Overproduction of H2S, generated by CBS, inhibits mitochondrial Complex IV and suppresses oxidative phosphorylation in Down syndrome

Abstract

Down syndrome (DS) is associated with significant perturbances in mitochondrial function. Here we tested the hypothesis that the suppression of mitochondrial electron transport in DS cells is due to high expression of cystathionine-β-synthase (CBS) and subsequent overproduction of the gaseous transmitter hydrogen sulfide (H₂S). Fibroblasts from DS individuals showed higher CBS expression than control cells; CBS localization was both cytosolic and mitochondrial. DS cells produced significantly more H₂S and polysulfide and exhibited a profound suppression of mitochondrial electron transport, oxygen consumption, and ATP generation. DS cells also exhibited slower proliferation rates. In DS cells, pharmacological inhibition of CBS activity with aminooxyacetate or siRNA-mediated silencing of CBS normalized cellular H₂S levels, restored Complex IV activity, improved mitochondrial electron transport and ATP synthesis, and restored cell proliferation. Thus, CBS-derived H₂S is responsible for the suppression of mitochondrial function in DS cells. When H₂S overproduction is corrected, the tonic suppression of Complex IV is lifted, and mitochondrial electron transport is restored. CBS inhibition offers a potential approach for the pharmacological correction of DS-associated mitochondrial dysfunction.

Keywords: H2S; bioenergetics; metabolism; mitochondria.

Fig. 1.

CBS-derived H₂S is responsible for the suppression of mitochondrial function in DSCs. DSCs exhibit markedly higher CBS expression—which is, in part, localized to the mitochondria—than healthy CCs, shown by (A) Western blotting and (C) confocal microscopy. (Scale bar: 5 µm.) (B) TST and ETHE1 expression was similar in DSCs and CCs. (D) DSCs contain increased amounts of intracellular H₂S and polysulfide. Top photomicrographs: AzMC-based H₂S live cell imaging. (Scale bar: 20 µm.) (E) DSCs exhibit reduced cell proliferation rate; this is normalized by AOAA (3 µM); treatment of CCs with the H₂S donor GYY4137 (3 mM) phenocopies the inhibition of proliferation seen in DSCs. (F) DSCs exhibit low basal and maximal mitochondrial oxygen consumption rates (OCR); these parameters are improved by AOAA; exposure of CCs to GYY4137 phenocopies the metabolic suppression seen in DSCs. (G) Complex IV is blocked in DSCs but it is restored by AOAA. (H–L) SiRNA-mediated silencing of CBS in DSCs (H) suppresses H₂S production (I), normalizes DSC proliferation (J), and enhances OCR (K) and Complex IV activity (L). (M) Increased expression of various mitochondrial proteins belonging to electron chain transport Complexes II, III, and IV in DSCs. (N) Bell-shaped cellular bioenergetic role of H₂S in DSCs vs. CCs; effect of CBS inhibition. *P < 0.05 and **P < 0.01, significant difference between CCs and DSCs; ^#P < 0.05 and ^##P < 0.01, significant effect of AOAA in DSCs; ^§P < 0.05, significant effect of GYY4137 in CCs or significant difference between AOAA and GYY4137+AOAA in DSCs. Data are shown as mean ± SEM of at least 3 experiments. One- and 2-way ANOVAs were performed, followed by a post hoc Bonferroni test.