Developing rat models of HIV-associated neurotoxicity and neuroinflammation

National Institute On Drug Abuse

Investigators

Abstract

Despite the effectiveness of anti-retroviral therapy for HIV, infected cells harboring provirus within the central nervous system contribute to HIV-associated neurocognitive disorder (HAND). Brain-resident glial cell populations, primarily microglia, are infected by the virus and negatively impact the functionality and viability of surrounding neurons through a variety of mechanisms including pro-inflammatory cytokine release, neurotrophic dysregulation, aberrant glutamate homeostasis, and direct damage from viral protein production. Furthermore, it has been established that microglia harbor latent provirus, which can be activated by pro-inflammatory stimuli leading to productive viral replication. Thus, the microglia within the CNS may serve as viral reservoirs, allowing for viral compartmentalization, evolution, and escape. Understanding the pathobiology of HIV-infected microglia is paramount to the development of novel therapeutic approaches that delay or cure HIV-mediated pathogenesis in the CNS. We are developing novel rat models of HIV pathogenesis that allow tissue specific and temporally-regulated expression of an HIV provirus in the rat brain. For example, we have integrated a modified HIV provirus into the rat genome and this provirus has been rendered replication-defective by the replacing the gag-pol region with a transcription termination signal that is flanked by Cre-dependent recombination sites. This transgenic provirus has several advantages over the previously established HIV-1 Tg rat that has been used in numerous studies focused on HIV-related neuropathogenesis. The most notable difference is that in the HIV-1Tg rat model, the HIV proviral genomes are transcriptionally competent in ALL cells of the rat, and the transgene can be expressed throughout its lifetime including embryogenesis. The systemic/ubiquitous and constitutive expression of viral proteins may interfere with normal development and otherwise produce pathologies that are not relevant to a disease typically confined to T cells and microglia. In our iHIV model, the default state of the provirus expression is OFF but upon exposure to the activity of Cre recombinase, which is delivered to a defined location by an injected virus (or nanoparticle) or inherited by crossing with a second rat expressing Cre from a microglia specific promoter, the provirus can express when the HIV promoter is activated. With our new models, we have the potential to activate HIV gene expression in a temporal, spatial and cell specific manner which we will use for microglial expression. Overall, we are using novel rat models to study HIV-associated neurocognitive impairment and comorbidity of HIV and drugs of abuse in context of microglial reservoirs of HIV in the brain. During this first year of funding, we have initiated the construction of new transgenic rats for creating a new model of HAND by selectively expressing a modified HIV genome in rat microglia. We have also established the methods to isolated nuclei from the rat brain to perform single nuclei RNA sequencing. We have also established dPCR-based assays to quantify expression of HIV RNA including the relative expression of the various splice products of the HIV RNA.