Histone decacetylase inhibitors prevent mitochondrial fragmentation and elicit early neuroprotection against MPP+

Abstract

Background: Parkinson's disease (PD) is a common neurodegenerative disease, characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra. Recent investigations have shown that mitochondrial fragmentation, an early event during apoptosis, is implicated in the degeneration of DA neurons in PD, and more importantly, preventing mitochondrial fragmentation could rescue cell death in several PD models. Therefore, mitochondrial dynamics may be a therapeutic target for early intervention in PD. However, much remains unknown about the mechanism underlying mitochondrial fragmentation in PD.

Methods: The alterations in mitochondrial morphology, cell apoptosis, and mitochondrial shaping protein levels were detected after SH-SY5Y cells were treated with various doses of MPP+ or rotenone.

Results: Mitochondrial fragmentation is an early event during apoptosis caused by MPP+ but not rotenone, and Trichostatin A (TSA), a commonly used histone deacetylase (HDAC) inhibitor, selectively rescues mitochondrial fragmentation and cell death induced by lower doses of MPP+. Mitochondrial fragmentation triggered by lower doses of MPP+ may be a result of Mfn2 down-regulation, which could be completely reversed by TSA. Further investigation suggests that TSA prevents MPP+-induced Mfn2 down-regulation via inhibiting histone deacetylation over Mfn2 promoter and alleviating its transcriptional dysfunction.

Conclusions: Histone deacetylase inhibitors prevent mitochondrial fragmentation and elicit early neuroprotection in PD cell model induced by MPP+. Hence, HDAC inhibitors may be a potential early treatment for PD.

Keywords: Early neuroprotection; Histone deacetylase inhibitors; Mitochondrial fragmentation; Parkinson's disease.

Figure 1

The effects of histone deacetylase inhibitors on mitochondrial fragmentation and cell apoptosis in MPP+ or rotenone cell model. After transfected with a mitochondria‐targeted RFP for 24 h, SH‐SY5Y cells were treated with different doses of MPP+ (1 mM, M1; 2 mM, M2; 3 mM, M3) or rotenone (1 μM, R1; 2 μM, R2; 3 μM, R3). Representative images taken by live cell imaging system (A) and quantitative analysis of mitochondrial length (C) show that MPP+ and rotenone both induce mitochondrial fragmentation in SH‐SY5Y cells in a dose‐ and time‐dependent fashion, except that mitochondria are not shortened but even slightly elongated in R1 group. Flow cytometry results (D) demonstrate that mitochondrial fragmentation is triggered prior to cell apoptosis in the MPP+ but not yet in the rotenone model. Co‐treatment with 0.1 nM Trichostatin A (T) or 0.1 μM suberoylanilide hydroxamic acid (SAHA, S) for 8 h selectively rescues mitochondrial fragmentation (B, E, G) and cell apoptosis (F, H) in the cells exposed to 1 or 2 mM MPP+, but does not have protective effect in other groups. n = 4; *P < 0.05, **P < 0.01 versus control; ^# P < 0.05 T + M/S + M group versus M group of the same dose. Scale bars for 10 μm.

Figure 2

Trichostatin A (TSA) blocks the down‐regulation of Mfn1 and Mfn2 in MPP+ cell model. SH‐SY5Y cells were treated with different doses of MPP+ (1 mM, M1; 2 mM, M2; 3 mM, M3) or rotenone (1 μM, R1; 2 μM, R2; 3 μM, R3). Representative immunoblotting images and semi‐quantitative analysis of mitochondrial shaping protein levels (Drp1, Mfn1, Mfn2, Opa1, and Fis1) (A, B) show that Mfn1 and Mfn2 levels are both decreased in a dose‐ and time‐dependent fashion in MPP+‐exposed cells. Total Drp1 levels are not affected during drug exposure, while mitochondrial Drp1 levels are elevated in M3, R2, and R3 groups. Co‐treatment with 0.1 nM TSA (T) for 8 h entirely blocks the down‐regulation of Mfn1 and Mfn2 induced by MPP+, yet has no influence on Drp1 mitochondrial recruitment in the MPP+ or rotenone cell model. n = 4; *P < 0.05, **P < 0.01 versus control; ^# P < 0.05 T + M group versus M group of the same dose or T + R group versus R group of the same dose.

Figure 3

Trichostatin A (TSA) inhibits histone deacetylation over Mfn1 and Mfn2 promoters and alleviates their transcriptional dysfunction induced by MPP+. (A) Representative semi‐quantitative PCR analysis and quantitative real‐time PCR analysis show that Mfn1 and Mfn2 mRNA levels in SH‐SY5Y cells are both reduced after exposure to 2 mM MPP+ for 8 h, which are reversed by co‐treatment with 0.1 nM TSA. (B) Representative semi‐quantitative PCR analysis and quantitative real‐time PCR analysis of ChIP samples show that the acetylation levels of H3 and H4 over Mfn1 and Mfn2 promoter in SH‐SY5Y cells are both decreased after exposure to 2 mM MPP+ for 8 h, which is completely inhibited by TSA. n = 4; *P < 0.05 versus control; ^# P < 0.05 T + M group versus M group.

Figure 4

The effects of Mfn1/2 overexpression on mitochondrial fragmentation and cell apoptosis in the MPP+ cell model. SH‐SY5Y cells were first transfected with constructed plasmids containing human Mfn1 or Mfn2 cDNA followed by IRES‐GFP, and 2 days later transfected with a mitochondria‐targeted RFP. After 24 h, cells were treated with 2 or 3 mM MPP+ (M2, M3) for 8 h. Representative images taken by live cell imaging system (A) and quantitative analysis of mitochondrial length (B) show that MPP+ induces significant mitochondrial fragmentation in the cells transfected with an empty vector. Mfn2 overexpression selectively prevents mitochondrial fragmentation in M2 yet not M3 group, and Mfn1 overexpression has no effect on mitochondrial fragmentation induced by 2 or 3 mM MPP+. (C) Flow cytometric analysis shows that MPP+ induces severe cell apoptosis in the cells transfected with an empty vector. Mfn2 overexpression selectively attenuates cell apoptosis in M2 but not M3 group, while Mfn1 overexpression has no effect on cell damage caused by 2 or 3 mM MPP+. (D, E) Representative immunoblotting images and semi‐quantitative analysis show that Mfn1 or Mfn2 protein levels are robustly elevated after transfection for 2–4 days. n = 4; *P < 0.05, **P < 0.01 versus control; ^# P < 0.05, ^## P < 0.01 Mfn2 group versus vector group of the same dose of MPP+. Scale bars for 10 μm.

Figure 5

Blocking Bax translocation to mitochondria does not prevent mitochondrial fragmentation in the MPP+ cell model. (A) Representative immunoblotting image and semi‐quantitative analysis show that mitochondrial Bax levels are robustly increased after exposed to 2 or 3 mM MPP+ for 8 h, which is entirely blocked by Bax inhibiting peptide V5 (V5, 100 μM). Trichostatin A selectively reverses the elevation of mitochondrial Bax levels in the cells treated with 2 mM MPP+. (B) Representative images taken by live cell imaging system shows mitochondrial morphology in SH‐SY5Y cells. Co‐treatment with peptide V5 has no impact on mitochondrial fragmentation in the cells exposed to MPP+. (C) Quantitative analysis of changes in mitochondrial length per cell. Peptide V5 has no impact on the decrease of mitochondrial length in the cells exposed to MPP+. (D) Flow cytometry results show that Peptide V5 remarkably inhibits cell apoptosis in the cells exposed to MPP+. n = 4; *P < 0.05, **P < 0.01 versus control; ^# P < 0.05 T + M/V + M group versus M group of the same dose. Scale bars for 10 μm.