Rare PMP22 variants in mild to severe neuropathy uncorrelated to plasma GDF15 or neurofilament light.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous set of hereditary neuropathies whose genetic causes are not fully understood. Here, we characterize three previously unknown variants in PMP22 and assess their effect on the recently described potential CMT biomarkers' growth differentiation factor 15 (GDF15) and neurofilament light (NFL): first, a heterozygous PMP22 c.178G > A (p.Glu60Lys) in one mother-son pair with adult-onset mild axonal neuropathy. The variant led to abnormal splicing, confirmed in fibroblasts by reverse transcription PCR. Second, a de novo PMP22 c.35A > C (p.His12Pro), and third, a heterozygous 3.2 kb deletion predicting loss of exon 4. The latter two had severe CMT and ultrasonography showing strong nerve enlargement similar to a previous case of exon 4 loss due to a larger deletion. We further studied patients with PMP22 duplication (CMT1A) finding slightly elevated plasma NFL, as measured by the single molecule array immunoassay (SIMOA). In addition, plasma GDF15, as measured by ELISA, correlated with symptom severity for CMT1A. However, in the severely affected individuals with PMP22 exon 4 deletion or p.His12Pro, these biomarkers were within the range of variability of CMT1A and controls, although they had more pronounced nerve hypertrophy. This study adds p.His12Pro and confirms PMP22 exon 4 deletion as causes of severe CMT, whereas the previously unknown splice variant p.Glu60Lys leads to mild axonal neuropathy. Our results suggest that GDF15 and NFL do not distinguish CMT1A from advanced hypertrophic neuropathy caused by rare PMP22 variants.

Human IP3 receptor triple knockout stem cells remain pluripotent despite altered mitochondrial metabolism.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ER Ca2+-release channels that control a broad set of cellular processes. Animal models lacking IP3Rs in different combinations display severe developmental phenotypes. Given the importance of IP3Rs in human diseases, we investigated their role in human induced pluripotent stem cells (hiPSC) by developing single IP3R and triple IP3R knockouts (TKO). Genome edited TKO-hiPSC lacking all three IP3R isoforms, IP3R1, IP3R2, IP3R3, failed to generate Ca2+ signals in response to agonists activating GPCRs, but retained stemness and pluripotency. Steady state metabolite profiling and flux analysis of TKO-hiPSC indicated distinct alterations in tricarboxylic acid cycle metabolites consistent with a deficiency in their pyruvate utilization via pyruvate dehydrogenase, shifting towards pyruvate carboxylase pathway. These results demonstrate that IP3Rs are not essential for hiPSC identity and pluripotency but regulate mitochondrial metabolism. This set of knockout hiPSC is a valuable resource for investigating IP3Rs in human cell types of interest.

Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism.

Mutations in mitochondrial DNA encoded subunit of ATP synthase, MT-ATP6, are frequent causes of neurological mitochondrial diseases with a range of phenotypes from Leigh syndrome and NARP to ataxias and neuropathies. Here we investigated the functional consequences of an unusual heteroplasmic truncating mutation m.9154C>T in MT-ATP6, which caused peripheral neuropathy, ataxia and IgA nephropathy. ATP synthase not only generates cellular ATP, but its dimerization is required for mitochondrial cristae formation. Accordingly, the MT-ATP6 truncating mutation impaired the assembly of ATP synthase and disrupted cristae morphology, supporting our molecular dynamics simulations that predicted destabilized a/c subunit subcomplex. Next, we modeled the effects of the truncating mutation using patient-specific induced pluripotent stem cells. Unexpectedly, depending on mutation heteroplasmy level, the truncation showed multiple threshold effects in cellular reprogramming, neurogenesis and in metabolism of mature motor neurons (MN). Interestingly, MN differentiation beyond progenitor stage was impaired by Notch hyperactivation in the MT-ATP6 mutant, but not by rotenone-induced inhibition of mitochondrial respiration, suggesting that altered mitochondrial morphology contributed to Notch hyperactivation. Finally, we also identified a lower mutation threshold for a metabolic shift in mature MN, affecting lactate utilization, which may be relevant for understanding the mechanisms of mitochondrial involvement in peripheral motor neuropathies. These results establish a critical and disease-relevant role for ATP synthase in human cell fate decisions and neuronal metabolism.

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.

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.

LIN28B affects gene expression at the hypothalamic-pituitary axis and serum testosterone levels.

Genome-wide association studies (GWAS) have recurrently associated sequence variation nearby LIN28B with pubertal timing, growth and disease. However, the biology linking LIN28B with these traits is still poorly understood. With our study, we sought to elucidate the mechanisms behind the LIN28B associations, with a special focus on studying LIN28B function at the hypothalamic-pituitary (HP) axis that is ultimately responsible for pubertal onset. Using CRISPR-Cas9 technology, we first generated lin28b knockout (KO) zebrafish. Compared to controls, the lin28b KO fish showed both accelerated growth tempo, reduced adult size and increased expression of mitochondrial genes during larval development. Importantly, data from the knockout zebrafish models and adult humans imply that LIN28B expression has potential to affect gene expression in the HP axis. Specifically, our results suggest that LIN28B expression correlates positively with the expression of ESR1 in the hypothalamus and POMC in the pituitary. Moreover, we show how the pubertal timing advancing allele (T) for rs7759938 at the LIN28B locus associates with higher testosterone levels in the UK Biobank data. Overall, we provide novel evidence that LIN28B contributes to the regulation of sex hormone pathways, which might help explain why the gene associates with several distinct traits.