Nirmal Kumar

Date of Award

August 2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Jonathan D. Geiger

Abstract

Altered brain iron metabolism, high reactive species production, and endolysosome dysfunction are key pathogenic processes in neurodegenerative disorders including HIV-1-associated neurocognitive disorders (HAND). People living with HIV-1 and ingesting opioids are at a higher risk of developing HAND and HIV-1 proteins including HIV-1 gp120 as well as opioids can dysregulate levels of endolysosome iron, increase levels of reactive oxygen species (ROS), and cause neural cell death. Currently, little is known about the involvement of the newly defined reactive species interactome (RSI) in HIV-1 gp120- and morphine-induced neurotoxicity. Endolysosomes are subcellular acidic organelles known for regulating cellular iron metabolism and redox homeostasis, and HIV-1 gp120 and morphine increase the release of iron from endolysosomes in sufficient levels to increase levels of iron and ROS in the cytosol and in mitochondria as well as induce neurotoxicity. Furthermore, endolysosome-resident divalent cation channels including transient receptor potential mucolipin 1 (TRPML1) that function as redox sensors and regulate Fe2+ signaling are implicated in the neurotoxic action of HIV-1 gp120. Because of the importance of iron in influencing redox homeostasis in HAND patients and in patients suffering from iron overload, this dissertation work was aimed to examine the effects of gp120 and morphine alone and in combination, and of chemically induced iron overload on levels of endolysosome Fe2+ and RSI in endolysosome, cytosol, and mitochondria, as well as cell death (Study #1). Using SH-SY5Y human neuroblastoma and U87MG human astrocytoma cell lines we found that HIV-1 gp120, morphine, and chemically-induced iron overload with ferric ammonium citrate (FAC) (1) de-acidified endolysosomes, (2) decreased endolysosome Fe2+ levels, (3) increased levels of endolysosome ROS, lipid peroxidation (LPO) and nitric oxide (NO), and decreased levels of endolysosome hydrogen sulfide (H2S), (4) increased levels of cytosolic Fe2+ and ROS, and decreased cytosolic H2S levels, (5) increased levels of mitochondrial Fe2+, ROS, LPO, and NO and decreased mitochondrial H2S levels, and (6) induced cell death. All the above effects were blocked by the endolysosome-specific iron chelator deferoxamine. To examine mechanisms by which HIV-1 gp120 impairs endolysosome function and triggers disruption in cellular iron and the RSI (Study #2), we focused on the role of the endolysosome-resident divalent cation channel TRPML1. We found that HIV-1 gp120-induced increases in intracellular reactive species levels activated TRPML1, and that TRPML1 activation increased oxidation-induced release of endolysosome Fe2+, increases in cytosolic Fe2+ and ROS levels, and decreases in cytosolic H2S levels. Moreover, TRPML1 redox activation induced by HIV-1 gp120 increased levels of endolysosome ROS, LPO, NO, sulfane sulfur (S0), and protein cysteine oxidation (PCO), and decreased levels of endolysosome H2S, glutathione (GSH), Fe2+, and increased endolysosome pH. gp120-induced TRPML1-mediated release of endolysosome Fe2+ into the cytosol is a secondary consequence of endolysosome GSH depletion, and disruption in endolysosome Fe-GSH complexes caused TRPML1 redox activation-induced increases in endolysosome reactive species. Taken together, the endolysosome Fe2+ pool appears sufficient to account for HIV-1 gp120, morphine, and iron overload-induced disruption of inter-organellar iron signaling and the RSI; potential targets for therapeutics against HAND and other diseases associated with iron overload and redox imbalance. Further, our studies suggest that TRPML1 is a key modulator of endolysosome and cytosolic iron and RSI homeostasis.

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