HIV-1 infection and opioid drug abuse are known to cause neurodegeneration and disease progression. It is known that HIV usually enters the CMS very soon after infection and people can develop various neurological problems such as encephalitis, meningitis or neuropathy. These syndromes are thought to be due either to HIV itself infecting cells, or to the immune system's response to HIV infection. HIV-Tat protein through cytokine upregulation may play a direct role in neuropathogenesis which is related to monocyte infiltration into the CMS. Opioid drugs such as morphine and heroin may also contribute to alterations in immune function. There is a synergy between HIV Tat and opioids in altering cytokine production but the mechanism is still unknown. Our hypothesis is that Tat and opioids may synergistically alter astrocyte cytokine production that then contributes directly to neuronal loss of function and apoptosis. We want to determine the role of HIV Tat full length compared to the one exon Tat in the synergy with opioids in astrocyte and neuroblastoma cell lines by completing three aims:
Aim 1 - Determine the role of the full length Tat protein in synergy with morphine in altering cytokine production in astrocyte and neuroblastoma cell cultures.
Aim 2 - Determine the response of neuronal cells to astrocytes treated with Tat/morphine that may be related to neuropathology, specifically an increase in apoptosis and altered NMD A receptor (NMDAR) current.
Aim 3 - Test the role of the transcription factor NF-kB in the synergy between Tat and opioids on changes in cytokine gene expression. Astrocyte and neuroblast cell lines will be treated with Tat by transfection with and without morphine. Cytokine mRNA and protein levels will be measured by real time RT-PCR and ELISA/cytokine bead array. Changes in apoptosis and neuron function will be measured by annexin V staining, calcium imaging and voltage clamp for NMDAR sensitivity. Finally, NF-kB involvement will be assessed by gel shift assay and also using a dominant negative IkB plasmid. We expect to find that the synergy between Tat and morphine is dependent both on the form of Tat used as well as the transcription factor NF-kB. The work proposed will offer a better understanding of how this very important viral protein contributes to HIV neuropathology. The purpose of this project is to establish the mechanism by which HIV-1 Tat and opioids use can exacerbate the HIV related neuropathology. This study will be important to understand the role of HIV Tat in disease progression of HIV infected patients and opioid users.