In this application the Principal Investigator focuses on the activity of platelet activating factor (PAF) in mediating neuronal damage and death in HIV-associated dementia. His rationale is based on studies that have correlated levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in brain tissue and PAF in cerebrospinal fluid (CSF) with neurologic disease in patients infected with HIV. Studies have demonstrated that activated HIV-infected monocytes secrete increased amounts of TNF-alpha and PAF. Exogenous TNF-alpha and PAF induce neuronal apoptosis. TNF-induced neuronal apoptosis can be blocked by PAF receptor antagonism, and neurotoxicity of conditioned media from HIV-infected monocytes can be ameliorated by enzymatic hydrolysis of PAF. PAF-induced neurotoxicity, in turn, can be blocked by NMDA receptor antagonists. In the proposed studies the researchers will test the hypotheses that (1) the HIV glycoprotein gpl20 induces PAF production by monocytes, and (2) subsequent PAF receptor activation increases intracellular oxidative stress and NMDA receptor over-activation (""""""""weak"""""""" excitotoxicity), resulting in neuronal apoptosis. In in vitro studies the researchers will test whether treatment with different species of the HIV glycoprotein gpl20 affect PAF production from uninfected monocytes. They will apply conditioned media from PAF-treated microglia or astrocytes to neuronal cultures depleted of glia to determine whether PAF receptor activation in glia is necessary for neurotoxicity. In studies with PAF-treated neuronal cultures they will test the hypothesis that mitochondrial dysfunction and increased oxidative stress are necessary prerequisites for lethal NMDA receptor activation in vulnerable neurons by measuring mitochondrial membrane potential and reactive oxygen species with confocal and fluorescence microscopy, as well as measuring NMDA receptor channels with cellular autoradiography. They will measure neuronal apoptosis in cultures treated with PAF and antioxidants. To determine which G protein and intracellular effector system mediates PAF receptor activation in neural cells, the researchers will measure intracellular calcium and neuronal apoptosis in PAF- or vehicle-treated neuronal cultures in the presence and absence of specific agents that stimulate or inhibit G protein subtypes/second messenger enzymes. To determine whether PAF induces """"""""weak"""""""" excitotoxicity in vivo, they will inject stereotactically varying doses of PAF plus mitochondrial toxin (malonate) into subcortical brain regions of rats, and will examine brain tissue for changes in glutamate receptors and neuronal apoptosis. These studies will help provide insights into the mechanisms of PAF-induced neuronal apoptosis.
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