Bovine and murine models highlight novel roles for SLC25A46 in mitochondrial dynamics and metabolism, with implications for human and animal health
[['Mitochondria are eukaryotic organelles with a wide range of functions .', 'In addition to delivery of energy to cells via oxidative phosphorylation ( OXPHOS ) , they are involved in various other bioenergetic reactions , including Krebs cycle , β-oxidation of fatty acids and heme biosynthesis .', 'Furthermore , they have roles in calcium signaling , stress response and apoptosis [1–3] .', 'Consequently , they are a vital organelle .', 'Not surprisingly , mitochondrial dysfunction is shown to be responsible for an increasing number of diseases , inherited or not [2 , 4] .', 'To enable a variety of cells to respond to variable physiological conditions , particularly to adapt to varying energy demands , mitochondrial morphology is highly dynamic , with three main mechanisms: fusion , fission and cristae remodeling [5–7] .', 'The balance between fusion and fission is particularly critical to regulate mitochondrial shape , size and number .', 'In mammals , mitochondrial morphology is regulated by the following GTPase proteins: DRP1 ( Dynamin related protein 1 ) for fission , mitofusin MFN1 and MFN2 , and OPA1 ( Optic atrophy 1 ) for fusion .', 'All these proteins are essential for development [8–10] and despite ubiquitous expression , their mutations primarily cause neurological diseases , as is common for proteins involved in mitochondrial dynamics [11–13] , probably due to neurons being energy-intensive cells [14] .', 'Fusion proteins , for example , are involved in diverse syndromes .', 'Dominant mutations of OPA1 cause Autosomal Dominant Optic Atrophy ( ADOA ) , affecting mitochondrial morphology ( aggregated and fragmented ) and content ( reduced content of mitochondrial DNA ( mtDNA ) and reduced ATP production ) [15–18] .', 'Mutations in MFN2 cause Charcot-Marie-Tooth type 2A ( CMT2A ) disease in humans , a sensorimotor axonopathy with aggregated swollen mitochondria and altered structural integrity of inner and outer mitochondrial membranes [19 , 20] .', 'Mutations of orthologous genes cause neurodegenerative diseases in other mammals , with for example , different mutations of MFN2 causing respectively an early axonopathy in Tyrolean Grey breed [21] and fetal-onset neuroaxonal dystrophy in dog [22] .', 'Recently , human patients with combined ADOA and CMT2 phenotypes were identified as having recessive mutations in SLC25A46 [23] .', 'This gene encodes a protein belonging to the mitochondrial carrier transporter family [24] , anchored on the outer mitochondrial membrane [23] .', '53 proteins belong to this family .', 'Most of them are responsible for the transport of a quantity of diverse metabolites across the inner mitochondrial membrane , which are necessary for all the metabolic pathways taking place in mitochondria [25–27] However , the observed phenotypes linked to SLC25A46 dysfunction suggested that SLC25A46 is rather involved in mitochondrial dynamics , and particularly may act as a pro-fission factor [23] .', 'In cattle , due to massive inbreeding and bottlenecks effects in each selected breed , recessive mutations are likely to be transmitted to a large proportion of the population , leading to emergences of hereditary diseases [28] .', 'In the late 2000’s , such an outbreak was described in the French Rouge-des-Prés breed with a new sensorimotor polyneuropathy named “Syndrome des veaux tourneurs” ( “Turning calves syndrome” ) because of a propensity of the affected calves to turn around themselves before falling down [29] .', 'This neurodegenerative disease is characterized by an early onset of ataxia , especially of hindlimbs , and paraparesia affecting young calves ( 2–6 weeks old ) .', 'Despite symptomatic care , nervous symptoms progress over the next months , leading to repetitive falls and ultimately resulting in permanent recumbency and inevitably euthanasia .', 'Degenerative lesions involve both the general proprioceptive sensory and upper motor neuron motor systems [29] .', 'The number of cases in this breed has rapidly increased in a few years ( based on statistics from the French National Observatory for Bovine Genetic Diseases ) , prompting a genetic study to identify the causal mutation .', 'We identified herein by homozygosity mapping the 3Mb haplotype associated to this disease on bovine chromosome 7 .', 'Further examination of this genetic interval allowed us to determine that this disease resulted from a single nucleotide polymorphism in the coding region of the SLC25A46 gene , leading to an apparently damaging amino acid substitution .', 'The eradication of the “Turning calves syndrome” was undertaken , through the selection of non-carrier males so the number of reported affected calves rapidly dropped to zero .', 'Therefore , a novel mouse knockout model of Slc25a46 was produced to elucidate the function of the encoded protein .', 'The resulting phenotype described below included nervous symptoms but had more widespread effects , including alterations in mitochondrial dynamics and metabolism that caused premature death , thus extending the range of phenotypes associated with polymorphisms of this gene .'], ['Calves from the Rouge-des-Prés breed presenting an ataxic gait and paraparesis of hindlimbs as described in [29] were examined by a veterinarian , and diagnosis was confirmed by histopathology .', 'Pedigree analysis of 11 of them confirmed the autosomal recessive determinism of the “Turning calves syndrome” and the involvement of a predominant founder ancestor ( S1 Fig ) .', 'This bull , born in 1973 , was a historical sire of the Rouge-des-Prés breed ( contributing 6% of the breed ) .', 'Genotyping of 12 affected calves followed by homozygosity mapping identified a single 3 . 1 Mb homozygous interval at the telomeric end of bovine chromosome 7 ( S1 Table ) .', 'This information was used to design a genetic indirect test , based on the haplotype associated to the disease , allowing to begin the selection against the “Turning calves syndrome” of the Rouge-des-Prés breed .', 'To identify the causative mutation , whole-genome sequencing was performed on two affected cattle , one heterozygous carrier and one wild-type ( WT ) .', 'The detected polymorphisms ( SNP and small indels ) were filtered in several steps .', 'First , the genotype/phenotype correlation had to be perfect , i . e . affected cattle had to be homozygous for the polymorphism , and the WT and carrier cattle had to be homozygous or heterozygous , respectively , for the WT allele .', 'Second , since this mutation is supposedly specific to the Rouge-des-Prés breed , with relatively recent emergence , polymorphisms were discarded if they were already present in the dbSNP database and/or in the Illumina SNP chip .', 'Finally , polymorphisms were filtered according to their predicted effects on transcript and/or protein , based on the hypothesis that this mutation is very deleterious ( Table 1 ) .', 'The two remaining putative causal SNPs were a single substitution in exon 15 of MAN2A1 gene and a single substitution in exon 4 of SLC25A46 gene ( Table 2 ) .', 'These two polymorphisms were further tested ( Sanger sequencing and Taqman assay ) on an extended DNA multibreed panel , including 93 Rouge-des-Prés cattle , and 321 samples from 12 French cattle breeds .', 'The MAN2A1 polymorphism was discarded because the genotype/phenotype association was not always found , and because it was present in other breeds .', 'The SLC25A46 polymorphism had a perfect genotype/phenotype association .', 'All the affected animals were homozygous for this mutation .', 'All the proteins from the mitochondrial carrier family share a common structure with three tandemly repeated homologous domains about 100 amino acids long .', 'Each domain contains two transmembrane alpha-helices forming a funnel-shaped cavity allowing the binding and the transport of the substrate from the intermembrane space to the matrix by a conformational transition [26 , 27 , 30] .', 'The C/T SLC25A46 substitution leads to replacement of an arginine by a cysteine , in the first transmembrane helix of the protein ( Fig 1A–1C ) .', 'This amino acid is highly conserved throughout evolution in SLC25A46 proteins ( Fig 1D ) .', 'When compared to the other mitochondrial carriers , as described in [30] , it appears that this amino-acid is not conserved across this family .', 'The most frequent amino-acid at this position is a threonine , and is shared by only 19 of the 53 mitochondrial carriers .', 'This suggests a role more related to the specific biological function ( s ) of SLC25A46 .', 'Based on SIFT software [31] , this substitution was expected to affect the function of SLC25A46 .', 'The mutated protein was expressed normally in brain and liver of affected animals and was present in mitochondrial-enriched protein extracts , consistent with a typical mitochondrial localization ( Fig 1E ) .', 'Affected calves have characteristic degenerative microscopic lesions in the central nervous system ( CNS ) and peripheral nervous system ( PNS ) , both in grey matter ( brain stem lateral vestibular nuclei and spinal cord thoracic nuclei ) and white matter ( dorsolateral and ventromedial funiculi of the spinal cord ) , in addition to demyelination in certain peripheral nerves [29] .', 'Electron microscopy confirmed this neuropathy phenotype , with discrete lesions of demyelination and a few enlarged nodes of Ranvier ( Fig 1F ) .', 'As mentioned above , selection against this disease in the affected breed was undertaken for obvious economic reasons as soon as the genetic test was commercially available .', 'Thus , affected animals were rapidly unavailable , limiting the range of phenotypic investigations that could be performed to analysis of previously collected tissue samples .', 'To better characterize the function of SLC25A46 , construction of mouse models was initiated .', 'SLC25A46 mouse models were constructed , using TALEN ( Translation Activator-Like Effector Nuclease ) technology , by targeting mouse exon 3 , the exon homologous to the one mutated in the bovine gene .', 'Following microinjection of the TALEN mRNA and screening of the resulting mice , two transgenic lines were established in a pure FVB/N genetic background:', '1 ) Tg26 line with a 75 bp DNA deletion inducing exon 3 aberrant splicing and resulting in a truncated protein of 159 amino acids; and', '2 ) Tg18 line with a 15 bp insertion / 3 bp deletion , causing replacement of 2 amino acids from the first transmembrane domain by six modified amino-acids ( Fig 2A , S2A Fig ) .', 'Heterozygous mice were viable and appeared as fit as their WT counterparts ( they were monitored for at least 12 months ) .', 'Transmission of the mutated allele followed Mendelian inheritance .', 'In both lines , Slc25a46 was undetectable by western blot analysis , on both total protein and on protein extracts enriched for mitochondrial proteins ( Fig 2B and 2C ) .', 'However , in Tg18 line , Slc25a46 mRNA levels were unchanged in homozygous mutant animals , except in peripheral nerves ( S2B Fig ) .', 'It suggests that a repression of the translation of the Slc25a46 mRNA occurred in Tg18 mice and/or more likely that the mutated protein was not properly associated with the mitochondrial membrane and consequently was rapidly degraded .', 'A degradation mechanism must also occur in Tg26 mice for the putatively translated truncated protein , in addition to a noticeable reduction in amount of Slc25a46 mRNA , probably due to mRNA decay ( S2C Fig ) .', 'Thus , homozygous mutants from both lines were regarded as functional knock-outs and will now be referred to as Tg-/- mice .', 'At birth , Tg-/- pups from the two lines were indistinguishable from each other and from their WT and heterozygous littermates , despite reported expression of the Slc25a46 gene early during mouse embryogenesis in various EST databases .', 'However , their growth was reduced compared to the WT pups from the end of the 1st week of life , and from the 2nd week , they stopped gaining weight ( Fig 3A and 3B ) .', 'The observed reduced growth rate started despite a normal feeding behavior during the first weeks , while the pups were still nursed by their mother , as evidenced by the presence of milk in their stomach ( Fig 3C ) and by a normal behavior in the cage ( i . e . all the pups were regularly seen under their mother , and none of them was left alone in the cage ) .', 'Yet , at 3 weeks of age , intestinal tracts of Tg-/-mice were less filled than their WT counterparts , with reduced feces ( Fig 3D ) , consistent with their cachectic state .', 'Furthermore , there were intestinal hemorrhages in the oldest Tg-/- animals ( Fig 3E ) .', 'Intestinal length and diameter were smaller in Tg-/- than in WT mice .', 'However , histological staining did not reveal any obvious change in the intestine from the Tg-/- mice which could explain their reduced growth ( S3A Fig ) .', 'An ataxic gait was apparent from the 2nd week of life , especially on the hind limbs ( Fig 3F and 3G ) .', 'Tg-/- mice walked on the tip of their toe , instead of putting the whole foot sole on the soil ( Fig 3F and 3G ) .', 'This was evocative of a proprioception defect , as it is described in the “Turning calves syndrome” [29] .', 'However , it did not evolve to permanent recumbency , perhaps due to the short lifespan of the Tg-/- mice ( see below ) .', 'Moderate hyperreflexia was also evidenced on hindlimbs when pinching the mice’s toes .', 'An epileptic-like phenotype was also noticed from the 2nd week of life ( S1 Movie ) .', 'All Tg-/- mice died between the 3rd and 4th weeks of life , either spontaneously in the cage , or by euthanasia for evident ethic reasons ( Fig 3H ) .', 'Post-mortem examination revealed that several other organs were affected in Tg-/-mice .', 'Thymus and spleen were significantly smaller relatively to the body mass ( Fig 3I and 3J ) .', 'This was expected as they are described as metabolic state sensors , with rapid atrophy in case of malnutrition [32] .', 'Liver was also significantly smaller ( Fig 3I and 3J ) , and biochemical blood analyses showing increased biliary acids , bilirubin and cholesterol in Tg-/- mice were indicative of a cholestasis , and consistent with a stress of the liver ( Table 3 ) .', 'Liver histology was nonetheless almost normal ( S3B Fig ) .', 'Muscle damage was also suspected , based on a general decrease of muscle mass combined with increased creatine kinase and aspartate amino transferase ( Table 3 ) , but muscle histopathology was also unchanged in Tg-/- mice ( S3C Fig ) .', 'Biochemical analysis revealed a highly-disturbed metabolism in Tg-/- mice , confirming the general alteration of their state ( Table 3 ) .', 'Severe hypoglycemia was noted , which may be linked to the observed growth defect .', 'Low plasma iron concentrations combined with high ferritin were indicative of defective iron metabolism and/or storage .', 'Therefore , the phenotype of the Tg-/- mice was distinctly different from that of the above-mentioned bovine sensorimotor polyneuropathy , presenting a wider range of symptoms .', 'Since Tg-/- mice displayed symptoms evocative of proprioception and motor involvement , investigations were then undertaken on the nervous system of Tg-/- mice .', 'However , no major defect of the CNS was detected in Tg-/- mice ( based on HES and Luxol blue staining ) , with only minimal lesions consisting of rare vacuolated neurons in the lateral vestibular nuclei ( Fig 4A ) .', 'Peripheral nerves lacked visible degenerative lesions , although the presence of macrophages containing lipid debris suggested a possible degenerative process ( Fig 4B ) .', 'Axon diameters and myelin sheath thickness was comparable in both genotypes ( Fig 4C ) .', 'Thus , even if it was not possible to exclude a peripheral neuropathy in Tg-/- mice , the fast evolution of the disease up to death may limit it to an early very mild form .', 'Furthermore , the study of the optic nerve could not highlight any difference between WT and Tg-/- pups ( Fig 4D ) , nor degenerative lesions in the Tg-/- axons , an observation recalling the lack of reported vision defect in the “Turning calves” [29] but contrasting with consistency of this phenotype in recently reported human cases [23 , 33 , 34] .', 'However , axons from CNS and PNS had abnormal round , small and aggregated mitochondria as evidenced in myelinated and non-myelinated fibers ( Fig 5A and 5B ) , indicating a fusion/fission imbalance , an observation also noticed in tissues from affected “Turning calves” ( Fig 5C ) .', 'Moreover , most mitochondria in Tg-/- mice had abnormalities of their internal architecture , namely abnormal membranes and cristae .', 'These abnormal mitochondria were also detected in the enteric nervous system of Tg-/- mice , in intestinal Auerbach plexus cells ( Fig 5D ) .', 'In accordance with ubiquitous expression of Slc25a46 in mice ( S2B and S2C Fig ) , there were abnormal mitochondria in other organs from Tg-/- mice , indicating a generalized mitochondrial defect .', 'Skeletal muscles also had numerous aggregated mitochondria , although their morphology generally remained normal ( Fig 5E ) , as well as muscular layers in the intestinal tract .', 'Hepatocytes had numerous and smaller mitochondria with vesicular cristae , rarely attached to the inner mitochondrial membrane ( Fig 5F–5H ) .', 'Since mitochondrial internal architecture and morphology were altered in several tissues , we searched for effects on mitochondrial metabolism .', 'Regarding activity of each respiratory chain complex in mice , there were significant decreases for complexes I , III , and IV in brain and muscle from Tg-/- mice ( Fig 6A ) .', 'However , there was an opposite trend in liver from Tg-/- mice , with a significant increase for complexes III and IV activities .', 'This particular response could be explained by the specific effect of physiological stresses on the mitochondrial metabolism in various tissues and specifically the liver [35 , 36] .', 'Krebs cycle enzymes , localized in the mitochondrial matrix , generally had no change in activities , except aconitase which was increased in muscles from Tg-/- mice , and fumarase which was increased in liver ( Fig 6B ) .', 'Proper fusion/fission equilibrium is necessary to maintain a homogeneous and healthy population of mitochondria [37] .', 'For example , several missense mutations of MFN2 causing autosomal dominant optic atrophy ‘plus’ phenotype induce a respiratory chain defect and mtDNA deletions and eventually mtDNA depletion in muscle cells [38 , 39] .', 'Moreover , loss of mtDNA is also found in Ugo1p depleted cells , Ugo1p being SLC25A46’s homolog in yeast [40] .', 'However , there was no significant mtDNA depletion or large deletion in liver , muscle or brain from homozygous mutant mice ( Fig 6C ) .', 'Comparative MS-MS analysis was conducted in brain protein extracts after enrichment of mitochondrial proteins , in order to detect changes in protein expression induced by disruption of Slc25a46 in mouse .', 'Amongst the detected proteins , only 26 were significantly up- or downregulated ( Table 4 ) .', 'Interestingly , five downregulated proteins belonged to the Hsp70 ( 70 kDa Heat-shock protein ) family ( Grp78 , Hs71l , Hs71b , Hsp7c , Hs74 ) , as well as three others for which the p-value nearly reached the significance threshold ( Hsp72 , Grp75 , Hs90b ) .', 'Such observations might suggest a role , direct or indirect , of Slc25a46 in the mitochondrial-Endoplasmic Reticulum ( ER ) contact sites ( see below ) .', 'Alterations were also noted in mitochondrial membrane proteins associated with glucose transport ( Hk1 , Hk1-sb ) , and fatty acid metabolism ( Gpdm , Echa ) .', 'Notably , hemoglobin subunits were significantly upregulated ( Hba , Hbb1 , Hbb2 ) .', 'This upregulation may be linked to an iron dysregulation , as evidenced by biochemical analyses on Tg-/- mice .', 'While this manuscript was first submitted , a paper was published , with evidence of interaction between SLC25A46 and fusion proteins MFN2 and OPA1 , and MIC60 and MIC19 proteins belonging to the MICOS complex [34] .', 'Notably MS-MS results did not show any significant change of expression for these proteins , their level was then monitored by western blot on brain extracts ( S4A Fig ) .', 'Tg-/- mice did not display significant expression level for these proteins .', 'Thus the knock-out of Slc25a46 in mouse does not lead to a reduction of Mic60 and a potential disruption of the MICOS complex , contrary to the fibroblasts treated with siRNA , as described in [34] .', 'Moreover , it is not compensated by a change in the expression of fusion proteins Opa1 and Mfn2 .', 'Expression of OPA1 , MFN2 and MIC60 was also monitored by Western blot on bovine brain and liver protein extracts , but we could not infer a significant change in the expression of these proteins , especially because the number of biological samples was very low ( S4B Fig ) .'], ['In the present study , we provided reliable evidence that the “Turning calves syndrome” , a recessive sensorimotor polyneuropathy reported in the French Rouge-des-Prés breed in the late 2000’s , was caused by a point mutation in SLC25A46 gene .', 'The single amino acid substitution did not affect protein expression nor its proper location within the mitochondria ( based on western blot ) .', 'However , it affected a highly conserved amino acid , in the first transmembrane helix of the protein .', 'Amongst the mitochondrial carrier proteins , 14 are known to be associated to rare metabolic diseases [27 , 41] .', 'Mutations are mostly located in functional domains of the proteins , including the substrate binding sites and the matrix and cytosolic gates ( which respectively open/close the carrier to the mitochondrial matrix and towards the cytosol ) [41] Interestingly , even the mutated amino-acid is not conserved amongst the mitochondrial carrier family , it is located just in the matrix gate area [30] , which is known to be critical for the conformational change .', 'Electron microscopy confirmed axonal lesions in affected cattle and identified abnormal round and aggregated mitochondria in axons .', 'This phenotype is reminiscent of mutations in mitochondrial fusion proteins such as MFN2 in CTM2A disease [20] , consistent with an fusion/fission imbalance .', 'However , SLC25A46 function in fusion or fission remains elusive .', 'Ugo1p , which is SLC25A46 homolog in yeast , plays a crucial role in fusion , in close interaction with Fzo1p and Mgm1p ( MFN1/2 and OPA1 homologs , respectively ) [40 , 42 , 43] .', 'Ugo1p mutants had fragmented mitochondria , and a loss of mtDNA [40] .', 'In humans , a pro-fission role of SLC25A46 was proposed , due to an increase of mitochondrial branching in fibroblasts derived from a patient carrying a homozygous missense mutation in the carrier domain of the protein [23] .', 'In contrast , there was another report of a SLC25A46 mutation in a splice site , leading to a truncated transcript and perhaps to a knock-out [33] .', 'In this case , the mitochondrial network was fragmented , suggesting a fusion role for SLC25A46 .', 'To better understand SLC25A46 function , and because the selection against “Turning calves syndrome” made new biological material collection difficult in cattle , mouse knock-out models were constructed .', 'In Tg-/- mice , nervous degenerative phenotypes ( ataxic gait and epilepsy ) were apparent from the 2nd week of life .', 'Furthermore , Tg-/- mice also had pronounced weight loss and metabolic defects leading to premature death around weaning .', 'Although histopathology did not account for this drastic phenotype , electron microscopy implicated involvement of mitochondria in several tissues .', 'There was a fusion/fission imbalance ( similar to cattle ) , with numerous round aggregated mitochondria in the central and peripheral nervous systems , including the enteric nervous system .', 'Abnormal mitochondria were present in Auerbach plexus cells .', 'This may have contributed to dysmotility of the intestinal tract , and the subsequent observed weight loss , at least partially , as often described for multi-systemic mitochondrial diseases such as Mitochondrial Neurogastrointestinal Encephalopathy Syndrome .', '[44] These small and numerous mitochondria were also detected in muscle and liver .', 'Clearly , effects of disruption of SLC25A46 were not restricted to the nervous system , consistent with ubiquitous expression of the gene .', 'Mitochondria regulate their shape in accordance with the metabolic state of the cell .', 'In case of starvation , mitochondrial length is increased , by phosphorylation of the fission protein Drp1 , leading to decreased fission [45] and/or by oligomerization of fusion protein OPA1 [46] .', 'In mitochondrial fusion-incompetent cells , mitochondria cannot fuse and are degraded , leading to cell death .', 'Tg-/- mice which experience weight loss , are in a metabolic state mimicking starvation .', 'Consequently , the absence of elongated mitochondria suggests an impaired fusion in these animals .', 'In addition to the fusion/fission imbalance , mitochondria from Tg-/- mice had disturbed internal architecture , with distorted and vesicular-like cristae , and cristae less frequently attached to the membrane .', 'Cristae morphology is maintained and regulated mainly by OPA1 and by the MICOS complex [1 , 47] .', 'This complex is composed of six subunits in yeast , with all of them inserted in the inner mitochondrial membrane [48] .', 'Mutations in genes encoding these subunits result in an altered internal architecture , i . e . loss of cristae junctions , and cristae organized as membrane stacks [48–50] .', 'MIC60 , also known as Mitofilin , is one of the key players of the MICOS complex .', 'In yeast , the MIC60 homolog Fcj1p interacts with SLC25A46 homolog Ugo1p , forming close contact sites between outer and inner mitochondrial membranes [51] .', 'The interaction between MIC60 and SLC25A46 has been recently documented in human [23 , 34] .', 'In the report from Janer et al . , the absence of SLC25A46 resulted in a marked decrease in the steady-state level of MIC60 in studied human fibroblasts [34] .', 'Based on abnormalities of mitochondrial architecture detected in Tg-/- mice , we inferred that Slc25a46 ( potentially in association with Mic60 ) , may contribute to establishment of a proper contact between outer and inner mitochondrial membranes in mammals .', 'However , Mic60 was only marginally downregulated in Tg-/- mice ( MS/MS analysis ) , with the p-value nearly reaching the threshold of significance , and this downregulation could not be observed by Western blot analysis .', 'Furthermore , MS/MS analysis did not highlight any downregulation of fusion factors interacting with Slc25a46 , such as OPA1 and MFN2 , nor did specific analysis of these proteins by Western blotting .', 'These differences suggest either cell-type ( fibroblast vs brain cells ) and/or species’ specificities .', 'Since cristae contain OXPHOS subunits ( i . e . respiratory complexes I to V ) , disorganization of cristae often decreases activity of OXPHOS subunits [49 , 52 , 53] and disturbs assembly of respiratory supercomplexes , with profound reduction in respiration efficiency [54] .', 'Mitochondrial metabolism is indeed affected in Tg-/- mice , with a marked decrease in complexes I , III , IV activities in brain and muscle , and an increase in complexes III and IV activities in liver .', 'This discrepancy is not unlikely , given the specificity of each tissue and each cell type in the response to physiological stresses [35 , 36] or to mutations [55 , 56] .', 'Our proteomic analysis highlighted a potential interaction between Slc25a46 and Hsp70 proteins; eight of the latter were down-regulated in Tg-/- mice .', 'These chaperone proteins , participate in the protein folding [57–59] and in the protein import across the outer mitochondrial membrane [60 , 61] in close interaction with Tom70 , which is also significantly downregulated .', 'Thus , in Tg-/- mice , importation of proteins may be downregulated , either by a direct interaction between Slc25a46 and the import machinery ( interactions between Slc25a46 and Hsp90 , Grp75 and Grp78 were recently evidenced by immunoprecipitation [23] ) , or by a general alteration of the outer mitochondrial membrane structure .', 'Interestingly , Grp78 also known as BiP , which is one of the most significantly downregulated protein in the Tg-/- mice , is considered as a major regulator of the ER , due to its multiple roles in the ER function [62] , and is shown to act at the ER-mitochondria interface under stress conditions [63 , 64] .', 'The recent observation that SLC25A46 interacts with the Endoplasmic Reticulum Membrane Complex ( EMC ) and may participate to the regulation of the phospholipid flux between ER and mitochondria appears to support the pivotal role of SLC25A46 between ER and mitochondria [34] .', 'Alternatively , since all these Hsp70 proteins function under the dependence of ATP , the affected mitochondrial metabolism may be insufficient to provide enough ATP , which could downregulate Hsp70 protein expression .', 'Collectively , there was good evidence for a pivotal function of SLC25A46 between the outer and inner mitochondrial membranes .', 'Disruption of Slc25a46 in mouse not only affected the subtle equilibrium between fusion and fission , but also disturbed the internal architecture and the link with a pool of Hsp70 chaperone proteins and potentially the mitochondrial-ER trafficking .', 'Consequently , mitochondrial and general metabolisms were severely impacted , leading to premature death .', 'This model seemed similar to an affected infant that died seven days after birth [33] .', 'In contrast , in cattle affected by the “Turning calves syndrome” , the mutated protein was still present and we inferred that it retained a portion of its activity , as in humans carrying homozygous missense mutations .', 'According to the localization of the mutations ( in cytosolic , transmembrane or inter mitochondrial membrane domains ) , interactions with various proteins could be disturbed , affecting only a part of SLC25A46’s functions .', 'Furthermore , alteration of SLC25A46’s functions might also result in species-specific phenotype , as all human cases reported so far suffer from optic atrophy , which is not observed in the bovine [29] and mouse models reported here ( Table 5 ) .', 'However , it should be mentioned that FVB/N mice carry two mutations which result in severe vision impairment: a mutation in the tyrosinase gene ( TyrC ) causing an albino phenotype and the retinal degeneration mutation ( Pde6brd/rd11 ) [65] .', 'Consequently , FVB/N mice suffer from early onset retinal degeneration and blindness around weaning [66] which might interfere with the observation of the phenotype .', 'Overall , our data in both models provided a basis for the wide range of human phenotypes described for SLC25A46 mutations .', 'Furthermore , there was clear evidence that SLC25A46 should be added to the list of candidate genes causing premature neonatal death , with a potential link between early deaths and SLC25A46 mutations that result in the absence or in a drastic reduction of the amount of the protein ( see Table 5 ) .', 'Finally , we produced the first Slc25a46 knock-out mouse model , which should be useful to further elucidate the function of SLC25A46 in mitochondrial dynamics .'], ['All procedures involving animals conformed to the Guide for the Care and Use of Laboratory Animals ( NIH Publication No . 85-23 , revised 1996 ) .', 'All efforts were made to minimize suffering .', 'Blood samples were collected from cattle by veterinarians or by trained and licensed technicians during routine blood sampling for paternity testing , genomic selection or annual prophylaxis .', 'Affected calves were euthanized for ethical reasons , due to the absence of effective treatment .', 'All samples and data were obtained with permission of breeders or breed organizations .', 'For mice , protocols were approved by the Animal Experimentation Ethics Committee and the French Ministry of Research ( APAFIS#1227–2015100516164803 v3 ) , and the Haut Conseil des Biotechnologies ( HCB n°6461 ) .', 'Twelve affected calves were examined clinically and confirmed to have the disease [29] .', 'Blood samples were collected from these calves and their parents , and DNA was extracted with a Genisol Maxi-Prep kit .', 'Blood samples were also collected from control animals known to be unaffected based on our ge
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['Neuropathies are neurodegenerative diseases affecting humans and other mammals .', 'Many genetic causes have been identified so far , including mutations of genes encoding proteins involved in mitochondrial dynamics .', 'Recently , the “Turning calves syndrome” , a novel sensorimotor polyneuropathy was described in the French Rouge-des-Prés cattle breed .', 'In the present study , we determined that this hereditary disease resulted from a single nucleotide substitution in SLC25A46 , a gene encoding a protein of the mitochondrial carrier family .', 'This mutation caused an apparent damaging amino-acid substitution .', 'To better understand the function of this protein , we knocked out the Slc25a46 gene in a mouse model .', 'This alteration affected not only the nervous system but also altered general metabolism , resulting in premature mortality .', 'Based on optic microscopy examination , electron microscopy and on biochemical , metabolic and proteomic analyses , we showed that the Slc25a46 disruption caused a fusion/fission imbalance and an abnormal mitochondrial architecture that disturbed mitochondrial metabolism .', 'These data extended the range of phenotypes associated with Slc25a46 dysfunction .', 'Moreover , this Slc25a46 knock-out mouse model should be useful to further elucidate the role of SLC25A46 in mitochondrial dynamics .']
['Mitochondria are essential organelles , the site of numerous biochemical reactions , with a critical role in delivering energy to cells , particularly in the nervous system .', 'Consequently , disrupted mitochondrial function often results in neurodegenerative diseases , in humans and in other mammals .', 'Herein , we determined that the “Turning calves syndrome” , a new hereditary sensorimotor polyneuropathy in the French Rouge-des-Prés cattle breed was due to a single substitution in SLC25A46 , a gene encoding a protein of the mitochondrial carrier family .', 'We created a mouse knock-out model and determined that disruption of this gene dramatically disturbed mitochondrial dynamics in various organs that resulted in altered metabolism and early death , indirectly confirming the gene identification in cattle .', 'Moreover , our novel findings extended the range of phenotypes associated with polymorphisms of this gene and help to elucidate the role of SLC25A46 in mitochondrial function .']
['protein metabolism', 'ruminants', 'mitochondrial dna', 'vertebrates', 'animals', 'mammals', 'animal models', 'model organisms', 'experimental organism systems', 'forms of dna', 'mitochondria', 'dna', 'bioenergetics', 'molecular biology techniques', 'cellular structures and organelles', 'extraction techniques', 'research and analysis methods', 'mitochondrial membrane', 'protein extraction', 'artificial gene amplification and extension', 'mouse models', 'metabolism', 'molecular biology', 'biochemistry', 'cattle', 'cell biology', 'nucleic acids', 'polymerase chain reaction', 'genetics', 'biology and life sciences', 'energy-producing organelles', 'amniotes', 'bovines', 'organisms']