Effect of TIMBD on Neurotoxicity Associated with HIV-1 Gp120 and Tat

Abstract

Human Immunodeficiency Virus (HIV)-associated neurocognitive disorder (HAND) is one of the undermining disorders that affects majority of HIV infected patients. Patients with HAND suffer from both cognitive and motor dysfunction which is characterized by memory loss and changes in personality. HIV-1 gp120 and Tat are essential proteins that are involved in neurotoxicity. HIV-1 gp120 and Tat play a role in HAND associated oxidative stress and neuroinflammation produced in astrocytes, which is considered a major contributor for damage in CNS. HIV-1 Tat is able to affect behavior by modifying the neurotransmission genes. Natural compounds have been studied extensively as therapeutic intervention agents in neurodegenerative diseases. However, poor bioavailability and limited potency of natural compounds limited their successful use in humans. In an effort to develop a new chemical that can overcome the pitfalls associated with natural compounds, our laboratory has synthesized an analog of resveratrol 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD). TIMBD is suggested to have powerful neuroprotective effects based on our preliminary findings. The following study was based on the hypothesis that our newly synthesized compound TIMBD is able to decrease oxidative stress and neuroinflammation associated with gp120 in astrocytes and protects astrocytes from cell death. Furthermore, it is hypothesized that TIMBD improves the behavioral deficits associated with HIV-1 Tat using Tat-tg mice and the mechanism is through modulation of neurotransmitter genes. Additionally, TIMBD is hypothesized to decrease oxidative stress and neuroinflammation associated with HIV-1 Tat in astrocytes in vitro. We investigated the effect of TIMBD on oxidative stress produced by gp120 in astrocytes. We measured oxidative stress markers in SVG astrocytes in response to gp120. We also measured levels of pro-oxidant and anti-oxidant enzymes in response to gp120 and TIMBD in SVG and primary astrocytes. We confirmed our results by measuring enzymes expression levels of pro- and anti-oxidant genes in vivo using protein extracted from mice brain tissues, which were gp120 gp120-transgenic (tg) and treated with TIMBD. We also determined the involvement of NRF2 transcription factor by measuring its expression in nuclear and cytoplasmic fractions in SVG astrocytes. We further measured cell death following TIMBD treatment on gp120- affected SVG astrocytes. We investigated the effect of TIMBD on neuroinflammatory cytokines produced by gp120 in astrocytes. We measured expression of IL6, IL8 and CCL5 at RNA and protein levels following TIMBD or RES treatment on gp120-treated SVG astrocytes. The results were further confirmed by using immunocytochemistry technique that involved measuring inflammatory proteins in astrocytes using confocal microscopy. Next, the mechanism by which TIMBD decreased neuroinflammation associated with gp120 was determined. The expression of signaling proteins involved in AP1, STAT3 and NFĸB pathways were measured at protein expression levels. The upstream signaling was further identified by measuring protein expression levels of p38MAPK, pAKT and pIKKs. We investigated the effect of TIMBD on the behavioral deficits associated with HIV-1 Tat using Tat-transgenic (tg) mice. The anxiety-like behavior was assessed using open field test and light/dark box test following TIMBD treatment for wild type (WT)-control mice and Tat- tg mice. The learning and spatial memory were determined using Morris water maze test which involved acquisition trials of 5 days and 1 day of probe trial. Next, the mechanism by which TIMBD improves behavioral deficits was determined by measuring protein expression levels of many neurotransmission proteins. These include neurotrophic factors BDNF and CNTF, the pre-synaptic proteins synapsin and synaptophysin, the post-synaptic proteins PSD95 and Arg3.1, and calcium signaling molecule pCAMK-II. We studied the effect of TIMBD on HIV-1 Tat associated oxidative stress and neuroinflammation in astrocytes. We measured ROS production in SVG and primary astrocytes as response to HIV-1 Tat and TIMBD. We also measured levels of pro-oxidant and anti-oxidant enzymes in response to TIMBD and Tat in SVG astrocytes. Additionally, we measured expression of IL6 and IL8 RNA levels following TIMBD or RES treatment on Tat-transfected SVG astrocytes. In conclusion, we demonstrated that TIMBD was able to decrease inflammation and oxidative stress associated with HIV-1 viral proteins in astrocytes. It is suggested that TIMBD provides the potential of being used as a neurotherapeutic candidate to prevent HIV associated neurocognitive deficits.