Date of Award

January 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Jonathan D. Geiger

Abstract

Approximately 1.2 million people in the U.S.A are living with HIV-1. The development of antiretroviral therapeutics has increased the life expectancy of people living with HIV-1 (PLWH). Yet, there is a growing concern for aging HIV-positive individuals because about 50% of PLWH develop neurological impairments, termed HIV-associated neurocognitive disorders (HAND). There are no treatments for HAND and the mechanisms underlying its pathogenesis remain unclear. Several factors are implicated in HAND pathogenesis, such as HIV-1 coat protein gp120, endolysosome and mitochondrial dysfunction, and elevated levels of reactive oxygen species (ROS). Endolysosomes are acidic organelles that contain readily releasable stores of cations, such as iron, which can be released upon de-acidification. Mitochondria are energy producing organelles, which can also act as cellular sinks for iron when levels of cytosolic iron are elevated. Iron is an essential metal for human life, but it is tightly regulated due to its ability to generate ROS through Fenton or Fenton-like reactions. HIV-1 gp120 increases ROS production, de-acidifies endolysosomes and reduces mitochondrial respiration. We used U87MG astrocytoma cells, confocal microscopy, flowcytometry, filter-based imaging, and western blotting to investigate mechanisms by which HIV-1 gp120 affects endolysosome iron stores, levels of ROS, and the effects of gp120 on expression levels of iron related proteins. In Chapter 2, we describe findings that HIV-1 gp120 de-acidifies endolysosomes, induced iron release from endolysosomes, increased iron levels in mitochondria, and that iron from endolysosomes led to increases in levels of cytosolic and mitochondrial ROS levels. Our findings suggest that endolysosome iron stores can be upstream of HIV-1 gp120 induced increases in ROS production. In Chapter 3, we described findings about the responsiveness of our cells to iron supplementation and chelation, as well as how HIV-1 gp120 affects expression levels of iron related proteins. We found that 24 h treatment with HIV-1 gp120 increased the cell surface expression levels of ferroportin (FPN), an iron export protein, but we did not observe any changes in other iron related proteins. Our findings might suggest that U87MG cells increase cell surface FPN in order to regulate cytosolic iron levels. We considered our findings from Chapter 2 and 3 as evidence that HIV-1 gp120 induces lysosomal stress. In Chapter 4, we outlined a new concept in understanding how endolysosomes adapt to their environment during different stress inducing events; this was termed “lysosome stress response” (LSR). We provided criteria and a working definition for LSR. We also highlighted how LSR is observed in other organellar stress responses. Taken together, these findings provide a mechanism by which HIV-1 gp120 affects ROS production. Moreover, this work highlights endolysosomes as a potential upstream therapeutic target for any disease in which elevated ROS is implicated and broadens our understanding of cellular biology.

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