AIDS can produce substantial dysfunction in the central nervous system (CNS), including encephalopathy/dementia by affecting the brain, myelopathy by affecting the spinal cord, and visual loss by affecting the retina. These adverse effects can occur in the apparent absence of infectivity of neurons by HIV-1, and even in the absence of superinfection with other opportunistic organisms. Therefore, one possibility is that HIV-1 somehow produces a toxic agent that injures neurons. One molecule that is a candidate as a toxic product of HIV-1 is the envelope protein gpl20, since it is shed from the virus. For the current proposal, 3 tissue culture models of CNS injury will be used, comprised of (i) postnatal rodent retinal ganglion cells, (ii) rodent hippocampal cortex neurons, and (iii) human cortical neurons (obtained from neurosurgical specimens). In preliminary studies on rat retinal ganglion cell and hippocampal neurons, gpl2O, in picomolar amounts and in a dose-dependent fashion, induces a dramatic increase in intracellular [Ca2+]; in retinal ganglion cells this rise in [Ca2+] is associated with neuronal injury and even cell death. Additional preliminary experiments have demonstrated that calcium channel antagonists are capable of totally preventing this form of neurotoxicity caused by the HIV-1 envelope protein. Studies are proposed to test if these toxic effects apply to other types of neurons, including human cortical neurons, and to elucidate further the mode of action of gpl2O on intracellular [Ca2+] using calcium imaging with the dye fura-2, patch-clamp recording, and concomitant viability assays.
The specific aims of the proposed study are -- 1 . To monitor the level of free calcium in retinal ganglion cell and cortical neurons in vitro after exposure to HIV-1 envelope protein gpl 20; rodent and human neurons will be used. 2. To assess injury to these neurons after exposure to gpl20 in vitro using a well-developed test of viability, the uptake and cleavage of fluorescein diacetate; to assess the protective effects from gpl 20 neurotoxicity of a variety of calcium channel antagonists by generating dose-response curves. 3. To perform patch-clamp recordings of these neurons at both the whole-cell and single-channel levels to determine, as suggested in preliminary experiments, if gpl20 affects a specific type of calcium channel; to study the mechanism of this effect.
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