ETFDH mutation involves excessive apoptosis and neurite outgrowth defect via Bcl2 pathway

Abstract

The most common mutation in southern Chinese individuals with late-onset multiple acyl-coenzyme A dehydrogenase deficiency (MADD; a fatty acid metabolism disorder) is c.250G > A (p.Ala84Thr) in the electron transfer flavoprotein dehydrogenase gene (ETFDH). Various phenotypes, including episodic weakness or rhabdomyolysis, exercise intolerance, and peripheral neuropathy, have been reported in both muscular and neuronal contexts. Our cellular models of MADD exhibit neurite growth defects and excessive apoptosis. Given that axonal degeneration and neuronal apoptosis may be regulated by B-cell lymphoma (BCL)-2 family proteins and mitochondrial outer membrane permeabilization through the activation of proapoptotic molecules, we measured the expression levels of proapoptotic BCL-2 family proteins (e.g., BCL-2-associated X protein and p53-upregulated modulator of apoptosis), cytochrome c, caspase-3, and caspase-9 in NSC-34 cells carrying the most common ETFDH mutation. The levels of these proteins were higher in the mutant cells than in the wide-type cells. Subsequent treatment of the mutant cells with coenzyme Q10 downregulated activated protein expression and mitigated neurite growth defects. These results suggest that the activation of the BCL-2/mitochondrial outer membrane permeabilization/apoptosis pathway promotes apoptosis in cellular models of MADD and that coenzyme Q10 can reverse this effect. Our findings aid the development of novel therapeutic strategies for reducing axonal degeneration and neuronal apoptosis in MADD.

Keywords: Apoptosis; Bcl-2; Carnitine; Coenzyme Q10; ETFDH; MOMP; Multiple acyl-coenzyme A dehydrogenase deficiency; Neuropathy; Riboflavin.

Fig. 1

The apoptosis assays. (A: wild type and B: Mutant) Flow cytometry showed the increased apoptosis in the mutant-type cells. In the diagram, the fourth quadrant (Q4) represents healthy living cells (FITC-/PI-), the third quadrant (Q3) represents early apoptotic cells (FITC+/PI-), the second quadrant (Q2) represents late apoptotic cells (FITC+/PI+), and the first quadrant (Q1) represents dead cells. (C) the cell apoptosis index of wild type and mutant cells. Apoptosis index (%) = early apoptosis rate (Q3) + late apoptosis rate (Q2). (n = 2). (D) The TUNEL stain was performed to detect the apoptotic cells. The scale bar is 50 μm in length. (E) The TUNEL positive cell counts of the wild type cells and the mutant cells. n = 11 ~ 14. The statistical significance of the different protein expressions was estimated by two-tailed student’s t-test analysis. (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Fig. 2

The Western blotting results of relative protein expression level of wild type and mutant. Including the Cytochrome-C, Caspase-3, Bax, Caspase-9 and Bcl-2. W: ETFDH-250G (wild type), M: ETFDH-250 A (mutant type). The statistical significance of the different protein expressions was estimated by two-tailed student’s t-test analysis. (*: p < 0.05, **: p < 0.01, ***: p < 0.001). n = 2 ~ 4.

Fig. 3

The effect of neurites outgrowth by coenzyme-Q10 treatment. (A) the morphology of neurites in wild type and mutant cells observed by phase contrast microscopy. The scale bar is 100 μm in length. (B) The lengths of neurites in wild type and mutant cells with coenzyme-Q10 treatment. W: ETFDH-250G (wild type), M: ETFDH-250 A (mutant type), W + Q10: wild type treated with coenzyme Q10, M + Q10: mutant type treated with coenzyme Q10. The statistical significance of the different protein expressions was estimated by one-way analysis of variance (ANOVA) followed by Tukey’s test (HSD). (*: p < 0.05, **: p < 0.01, ***: p < 0.001). n = 60.

Fig. 4

The Western blotting of BCL-2 regulated apoptosis signaling pathways that treated with coenzyme Q10. (A) The Western blotting results including PUMA, Bax, Cytochrome-C, Caspase-9, Caspase-3, FOXO3a, p-FOXO3a and Bcl-2. W: ETFDH-250G (wild type), M: ETFDH-250 A (mutant type), W + Q10: wild type treated with coenzyme Q10, M + Q10: mutant type treated with coenzyme Q10. (B, C, D, E, F, G, H and I) The quantification of the Western blot results. All the protein expression levels were normalized with GAPDH expression level. The statistical significance of the different protein expressions was estimated by one-way analysis of variance (ANOVA) followed by Tukey’s test (HSD). (*: p < 0.05, **: p < 0.01, ***: p < 0.001). n = 2 ~ 4.

Fig. 5

The Western blotting of BCL-2 regulated apoptosis signaling pathway that treated with riboflavin (B2). (A) The Western blotting results including the PUMA, Bax, Cytochrome-C, Caspase-9, Caspase-3, FOXO3a and p-FOXO3a. W: ETFDH-250G (wild type), M: ETFDH-250 A (mutant type), W + B2: wild type treated with riboflavin (B2), M + QB2: mutant type treated with riboflavin (B2). (B, C, D, E, F, G and H) All the protein expression levels were normalized with GAPDH expression level. The statistical significance of the different protein expressions was estimated by one-way analysis of variance (ANOVA) followed by Tukey’s test (HSD). (*: p < 0.05, **: p < 0.01, ***: p < 0.001). n = 2 ~ 4.