|dc.description.abstract||HIV associated neurocognitive disorder (HAND) remains a major concern for patients infected with HIV. The viral envelope protein, gp120 has been extensively studied and some of its neurotoxic effects are due to the increased expression of various
proinflammatory cytokines. Additionally, it has been well documented that various drugs of abuse can exacerbate HAND, but the mechanism by which this occurs is still poorly understood. The present study was based on the central hypothesis that HIV-1 gp120 and methamphetamine (MA) interact with each other to increase the cytotoxicity in the astrocytes, which is mediated via induction of various pro-inflammatory
cytokines/chemokines and oxidative stress. In order to test these hypothesis four different studies were designed. We also investigated the mechanism(s) and pathways involved in the functional interaction between gp120 and MA. Furthermore, in order to understand the functional implications of the interaction between MA and gp120, we examined the
combined effect of MA and gp120 to produce oxidative stress and apoptotic cell death. In the first study, we investigated the role of gp120 in the cytokine production in astrocytes. SVGA astrocytes and human fetal astrocytes were either transfected with a plasmid coding gp120 or treated with recombinant gp120 protein and the expression levels of various cytokines at RNA and protein levels were measured. In order to better explain the role of gp120 in the induction of proinflammatory cytokines/chemokines, 3 major and highly induced cytokines/chemokines were screened and further mechanistic studies were aimed with these 3 cytokines/chemokines. We investigated the role of NF-
κB pathway in the transcriptional regulation followed by studies to identify molecular mechanisms. Various MAPKs were assessed for their involvement in the gp120-mediated cytokine/chemokine production. Finally the involvement of PI3K/Akt was explored to
understand the signaling pathway upstream of NF-κB.
The second study was designed to address the role of MA in the cytokine production in astrocytes. As observed with gp120, MA also showed increase in the
expression of various proinflammatory cytokines. In particular, the induction of IL-6 and IL-8 was found to involve the NF-κB pathway. Furthermore, the activation of NF-κB was found to be independent of MAPKs. However, our studies with PI3K/Akt pathway and mGluR5 suggested that these signaling molecules could trigger the activation of NF-κB.
In the third part of the study, we demonstrated that astrocytes treated with both MA and gp120 increased the expression of IL-6 to levels that are much higher than when cells are treated with either agent alone. This suggested that MA and gp120 may act
synergistically to increase the expression of IL-6. Furthermore, the increase in the levels of IL-6 due to treatment with both gp120 and MA was found to involve the PI3K/Akt and NF-κB pathways. Inhibition of either of these pathways could abrogate the increased expression of IL-6 due to MA or gp120 alone, and the increased expression of IL-6 when
the astrocytes were treated with both gp120 and MA. Additionally, our results demonstrated that neither MA nor gp120 utilized the JNK-MAPK and ERK1/2 pathways to increase IL-6 expression. In the final chapter, we also demonstrated that gp120 and MA cause apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH
oxidase (NOX) pathways. The results showed that both MA and gp120 induced ROS production in concentration- and time-dependent manners. The combination of gp120 and MA also induced CYP2E1 expression at both mRNA and protein levels, suggesting the involvement of CYP2E1 in ROS production. This was further confirmed by using
selective inhibitors of ROS (Vitamin C), CYP2E1 (DAS), NOX, diphenyleneiodonium (DPI), and FWH reaction, deferoxamine (DFO), which significantly reduced ROS production. These findings were further confirmed using specific siRNAs against NOX2 and NOX4 (NADPH oxidase family). Furthermore, gp120 and MA both induced apoptosis (as evidenced by increased caspase-3 activity and DNA lesion via TUNEL assay) and cell death (measured using MTT assay). Additionally, Vitamin C, DAS, DPI, and DFO completely abolished apoptosis and cell death, suggesting the involvement of CYP and NOX pathways in ROS-mediated apoptotic cell death.
In conclusion, we showed that both MA and gp120 independently and in combination increased the production of pro-inflammatory cytokine/chemokines via different pathways. The functional consequences for the interaction between gp120 and MA led to oxidative stress and apoptotic cell death in astrocytes. Thus, our current study provides the evidence and underlying mechanism for the neurotoxic potential of HIV protein, gp120 and substance of abuse, methamphetamine||eng