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

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

Diagnosis of prostate cancer with magnetic resonance imaging in men treated with 5-alpha-reductase inhibitors

Purpose The primary aim of this study was to evaluate if exposure to 5-alpha-reductase inhibitors (5-ARIs) modifies the effect of MRI for the diagnosis of clinically significant Prostate Cancer (csPCa) (ISUP Gleason grade >= 2).Methods This study is a multicenter cohort study including patients undergoing prostate biopsy and MRI at 24 institutions between 2013 and 2022. Multivariable analysis predicting csPCa with an interaction term between 5-ARIs and PIRADS score was performed. Sensitivity, specificity, and negative (NPV) and positive (PPV) predictive values of MRI were compared in treated and untreated patients.Results 705 patients (9%) were treated with 5-ARIs [median age 69 years, Interquartile range (IQR): 65, 73; median PSA 6.3 ng/ml, IQR 4.0, 9.0; median prostate volume 53 ml, IQR 40, 72] and 6913 were 5-ARIs naive (age 66 years, IQR 60, 71; PSA 6.5 ng/ml, IQR 4.8, 9.0; prostate volume 50 ml, IQR 37, 65). MRI showed PIRADS 1-2, 3, 4, and 5 lesions in 141 (20%), 158 (22%), 258 (37%), and 148 (21%) patients treated with 5-ARIs, and 878 (13%), 1764 (25%), 2948 (43%), and 1323 (19%) of untreated patients (p < 0.0001). No difference was found in csPCa detection rates, but diagnosis of high-grade PCa (ISUP GG >= 3) was higher in treated patients (23% vs 19%, p = 0.013). We did not find any evidence of interaction between PIRADS score and 5-ARIs exposure in predicting csPCa. Sensitivity, specificity, PPV, and NPV of PIRADS >= 3 were 94%, 29%, 46%, and 88% in treated patients and 96%, 18%, 43%, and 88% in untreated patients, respectively.Conclusions Exposure to 5-ARIs does not affect the association of PIRADS score with csPCa. Higher rates of high-grade PCa were detected in treated patients, but most were clearly visible on MRI as PIRADS 4 and 5 lesions.Trial registration The present study was registered at ClinicalTrials.gov number: NCT05078359

Diseases Caused by Mutations in Mitochondrial Carrier Genes SLC25: A Review

In the 1980s, after the mitochondrial DNA (mtDNA) had been sequenced, several diseases resulting from mtDNA mutations emerged. Later, numerous disorders caused by mutations in the nuclear genes encoding mitochondrial proteins were found. A group of these diseases are due to defects of mitochondrial carriers, a family of proteins named solute carrier family 25 (SLC25), that transport a variety of solutes such as the reagents of ATP synthase (ATP, ADP, and phosphate), tricarboxylic acid cycle intermediates, cofactors, amino acids, and carnitine esters of fatty acids. The disease-causing mutations disclosed in mitochondrial carriers range from point mutations, which are often localized in the substrate translocation pore of the carrier, to large deletions and insertions. The biochemical consequences of deficient transport are the compartmentalized accumulation of the substrates and dysfunctional mitochondrial and cellular metabolism, which frequently develop into various forms of myopathy, encephalopathy, or neuropathy. Examples of diseases, due to mitochondrial carrier mutations are: combined D-2- and L-2-hydroxyglutaric aciduria, carnitine-acylcarnitine carrier deficiency, hyperornithinemia-hyperammonemia-homocitrillinuria (HHH) syndrome, early infantile epileptic encephalopathy type 3, Amish microcephaly, aspartate/glutamate isoform 1 deficiency, congenital sideroblastic anemia, Fontaine progeroid syndrome, and citrullinemia type II. Here, we review all the mitochondrial carrier-related diseases known until now, focusing on the connections between the molecular basis, altered metabolism, and phenotypes of these inherited disorders

THE HUMAN GENE SLC25A17 ENCODES A PEROXISOMAL TRANSPORTER OF COENZYME A, FAD AND NAD+.

The essential cofactors coenzyme A (CoA), FAD and NAD+ are synthesized outside the peroxisomes and therefore must be transported into the peroxisomal matrix where they are required for important processes. In this work we have functionally identified and characterized SLC25A17, which is the only member of the mitochondrial carrier family that has previously been shown to be localized in the peroxisomal membrane. Herein, recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN, AMP and to a lesser extent of NAD+, adenosine 3',5'-diphosphate (PAP) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and inhibited by pyridoxal-5'-phosphate and other mitochondrial carrier inhibitors. It was expressed to various degrees in all the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD+ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP. This is the first report describing the identification and characterization of a transporter for multiple free cofactors in peroxisomes

Three mitochondrial transporters of Saccharomyces cerevisiae are essential for ammonium fixation and lysine biosynthesis in synthetic minimal medium

The nuclear genes of Saccharomyces cerevisiae YHM2, ODC1 and ODC2 encode three transporters that are localized in the inner mitochondrial membrane. In this study, the roles of YHM2, ODC1 and ODC2 in the assimilation of nitrogen and in the biosynthesis of lysine have been investigated. Both the odc1δ. odc2δ double knockout and the yhm2δ mutant grew similarly as the YPH499 wild-type strain on synthetic minimal medium (SM) containing 2% glucose and ammonia as the main nitrogen source. In contrast, the yhm2δ. odc1δ. odc2δ triple knockout exhibited a marked growth defect under the same conditions. This defect was fully restored by the individual expression of YHM2, ODC1 or ODC2 in the triple deletion strain. Furthermore, the lack of growth of yhm2δ. odc1δ. odc2δ on 2% glucose SM was rescued by the addition of glutamate, but not glutamine, to the medium. Using lysine-prototroph YPH499-derived strains, the yhm2δ. odc1δ. odc2δ knockout (but not the odc1δ. odc2δ and yhm2δ mutants) also displayed a growth defect in lysine biosynthesis on 2% glucose SM, which was rescued by the addition of lysine and, to a lesser extent, by the addition of 2-aminoadipate. Additional analysis of the triple mutant showed that it is not respiratory-deficient and does not display mitochondrial DNA instability. These results provide evidence that only the simultaneous absence of YHM2, ODC1 and ODC2 impairs the export from the mitochondrial matrix of i) 2-oxoglutarate which is necessary for the synthesis of glutamate and ammonium fixation in the cytosol and ii) 2-oxoadipate which is required for lysine biosynthesis in the cytosol. Finally, the data presented allow one to suggest that the yhm2δ. odc1δ. odc2δ triple knockout is suitable in complementation studies aimed at assessing the pathogenic potential of human SLC25A21 (ODC) mutations

Human and plant peroxisomal cofactor transporters.

Peroxisomes are small organelles found in all eukaryotes, involved in a number of important metabolic pathways, including fatty acid α- and β-oxidation, biosynthesis of ether phospholipids and bile acids, and the degradation of purines, amino acids and polyamines. The functional role of the peroxisomal membrane as a permeability barrier to substrates and cofactors has been controversial for many years. The essential cofactors CoA, FAD and NAD+ are synthesized outside the peroxisomes and must be transported into the peroxisomal matrix where they are required for important processes. SLC25A17 (solute carrier family 25 member 17) is the only member of the mitochondrial carrier family that has been shown to be localized in the peroxisomal membrane. Recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN and AMP, and to a lesser extent of NAD+, PAP (adenosine 3',5'-diphosphate) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and was inhibited by pyridoxal 5'-phosphate and other mitochondrial carrier inhibitors. Moreover it was expressed to various degrees in all of the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD+ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP [1]. The plant homologue of SLC25A17 is the peroxisomal protein PXN encoded by the Arabidopsis gene At2g39970 which has recently been found to transport NAD+, NADH, AMP and ADP [2]. Upon heterologous expression of PXN in bacteria followed by purification and reconstitution in liposomes, uptake and efflux experiments revealed that PXN transports coenzyme A (CoA), dephospho-CoA, acetyl-CoA and adenosine 3', 5'-phosphate (PAP), besides NAD+, NADH, AMP and ADP. PXN catalyzed fast counter-exchange of substrates and much slower uniport. Transport was saturable with a submillimolar affinity for NAD+, CoA and other substrates. The physiological role of PXN is probably to provide the peroxisomes with the essential coenzymes NAD+ and CoA [3]. [1] G. Agrimi, A. Russo, P. Scarcia, F. Palmieri, The human gene SLC25A17 encodes a peroxisomal transporter of coenzyme A, FAD and NAD+, Biochem. J., 443 (2012) 241–247

Uridine Treatment of the First Known Case of SLC25A36 Deficiency

SLC25A36 is a pyrimidine nucleotide carrier playing an important role in maintaining mitochondrial biogenesis. Deficiencies in SLC25A36 in mouse embryonic stem cells have been associated with mtDNA depletion as well as mitochondrial dysfunction. In human beings, diseases triggered by SLC25A36 mutations have not been described yet. We report the first known case of SLC25A36 deficiency in a 12-year-old patient with hypothyroidism, hyperinsulinism, hyperammonemia, chronical obstipation, short stature, along with language and general developmental delay. Whole exome analysis identified the homozygous mutation c.803dupT, p.Ser269llefs*35 in the SLC25A36 gene. Functional analysis of mutant SLC25A36 protein in proteoliposomes showed a virtually abolished transport activity. Immunoblotting results suggest that the mutant SLC25A36 protein in the patient undergoes fast degradation. Supplementation with oral uridine led to an improvement of thyroid function and obstipation, increase of growth and developmental progress. Our findings suggest an important role of SLC25A36 in hormonal regulations and oral uridine as a safe and effective treatment

Altered calcium homeostasis and mitochondrial dysfunction in autism

Aims: Genetic variants of the brain isoform of the mitochondrial asp/glu carrier (AGC1), were previously found associated with autism. Our initial aim was to investigate asp/glu transport rates and AGC content in post-mortem brains of autistic patients. Methods: Temporocortical gray matter from matched patient-control pairs was used to measure reconstituted AGC1 activity from tissue homogenates or isolated mitochondria.. Protein content and oxidative damage were evaluated by western blotting.and oxyblotting, respectively. The activity of respiratory chain complexes was determined by standard diagnostic procedures. Results: AGC1 transport rates were significantly higher in tissue homogenates from autistic patients, including those with no history of seizures and with normal EEGs prior to death. This increase was consistently blunted by the Ca2+ chelator EGTA; no difference in AGC1 transport rates was found in isolated mitochondria from patients and controls; control mitochondria showed increased AGC1 activity when exposed to the post-mitochondrial supernatant of his/her matched patient than to his/her own supernatant. Complex IV and complex V activities were both increased in autistic patients but no significant difference in their protein abundance as well as in AGC1 content was detected by western blotting; oxidized mitochondrial proteins were markedly increased in the majority but not all of the patients tested. Interestingly, oxidative damage correlated with the reduction of complex I activity. Conclusions: Excessive Ca2+ levels boosts AGC activity in neurons and, to a more variable degree, oxidative stress and OXPHOS dysfunction in autistic brains. The modulation of AGC1 and/or Ca2+ homeostasis could provide new preventive and therapeutic strategies

Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter

The requirement for small molecule transport systems across the peroxisomal membrane has previously been postulated, but not directly proven. Here we report the identification and functional reconstitution of Ant1p (Ypr128cp), a peroxisomal transporter in the yeast Saccharomyces cerevisiae, which has the characteristic sequence features of the mitochondrial carrier family. Ant1p was found to be an integral protein of the peroxisomal membrane and expression of ANT1 was oleic acid inducible. Targeting of Ant1p to peroxisomes was dependent on Pex3p and Pex19p, two peroxins specifically required for peroxisomal membrane protein insertion. Ant1p was essential for growth on medium-chain fatty acids as the sole carbon source. Upon reconstitution of the overexpressed and purified protein into liposomes, specific transport of adenine nucleotides could be demonstrated. Remarkably, both the substrate and inhibitor specificity differed from those of the mitochondrial ADP/ATP transporter. The physiological role of Ant1p in S.cerevisiae is probably to transport cytoplasmic ATP into the peroxisomal lumen in exchange for AMP generated in the activation of fatty acids