Background:  Cell cycle proteins are thought to be involved in neuronal survival of post-mitotic neurons and alterations of their function have been associated with various neurodegenerative disorders, including neuroAIDS. One of these proteins is the transcription factor E2F1, which is negatively regulated by the retinoblastoma gene product Rb. In non-proliferating cells, E2F1 induces expression of apoptotic genes. We have been testing the hypothesis that transcriptional activity of E2F1 plays a crucial role in CXCR4-mediated neurotoxicity and HIV neuropathogenesis, by using in vitro and ex vivo approaches.

Methods:  Primary cultures of rat cortical neurons were exposed to HIVgp120_IIIB  (200 pM) to determine the effect of the viral protein on neuronal survival, E2F1 expression/activation, and level of E2F1-dependent genes. We used Western blot analysis, immunocytochemistry, electromobility shift assays, and gene reporter assays. Cultures from E2F1-deficient mice were used to evaluate the role of E2F1 in gp120-mediated neurotoxicity. Protein levels of E2F1 and its apoptotic targets were also studied in brain tissue, ie, frontal cortex, from HIV patients with or without neurological deficits by immunohistochemistry (tissue obtained from NNTC). Neurons were identified by MAP2 staining. Data are reported as meanąSEM of at least 3 independent experiments (p <0.05 using Student’s t-test or one-way ANOVA).

Results:  Treatment of neurons with gp120-induced a time-dependent increase of E2F1 in the nucleus of primary neurons, which preceded caspase-3 activation and cell death. Gene reporter assay indicated that gp120-treatment promotes transcriptional activity of E2F1 and increased levels of the E2F1-dependent apoptotic proteins Cdc2 and Puma. These effects of gp120 were inhibited by the CXCR4 antagonist AMD3100. Studies with cultures from E2F1-deficient mice showed that E2F1 is required for gp120-induced neurotoxicity and up-regulation of apoptotic proteins. Finally, levels of E2F1 protein were greater in the nucleus of neurons in brains of HIV-infected patients exhibiting dementia than in HIV-negative subjects or HIV-positive neurologically normal patients.

Conclusions:  Overall, these studies indicate that the transcription factor E2F1 is involved in CXCR4-mediated neurotoxicity and HIV neuropathogenesis.