SNJ-1945, a calpain inhibitor, protects SH-SY5Y cells against MPP(+) and rotenone

Abstract

Complex pathophysiology of Parkinson's disease involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in Parkinson's disease and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. To unravel these mechanisms SH-SY5Y neuroblastoma cells were differentiated into dopaminergic and cholinergic phenotypes, respectively, and used as cell culture model following exposure to two parkinsonian neurotoxicants MPP(+) and rotenone. SNJ-1945, a cell-permeable calpain inhibitor was tested for its neuroprotective efficacy. MPP(+) and rotenone dose-dependently elevated the levels of intracellular free Ca(2+) and induced a concomitant rise in the levels of active calpain. SNJ-1945 pre-treatment significantly protected cell viability and preserved cellular morphology following MPP(+) and rotenone exposure. The neurotoxicants elevated the levels of reactive oxygen species more profoundly in SH-SY5Y cells differentiated into dopaminergic phenotype, and this effect could be attenuated with SNJ-1945 pre-treatment. In contrast, significant levels of inflammatory mediators cyclooxygenase-2 (Cox-2 and cleaved p10 fragment of caspase-1) were up-regulated in the cholinergic phenotype, which could be dose-dependently attenuated by the calpain inhibitor. Overall, SNJ-1945 was efficacious against MPP(+) or rotenone-induced reactive oxygen species generation, inflammatory mediators, and proteolysis. A post-treatment regimen of SNJ-1945 was also examined in cells and partial protection was attained with calpain inhibitor administration 1-3 h after exposure to MPP(+) or rotenone. Taken together, these results indicate that calpain inhibition is a valid target for protection against parkinsonian neurotoxicants, and SNJ-1945 is an efficacious calpain inhibitor in this context. SH-SY5Y cells, differentiated as dopaminergic (TH positive) and cholinergic (ChAT positive), were used as in vitro models for Parkinson's disease. MPP+ and rotenone induced up-regulation of calpain, expression, and activity as a common mechanism of neurodegeneration. SNJ-1945, a novel calpain inhibitor, protected both the cell phenotypes against MPP+ and rotenone.

Keywords: calpain; cell viability; experimental parkinsonism; inflammation; neuroprotection; oxidative stress.

Fig. 1

A Elevation of free [Ca²⁺]_i levels in SH-SY5Y-DA cells. Following exposure to different concentrations of MPP⁺ (50, 100 or 500 µM) and rotenone (10, 50 or 100 nM) for 24 h, cells were loaded with ratiometric dye Fura-2AM. A dose-dependent rise in [Ca²⁺]_i was recorded by monitoring the emission at 510 nm and dual excitement at 340 and 380 nm respectively. Bar graphs represent the % increase of [Ca²⁺]_i compared to the control cells; data are expressed as mean ± SEM (n ≥ 3); *p ≤ 0.05, significantly different from control. B Upregulation of calpain in SH-SY5Y-DA cells. Representative images from three independent experiments (n = 3) illustrate that active µ-calpain IR (green) was significantly increased in SH-SY5Y-DA cells following MPP⁺ (100 µM) and rotenone (50 nM) exposure, compared to the control cells.

Fig. 2

A-B SNJ-1945-mediated protection of viability in SH-SY5Y-DA cells. MTT assay performed following 24 h exposure to MPP⁺ (50–500 µM) or rotenone (10–100 nM) showed reduction in cell viability in a dose-dependent manner. Pre-treatment with SNJ-1945 (100 or 250 µM) was found protective, whereas the lowest dose used (10 µM) was ineffective. Bar graphs represent data expressed as mean ± S.E.M. of cell viability (%), obtained from 4 to 6 independent assays; *p ≤ 0.05, significantly different from viability of control cells; ^@p ≤ 0.05 significantly different from viability of neurotoxicant-exposed cells respectively.

Fig. 2

A-B SNJ-1945-mediated protection of viability in SH-SY5Y-DA cells. MTT assay performed following 24 h exposure to MPP⁺ (50–500 µM) or rotenone (10–100 nM) showed reduction in cell viability in a dose-dependent manner. Pre-treatment with SNJ-1945 (100 or 250 µM) was found protective, whereas the lowest dose used (10 µM) was ineffective. Bar graphs represent data expressed as mean ± S.E.M. of cell viability (%), obtained from 4 to 6 independent assays; *p ≤ 0.05, significantly different from viability of control cells; ^@p ≤ 0.05 significantly different from viability of neurotoxicant-exposed cells respectively.

Fig. 3

SNJ-1945-mediated protection of SH-SY5Y-DA cell morphology. In situ Wright staining assay was used to distinguish apoptotic cells following 24 h exposure to MPP⁺ (100 µM) or rotenone (50 nM) with/without pre-treatment with SNJ-1945 (100 or 250 µM). Representative images show healthy control and cells treated with SNJ-1945 (250 µM), (A, B), which were significantly altered (shrunken) with MPP⁺ (100 µM), (C) or rotenone (50 nM), (D); Pre-treatment with SNJ-1945 (100 or 250 µM) attenuated MPP⁺-induced (D, E respectively) and rotenone-induced (G, H respectively) alterations in cell morphology. Images were captured by light microscope at 200× magnification.

Fig. 4

Rise in ROS in SH-SY5Y-DA cells. (A) In situ profiles of ROS in cells following MPP⁺ (100 µM) or rotenone (50 nM) exposure for 24 h with respect to control in upper panel and pre-treatment with SNJ-1945 (250 µM) in lower panel. (B) Cumulative levels of ROS after prolonged 72 h exposure to neurotoxicants and SNJ-1945-mediated protection against elevated levels of ROS in SH-SY5Y-DA cells.

Fig. 5

Immunofluorecent images of tyrosin hydroxylase (TH) staining in SH-SY5Y cells. The TH staining was performed to confirm dopaminergic phenotype of SH-SY5Y cells differentiated with RA and PMA. Greater levels of TH IR (red) were observed in SH-SY5Y cells differentiated with RA and PMA, compared to the low intensity in undifferentiated SH-SY5Y cells and cells differentiated with RA only. Images were captured by fluorescent microscope at 200× magnification.

Fig. 6

Induction of inflammatory mediators in SH-SY5Y-ChAT cells. Significantly elevated levels of caspase-1, p10 cleaved fragment of caspase-1 and Cox-2 was found in SH-SY5Y-ChAT cells; SNJ-1945 dose-dependently protected against the effects of both MPP⁺ (A, B) and rotenone (C, D); quantifications are represented in the right panels correspondingly (B, D). Representative immunoblots from three independent experiments (n = 3) and corresponding bar graphs showed significantly increased levels of Cox-2, caspase-1 and cleaved p10 fragment in MPP⁺ or rotenone-exposed SH-SY5Y-ChAT cells (*p ≤ 0.05, compared to control). SNJ-1945 relatively reduced levels of Cox-2, caspase-1, and cleaved p10 fragment in SH-SY5Y-ChAT cells (^@p ≤ 0.05, relative to neurotoxicant-exposed cells). SNJ-1945 (100 or 250 µM) was found effective against MPP⁺ or rotenone, whereas 50 µM SNJ-1945 had limited efficacy.

Fig. 7

Altered calpain activity but not caspase-3 in SH-SY5Y-DA cells. Immunoblot analysis of caspase-3 failed to discern any MPP⁺ (100 µM) or rotenone (50 nM)-induced alterations in the active bands of caspase-3; this was further confirmed by unaltered levels of 120 kDa SBDP amongst different groups. Calpain-specific 145 kDa SBDP showed distinct upregulation by MPP⁺ or rotenone (*p ≤ 0.05, compared to control); this effect could be significantly attenuated by SNJ-1945 pre-treatment (^@p ≤ 0.05, relative to neurotoxicant-exposed cells). Differentiation regimen (RA/PMA or RA/BDNF) had not influence either, (n = 3).

Fig. 8

Altered profiles of protease expression and activity in SH-SY5Y-ChAT cells. Upregulation of proteases (caspase-3, -8) levels were seen in MPP⁺ (100 µM) or rotenone (50 nM) exposed cells; SNJ-1945 dose-dependently attenuated this response as shown in (A) and the densitometric analysis in (B, C). MPP⁺ and rotenone also induced activation of calpain and caspase-3 observed as elevation of 145 kDa and 120 kDa SBDP respectively in (A) and the corresponding densitometric analysis in (D, E); (n = 3); (*p ≤ 0.05, compared to control) and (^@p ≤ 0.05, relative to neurotoxicant-exposed cells).