Mutations in the Mitochondrial Citrate Carrier SLC25A1 are Associated with Impaired Neuromuscular Transmission

Abstract

Background and objective: Congenital myasthenic syndromes are rare inherited disorders characterized by fatigable weakness caused by malfunction of the neuromuscular junction. We performed whole exome sequencing to unravel the genetic aetiology in an English sib pair with clinical features suggestive of congenital myasthenia.

Methods: We used homozygosity mapping and whole exome sequencing to identify the candidate gene variants. Mutant protein expression and function were assessed in vitro and a knockdown zebrafish model was generated to assess neuromuscular junction development.

Results: We identified a novel homozygous missense mutation in the SLC25A1 gene, encoding the mitochondrial citrate carrier. Mutant SLC25A1 showed abnormal carrier function. SLC25A1 has recently been linked to a severe, often lethal clinical phenotype. Our patients had a milder phenotype presenting primarily as a neuromuscular (NMJ) junction defect. Of note, a previously reported patient with different compound heterozygous missense mutations of SLC25A1 has since been shown to suffer from a neuromuscular transmission defect. Using knockdown of SLC25A1 expression in zebrafish, we were able to mirror the human disease in terms of variable brain, eye and cardiac involvement. Importantly, we show clear abnormalities in the neuromuscular junction, regardless of the severity of the phenotype.

Conclusions: Based on the axonal outgrowth defects seen in SLC25A1 knockdown zebrafish, we hypothesize that the neuromuscular junction impairment may be related to pre-synaptic nerve terminal abnormalities. Our findings highlight the complex machinery required to ensure efficient neuromuscular function, beyond the proteomes exclusive to the neuromuscular synapse.

Keywords: Congenital myasthenic syndrome; SLC25A1; mitochondrial citrate carrier; neuromuscular junction.

Fig. 1

Sequence alignment of mitochondrial citrate carrier (SLC25A1) from different organisms (panel A) and other members of mitochondrial carrier family (panel B). Accession numbers for each species/carrier is given. Panel C shows structural comparative model of human CIC and docking of citrate. The 3D comparative model of human CIC is reported in white cartoon representation. The six transmembrane helices are indicated by black labels (H1-H6). Two of the three helices parallel to the membrane planes are also labelled (h12 and h56). The citrate ligand is shown in cyan licorice. The pathogenic mutations p.R247Q (this work), p.G130D, p.R282H [17] are displayed in red surf representation.

Fig. 1

Sequence alignment of mitochondrial citrate carrier (SLC25A1) from different organisms (panel A) and other members of mitochondrial carrier family (panel B). Accession numbers for each species/carrier is given. Panel C shows structural comparative model of human CIC and docking of citrate. The 3D comparative model of human CIC is reported in white cartoon representation. The six transmembrane helices are indicated by black labels (H1-H6). Two of the three helices parallel to the membrane planes are also labelled (h12 and h56). The citrate ligand is shown in cyan licorice. The pathogenic mutations p.R247Q (this work), p.G130D, p.R282H [17] are displayed in red surf representation.

Fig. 2

Functional characterization of the wild-type (WT) and the p.R241Q Ctp1. The uptake rate of (¹⁴C) citrate was measured by adding 0.1 mM of (¹⁴C) citrate to proteoliposomes reconstituted with purified WT or with the mutated Ctp1 protein. The proteoliposomes were preloaded internally with 10 mM of citrate. The means and SDs from five independent experiments are shown (*p < 0.01, two-tailed unpaired Student’s t-test).

Fig. 3

Total lysates of fibroblast cells (patient and control) were subjected to Western blot analysis upon separation by SDS-PAGE and SLC25A1 (~31-kDa) and β-ATPase (~55-kDa) levels were determined by densitometric analysis. The relative ratio was calculated and the means and SDs from three independent experiments are shown (*p < 0.01, two-tailed unpaired Student’s t-test).

Fig. 4

Transport assay of reconstituted CTP1p wild type (WT) and mutated Ctp1p forms into liposomes. At time zero 0.1 mM (¹⁴C) citrate was added to liposomes reconstituted with the recombinant wild-type or mutants and containing 10 mM citrate. At the indicated times, the uptake of the labeled substrate was terminated by adding 20 mM pyridoxal 5′-phosphate and 20 mM bathophenanthroline. Similar results were obtained in four independent experiments.

Fig. 5

Live embryos imaged at 48hpf following co-injection of SLC25A1a (5 ng) and SLC25A1b (2.5 ng). Injected embryos demonstrate a range of phenotypes with mild to severe morphological abnormalities. Embryos exhibit a developmental delay with curvature and shortening of the tail and also oedema of the hindbrain, heart, yolk sac and tail. Scale bars: 500 μm.

Fig. 6

Neuromuscular junctions following injection with SLC25A1 MOs. Non-injected control Wild Type Golden embryos (left) and SLC25A1 MO injected embryos were stained for postsynaptic AChR (α-bungarotoxin, red staining) and presynaptic nerve endings (SV2 antibody, green staining). Combined SLC25A1 MO (a 5 ng & b 2.5 ng) injected 48hpf embryos demonstrate short motor axons and erratic outgrowth toward the muscle fibre. Scale bar: 50 μm.

Fig. 7

Muscle morphology following injection with SLC25A1 MOs. WT and control MO injected embryos (left) and SLC25A1 MO injected embryos (centre and right) were stained for F-actin with Alexa Fluor^® 594 conjugated phalloidin (5 μg/ml). WT embryos and those injected with a standard control MO showed normal muscle morphology. Combined SLC25A1 MO with and without the addition of anti-p53 MO also demonstrate normal muscle morphology. Scale bar: 50 μm.