The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease

Abstract

In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1 (SIAH-1) in PC12 cells. 1-Methyl-4-phenylpyridinium (MPP(+)) treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased mRNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 mRNA expression. It also increased cell viability. Combined treatment with MPP(+) and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson's disease.

Keywords: 1-methyl-4-phenylpyridinium; E3 ubiquitin ligase seven in absentia homolog 1; Parkinson's disease; autophagy; nerve regeneration; neural regeneration; neurodegeneration; rapamycin; ubiquitin-proteasome system.

Figure 1

MTT assay for cell viability after treatment with MPP⁺, RAPA and SIAH-1 antibody. RAPA and SIAH-1 antibody treatment had no significant effect on cell viability under normal growth conditions (P > 0.05). In the MPP⁺ group, RAPA and SIAH-1 antibody significantly increased cell viability (##P < 0.01, vs. MPP⁺ group; mean ± SD, n ≥ 6, one-way analysis of variance followed by Fisher's least significant difference post hoc test). I: Control group; II: RAPA group; III: anti-SIAH-1 group; IV: MPP⁺ group; V: MPP⁺ RAPA group; VI: MPP⁺ anti-SIAH-1 group. MTT: 3-(4,5-Cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MPP⁺: 1-methyl-4-phenylpyridinium; RAPA: rapamycin; SIAH-1: E3 ubiquitin ligase seven in absentia homolog 1.

Figure 2

Effects of SIAH-1 antibody treatment on α-synuclein and the autophagy degradation pathway in PC12 cells. Western blot assay for protein levels (upper panel) and statistical analysis of optical density measurements (target protein/β-actin; lower panel) in PC12 cells after treatment with MPP⁺, RAPA and SIAH-1 antibody. (A) SIAH-1 (37 kDa); (B) α-synuclein (19 kDa); (C) p53 (55 kDa); (D) LC3-II (17 kDa); (E) E1 (117 kDa). Values are represented as the mean ± SD (n = 5) (one-way analysis of variance followed by Fisher's least significant difference post hoc test). #P < 0.05, ##P < 0.01, vs. control group (MPP⁺, RAPA and anti-SIAH-1 are “–”). **P < 0.01, vs. MPP⁺ group (MPP⁺ is “+”; RAPA and anti-SIAH-1 are “–”). SIAH-1: E3 ubiquitin ligase seven in absentia homolog 1; LC3: light chain 3; MPP⁺: 1-methyl-4-phenylpyridinium; RAPA: rapamycin.

Figure 3

Change in α-synuclein and SIAH-1 in PC12 cells in response to MPP⁺. (A) Immunolabeling for α-synuclein (green) and SIAH-1 (red) or DAPI staining (blue) in PC12 cells. Image overlays are shown in the third column. MPP⁺ treatment caused an increase in SIAH-1 and α-synuclein fluorescence intensities (laser scanning confocal microscope; scale bars: 30 μm). (B) Quantification of fluorescence intensities of α-synuclein and SIAH-1. At least 30 cells were included for analysis from five images per group. Values are presented as the mean ± SD (n = 5; one-way analysis of variance followed by Fisher's least significant difference post hoc test). **P < 0.01, vs. control group (normal growth of cells). SIAH-1: E3 ubiquitin ligase seven in absentia homolog 1; MPP⁺: 1-methyl-4-phenylpyridinium; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 4

Colocalization of α-synuclein, SIAH-1 and microtubule-associated protein LC3 in PC12 cells after SIAH-1 antibody treatment. (A, C) Laser scanning confocal microscope; scale bars: 30 μm. Immunoreactivity for α-synuclein, SIAH-1 and LC3 decreased after treatment with SIAH-1 antibody, and colocalization of α-synuclein and LC3 was lost, but α-synuclein and SIAH-1 retained their colocalization. (A) Immunostaining for α-synuclein (green), microtubule-associated protein LC3 (red) and DAPI (blue). (B) Quantification of immunostaining results in A. (C) Immunostaining for α-synuclein (green), SIAH-1 (red) and DAPI (blue). (D) Quantification of immunostaining results in C. Values are presented as the mean ± SD (n = 5; one-way analysis of variance followed by Fisher's least significant difference post hoc test). **P < 0.01, vs. control group (normal growth of cells). SIAH-1: E3 ubiquitin ligase seven in absentia homolog 1; LC3: light chain 3; DAPI: 4′,6-diamidino-2-phenylindole.