The Effect of Drugs of Abuse and HIV-1 GP120 on Autophagy in Astrocytes
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Abstract
The life expectancy of individuals infected with HIV-1 has significantly improved since the introduction of highly active anti-retroviral therapy (HAART). However, this has led to an increase in the prevalence of HIV-associated neurocognitive disorders (HAND), especially the mild forms of HAND. Drugs of abuse are known to exacerbate neuropathological dysfunction in HIV-infected individuals. The neuropathological manifestation results from cell death in the CNS wherein autophagy is expected to play an important role. Autophagy is generally considered protective during deprivation or stress. However, excessive autophagy can be destructive, leading to autophagic cell death. The present study was based on the central hypothesis that drugs of abuse and HIV-1 viral proteins work synergistically in mediating autophagy in human astrocytes. The level of autophagy mediated by cocaine, methamphetamine, and HIV-1 gp120 is determined. Furthermore, we examined the cellular mechanisms responsible for cocaine and methamphetamine-mediated autophagy pathway, and the role of autophagy in drug-induced cytotoxicity. In the first chapter, we investigate the effect of cocaine on autophagy in SVGA astrocytes. We measured levels of the autophagic marker protein LC3II in SVGA astrocytes after exposure with cocaine. The results showed that cocaine caused an increase in LC3II level in a dose- and time-dependent manner. This result was also confirmed by detecting LC3II in SVGA astrocytes using confocal microscopy and transmission electron microscopy. Next, we sought to explore the mechanism by which cocaine induces the autophagic response. The involvement of sigma 1 receptor, and autophagy signaling proteins p-mTOR, Atg5, Atg7, and p-Bcl-2/Beclin-1 were determined by using selective inhibitors and siRNAs. In addition, we determined the role of autophagy in chronic cocaine exposure using MTT assay and western blot analysis. In the second chapter, the effect of methamphetamine on autophagy in SVGA astrocytes, as well as human fetal astrocytes were examined. The mechanistic pathway of methamphetamine-mediated autophagy was determined. In the third chapter, the synergistic effect of methamphetamine and HIV-1 gp120 IIIb on autophagy in SVGA astrocytic cell line and human astrocytes was determined. The signaling mechanism of methamphetamine- and gp120- mediated autophagy is investigated by utilization of specific inhibitors and siRNAs. Furthermore, the role of autophagy during methamphetamine- and gp120- induced cytotoxicity is examined by MTT assay and PI staining. In conclusion, we showed that drugs of abuse and HIV-1 viral protein gp120 were able to induce autophagy in astrocytes. The underlying mechanism provides potential therapeutic target for neuroAIDS.
Table of Contents
General introduction -- General materials and methods -- The effects of cocaine on autophagy in astrocytes -- Methamphetamine potentiates HIV-1 GP120-mediated autophagy via beclin-1 and ATG5/7 as a pro-survival response in astrocytes -- Effect of antioxidants on autophagy mediated by methamphetamine and GP120 -- Summary and future directions -- Appendix
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Ph.D.