ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons.

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.

Inter-organellar and systemic responses to impaired mitochondrial matrix protein import in skeletal muscle.

Effective protein import from cytosol is critical for mitochondrial functions and metabolic regulation. We describe here the mammalian muscle-specific and systemic consequences to disrupted mitochondrial matrix protein import by targeted deletion of the mitochondrial HSP70 co-chaperone GRPEL1. Muscle-specific loss of GRPEL1 caused rapid muscle atrophy, accompanied by shut down of oxidative phosphorylation and mitochondrial fatty acid oxidation, and excessive triggering of proteotoxic stress responses. Transcriptome analysis identified new responders to mitochondrial protein import toxicity, such as the neurological disease-linked intermembrane space protein CHCHD10. Besides communication with ER and nucleus, we identified crosstalk of distressed mitochondria with peroxisomes, in particular the induction of peroxisomal Acyl-CoA oxidase 2 (ACOX2), which we propose as an ATF4-regulated peroxisomal marker of integrated stress response. Metabolic profiling indicated fatty acid enrichment in muscle, a shift in TCA cycle intermediates in serum and muscle, and dysregulated bile acids. Our results demonstrate the fundamental importance of GRPEL1 and provide a robust model for detecting mammalian inter-organellar and systemic responses to impaired mitochondrial matrix protein import and folding.

Serum Creatine, Not Neurofilament Light, Is Elevated in CHCHD10-Linked Spinal Muscular Atrophy.

OBJECTIVE: To characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular atrophy Jokela (SMAJ) type for disease monitoring and for the understanding of pathogenic mechanisms. METHODS: We collected serum samples from a cohort of 49 patients with SMAJ, all carriers of the heterozygous c.197G>T p.G66V variant in CHCHD10. As controls, we used age- and sex-matched serum samples obtained from Helsinki Biobank. Creatine kinase and creatinine were measured by standard methods. Neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) were measured with single molecule array (Simoa), fibroblast growth factor 21 (FGF-21), and growth differentiation factor 15 (GDF-15) with an enzyme-linked immunosorbent assay. For non-targeted plasma metabolite profiling, samples were analyzed with liquid chromatography high-resolution mass spectrometry. Disease severity was evaluated retrospectively by calculating a symptom-based score. RESULTS: Axon degeneration marker, NfL, was unexpectedly not altered in the serum of patients with SMAJ, whereas astrocytic activation marker, GFAP, was slightly decreased. Creatine kinase was elevated in most patients, particularly men. We identified six metabolites that were significantly altered in serum of patients with SMAJ in comparison to controls: increased creatine and pyruvate, and decreased creatinine, taurine, N-acetyl-carnosine, and succinate. Creatine correlated with disease severity. Altered pyruvate and succinate indicated a metabolic response to mitochondrial dysfunction; however, lactate or mitochondrial myopathy markers FGF-21 or GDF-15 was not changed. CONCLUSIONS: Biomarkers of muscle mass and damage are altered in SMAJ serum, indicating a role for skeletal muscle in disease pathogenesis in addition to neurogenic damage. Despite the minimal mitochondrial pathology in skeletal muscle, signs of a metabolic shift can be detected.

Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons.

Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot-Marie-Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.