Erinacine A-Enriched Hericium erinaceus Mycelium Ethanol Extract Lessens Cellular Damage in Cell and Drosophila Models of Spinocerebellar Ataxia Type 3 by Improvement of Nrf2 Activation

Abstract

Spinocerebellar ataxia type 3 (SCA3), caused by the abnormal expansion of polyglutamine (polyQ) in the ataxin-3 protein, is one of the inherited polyQ neurodegenerative diseases that share similar genetic and molecular features. Mutant polyQ-expanded ataxin-3 protein is prone to aggregation in affected neurons and is predominantly degraded by autophagy, which is beneficial for neurodegenerative disease treatment. Not only does mutant polyQ-expanded ataxin-3 increase susceptibility to oxidative cytotoxicity, but it also hampers antioxidant potency in neuronal cells. Nuclear factor erythroid-derived 2-like 2 (Nrf2), a master transcription factor that controls antioxidant and detoxification gene expression, plays a crucial role in neuroprotection in SCA3 and other neurodegenerative diseases. The present data showed that treatment with erinacine A-enriched Hericium erinaceus mycelium ethanol extract (HEME) extended longevity and improved locomotor activity in ELAV-SCA3tr-Q78 transgenic Drosophila. Moreover, HEME treatment enhanced antioxidant potency and autophagy, which, in turn, corrected levels of mutant polyQ-expanded ataxin-3 and restrained protein aggregation in both cell and Drosophila models of SCA3. Markedly, HEME increased the activation of Nrf2. Silencing Nrf2 protein expression negated most of the promising effects of HEME on SK-N-SH-MJD78 cells, highlighting the critical role of increased Nrf2 activation in the efficacy of HEME treatment. These findings suggest that HEME has therapeutic potential in SCA3 by enhancing autophagic and Nrf2-mediated antioxidant pathways, which may also influence neurodegenerative progression in other polyQ diseases.

Keywords: Nrf2; PolyQ diseases; autophagy; erinacine A-enriched Hericium erinaceus mycelium; mutant polyQ-expanded ataxin-3; oxidative stress; spinocerebellar ataxia type 3.

Figure 1

Effects of HEME on longevity in ELAV-SCA3tr-Q78 transgenic Drosophila. Longevity was compared across groups by Kaplan–Meier log rank analysis. The X-axis represents the mean life-span, and SDs are shown, * p < 0.001 (n = 320).

Figure 2

Effects of HEME on locomotor activity in ELAV-SCA3tr-Q78 transgenic Drosophila. * Values are means ± SD, n = 40 flies in three separate experiments. An asterisk (*) indicates a significant difference between ELAV-SCA3tr-Q27 flies and ELAV-SCA3tr-Q78 flies. In ELAV-SCA3tr-Q78 flies within the same age group, values not sharing the same letter are significantly different (p < 0.001).

Figure 3

Effects of HEME on ELAV-SCA3tr-Q78 transgenic Drosophila. (A,B) Mutant and normal ataxin-3, Hsp27, p62, Beclin-1, LC3-II, Nrf2, HO-1, NQO1, GR, catalase, GPx1, GPx2, SOD1, and SOD2 protein expression in 19-day-old ELAV-SCA3tr-Q27 and ELAV-SCA3tr-Q78 flies. Values are means ± SD, n = 50 flies in three separate experiments. The values are expressed as the percentage of ELAV-SCA3tr-Q78 flies treated with the vehicle control. An asterisk (*) indicates a significant difference between ELAV-SCA3tr-Q27 flies and ELAV-SCA3tr-Q78 flies. In ELAV-SCA3tr-Q78 fly groups, values not sharing the same letter are significantly different (p < 0.001).

Figure 4

Effect of HEME on protein expression of mutant ataxin-3 and Hsp27 in SK-N-SH-MJD78 cells. Cells were treated with or without DMSO vehicle control or HEME for 24 h. Data are means ± SDs of at least three independent experiments and are expressed as the percentage of SK-N-SH-MJD78 cells treated with the vehicle control. Values not sharing the same letter are significantly different (p < 0.0001).

Figure 5

Effect of HEME on autophagy induction in SK-N-SH-MJD78 cells. Cells were treated with or without DMSO vehicle control or with 1.25–5 µg/mL HEME for 24 h. (A) Protein expression of p62, Beclin-1, and LC3-II. (B) Autophagic cells were assessed using acridine orange staining. Data are means ± SDs of at least three independent experiments and are expressed as the percentage of SK-N-SH-MJD78 cells treated with the vehicle control. Values not sharing the same letter are significantly different (p ≤ 0.0001).

Figure 6

Effects of HEME on ROS, GSH levels, catalase, GPx and SOD activity in SK-N-SH-MJD78 cells. SK-N-SH-MJD78 cells were treated with or without DMSO vehicle control or HEME for 24 h, except for measurement of H2DCFDA (30 h treatment). Values are expressed as the percentage of SK-N-SH-MJD78 cells treated with the vehicle control. Data are means ± SD of at least three separate experiments and not sharing the same letter are significantly different (p < 0.01 and H2DCFDA; p = 0.012).

Figure 7

Effect of HEME on antioxidant enzyme expression and Nrf2 activation in SK-N-SH-MJD78 cells. (A,B) Cells were treated with or without DMSO vehicle control or with HEME for 24 h. Protein expression of HO-1, NQO1, GPx1, GPx2, GR, catalase, SOD1, SOD2, and nuclear Nrf2. (C) Before treatment with HEME, cells were transfected with ARE-luciferase reporter construct for 16 h. Data are means ± SD of at least three independent experiments and are expressed as the percentage of SK-N-SH-MJD78 cells treated with the vehicle control. Values not sharing the same letter are significantly different (p < 0.0001).

Figure 7

Effect of HEME on antioxidant enzyme expression and Nrf2 activation in SK-N-SH-MJD78 cells. (A,B) Cells were treated with or without DMSO vehicle control or with HEME for 24 h. Protein expression of HO-1, NQO1, GPx1, GPx2, GR, catalase, SOD1, SOD2, and nuclear Nrf2. (C) Before treatment with HEME, cells were transfected with ARE-luciferase reporter construct for 16 h. Data are means ± SD of at least three independent experiments and are expressed as the percentage of SK-N-SH-MJD78 cells treated with the vehicle control. Values not sharing the same letter are significantly different (p < 0.0001).

Figure 8

Effect of HEME in SK-N-SH-MJD78 cells transiently transfected with si-Nrf2. Cells were transiently transfected with si-NTC or si-Nrf2 as well as with or without ARE-luciferase reporter construct for 16 h and then treated with or without DMSO vehicle control or 1.25–5 µg/mL HEME for 24 h. Protein expression of (A) Nrf2, mutant and normal ataxin-3, and Hsp27, (B) p62, Beclin-1, and LC3-II. (C) Levels of ARE–luciferase reporter gene activities, GSH, and protein aggregations, as well as activity of catalase, GPx, and SOD. Data are means ± SD of at least three separate experiments. Within treatments with the same plasmid transfection, values are expressed as the percentage of SK-N-SH-MJD78 cells treated with the vehicle control, and values not having the same letter are significantly different (p < 0.0001).

Figure 8

Effect of HEME in SK-N-SH-MJD78 cells transiently transfected with si-Nrf2. Cells were transiently transfected with si-NTC or si-Nrf2 as well as with or without ARE-luciferase reporter construct for 16 h and then treated with or without DMSO vehicle control or 1.25–5 µg/mL HEME for 24 h. Protein expression of (A) Nrf2, mutant and normal ataxin-3, and Hsp27, (B) p62, Beclin-1, and LC3-II. (C) Levels of ARE–luciferase reporter gene activities, GSH, and protein aggregations, as well as activity of catalase, GPx, and SOD. Data are means ± SD of at least three separate experiments. Within treatments with the same plasmid transfection, values are expressed as the percentage of SK-N-SH-MJD78 cells treated with the vehicle control, and values not having the same letter are significantly different (p < 0.0001).

Figure 8

Effect of HEME in SK-N-SH-MJD78 cells transiently transfected with si-Nrf2. Cells were transiently transfected with si-NTC or si-Nrf2 as well as with or without ARE-luciferase reporter construct for 16 h and then treated with or without DMSO vehicle control or 1.25–5 µg/mL HEME for 24 h. Protein expression of (A) Nrf2, mutant and normal ataxin-3, and Hsp27, (B) p62, Beclin-1, and LC3-II. (C) Levels of ARE–luciferase reporter gene activities, GSH, and protein aggregations, as well as activity of catalase, GPx, and SOD. Data are means ± SD of at least three separate experiments. Within treatments with the same plasmid transfection, values are expressed as the percentage of SK-N-SH-MJD78 cells treated with the vehicle control, and values not having the same letter are significantly different (p < 0.0001).