p-Coumaric Acid Has Protective Effects against Mutant Copper-Zinc Superoxide Dismutase 1 via the Activation of Autophagy in N2a Cells

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motor neurons. In previous our study, an ethanol extract of Brazilian green propolis (EBGP) prevented mutant copper-zinc superoxide dismutase 1 (SOD1^mut)-induced neurotoxicity. This paper aims to reveal the effects of p-coumaric acid (p-CA), an active ingredient contained in EBGP, against SOD1^mut-induced neurotoxicity. We found that p-CA reduced the accumulation of SOD1^mut subcellular aggregation and prevented SOD1^mut-associated neurotoxicity. Moreover, p-CA attenuated SOD1^mut-induced oxidative stress and endoplasmic reticulum stress, which are significant features in ALS pathology. To examine the mechanism of neuroprotective effects, we focused on autophagy, and we found that p-CA induced autophagy. Additionally, the neuroprotective effects of p-CA were inhibited by chloroquine, an autophagy inhibiter. Therefore, these results obtained in this paper suggest that p-CA prevents SOD1^mut-induced neurotoxicity through the activation of autophagy and provides a potential therapeutic approach for ALS.

Keywords: amyotrophic lateral sclerosis; autophagy; copper–zinc superoxide dismutase 1; endoplasmic reticulum stress; oxidative stress; p-coumaric acid.

Figure 1

p-Coumaric acid (p-CA) reduced subcellular aggregation of mutant copper–zinc superoxide dismutase 1 (SOD1^mut) and protected against SOD1^mut-induced neurotoxicity. (A) Imaging of cytoplasmic mCherry–SOD1 aggregates (white arrowheads) in the presence or absence of p-CA in N2a cells. (B) Quantified analysis of imaging. (C) Immunoblot analysis of mCherry with a 1% Triton X-100 insoluble fraction. (D) Densitometric quantification of mCherry. (E) Cell viability was measured by MTT assay. (F) Cell toxicity was measured by LDH assay. Differences were evaluated by one-way ANOVA (mean ± SEM, n = 3). *** p < 0.001 vs. SOD1^WT, ^††† p < 0.001, ^† p < 0.05 vs. SOD1^G85R. p-CA: p-coumaric acid. Scale bar: 10 µm.

Figure 2

p-CA reduced mutant-SOD1-related oxidative stress. (A) Confocal imaging of CellROX in N2a cells transfected with mCherry–SOD1^G85R with p-CA for 24 h. (B) Quantified analysis of CellROX using Image J. (C) Confocal imaging of MitoSOX in N2a cells transfected with mCherry–SOD1^G85R with p-CA for 24 h. (D) Quantified analysis of MitoSOX using Image J. (E) Typical spectra of DMPO-OH spin generated from H₂O₂ plus Fe²⁺ in the absence (control) or presence of p-CA. (F) The amount of hydroxy radicals was semi-quantitatively measured as the formation of DMPO-OH spin adducts by ESR spectrometry. Differences were evaluated by one-way ANOVA (mean ± SEM, n = 3). *** p < 0.001 vs. SOD1^WT, ^†† p < 0.01, ^† p < 0.05 vs. SOD1^G85R. Scale bar: 10 µm. p-CA: p-coumaric acid.

Figure 3

p-CA reduced SOD1^mut-related ER stress. (A) Immunoblot analysis of BiP and CHOP relating to ER stress. (B,C) Densitometric quantification of BiP (B) and CHOP (C). Differences were evaluated by one-way ANOVA (mean ± SEM, n = 3). *** p < 0.001 vs. SOD1^WT, ^††† p < 0.001, ^† p < 0.05 vs. SOD1^G85R. p-CA: p-coumaric acid.

Figure 4

p-CA prevented SOD1^mut-associated neurotoxicity through autophagy. (A) Immunoblot analysis of LC3 and p62 relating to autophagy. (B) Densitometric quantification of LC3 and p62. ^††† p < 0.001 vs. control. (C) Immunoblot analysis of LC3 and p62 relating to autophagy with SOD1^G85R-expressing cells. (D) Densitometric quantification of LC3 and p62. ^††† p < 0.001 vs. control. (E) Imaging of cytoplasmic mCherry–SOD1 aggregates (white arrowheads) in the N2a cells with CQ (1 nM) before p-CA (100 nM) treatment. (F) Quantified analysis of imaging. (G) Cell viability was measured by MTT assay. (H) Cell toxicity was measured by LDH assay. Differences were evaluated by one-way ANOVA (mean ± SEM, n = 3). *** p < 0.001 vs. SOD1^WT, ^††† p < 0.001, ^†† p < 0.01, ^† p < 0.05. vs. SOD1^G85R, ^### p < 0.001, ^# p < 0.05. vs. SOD1^G85R + p-CA. Scale bar: 10 µm. p-CA: p-coumaric acid, CQ: chloroquine.