Richard J. Noel, PhD

Department of Biochemistry

Research Info

1. Area: HIV Neuropathology; with and without drug abuse
   Research questions: What are the synergistic neurotoxicities of HIV proteins and drugs of abuse? By what mechanisms do any synergy or simple combined effects occur?
   Currently our lab is working with a number of cell culture models to study the cellular effects of HIV neurotoxins (Tat, Vpr, Nef, and gp120, but not in combinations) with and without drugs of abuse (cocaine or morphine, plus combination = speedball). There is a fair amount of literature supporting synergy between virotoxins (predominantly Tat or gp120) and various single drugs of abuse (morphine, cocaine, methamphetamine); however, the mechanisms by which neuronal function is compromised and/or neuron loss occurs are somewhat less well known. The culture models include human cell lines (SVGA – astrocytoma, SH-SY5Y – neuroblastoma), rat primary cultures (astrocytes and we are working on primary neuronal cultures), and also rat tissue culture explants (culture of rat hippocampal slices, also under development). We treat these model systems with viral proteins using transfection. We are also developing the capacity to isolate recombinant proteins from E. coli using a his-tag expression system. We add drugs of abuse (morphine, cocaine, speedball) and then perform assays to measure cytokine gene expression (mRNA and protein), apoptosis, proliferation, oxidative stress. We also will look for morphological changes, particularly once we increase our proficiency with the explants, using immunofluorescence microscopy.
   Our results to date have shown involvement of a number of cytokines upregulated by Tat, Vpr, or Nef (IL-6, TNF-alpha, IL-1beta) in astrocytes. Drugs of abuse do not seem to have synergistic effects in our system, though in some cases morphine has shown combined effects (less than additive). In addition to filling out our ongoing studies, we hope to start using a co-culture system, particularly with the hippocampal explants to assess some of the same molecular parameters as in cell culture. In addition, we will collaborate with a neurophysiologist at PSM (Dr. James Porter) to assess neuronal plasticity and neurophysiological changes (receptor expression, sensitivity, long term potentiation) in these explants. These studies can then be moved to a rat model so that behavioral and learning deficits can be integrated to lend biological significance to the molecular and cell physiology components.
    
2. Area: Structure/function properties of HIV-1 Tat second exon
   Research questions: How does the C-terminus of Tat impart added function? Does full length Tat have altered effects on the virus (replication), the host (gene regulation) or both?
   Tat exists in two naturally occurring forms during the viral replication cycle – amino acids 1-101 from the spliced transcript and amino acids 1-72 from the unspliced transcript. It is generally considered the that second exon is conserved because of essential function, but only a few groups have shown differences in replication (T cells and macrophages) and apoptosis induction (T cells). We are interested in studying the contribution of the C-terminus on host gene regulation, particularly cytokine upregulation using deletion and alanine-scanning mutagenesis. We have recently begun to examine the different effects of mutation of critical amino acids in the context of Tat72 vs. Tat101 for LTR transactivation, as well.
    
3. Area: Phlyogenetic analysis of viral evolution and distribution
   We have looked at viral sequence distribution in a number of areas in the Caribbean as well as the continental US to try to discover patterns of the epidemic (which are more closely related, what areas seem to have shared pools of virus). We also have used a number of phylogenetic tools to examine how viral gene evolution (notably Tat, env, Vpr and Nef) is related to disease progression in a rhesus macaque model of morphine addiction and AIDS.