Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1) is a rare, autosomal recessive disorder caused by mutations in the NFU1 gene. NFU1 is responsible for delivery of iron-sulfur clusters (ISCs) to recipient proteins which require these metallic cofactors for their function. Pathogenic variants of NFU1 lead to dysfunction of its target proteins within mitochondria. To date, 20 NFU1 variants have been reported and the unique contributions of each variant to MMDS1 pathogenesis is unknown. Given that over half of MMDS1 individuals are compound heterozygous for different NFU1 variants, it is valuable to investigate individual variants in an isogenic background. In order to understand the shared and unique phenotypes of NFU1 variants, we used CRISPR/Cas9 gene editing to recreate exact patient variants of NFU1 in the orthologous gene, nfu-1 (formerly lpd-8), in C. elegans. Five mutant C. elegans alleles focused on the presumptive iron-sulfur cluster interaction domain were generated and analyzed for mitochondrial phenotypes including respiratory dysfunction and oxidative stress. Phenotypes were variable between the mutant nfu-1 alleles and generally presented as an allelic series indicating that not all variants have lost complete function. Furthermore, reactive iron within mitochondria was evident in some, but not all, nfu-1 mutants indicating that iron dyshomeostasis may contribute to disease pathogenesis in some MMDS1 individuals.

Neuromuscular dysfunction is a common feature of mitochondrial diseases and frequently presents as ataxia, spasticity, and/or dystonia all of which can severely impact individuals afflicted with mitochondrial diseases. Dystonia is one of the most common symptoms of Multiple Mitochondrial Dysfunctions Syndrome 1, a disease associated with mutations in the causative gene (NFU1) that impairs iron-sulfur cluster biogenesis. We have generated C. elegans strains that recreated patient-specific point variants in the C. elegans ortholog (NFU-1) that result in allele-specific dysfunction. Each of these mutants, Gly147Arg and Gly166Cys, have altered acetylcholine signaling at neuromuscular junctions, but opposite effects on activity and motility. We find that the Gly147Arg variant is hypersensitive to acetylcholine and that knockdown of acetylcholine release rescues nearly all neuromuscular phenotypes of this variant. In contrast, we find that the Gly166Cys variant causes predominantly postsynaptic acetylcholine hypersensitivity due to an unclear mechanism. These results are important for understanding the neuromuscular conditions of MMDS1 patients and potential avenues for therapeutic intervention.