP1 signaling supports viral replication in vivo within tissue compartments via an as yet uncharacterized mechanism that varies with sex. In addition, OPN/SPP1 expression is strongly correlated with the retention of immune cells in the peripheral organs. OPN/SPP1 supports increased trafficking of SIV monocytes to the brain, but whether the same is true for human cells is not known. We discovered using micro- PET neuroimaging, that when homeostasis is disrupted by HIV infection, OPN/SPP1 is a potent sensor and regulator of neuroinflammation in the brain. An additional novel insight gained was the detection not only of inflamed microglia, but of a unique subset of “activated” translocator protein (TSPO) and tyrosine hydroxylase (TH) reactive neurons in the striatum. We hypothesize that the conservation of OPN as a sensor/regulator of CNS homeostasis in adult mammals suggests that it plays a key central mechanistic role in neuroprotective pathways that modulate neuroinflammation. Moreover, the molecular mechanisms are tissue context-dependent and utilize ligand-receptor dynamics initiated by microglia and propagated by specific neurons in the striatum. Teasing out of the mechanistic details requires a combinatorial approach using basic biochemical and molecular tools, in vitro primary cell culture and the latest in vivo chimeric mouse models. Importantly, we have successfully demonstrated that the mouse-human chimera represented in the NSG-hCD34 system recapitulates key aspects of neuroinflammation similar to that seen in people with neuroHIV. Testing of the underlying hypotheses of the integrated aims of this proposal will determine which splice variants of OPN/SPP1 are active in the CNS, identify new mechanistic insights into why disruption of OPN/SPP1 increases neuroinflammatory signalin...