Deletion of gene coding for mitochondrial citrate carrier in C. elegans impairs the acetylcholine synthesis

The mitochondrial citrate carrier (CIC) encoded by the SLC25A1 gene, catalyzes the export of citrate from mitochondria to the cytosol where it is broken into acetyl-CoA and oxaloacetate. Acetyl-CoA can be used as building block for de novo synthesis of fatty acids or, in motoneurons, can be condensed to choline to give acetylcholine. Pathogenic SLC25A1 variants were identified in several patients (1, 2) that suffered from a severe neurodevelopmental syndrome (1) and CIC deficiency has been classified as an inborn disorder of metabolism (OMIM: 615182) whose hallmark is combined D-2- and L-2-hydroxyglutaric aciduria (2). More recently we reported a novel homozygous mutation in the SLC25A1 gene in an affected sib pair. Both patients presented with myasthenia and impaired neuromuscular junction (NMJ) transmission whilst no neurodevelopment disorder has been observed (3). Interestingly, the identified mutation in sib pair caused a milder activity impairment than the previously reported mutations suggesting a fundamental role of CIC in neuromuscular transmission whose defect was previously masked by the harsher phenotypes. Signal transmission at Neuromuscular junction (NMJ) relies on massive synthesis in motor neurons of acetylcholine. While the choline can be recycled after neurotransmitter release and breakdown in the synaptic cleft, the acetylCoA must be continuously generated by oxidative metabolism in mitochondria. Knocking down the SLC25A1 orthologues by injection of antisense morpholino oligonucleotides in zebrafish embryos showed short and erratic outgrowth of motor axons toward muscle fiber at NMJ suggesting that transmission impairment could be related to pre-synaptic nerve terminal abnormalities. Using the CRISPR/CAS9 approach we obtained stable lines of Caenorhabditis elegans knocked-out in the SLC25A1 ortholog that showed resistance to levamisole, a nicotinic acetylcholine receptor agonist, that causes continued stimulation of the worm muscles, leading to paralysis. This phenotype was, at least in part, rescued by the expression of wild-type SLC25A1 under the control of a neuron-specific promoter, strongly pointing towards an underlying pre-synaptic defect. qPCR analysis showed an up-regulation of genes involved in presynaptic acetylcholine biosynthesis and its release in the synaptic cleft, and a down-regulation of acetylcholine esterase. Furthermore, the amount of acetylcholine is decreased by about 50% in KO strain. Lipidomic analysis by mass spectrometry (HILIC-ESI-FTMS) showed no gross defect in lipogenesis. Altogether these data demonstrate a conserved role of CIC in neuromuscular transmission. Furthermore, our results validate the worm C. elegans as an animal model suitable for further study of the molecular and cellular underpinnings of the NMJ transmission defect associated to CIC deficiency 1. Edvardson et al. 2013 J Med Genet 50:240-5 2. Nota et al. 2013 Am J Hum Genet 92:627-31 3. Chaouch et al. 2014 J Neuromuscul Dis 1:75-90