Project 3 Abstract: Long-term antiretroviral therapy (ART) leads to continuous low-level human immunodeficiency viral infection and end organ disease that includes the central nervous system. This is often seen as subclinical cognitive impairments recorded only by neuropsychological examination. We posit that the long-acting nanoformulated ART (nanoART) developed in Projects 1 and 2 would lead to minimal if no toxicities when recorded by conventional immune, chemical, hematologic, biochemical and histologic endpoints (Project 2). However, what cannot be seen by such parameters are measures of subcellular function (mitochondria), cellular function (energy and membrane metabolism) and tissue structure and function (myelination, neurotransmitter and metabolic aberrations). Changes in these parameters may also be missed by standard behavioral and cognitive tests. To these ends, we wish to take nanotoxicology to yet another level. Here we seek to decipher subtle effects of the virus and nanoART therapy is through brain metabolomics. To accomplish this goal, we have replicated a portion of the behavioral, histological, and neuroimaging abnormalities observed in human neuroAIDS in a humanized mouse model of HIV-1 infection. The model is divergent from others available in that it supports sustained viral replication, results in spontaneous neural disease associated with CD4+ T cell loss and metabolomics change. We will use this model as a platform to determine long-term effects (measured in months) of aberrations in neural function and region specific brain biochemistry with untargeted metabolomics. Identified biochemical pathways will be validated by targeted measures. Such studies will be highly sensitive. Specifically, we will bridge these data sets with proton magnetic resonance spectroscopy, diffusion tensor magnetic resonance imaging and magnetization transfer magnetic resonance imaging. These linkages will serve to guide translational studies with an eye towards clinical utility. Validation of the experimental results will be made through histopathological tests (with Core C). Validation of magnetic resonance imaging using ligand coated magnetite particles in mouse and monkey experiments will be done through complete pharmacokinetic analyses (performed in conjunction with Project 2 and Core B), and confirmed by produced small magnetite antiretroviral particles and virologic and immunologic assays (with Project 1 and Core C).