PDGF-mediated protection of SH-SY5Y cells against Tat toxin involves regulation of extracellular glutamate and intracellular calcium

Abstract

The human immunodeficiency virus (HIV-1) protein Tat has been implicated in mediating neuronal apoptosis, one of the hallmark features of HIV-associated dementia (HAD). Mitigation of the toxic effects of Tat could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study we demonstrated that Tat-induced neurotoxicity was abolished by NMDA antagonist-MK801, suggesting the role of glutamate in this process. Furthermore, we also found that pretreatment of SH-SY5Y cells with PDGF exerted protection against Tat toxicity by decreasing extracellular glutamate levels. We also demonstrated that extracellular calcium chelator EGTA was able to abolish PDGF-mediated neuroprotection, thereby underscoring the role of calcium signaling in PDGF-mediated neuroprotection. We also showed that Erk signaling pathway was critical for PDGF-mediated protection of cells. Additionally, blocking calcium entry with EGTA resulted in suppression of PDGF-induced Erk activation. These findings thus underscore the role of PDGF-mediated calcium signaling and Erk phosphorylation in the protection of cells against HIV Tat toxicity.

Figure 1. Tat is toxic for SH-SY5Y cells

Effects of varying concentrations of Tat on the cell survival of SH-SY5Y cells using the MTT assay. *p<0.05 vs control group.

Figure 2. NMDA antagonist MK-801 alleviated the cytotoxicity of Tat on SH-SY5Y cells

SH-SY5Y cells were pretreated with MK801 for 60 min followed by exposure to Tat for 24 hrs and neuronal viability was measured using the MTT assay. **p<0.01 vs control group. #p<0.05 vs Tat (200 ng/ml) group.

Figure 3. PDGF exerts neuroprotection against Tat toxicity

PDGF protects SH-SY5Y cells against Tat neurotoxicity as shown by MTT assay (A) , Hoechst 33342 staining monitored by fluorescence microscopy (B) and caspase-3 activity assay (C). All the data in these figures are presented as mean±SEM of four individual experiments. *p<0.05; ***p<0.001 vs control group; #p<0.05 vs Tat (200 ng/ml) group.

Figure 4. Effect of PDGF on the extracellular glutamate levels in SH-SY5Y cell exposed to Tat

PDGF significantly decreased extracellular glutamate in SH-SY5Y cells. All the data in these figures are presented as mean±SEM of four individual experiments. *p<0.05 vs control group; #p<0.05 vs Tat (200 ng/ml) group.

Figure 5. Involvement of calcium influx in PDGF-mediated neuroprotection

Cell viability of SH-SY5Y cells exposed to Tat &/or PDGF in the absence or presence of EGTA (1 mM) was measured by MTT assay. All the data in these figures are presented as mean ± SEM of four individual experiments. *p<0.05 vs control group; #p<0.05 vs Tat-treated group; ⁺p<0.05 vs both PDGF &Tat treated group.

Figure 6. PDGF induced intracellular Ca²⁺ elevations in SH-SY5Y cells

(A) SH-SY5Y cells loaded with Fluo-4 [Ca²⁺]i sensitive fluorophores before and after PDGF treatment were recorded within a single field using a Fluoview 300 confocal microscope. Numbers in the panels indicate time in seconds. (B) Changes in intracellular [Ca²⁺]i levels in cells following PDGF treatment were measured using the Fluo-4/Fura Red ratio, and the change in ratio is illustrated from a typical neuron. All the experiments were repeated at least four times.

Figure 7. PDGF-induced Erk phosphorylation involves calcium influx

(A) SH-SY5Y cells exposed to PDGF in the presence of absence of EGTA were monitored for Erk activation. Densitometric analysis of prk/β-actin from a representative immunoblot is presented. Data are presented as mean ± SEM of four individual experiments. All of experiments were repeated at least for three times. *p<0.05 vs PDGF alone group.