Graduation Date

Summer 8-19-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmacology and Experimental Neuroscience

First Advisor

Jialin C. Zheng

Second Advisor

Yunlong Huang

Abstract

Glutamate serves as a crucial excitatory neurotransmitter that is essential for the proper functioning of the brain. However, excess levels of glutamate are neurotoxic and contribute to the pathogenesis of various neurodegenerative diseases, inducing HIV-1 associated neurocognitive disorders (HAND). Glutaminase 1 (GLS1) is an important mitochondrial enzyme responsible for producing glutamate from glutamine. GLS1 is upregulated during HAND and released from mitochondria to cytosol and extracellular space. However, why and how GLS1 is released remains unknown. In chapter II, we demonstrated that extracellular vesicles (EVs) carry GLS1 as cargos from cytosol to extracellular space during HIV infection and innate immune activation. The GLS-containing EVs induce neurotoxicity through the overproduction of glutamate, implicating the pathogenic role of EVs and GLS1 in HAND.

Regulation of EV remains to be fully elucidated in HAND. Interestingly, when carried as cargo, GLS1 showed a potential effect on the release of EV. Therefore, we hypothesize that the release of EV is dependent on GLS1-mediated glutamine and sphingolipid metabolism. In chapter III, we investigated the involvement of GLS1 in EV release in GLS1-overexpressing HeLa cells, HIV-1-infected macrophages, and immune-activated microglia through the use of GLS1 inhibitors. In the aforementioned cell types, GLS1 inhibitor significantly decreased the level of EVs, suggesting an important role of GLS1 in EV regulation. In chapter IV, we further investigated the mechanism of GLS1-mediated EV release. We identified that GLS1-mediated EV release is dependent upon the level of glutamine and α-ketoglutarate (α-KG). Because α-KG is an important metabolite of glutamate, these data suggest that α-KG is an essential GLS1 downstream factor that regulates the release of EVs. Biogenesis of EVs requires the sphingolipid ceramide. However, it is unknown whether GLS1-mediated EV release involves ceramide. We have demonstrated that the addition of ceramide rescued the suppression of EV release by GLS1 inhibitors, suggesting that GLS1 mediates EV release through ceramide.

In summary, our data revealed two interesting insights into the biology of EVs. First, GLS-containing EVs is a pathogenic component of neurodegeneration in HAND. Second, the release of EVs is dependent on GLS1-mediated glutamine and sphingolipid metabolism. Studies on both of these aspects in EVs could lead to potential novel therapeutic targets for HAND and other neurodegenerative diseases.

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