Platelet-derived growth factor protects neurons against gp120-mediated toxicity

Abstract

The human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 has been implicated in mediating neuronal apoptosis, a hallmark feature of HIV-associated dementia (HAD). Mitigation of the toxic effects of gp120 could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study the authors hypothesized that neurotrophic factor, such as platelet-derived growth factor (PDGF), could protect the neurons against gp120-mediated apoptosis. SH-SY5Y cells treated with gp120 exhibited increased cell death when measured by lactate dehydrogenase (LDH) and deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, with concomitant loss of neurites and increased cell rounding. Pretreatment with PDGF-BB, however, reduced gp120-associated neurotoxicity and rescued the neurite outgrowth. Additionally, gp120-mediated activation of caspase-3 was also significantly reduced in cells pretreated with PDGF-BB. Antiapoptotic effects of PDGF-BB were also confirmed by monitoring levels of anti- and proapoptotic genes, Bcl-xL and Bax, respectively. Furthermore, PDGF-mediated protection against gp120 involved the phosphoinositide (PI) 3-kinase/Akt pathway. Taken together these findings lead us to suggest that PDGF-BB could be considered as a therapeutic agent that can mitigate gp120-mediated neurotoxicity in HAD.

Figure 1

Decrease of PDGF-B chain in neurons of macaque brain with SHIV-encephalitis. Representative brain sections from the basal ganglia region of an uninfected (A) and SHIV-E (B) macaque immunostained with PDGF-B chain antibody. Red color represents PDGF-B chain positive cells. Degenerated neurons are shown in circle.

Figure 2

Down regulation of PDGF-B chain in neurons exposed to gp120. RT-PCR analysis for PDGF-B chain was done on total RNA extracted from SH-SY5Y cells (A) or primary rat neurons (B) exposed to 200ng/ml gp120 for 24h. C) Western blot analysis of PDGF-B chain on cell lysate obtained from SH-SY5Y cells treated with or without gp-120 (200ng/ml) for 24hrs. Figure shown is a representative of three different experiments.

Figure 3

PDGF protects SH-SY5Y cells against gp120-mediated toxicity. A) Phase contrast microscopy of SH-SY5Y cells treated with gp120 (200ng/ml) and/or PDGF-BB (20ng/ml) for 5 days. Cells treated with gp120 alone showed rounding (as shown by black arrows) with shortened neurites. Pre-treatment of gp120-treated neurons with PDGF-BB resulted in maintenance of cellular architecture with extended neuronal processes (indicated by white arrows). Scale bars, 50 μM. Neuronal viability in the presence of gp120 and/or PDGF-BB. SH-SY5Y cells (B) and rat primary neuronal culture (C) were exposed to gp120 (200ng/ml) with or without pre-treatment with PDGF-BB (20ng/ml). Supernatant fluids and cellular extracts were collected in 24h for LDH assay. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was performed using Student’s t-test. (*p < 0.01 gp120 vs control, #p<0.01 gp120 vs PDGF+gp120).

Figure 4

PDGF-BB mediates neuronal protection against gp120. A) SH-SY5Y cells treated with or without gp120 (200ng/ml) in the presence or absence of PDGF-BB (20ng/ml) for 18h were stained for TUNEL. Scale bar, 100 μM B) Histogram showing the percentage of TUNNEL positive SH-SY5Y cells relative to total number of neurons following various treatments. Six to eight images were randomly taken for counting positive signal in each experiment. Values are presented as mean ± SEM. (* p<0.01 gp120 vs control, #p<0.001 gp120 vs PDGF+gp120).

Figure 5

Neuronal apoptosis in SH-SY5Y cells exposed to gp120 with or without PDGF-BB pretreatment. Cells treated with or without gp120 IIIB in presence or absence of PDGF-BB pre-treatment were monitored for: A) caspase-3 activity assay in cell lysates and B) active caspase-3 staining by immunocytochemistry using the anti-cleaved caspase-3 antibody. The immunochemistry images showing nuclear (upper panel) and active caspase-3 staining (lower panel) were captured on Nikon inverted fluorescence microscope TE2000-E with a 20x and 100x (inset) objective (scale bar, 100 μM). Data for caspase-3 activity represents the mean ± SEM from seven independent experiments (*P<0.05 gp120 vs control, #P<0.01 gp120 vs PDGF+gp120).

Figure 6

Ratio of anti- and pro-apoptotic markers in PDGF-mediated neuroprotection of SH-SY5Y cells. A) Cells treated with or without gp120 in the absence or presence of PDGF-BB pre-treatment were lysed and assayed for levels of anti- and pro-apoptotic markers Bcl-xL and Bax, respectively by Western blot analysis in the same membrane. B) Densitometry scan of the ratio of band intensities of Bcl-xL/Bax from three different western blot experiments (*p<0.05, treatment vs control).

Figure 7

Involvement of PI3k/Akt pathway in PDGF-BB mediated protection against gp120 toxicity. A) LDH assay of SH-SY5Y cells pretreated with or without PI3K inhibitor (LY294002) or MEK1/2 inhibitor (U0126) followed by PDGF and/or gp120 exposure. Pre-treatment of cells with PI3K inhibitor resulted in significant (P value <0.05 PDGF+gp120+LY vs PDGF+gp120) reduction of PDGF-mediated protection. Pre-treatment with Erk1/2 inhibitor did not reverse the protective effect of PDGF. Data represents mean ± SEM from three independent experiments. B) Western blot analysis of lysates of phosphorylated Akt and Ek1/2 in SH-SY5Y cells pretreated with or without PI3K inhibitor (10uM LY294002) prior to PDGF and/or gp120 exposure. Blots were reprobed with antibody specific for β-actin for normalization.