PDGF suppresses oxidative stress induced Ca2+ overload and calpain activation in neurons

Abstract

Oxidative stress is crucially involved in the pathogenesis of neurological diseases such as stroke and degenerative diseases. We previously demonstrated that platelet-derived growth factors (PDGFs) protected neurons from H2O2-induced oxidative stress and indicated the involvement of PI3K-Akt and MAP kinases as an underlying mechanism. Ca(2+) overload has been shown to mediate the neurotoxic effects of oxidative stress and excitotoxicity. We examined the effects of PDGFs on H2O2-induced Ca(2+) overload in primary cultured neurons to further clarify their neuroprotective mechanism. H2O2-induced Ca(2+) overload in neurons in a dose-dependent manner, while pretreating neurons with PDGF-BB for 24 hours largely suppressed it. In a comparative study, the suppressive effects of PDGF-BB were more potent than those of PDGF-AA. We then evaluated calpain activation, which was induced by Ca(2+) overload and mediated both apoptotic and nonapoptotic cell death. H2O2-induced calpain activation in neurons in a dose-dependent manner. Pretreatment of PDGF-BB completely blocked H2O2-induced calpain activation. To the best of our knowledge, the present study is the first to demonstrate the mechanism underlying the neuroprotective effects of PDGF against oxidative stress via the suppression of Ca(2+) overload and inactivation of calpain and suggests that PDGF-BB may be a potential therapeutic target of neurological diseases.

Figure 1

Effects of PDGF-AA and PDGF-BB on the H₂O₂-induced increase in [Ca²⁺]_i. [Ca²⁺]_i was determined using Fura-2-AM fluorescent dye. (a) Pseudocolor images representing the relative [Ca²⁺]_i indicated by the fluorescence ratio between 340 and 380 nm (F340/380) in individual cortical neurons 15 and 30 min after the H₂O₂ treatment. Following a 24 h preincubation with 50 ng/mL PDGF-BB, neurons were loaded with Fura-2-AM and exposed to different concentrations of H₂O₂ (5, 10, and 20 μM). The inserted bar indicates the relationship between colors and fluorescent intensity ratios at 340 and 380 nm. (b) Histogram analyses of the mean F340/380 of (a). Three pictures were taken from each well, and the means of the F340/380 of each neuron were calculated. Data are expressed as means ± SEM derived from three different sets of experiments. (c) To compare the effects of PDGF-AA and PDGF-BB on [Ca²⁺]_i overload induced by H₂O₂, cells were pretreated with 50 ng/mL PDGF-AA or PDGF-BB for 24 h followed by exposure to 10 μM H₂O₂ for 15 and 30 min. (d) Histogram analysis of (c). Data are expressed as means ± SEM of three independent experiments. ^a1 P < 0.01 and ^a2 P < 0.05 versus the untreated control; ^b P < 0.01 versus the same H₂O₂ exposure without the PDGF pretreatment; ^c1 P < 0.01 and ^c2 P < 0.05 versus the same H₂O₂ exposure with the PDGF-AA pretreatment.

Figure 2

Effects of PDGF-BB on H₂O₂-induced calpain activation. Calpain-1 activity was measured 24 h after exposure to H₂O₂ as described in the Experimental procedures. (a) Calpain activity induced by the indicated amounts of H₂O₂ (5, 10, 20, 40, and 80 μM) exposure increased calpain activation in cortical neurons in a dose-dependent manner from 5–20 μM. ((b), (c)) Calpain activity in neuronal cultures pretreated with 50 ng/mL PDGF-BB 24 h (b) and 48 h (c) before exposure to H₂O₂ PDGF-BB completely blocked calpain activity induced by H₂O₂. Data are expressed as means ± SEM of three independent experiments. ^a1 P < 0.01 and ^a2 P < 0.05 versus the untreated control; ^b1 P < 0.01 and ^b2 P < 0.05 versus the same H₂O₂ exposure without the PDGF-BB pretreatment.