M- and T-tropic HIVs promote apoptosis in rat neurons

Abstract

Neuronal loss, reactive astrocytes, and other abnormalities are seen in the brain of individuals with acquired immune deficiency syndrome-associated Dementia Complex (ADC). Human immunodeficiency virus-1 (HIV-1) is believed to be the main agent causing ADC. However, little is known about the molecular and cellular mechanisms of HIV-1 neurotoxicity considering that HIV-1 does not infect post-mitotic neurons and that viral load does not necessarily correlate with ADC. Various viral proteins, such as the envelope protein gp120 and the transcription activator Tat, have been shown to induce neuronal apoptosis through direct and indirect mechanisms both in vitro and in vivo. Progeny HIV-1 virions can also cause neuronal death. However, it has not been fully established yet whether HIV-1 promotes neuronal apoptosis by a direct mechanism. To explore the neurotoxic effect of HIV-1, we exposed rat cerebellar granule cells and cortical neurons in culture to two different strains of HIV-1, IIIB and BaL, T- and M-tropic strains that utilize CXCR4 and CCR5 coreceptors, respectively, to infect cells. We observed that both viruses elicit a time-dependent apoptotic cell death in these cultures without inducing a productive infection as determined by the absence of the core protein of HIV-1, p24, in cell lysates. Instead, neurons were gp120 positive, suggesting that the envelope protein is shed by the virus and then subsequently internalized by neurons. The CXCR4 receptor antagonist AMD3100 or the CCR5 receptor inhibitor D-Ala-peptide T-amide blocked HIV IIIB and HIV Bal neurotoxicity, respectively. In contrast, the N-methyl-D-aspartate receptor blocker MK801 failed to protect neurons from HIV-mediated apoptosis, suggesting that HIV-1 neurotoxicity can be initiated by the viral protein gp120 binding to neuronal chemokine receptors.

Fig. 1

HIVs decrease neuronal survival. CGC (a) or cortical neurons (b) were exposed to boiled HIVs (control), IIIB, or BaL (1.5 ng/ml of p24, each) for the indicated time points. Cell survival was determined by MTT assay. Data are the mean±SEM of five independent determinations each point. Pound signs p<0.01 vs control; asterisks p<0.001 vs control

Fig. 2

HIVs activate caspase-3. CGC (a) or cortical neurons (b) were exposed to IIIB or BaL for the indicated time points. The number of caspase-3-positive neurons was determined as described in “Materials and methods.” Data, expressed as percent above control (boiled HIVs), are the mean±SEM of five independent determinations each point. Pound sign p<0.05 vs control; asterisks p<0.001 vs control

Fig. 3

Activation of chemokine receptors underlies the toxic effect of HIVs. CGC were exposed to medium control (heat-inactivatcd HIVs) or medium containing HIVs alone or in combination with AMD3100 (AMD) or DAPTA. AMD or DAPTA were added 15 min prior to HIVs. The number of caspase-3-positive neurons was then determined 12 and 24 h later. Data are the mean±SEM of five independent determinations each point. Pound sign p<0.05, asterisks p<0.01 vs control; carel sign p<0.05 vs HIVs

Fig. 4

MK801 does not prevent HIV-mediated neuronal apoptosis. CGC were exposed to medium control (boiled HIVs) or medium containing IIIB or BaL, alone or in combination with MK801 (10 μM). Neuronal survival was then determined at the indicated times. Data are the mean±SEM of five independent determinations each point. Pound signs p<0.001 vs control BaL, asterisks p<0.05 vs control IIIB

Fig. 5

Neurons exposed to IIIIB internalize gp120. CGC (a) or cortical cultures (b) were exposed to IIIB for 6 h. Cells were then washed, fixed, and gp120 IR determined by histological analysis as described in “Materials and methods.” The photomicrograph shows example of a neurofilament (red) and gp120- (green, appears as yellow due to the overlay of red and green) positive cells. Arrows indicate gp120 in cell bodies, while arrowheads indicate gp120 in processes. Blue DAPI. Scale bar 25 μm in a, 50 μm in b

Fig. 6

Time-dependent internalization of gp 120 in HIV-treated neurons. CGC (a) or cortical cultures (b) were exposed to medium control (boiled HIVs) or medium containing IIIB alone or in combination with AMD3100 for the indicated time points. Cells were then fixed, and the number of gp120-positive neurons was determined as described in Fig. 5. Data are the mean±SEM of five independent determinations each point. Pound signs p<0.05, double asterisks p<0.001 vs control; asterisks p<0.05, carel signs p<0.001 vs IIIB