Role of the transcription factor E2F1 in CXCR4-mediated neurotoxicity and HIV neuropathology

Abstract

This study sought to determine the role of the transcription factor E2F1 in CXCR4-mediated neurotoxicity and HIV neuropathology. We studied the effect of the HIV envelope protein gp120 on the expression of E2F1-dependent apoptotic proteins in human and rodent neurons and examined the expression pattern of E2F1 in the brain of HIV-infected individuals. Our findings suggest that in cultured neurons gp120 increased E2F1 levels in the nucleus, stimulated its transcriptional activity and enhanced the expression of the E2F1 target proteins Cdc2 and Puma. Studies with neuronal cultures from E2F1 deficient mice demonstrated that the transcription factor is required for gp120-induced neurotoxicity and up-regulation of Cdc2 and Puma. Levels of E2F1 protein were greater in the nucleus of neurons in brains of HIV-infected patients exhibiting dementia when compared to HIV-negative subjects or HIV-positive neurologically normal patients. Overall, these studies indicate that E2F1 is primarily involved in CXCR4-mediated neurotoxicity and HIV neuropathogenesis.

Figure 1. Expression of E2F1 in the nucleus of cortical neuron in HIV/HAD patients

Frontal cortices from HIV-positive patients (with or w/o neurological deficits) and HIV-negative control subjects were used to examine E2F1 expression by immunohistochemistry (as described in detail in the method section). Low levels of E2F1 were detected in the brains of neurologically normal HIV patients (A) or control subjects (graph), while E2F1 was up-regulated in the nucleus of cortical neurons in HIV/HAD brains (arrow head in B; 40X in C). Double-staining with the neuronal marker MAP2 was performed to identify E2F1-positive neurons (D) and quantify differences among the three groups of patients (E, ten fields/brain); *p<0.05 vs HIV; Scale bars 5.0 um.

Figure 2. Effect of HIV gp120 on E2F1 expression in rat primary neurons

Western blot analysis (A-B) and immunocytochemistry studies (C) indicate that E2F1 is up-regulated in the nucleus of rat primary cortical neurons after treatment with gp120_IIIB (200 pM) for the indicated time. The viral protein was added to the co-cultures at 7DIV. Antibodies against the neuronal-specific nuclear protein NeuN or β-actin, were used as markers in the immunoblots with nuclear (NE) and cytosolic (CE) extracts, respectively; Hoechst 33442 was used for nuclear staining in the immunocytochemistry studies. A significant increase in E2F1 protein levels was observed after a few hours of treatment with gp120_IIIB (generally 3 h) as shown by the graph in B, which reports the average of three independent experiments using total cell extracts from untreated (control) and gp120-treated neurons (mean ± SEM; *p<0.05). Each scatter plot in graph D shows the average intensity of E2F1 nuclear staining in individual neurons at each time points, as determined by immunostaining (bars at each time represent mean values). E2F1 positive pre-apoptotic neurons, i.e. cleaved caspase-3 positive neurons showing healthy morphology, were also observed after short term treatments with gp120 (arrows in C; blue bars in graph E, ^p<0.001 vs control). Apoptotic neurons, i.e. cleaved caspase-3 positive neurons with condensed/fragmented nuclei, were detected at later time (white bars in graph E; *p<0.001 vs control).

Figure 3. HIV gp120 up-regulates E2F1 pro-apoptotic targets in rat neurons

Treatment with gp120_IIIB (200 pM) increased transcriptional activity in rat primary neurons as determined by gene reporter assays (A, mean ± SEM of one representative experiment; *p<0.01 vs control). E2F1 over-expression was used as positive control for the assay. Elevated levels of transcriptional targets of E2F1 (Puma and Cdc2) were also found in neuronal extracts from primary rat cultures treated with gp120 (B–C) as well as in neurons transfected with the E2F1 expression plasmid (D). The effect of gp120_IIIB was blocked by pre-treatment of cultures with AMD3100 (100 ng/ml) 15 min before the addition of the gp120.

Figure 4. Effect of HIV gp120 on E2F1-dependent proteins in human neurons

E2F1 pro-apoptotic targets were increased after gp120_IIIB (200 pM) treatment in differentiated SH-SY5Y neuroblastoma cells (A). Nuclear extracts from differentiated SH-SY5Y cells treated with the gp120 (200 pM) were used to assess binding activity to E2F1 consensus sequences (B; arrow represents DNA-protein complex). Panels C and D show over-expression of E2F1-dependent proteins, namely Cdc2, in the cytoplasm of frontal cortical neurons in a HIV/HAD patient; higher magnification of one of these neurons is shown in E (Scale bar: 5.0 μm).

Figure 5. E2F1 is necessary for HIVgp120-induced cell death

Neuronal cultures from E2F1 KO and wild type mice were used to determine the role of E2F1 in gp120-induced apoptosis. DNA was extracted from the cerebellum of each animal for PCR analysis (A, bands 172 bp and 227 bp correspond to wild type and KO respectively, as expected). Cloned E2F1 was used as positive control. Survival assays performed as reported in the method section, show that KO cultures are resistant to gp120_IIIB (B/C, *p<0.0001 vs control in wild type and vs gp120 treatment in KO mice). E2F1 targets, Cdc2 and Puma were up-regulated after gp120 treatment in wild type but not in KO mice - as determined by Western blot analysis with total neuronal cell extracts. No difference in CXCR4 protein levels were observed between WT and KO mice (D).