Description of Research Expertise

Research Interests
Mechansims and determinants of HIV induced neuronal injury. Neuronal cell responses to virus induced injury.

Key words: neuron, hippocampus, apoptosis, gene expression, single-cell mRNA, HIV, chemokine receptor, NMDA receptor.

Description of Research
My laboratory is focused upon pathogenesis of HIV-1-infection of the central nervous system (CNS). We utilize techniques of single-cell microdissection and mRNA expression analysis, and standard molecular biological approaches in our unique in vitro model to study mechanisms of neurodegeneration caused by HIV. Within the CNS, HIV productively infects macrophages and microglia, with subsequent neuronal loss by apoptosis. In vitro modeling shows that such infection results in the release of soluble neurotoxins that act in part through activation of neuronal NMDA receptors with subsequent activation of cell death pathways, including apoptosis cascades and calpain activation. Specific NMDA receptor subunits (NR2A, NR2B) are critical for such degeneration. In addition, dysregulation of glial (astrocyte, macrophage) neurotransmitter and oxidative functions may subserve both apoptotic and anti-apoptotic roles. Major unanswered questions include:

1. What pathways are reponsible for neuronal apoptosis/damage and glial cell dysfunction in HIV infection?
2. How can NMDA receptor modulation alter neuronal susceptibility to HIV-induced damage?
3. Are specific genes turned on/off in neurons that are particularly vulnerable/resistant to HIV-induced injury?
4. What is the role of endogenous neuronal survival pathways in preventing HIV-induced damage?
5. How can in vitro modeling be used to define targets for neuroprotection against HIV-1?
6. How can single-cell mRNA analysis be used to define patterns of gene expression in the brain to define mechanisms of neuronal death/damage and survival that may identify unique targets for neuroprotective strategies against HIV?

To address these questions, we have developed several in vitro neuronal cell culture systems. We utilize primary rodent hippocampal and neocortical cell cultures as well as a unique human neuronal cell system utilizing NT2.N neurons in mixed neuronal/glial cell cultures to model HIV-1-induced neurodegeneration. We have developed a novel in vitro model using mixed cultures of human monocyte-derived macrophages with rodent neurons and astrocytes to analyze effects of HIV infection in the central nervous system. We have found that developmental susceptibility to HIV-1-induced neurodegeneration is determined by NMDA receptor subunit expression, and that the macrophage kynurenine metabolic pathway is a major contributor to the production of HIV-1-induced excitotoxin expression. We have utilized single-cell microdissection and mRNA expression analyses to develop gene expression profiles in individual neurons in archival human brain and in cell cultures. Our current projects involve in vitro and in vivo analysis of cell death pathways in neurons that are induced by HIV-infected macrophages, analysis of the role of specific NMDA receptor subunits in determining neuronal vulnerability to HIV-induced damage, analysis of the modulation of the kynurenine metabolic pathway in macrophages by HIV-1, and in vitro analysis of neuronal gene expression in hippocampal and neocortical cell cultures through single-cell mRNA analysis.

Rotation Projects for 2006-2007
1. Analysis of NMDA receptor subunit expression and phosphorylation patterns in hippocampal and cortical neurons susceptible to HIV neurotoxicity

2. Analysis of neuronal gene expression,in HIV-injured hippocampal and neocortical cells utilizing single-cell microdissection and mRNA analysis.

3. Identification of the downstream pathways of death in neurons by HIV-1, including the role of calpain activation.

Lab personnel:
Praveena Sabnekar, PhD - Research Specialist
Lorraine Kolson, BS - Research Specialist
Rebecca Chodroff, BS - Research Specialist
Lauren O'Donnell - CAMB Graduate student
Stephan Kadauke – CAMB MD/PhD student