Astrocyte TIMP-1: Regulation and Gene Delivery in HAND
Date of Award
Doctor of Philosophy
Field of Study
Graduate School of Biomedical Sciences
Abbot F. Clark
Johnny J. He
Despite antiretroviral therapy, HIV-associated neurocognitive disorders (HAND) persist in 30-70% of patients. During HAND, elevated matrix metalloproteinases (MMPs) in the brain exacerbate the disease by blood-brain barrier breakdown, neuroinflammation and direct neurotoxicity. Tissue inhibitors of metalloproteinases (TIMPs) counter MMP activity. In the brain, TIMP-1 is primarily produced by astrocytes in response to injury or inflammation. However, TIMP-1 is downregulated during chronic inflammation in astrocytes and in HIV encephalitis brain tissues. We propose that restoring astrocyte TIMP-1 levels could mitigate neurodegeneration due to its MMP-inhibitory and -independent neuroprotective functions. HIV-1 non-productively infects astrocytes, which express viral proteins such as transactivator of transcription (Tat). As Tat mimics aspects of HAND by direct and indirect mechanisms, glial fibrillary acidic protein (GFAP) promoter-restricted Tat expressing (GT-Tg) mice were used to model HAND in our studies. Prolonged astrocyte Tat expression in GT-Tg mice resulted in HAND-relevant behavioral impairments characterized by higher anxiety, lower ambulation, impaired spatial learning, and memory. Importantly, behavioral deficits were accompanied by altered brain MMP/TIMP balance. Our data from GT-Tg mouse model confirmed neurocognitive decline and TIMP-1 dysregulation in the context of HAND. As TIMP-1 was downregulated with prolonged Tat expression in mice, we focused on replenishing TIMP-1 via gene delivery to the brain using cationic polymers. Polyethylenimine (PEI) is a highly efficient polymer for transfecting mammalian cells, however, high cytotoxicity restricts its use. Hence, PEI was modified using arginine (A) and stabilized with polyethylene glycol (P) to produce multiple AnPn analogues. AnPn analogues were biocompatible and successfully delivered reporter genes to primary neural cells. Select AnPn led to sustained reporter gene expression in human astrocytes and in mouse brains. In order to restrict gene expression to astrocytes, truncated GFAP promoters were used to drive gene expression. Subsequently, GFAP promoters were modified enhancing their activity and increasing gene expression. Lastly, successful polymer-mediated GFAP promoter-driven TIMP-1 gene delivery was demonstrated in human astrocytes. Overall, these findings enhance our understanding HIV-1 Tat-mediated TIMP-1 regulation, provide a novel therapeutic TIMP-1 gene delivery system, and pave the way for future investigations geared towards preclinical translation of TIMP-1-based HAND therapy.
Joshi, C. R.
"Astrocyte TIMP-1: Regulation and Gene Delivery in HAND" Fort Worth, Tx: University of North Texas Health Science Center;