Brain-derived neurotrophic factor inhibits human immunodeficiency virus-1/gp120-mediated cerebellar granule cell death by preventing gp120 internalization

Abstract

The human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 has been implicated in the pathogenesis of HIV-1 dementia. Thus, inhibition of gp120 activity could reduce HIV toxicity in the brain. We have used primary cultures of rat cerebellar granule cells to examine mechanisms whereby gp120 causes cell death and to characterize neuroprotective agents. gp120 induced a time- and concentration-dependent apoptotic cell death, which was caspase-3-mediated but caspase-1 independent, and was totally blocked by the irreversible caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-chloromethylketone. Caspase-3 activation was observed only in neurons that internalize gp120, indicating that internalization is key to gp120 toxicity. Because brain-derived neurotrophic factor (BDNF) prevents caspase-3-mediated neuronal cell death, we examined whether BDNF could prevent gp120-mediated apoptosis. Preincubation of neurons with BDNF before the addition of gp120 reduced caspase-3 activation, and consequently rescued 80% of neurons from apoptosis. Most importantly, BDNF reduced the levels of CXC chemokine receptor-4 (CXCR4), a receptor that mediates HIV-1 gp120-induced apoptosis. This effect correlated with the ability of BDNF to reduce gp120 internalization and apoptosis. Moreover, BDNF blocked the neurotoxic effect of stromal-derived factor-1alpha, a natural ligand for CXCR4, further establishing a correlation between neuroprotection and downregulation of CXCR4. We propose that BDNF may be a valid therapy to slow down the progression of HIV/gp120-mediated neurotoxicity.

Figure 1.

gp120 induces neuronal cell death. Cerebellar granule cells were exposed for 24 hr to gp120 at the indicated doses (A) or to 5 nm gp120 for the indicated times (B). Control cells were exposed to heat-inactivated gp120. Cell survival was analyzed by MTT and TUNEL assays, and the number of dead cells was determined by Hoechst/PI staining. Neurons exposed to medium alone or to inactivated gp120 showed a similar number of cell deaths (5%). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control (n = 12 each point).

Figure 2.

Caspase-3 but not caspase-1 is induced by gp120. A, Cerebellar granule cells were exposed to gp120 (5 nm) and then caspase-3 and caspase-1 activity were measured at the indicated times. B, Neurons were exposed to DEVDK 5 min before gp120 and cell survival measured by counting Hoechst/PI-positive cells at the indicated times. *p < 0.05 and **p < 0.001 versus control (n = 9 each point).

Figure 3.

gp120 is internalized by neurons. Confocal microscopic analysis of cerebellar granule cells exposed to bcgp 120.A, Cultures were exposed to bcgp 120 for 12 hr and then stained for GPAF (blue) and Nissl (red). gp120 was visualized using fluorescein streptavidin (green). gp120 is only in Nissl-positive cells (20× magnification). B, Cerebellar granule cells were exposed to bcgp120 for 3 hr. Cells were stained for MAP-2 (red). Coverslips were the mounted using DAPI (blue). gp120 was visualized using fluorescein streptavidin (green). gp120 (yellow, overlay red and green) is found primarily in the cytoplasm around the nucleus (60× magnification).

Figure 4.

gp120 internalization precedes caspase-3 activation. Cerebellar granule cells were exposed to inactivated gp120 (A) or gp120 (B) for 6 hr and then stained for gp120 (green) and cleaved caspase-3 (blue). After that, Nissl staining was performed (red). Purple (overlay blue and red) indicates caspase-3-positive neurons (20× magnification). C, Graphic representation of the number of caspase-3- and gp120-positive cells. Cultures were exposed to gp120 for the indicated times and then analyzed for cleaved-caspase-3 and gp120 immunoreactivity. The images were captured and the number of gp120- and caspase-3 positive cells counted. The percentage of caspase-3-positive neurons increases with time after exposure to gp120. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control (n = 12 each time point).

Figure 5.

BDNF prevents gp120 activation of caspase-3. Cultures were exposed to inactivated gp120 for 6 hr (A), gp120 for 6 hr (B), or BDNF (50 ng/ml) 12 hr (C) before gp120. Cells were then fixed and stained for cleaved caspase-3 (red) and neurofilament (green). Cells were counterstained using DAPI (blue). Purple (overlay blue and red) indicates caspase-3 positive neurons. Scale bar, 35 μm. D, Cultures were exposed to BDNF concomitantly (0) or before gp120 for the indicated times. Caspase-3 activity was measured in cell lysates 6 hr after gp120. *p < 0.05 and **p < 0.001 versus gp120 (n = 9 each time point).

Figure 6.

BDNF inhibits gp120 neurotoxicity. Cultures were exposed to BDNF (50 ng/ml) concomitantly (0) or before gp120 for the indicated times. Cell survival was measured by Hoechst/PI staining 24 hr after gp120. *p < 0.05 and **p < 0.001 versus gp120 (n = 9 each time point).

Figure 7.

BDNF prevents gp120 internalization. Neurons were exposed to bcgp120 for 6 hr (A) or BDNF for 12 hr (B) before the addition of bcgp120. gp120 internalization (green) and cleaved caspase-3 immunoreactivity (red) were determined 6 hr later. Cells were counterstained using DAPI (blue). In BDNF-treated cultures the number of neurons positive for caspase-3 (purple, overlay blue and red) and gp120 is drastically reduced. Scale bar, 25 μm.

Figure 8.

BDNF decreases CXCR4 levels. Neurons were exposed to BDNF for the indicated times before the addition of gp120 and lysates prepared 6 hr later. A, Example of Western blot analysis of CXCR4 and CCR5 immunoreactivity in control and BDNF-treated (12 hr) neurons. B, Time course analysis of BDNF effect on CXCR4 and CCR5 levels. *p < 0.05, ** p < 0.01, and ***p < 0.001 (n = 6 per time point).

Figure 9.

CXCR4 receptor mediates gp120-induced cell death. Cultures were exposed to AMD 30 min before gp120 or SDF-1α. BDNF was added 12 hr before SDF-1α. Cell death was measured 24 hr later by Hoechst/PI. ^#p < 0.001 versus control; *p < 0.001 versus gp120 or SDF-1α'3b n = 9 each point.