TRPC1 protects human SH-SY5Y cells against salsolinol-induced cytotoxicity by inhibiting apoptosis

Abstract

Salsolinol, an endogenous neurotoxin, may be involved in the pathogenesis of Parkinson's disease. In this study, we sought to determine whether salsolinol-induced cytotoxicity in SH-SY5Y human neuroblastoma cells, a cloned cell line which expresses dopaminergic activity, could be prevented by overexpressing a Ca(2+) channel, transient receptor potential (TRPC1) protein. Exposure of SH-SY5Y cells to 500 microM salsolinol for 12 h resulted in a significant decrease in thapsigargin or carbachol-mediated Ca(2+) influx. Consistent with these results, SH-SY5Y cells treated with salsolinol showed approximately 60% reduction in TRPC1 protein levels. Confocal microscopy also showed that SH-SY5Y cells treated with salsolinol had a significant decrease in the plasma membrane staining of the TRPC1 protein. Interestingly, overexpression of TRPC1 increases TRPC1 protein levels and also protected SH-SY5Y neuroblastoma cells against salsolinol-mediated cytotoxicity as determined by 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The protective effect of TRPC1 was blocked by the addition of TRPC1 blockers lanthanum, or 2APB. Activation of TRPC1 protein by either thapsigargin or carbachol further protected SH-SY5Y cells from salsolinol treatments. Staining of SH-SY5Y cells with an apoptotic marker (YO-PRO-1) showed that TRPC1 protein protects against apoptosis. Furthermore, TRPC1 overexpression also inhibited cytochrome c release and decreased BAX protein levels required for apoptosis. Taken together, these findings suggest that the reduction in cell surface TRPC1 protein expression in response to salsolinol may be a contributory factor in cellular toxicity of the dopaminergic neurons. Furthermore, overexpression of TRPC1 could inhibit apoptotic complex thereby increasing neuronal cell survivability in Parkinson's disease.

Fig. 1

MPP⁺ and salsolinol decrease CCh- and Tg-stimulated TRPC1 activity. Ca²⁺ influx was measured in thapsigargin (Tg)- and carbachol (CCh)-stimulated SH-SY5Y cells with or without salsolinol treatment (12 h). (A, C) Fluorescence traces in either Tg (A)- or CCh (C)-treated cells in Ca²⁺ containing media. (B) Release of Ca²⁺ from intracellular stores in control and salsolinol-treated cells. (D) Average values. *Values that are significantly different from that of the respective control condition (P < 0.05, number of cells is indicated in each case). (E) Fluorescence traces of Ba²⁺ influx in control and salsolinol-treated cells.

Fig. 2

Salsolinol treatment decreases TRPC1 levels whereas overexpression of TRPC1 protects SH-SY5Y cells. (A) Western blot on crude membranes prepared from SH-SY5Y cells treated with salsolinol (500 μM) in a time-dependent manner (0, 12 and 24 h). Upper blot was probed using anti-TRPC1 antibody and the lower portion with anti-actin antibodies. (B) Anti-TRPC1 antibody and rhodamine-conjugated secondary antibody were used to detect endogenous TRPC1 protein in control- and salsolinol-treated cells. (C) Western blot on crude membranes prepared from control SH-SY5Y cells, or TRPC1 overexpressing cells treated with 500 μM of salsolinol for 12 h. (D) Bar graph indicating quantitative analysis of TRPC1 and actin proteins.

Fig. 3

Activation and overexpression of TRPC1 increases protection against salsolinol. (A) MTT assays were performed on control and TRPC1 overexpressing cells treated with 500 μM of salsolinol. (B) Represents bar graph showing percent cell survival as detected via MTT assay. These data are an average of 3 independent experiments performed in triplicate. *Significant values (P < 0.05). Cells were transiently transfected (for 24 h) with Ad-TRPC1 encoding virus. Other details of the experiment are provided in the Experimental procedures section. CCh, Tg, La³⁺ and 2APB were added 10 min prior to the addition of salsolinol. Marker for necrosis (PI staining) and apoptosis (YO-PRO-1) were used for staining of control or cell overexpressing TRPC1 with or without salsolinol treatment (C). Rhodamine-conjugated propidium iodide and FITC-conjugated YO-PRO-1 was added to control cells or cells treated with salsolinol for 12 h. Fluorescence images were taken immediately using either a 10× or 40× objective and the red and green cells were counted. (D) Represents mean bar graph from 700 to 900 cells in each group. *Values significantly different from its counterpart (P < 0.05).

Fig. 4

Overexpression of TRPC1 decreases protein required for apoptotic pathway. (A) Western blot performed on mitochondrial membrane fractions. Membrane fractions (25 μg) were resolved on a 4–20% SDS gel and probed with various antibodies as indicated. Bound cytochrome c in the mitochondria was assayed using anti-cytochrome c antibody (upper panel). Bax protein was identified using anti-Bax antibody. Similarly, Apaf1 and Actin antibodies were used to detect individual proteins, respectively. Details about the antibodies and method are described in the Experimental procedures section. (B) The proposed model for the neuroprotective role of TRPC1 in PD.