Infection with HIV- 1 can induce dementia in children and adults. Due to poor brain penetrance, current antiretroviral therapies do not sufficiently treat this problem. The HIV-1 envelope protein gpl20 binds to macrophages, microglia and T-cells via the chemokine receptors CCR5 and CXCR4, which serve besides CD4 as co-receptors for the virus. However, these chemokine receptors are also present on neurons and astrocytes. The HIV-1 gpl20 produces, in vitro and in vivo, injury and apoptosis in neurons. Moreover, transgenic mice expressing gp 120 in their central nervous system develop neuropathological features observed in AIDS brains. Macrophages and microglia seem to play a crucial role in HIV neuropathology by releasing toxic factors, but neuronal and astrocytic chemokine receptors could also contribute to injury of neurons, via direct interaction with gpl20. Against this background, the long-term objectives are to find future therapeutic targets and thus new potential treatments for HIV-associated dementia via the analysis of intercellular and intracellular signaling mechanisms that are affected by I/IV-l/gp120 and chemokines.
The specific aims are: (1) To analyze whether the combined knockout of the two HIV coreceptors, CXCR4 and CCR5, suffices to prevent neuronal injury induced by HIV/gpll20 using mixed neuronal-glial cerebrocortical cultures derived from combined CXCR4/CCR5-deficient (CXCR4 -/-/CCR5-/-) and wild-type mouse embryos. (2) To investigate in an in vivo model, in crosses of transgenic mice expressing HIV-1 gp120 with knock out mice deficient in CCR5, if the selective lack of this beta-chemokine receptor, which confers neuroprotection when activated by its natural ligands, aggravates gpl20-induced neuronal damage. (3) To dissect the signal transduction underlying the neuroprotection against the toxicity of gpl20, SDF-1 and NMDA by beta-chemokines MIP-1beta and RANTES via CCR5 using mixed cerebrocortical cultures from rodents, including CCR5-deficient and wild-type mice, and human cells.
For specific aim 1, neuronal death occurring in cerebrocortical cultures exposed to gpl20 or chemokines will be monitored using several methods. These include staining with propidium iodide or Hoechst 33342 dye of condensed nuclei, and TUNEL staining of nicked DNA, in combination with immunostaining for neuronal markers. Final analysis will be accomplished by microscopy.
For specific aim 2, neuronal injury will be immunohistologically evaluated at 1.5, 3, 6 and 12 month of age using confocal laser scanning microscopy and deconvolution microscopy.
For specific aim 3, [Ca2+]i levels will be assessed with the fluorescent dye Fura-2/AM in cerebrocortical cultures, and isolated microglia/macrophages, astrocytes and neuronal cells exposed to gpl20, chemokines, or combinations thereof. Activation of p38 MAPK, JNK, ERK, Akt and STAT1 will be monitored by immunoblotting, immunocomplex protein kinase assay, and immunofluorescence. The study of the potential role of protein kinases in the effect of gpl20 and chemokines will include the use of pharmacological kinase inhibitors and adenoviral vectors. Since signal transduction via p38 MAPK seems to be crucial to the neurotoxicity of gpl20 and SDF- 1, we will study the effect on this pathway of a dominant negative mutant of p38 and also of a constitutive activator of p38, MKK6b(E). ? ?
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