Interplay of CHCHD2 with mutant CHCHD10 in stem cell-based modeling of late-onset spinal muscular atrophy

Abstract

A mitochondrial intermembrane space protein that is encoded by the nucleus, Coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) is without an exact known function. Mutations in CHCHD10 cause neuromuscular disorders, such as amyotrophic lateral sclerosis, late-onset spinal muscular atrophy (SMAJ) and mitochondrial myopathy. Although its precise purpose is unknown, CHCHD10 shapes a complex with its homologous protein CHCHD2, and is partially redundant in function. Moreover, it is seen that CHCHD2 mutations are related to causing Parkinson's disease. Detrimental mutations in CHCHD10/CHCHD2 cause changes in mitochondrial structure and function, and activation of cellular stress responses. These proteins have been hypothesised to be included in mitochondrial cristae maintenance, oxidative phosphorylation (OXPHOS) function, or have roles in responding to stress. CHCHD10 and CHCHD2 seem to be important for the proper functioning of the nerves. It is yet to be investigated how pathogenic mutations in CHCHD10 affect the complex formation with CHCHD2 in affected tissues. Our study focuses on SMAJ, which is a progressive and late-onset neuromuscular disease due to dominant p.G66V mutation in CHCHD10, affecting the lower motor neurons. By utilizing CRISPR/Cas9 techniques to knockout CHCHD2 from SMAJ patient-specific induced pluripotent stem cells (iPSCs) carrying p.G66V variant in CHCHD10, as well as from previously created CHCHD10 knockout iPSC, thus creating a double knockout iPSC line. These genome-edited iPSCs are further differentiated into spinal motor neurons and the effects of CHCHD2 knockout on mitochondrial function and the stability of mutant CHCHD10 are investigated. The aim is to interpret the function of CHCHD10 in motor neurons and how CHCHD2 availability affects CHCHD10/CHCHD2 complex function and mitochondrial homeostasis. The results showed successful knocking out of CHCHD2, with western blot showing absence of the protein and RT-qPCR confirming low CHCHD2 mRNA expression in knockout lines, validating the editing efficiency. The stability of CHCHD10 was observed using western blotting and found to be stable with comparable CHCHD10 protein levels observed in unedited and CHCHD2 knockout iPSC lines. Seahorse metabolic profiling revealed that CHCHD2 knockout in the CHCHD10 mutant caused increased basal and maximal respiration, ECAR, and proton leak, pointing toward metabolic stress and compensatory shifts toward glycolysis. Interestingly, double knockout lines exhibited restored metabolic balance, with OCR/ECAR ratios and ATP production returning to control-like levels, suggesting a compensatory adaptation when both proteins are absent. This work provides new insights into mitochondrial dysfunction in SMAJ and highlights the importance of CHCHD10/CHCHD2 balance.