Date of Award

December 2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Xuesong Chen

Abstract

Although combined antiretroviral therapy (cART) has dramatically increased the lifespan of people with Human Immunodeficiency Virus type 1 (HIV-1) (PWH), up to 50% of PWH develop HIV-associated neurocognitive disorder (HAND) even with cART, for which no effective treatment is currently available. Chronic neuroinflammation and accelerated aging play a critical role in the pathogenesis of HAND. Emerging evidence indicates that the HIV-1 brain exhibits molecular signatures of brain aging and cellular senescence. In the process of cellular senescence, senescent cells secrete pro-inflammatory factors called senescence-associated secretory phenotype (SASP) that can elicit deleterious paracrine-like effects on neighboring cells, contributing to brain aging and neurodegeneration. The development of premature aging in HAND during the post-cART era is complex and not yet fully understood. However, the chronic presence of viral proteins, such as Transactivator of Transcription (Tat), may disrupt neuronal homeostasis in the central nervous system (CNS), potentially leading to senescence and functional alterations of neurons.A critical mechanism disrupted in HAND is altered endolysosome function. Endolysosomes play a crucial role in maintaining cellular homeostasis, and endolysosome is observed in various neurodegenerative disorders. Our research has shown that various HIV-related factors including HIV-1 viral proteins (gp120 and Tat), certain antiretroviral drugs, and abusive drugs, induce alterations in the structure and function of endolysosomes in CNS cells. In astrocytes, endolysosomes are involved in releasing gliotransmitters, modulating membrane plasticity, pruning synapses, regulating energy metabolism, and clearing waste. Moreover, the endolysosomal pathway is essential for the turnover of cellular components and the regulation of cytokine and chemokine secretion, which modulate the inflammatory response. Thus, endolysosome dysfunction may contribute to impaired astrocytic function, reducing astrocyte’s ability to manage cellular stress and contributing to neurodegenerative processes in HAND. The development of HAND in the ART era is attributed, at least in part, to the HIV-1 Tat protein, which is actively secreted from HIV-1 infected cells. Significantly, current anti-HIV strategies do not block the secretion of Tat, and brain levels of Tat remain elevated even when HIV-1 levels are below detectable levels. In the brain, HIV-1 Tat could exert a direct neurotoxic effect or induce neuroinflammation by interacting with astrocytes, macrophage/microglia, and other CNS cells. Furthermore, HIV-1 Tat has been shown to induce cellular senescence in CNS cells. Thus, HIV-1 Tat in the brain could induce cellular senescence and the secretion of SASP that can elicit deleterious paracrine-like effects on neighboring cells, contributing to accelerated aging and neurodegeneration in HAND. However, it is not clear how HIV-1 Tat induces cellular senescence. Identifying the underlying mechanisms whereby HIV-1 Tat induces cellular senescence would provide insights into the pathogenesis of HAND and may lead to the development of new therapeutic strategies. Given that endolysosomes play an important role in the development of cellular senescence, we hypothesize that HIV-1 Tat induces the development of senescence-like phenotype in astrocytes via an endolysosome-dependent mechanism; Specifically, internalized HIV-1 Tat may interact with endolysosome-resident proteins to induce cellular senescence. The present studies were conducted in primary human astrocytes, utilizing various methods including Enzyme-linked Immunosorbent Assay (ELISA) for the secretion of pro-inflammatory factors, ratiometric measurement of endolysosome pH, galectin-3 puncta assay for assessing endolysosome membrane leakage, released LDH activity for assessing cytotoxicity, live-cell imaging to assess colocalization, immunoprecipitation for assessing protein-protein interaction, senescence-associated β-galactosidase (SA-β-Gal) activity assay for cellular senescence, bromodeoxyuridine (BrdU) incorporation assay for cell proliferation, immunoblotting to assess change in protein involved in cell cycle arrest. In addition, siRNA knockdown and pharmacological approaches were used to modulate identified target proteins that could interact with HIV-1 Tat. Our findings showed that HIV-1 Tat induces a pronounced inflammatory response in human astrocytes, evidenced by elevated levels of Interleukin 6 (IL-6), Interleukin 8 (IL-8), and C-C Motif Chemokine Ligand 2 (CCL2). Additionally, HIV-1 Tat caused endolysosomal de-acidification, formation of galectin-3 puncta, and increased release of galectin-3 and cathepsin B from damaged endolysosomes. Furthermore, HIV-1 Tat induced a senescence-like phenotype in astrocytes, characterized by reduced cell proliferation, cell cycle arrest, increased p16^INK4a/Rb and p21^CIP1 protein levels, and elevated SA-β-gal activity and expression. Significantly, we demonstrated that the arginine-rich domain of HIV-1 Tat is crucial for these effects. Furthermore, we identified two endolysosome-resident proteins that interact with HIV-1 Tat; namely TLR7 and SLC38A9. Using pharmacological approaches and siRNA knockdown technique, we demonstrated that both TLR7 and SLC38A9 are involved in HIV-1 Tat-induced inflammatory response, endolysosome damage, and cellular senescence. Our findings support our hypothesis that HIV-1 Tat contributes to inflammatory response and development of a senescence-like phenotype in primary human astrocytes through its interaction with TLR7 and SLC38A9. Our findings suggest that targeting TLR7 and SLC38A9 may represent promising therapeutic strategies to alleviate the development of neuroinflammation and senescence in HAND.

Available for download on Sunday, January 17, 2027

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