Nicotinamide riboside alleviates exercise intolerance in ANT1-deficient mice.

OBJECTIVE: Mitochondrial disorders are often characterized by muscle weakness and fatigue. Null mutations in the heart-muscle adenine nucleotide translocator isoform 1 (ANT1) of both humans and mice cause cardiomyopathy and myopathy associated with exercise intolerance and muscle weakness. Here we decipher the molecular underpinnings of ANT1-deficiency-mediated exercise intolerance. METHODS: This was achieved by correlating exercise physiology, mitochondrial function and metabolomics of mice deficient in ANT1 and comparing this to control mice. RESULTS: We demonstrate a peripheral limitation of skeletal muscle mitochondrial respiration and a reduced complex I respiration in ANT1-deficient mice. Upon exercise, this results in a lack of NAD+ leading to a substrate limitation and stalling of the TCA cycle and mitochondrial respiration, further limiting skeletal muscle mitochondrial respiration. Treatment of ANT1-deficient mice with nicotinamide riboside increased NAD+ levels in skeletal muscle and liver, which increased the exercise capacity and the mitochondrial respiration. CONCLUSION: Increasing NAD+ levels with nicotinamide riboside can alleviate the exercise intolerance associated to ANT1-deficiency, indicating the therapeutic potential of NAD+-stimulating compounds in mitochondrial myopathies.

ANT1 overexpression models: Some similarities with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by progressive muscle weakness. Adenine nucleotide translocator 1 (ANT1), the only 4q35 gene involved in mitochondrial function, is strongly expressed in FSHD skeletal muscle biopsies. However, its role in FSHD is unclear. In this study, we evaluated ANT1 overexpression effects in primary myoblasts from healthy controls and during Xenopus laevis organogenesis. We also compared ANT1 overexpression effects with the phenotype of FSHD muscle cells and biopsies. Here, we report that the ANT1 overexpression-induced phenotype presents some similarities with FSHD muscle cells and biopsies. ANT1-overexpressing muscle cells showed disorganized morphology, altered cytoskeletal arrangement, enhanced mitochondrial respiration/glycolysis, ROS production, oxidative stress, mitochondrial fragmentation and ultrastructure alteration, as observed in FSHD muscle cells. ANT1 overexpression in Xenopus laevis embryos affected skeletal muscle development, impaired skeletal muscle, altered mitochondrial ultrastructure and led to oxidative stress as observed in FSHD muscle biopsies. Moreover, ANT1 overexpression in X. laevis embryos affected heart structure and mitochondrial ultrastructure leading to cardiac arrhythmia, as described in some patients with FSHD. Overall our data suggest that ANT1 could contribute to mitochondria dysfunction and oxidative stress in FSHD muscle cells by modifying their bioenergetic profile associated with ROS production. Such interplay between energy metabolism and ROS production in FSHD will be of significant interest for future prospects.

Kearns-Sayre syndrome with rare imaging finding of SLC25A4 Mutation.

Kearns-Sayre Syndrome (KSS) is a subtype of chronic progressive external ophthalmoplegia (CPEO). In this case, A 21-year-old man diagnosed with KSS, and presented with chronic progressive blepharoptosis (ptosis) and external ophthalmoplegia, diffuse depigmentation of the retinal pigment epithelium, and cerebellar ataxia, with a cerebrospinal fluid protein of 254 mg/dL, was reported. Genetic screening revealed a novel mutated gene in SLC25A4 in the patient as well as in his mother: NM_001151:c.170G>C in exon 2. Its imaging finding is a characteristic progressive atrophy of the right cerebellar hemisphere. In conclusion, we found a case of KSS with a novel mutated gene in SLC25A4: NM_001151:c.170G>C in exon 2 as the pathogenic mechanism, and found that KSS can be caused only when the proportion of mutations in the SLC25A4 gene reach a certain degree, and the patient with KSS showed a unique cranial imaging feature of unilateral progressive cerebellar atrophy.

Mitochondrial DNA depletion syndrome with a mutation in SLC25A4 developing epileptic encephalopathy: A case report.

INTRODUCTION: Autosomal dominant mitochondrial DNA depletion syndrome (MTDPS-12A) is characterized by severe hypotonia from birth due to a mutation in the adenine nucleotide translocator 1 (ANT1). CASE REPORT: A 4-year-old female patient diagnosed with neonatal-onset mitochondrial disease, who had good cognitive function while receiving antiepileptic treatment, presented with sudden-onset status epilepticus with facial and limb myoclonus persisting for more than 30 min. Subsequently, she developed epileptic encephalopathy. Brain MRI showed progressive ventricular enlargement and marked white matter atrophy. She was unable to perform verbal communication or make eye contact and fingertip movements. She lacked any signs of cardiomyopathy. Sanger sequencing demonstrated a heterozygous de novo mutation of c.239G>A (p.Arg80His) in SLC25A4. Her right quadriceps muscle tissue showed lowered complexes I, III, and IV activities and mitochondria DNA depletion (mitochondria/nuclear DNA: 14.6 ± 2.2%) through the quantitative polymerase chain reaction. She was definitively diagnosed with MTDPS-12A. CONCLUSION: Status epilepticus causes encephalopathy in patients with MTDPS-12A. Reducing the energy requirement on the cardiac muscle and brain may be a treatment strategy for patients with MTDPS-12A. Therefore, seizure management and preventive treatment of status epilepticus are considered to be important for maintaining neurodevelopmental outcomes.

RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts.

RBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression in muscle cells, contributing to heart disease. Here, we employ both 3'-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. RBFOX2-mediated APA modulates mRNA levels and/or isoform expression of a collection of genes, including contractile and mitochondrial genes. Depletion of RBFOX2 adversely affects mitochondrial health in myoblasts, correlating with disrupted APA of mitochondrial gene Slc25a4. Mechanistically, RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site, enforcing the use of the proximal polyadenylation site. In sum, our results unveil a role for RBFOX2 in fine-tuning expression of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases.

A Yeast-Based Screening Unravels Potential Therapeutic Molecules for Mitochondrial Diseases Associated with Dominant ANT1 Mutations.

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.

Drosophila melanogaster Mitochondrial Carriers: Similarities and Differences with the Human Carriers.

Mitochondrial carriers are a family of structurally related proteins responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. The in silico analysis of the Drosophila melanogaster genome has highlighted the presence of 48 genes encoding putative mitochondrial carriers, but only 20 have been functionally characterized. Despite most Drosophila mitochondrial carrier genes having human homologs and sharing with them 50% or higher sequence identity, D. melanogaster genes display peculiar differences from their human counterparts: (1) in the fruit fly, many genes encode more transcript isoforms or are duplicated, resulting in the presence of numerous subfamilies in the genome; (2) the expression of the energy-producing genes in D. melanogaster is coordinated from a motif known as Nuclear Respiratory Gene (NRG), a palindromic 8-bp sequence; (3) fruit-fly duplicated genes encoding mitochondrial carriers show a testis-biased expression pattern, probably in order to keep a duplicate copy in the genome. Here, we review the main features, biological activities and role in the metabolism of the D. melanogaster mitochondrial carriers characterized to date, highlighting similarities and differences with their human counterparts. Such knowledge is very important for obtaining an integrated view of mitochondrial function in D. melanogaster metabolism.

Consequences of inner mitochondrial membrane protein misfolding.

Proteins embedded in the inner mitochondrial membrane (IMM) perform essential cellular functions. Maintaining the folding state of these proteins is therefore of the utmost importance, and this is ensured by IMM chaperones and proteases that refold and degrade unassembled and misfolded proteins. However, the physiological consequences specific to IMM protein misfolding remain obscure because deletion of these chaperones/proteases (the typical experimental strategy) often affects many mitochondrial processes other than protein folding and turnover. Thus, novel experimental systems are needed to evaluate the direct effects of misfolded protein on the membrane. Such a system has been developed in recent years. Studies suggest that numerous pathogenic mutations in isoform 1 of adenine nucleotide translocase (Ant1) cause its misfolding on the IMM. In this review, we first discuss potential mechanisms by which dominant Ant1 mutations may cause disease, highlighting IMM protein misfolding, per se, as a likely pathological factor. Then we discuss the intramitochondrial effects of Ant1 misfolding such as IMM proteostatic stress, respiratory chain dysfunction, and mtDNA instability. Finally, we summarize the mounting evidence that IMM proteostatic stress can perturb mitochondrial protein import to cause the toxic accumulation of mitochondrial proteins in the cytosol: a cell stress mechanism termed mitochondrial Precursor Overaccumulation Stress (mPOS).

Protective effects of higenamine combined with [6]-gingerol against doxorubicin-induced mitochondrial dysfunction and toxicity in H9c2 cells and potential mechanisms.

Higenamine (HG) is a well-known selective activator of beta2-adrenergic receptor (ß2-AR) with a positive inotropic effect. The present study showed that HG combined with [6]-gingerol (HG/[6]-GR) protects H9c2 cells from doxorubicin (DOX)-induced mitochondrial energy metabolism disorder and respiratory dysfunction. H9c2 cells were pretreated with HG/[6]-GR for 2 h before DOX treatment in all procedures. Cell viability was quantified by a cell counting kit­8 assay. Cardiomyocyte morphology, proliferation, and mitochondrial function were detected by a high content screening (HCS) assay. Cell mitochondrial stress was measured by a Seahorse XFp analyzer. To further investigate the protective mechanism of HG/[6]-GR, mRNA and protein expression levels of PPARα/PGC-1α/Sirt3 pathway-related molecules were detected. The present data demonstrated that protective effects of HG/[6]-GR combination were presented in mitochondria, which increased cell viability, ameliorated DOX-induced mitochondrial dysfunction, increased mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Most importantly, the protective effects were abrogated by GW6471 (a PPARα inhibitor) and ameliorated by Wy14643 (a PPARα agonist). Moreover, the combined use of HG and [6]-GR exerted more profound protective effects than either drug as a single agent. In conclusion, the results suggested that HG/[6]-GR ameliorates DOX-induced mitochondrial energy metabolism disorder and respiratory function impairment in H9c2 cells, and it indicated that the protective mechanism may be related to upregulation of the PPARα/PGC-1α/Sirt3 pathway, which promotes mitochondrial energy metabolism and protects against heart failure.

The inner mitochondrial membrane protein ANT1 modulates IL-6 expression via the JNK pathway in macrophages.

Mitochondria are increasingly associated with inflammation. Here, we focus on the relationship between inflammation and adenine nucleotide translocator type 1 (ANT1), which is localized in the mitochondrial inner membrane. ANT1 plays an important role in oxidative phosphorylation, and mutations in the ANT1 gene are responsible for mitochondrial diseases. Ample studies have demonstrated that ANT1 has a critical role in cardiomyocytes and neurons, but little has been reported on its functions in immune cells. We knocked down ANT1 expression in macrophages and examined inflammatory cytokine expression after lipopolysaccharide stimulation. ANT1 knockdown reduces the expression of IL-6. JNK, upstream of IL-6, is downregulated, but other MAP kinases and the NF-κB signaling remain unchanged. These results suggest that ANT1 modulates IL-6 expression through JNK in macrophages.

Molecular analysis of inherited cardiomyopathy using next generation semiconductor sequencing technologies.

BACKGROUND: Cardiomyopathies are the most common clinical and genetic heterogeneity cardiac diseases, and genetic contribution in particular plays a major role in patients with primary cardiomyopathies. The aim of this study is to investigate cases of inherited cardiomyopathy (IC) for potential disease-causing mutations in 64 genes reported to be associated with IC. METHODS: A total of 110 independent cases or families diagnosed with various primary cardiomyopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, left ventricular non-compaction, and undefined cardiomyopathy, were collected after informed consent. A custom designed panel, including 64 genes, was screened using next generation sequencing on the Ion Torrent PGM platform. The best candidate disease-causing variants were verified by Sanger sequencing. RESULTS: A total of 78 variants in 73 patients were identified. After excluding the variants predicted to be benign and VUS, 26 pathogenic or likely pathogenic variants were verified in 26 probands (23.6%), including a homozygous variant in the SLC25A4 gene. Of these variants, 15 have been reported in the Human Gene Mutation Database or ClinVar database, while 11 are novel. The majority of variants were observed in the MYH7 (8/26) and MYBPC3 (6/26) gene. Titin (TTN) truncating mutations account for 13% in our dilated cardiomyopathy cases (3/23). CONCLUSIONS: This study provides an overview of the genetic aberrations in this cohort of Chinese IC patients and demonstrates the power of next generation sequencing in IC. Genetic results can provide precise clinical diagnosis and guidance regarding medical care for some individuals.

Ant1 mutant mice bridge the mitochondrial and serotonergic dysfunctions in bipolar disorder.

Although mitochondrial and serotonergic dysfunctions have been implicated in the etiology of bipolar disorder (BD), the relationship between these unrelated pathways has not been elucidated. A family of BD and chronic progressive external ophthalmoplegia (CPEO) caused by a mutation of the mitochondrial adenine nucleotide translocator 1 (ANT1, SLC25A4) implicated that ANT1 mutations confer a risk of BD. Here, we sequenced ANT1 in 324 probands of NIMH bipolar disorder pedigrees and identified two BD patients carrying heterozygous loss-of-function mutations. Behavioral analysis of brain specific Ant1 heterozygous conditional knockout (cKO) mice using lntelliCage showed a selective diminution in delay discounting. Delay discounting is the choice of smaller but immediate reward than larger but delayed reward and an index of impulsivity. Diminution of delay discounting suggests an increase in serotonergic activity. This finding was replicated by a 5-choice serial reaction time test. An anatomical screen showed accumulation of COX (cytochrome c oxidase) negative cells in dorsal raphe. Dorsal raphe neurons in the heterozygous cKO showed hyperexcitability, along with enhanced serotonin turnover in the nucleus accumbens and upregulation of Maob in dorsal raphe. These findings altogether suggest that mitochondrial dysfunction as the genetic risk of BD may cause vulnerability to BD by altering serotonergic neurotransmission.

Xenotopic expression of alternative electron transport enzymes in animal mitochondria and their impact in health and disease.

The mitochondrial respiratory chain in vertebrates and arthropods is different from that of most other eukaryotes because they lack alternative enzymes that provide electron transfer pathways additional to the oxidative phosphorylation (OXPHOS) system. However, the use of diverse experimental models, such as human cells in culture, Drosophila melanogaster and the mouse, has demonstrated that the transgenic expression of these alternative enzymes can impact positively many phenotypes associated with human mitochondrial and other cellular dysfunction, including those typically presented in complex IV deficiencies, Parkinson's, and Alzheimer's. In addition, these enzymes have recently provided extremely valuable data on how, when, and where reactive oxygen species, considered by many as "by-products" of OXPHOS, can contribute to animal longevity. It has also been shown that the expression of the alternative enzymes is thermogenic in cultured cells, causes reproductive defects in flies, and enhances the deleterious phenotype of some mitochondrial disease models. Therefore, all the reported beneficial effects must be considered with caution, as these enzymes have been proposed to be deployed in putative gene therapies to treat human diseases. Here, we present a brief review of the scientific data accumulated over the past decade that show the benefits and the risks of introducing alternative branches of the electron transport into mammalian and insect mitochondria, and we provide a perspective on the future of this research field.

Movement disorders in genetically confirmed mitochondrial disease and the putative role of the cerebellum.

BACKGROUND: Mitochondrial disease can present as a movement disorder. Data on this entity's epidemiology, genetics, and underlying pathophysiology, however, is scarce. OBJECTIVE: The objective of this study was to describe the clinical, genetic, and volumetric imaging data from patients with mitochondrial disease who presented with movement disorders. METHODS: In this retrospective analysis of all genetically confirmed mitochondrial disease cases from three centers (n = 50), the prevalence and clinical presentation of video-documented movement disorders was assessed. Voxel-based morphometry from high-resolution MRI was employed to compare cerebral and cerebellar gray matter volume between mitochondrial disease patients with and without movement disorders and healthy controls. RESULTS: Of the 50 (30%) patients with genetically confirmed mitochondrial disease, 15 presented with hypokinesia (parkinsonism 3/15), hyperkinesia (dystonia 5/15, myoclonus 3/15, chorea 2/15), and ataxia (3/15). In 3 patients, mitochondrial disease presented as adult-onset isolated dystonia. In comparison to healthy controls and mitochondrial disease patients without movement disorders, patients with hypo- and hyperkinetic movement disorders had significantly more cerebellar atrophy and an atrophy pattern predominantly involving cerebellar lobules VI and VII. CONCLUSION: This series provides clinical, genetic, volumetric imaging, and histologic data that indicate major involvement of the cerebellum in mitochondrial disease when it presents with hyper- and hypokinetic movement disorders. As a working hypothesis addressing the particular vulnerability of the cerebellum to energy deficiency, this adds substantially to the pathophysiological understanding of movement disorders in mitochondrial disease. Furthermore, it provides evidence that mitochondrial disease can present as adult-onset isolated dystonia. © 2017 International Parkinson and Movement Disorder Society.

Two new cases of mitochondrial myopathy with exercise intolerance, hyperlactatemia and cardiomyopathy, caused by recessive SLC25A4 mutations.

We report the clinical, morphological and molecular features of two patients with autosomal recessive SLC25A4 (ANT1) gene mutations. Furthermore, all previously published cases are reviewed to identify valuable features for future diagnosis. Patients present a common phenotype with exercise intolerance, hyperlactatemia, and hypertrophic cardiomyopathy. Muscle biopsies show wide sub-sarcolemmal mitochondrial aggregates, and increased activities of all respiratory chain complexes. The phenotype of recessive SLC25A4 (ANT1) mutations although rare, is homogenous and easily recognizable and could help orientate the molecular analysis in adults with exercise intolerance associated with hyperlactatemia.

Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis.

Aberrant activation of NLRP3 inflammasome has an important function in the pathogenesis of various inflammatory diseases. Although many components and mediators of inflammasome activation have been identified, how NLRP3 inflammasome is regulated to prevent excessive inflammation is unclear. Here we show NLRP3 inflammasome stimulators trigger Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) translocation to the mitochondria, to interact with and dephosphorylate adenine nucleotide translocase 1 (ANT1), a central molecule controlling mitochondrial permeability transition. This mechanism prevents collapse of mitochondrial membrane potential and the subsequent release of mitochondrial DNA and reactive oxygen species, thus preventing hyperactivation of NLRP3 inflammasome. Ablation or inhibition of SHP2 in macrophages causes intensified NLRP3 activation, overproduction of proinflammatory cytokines IL-1ß and IL-18, and increased sensitivity to peritonitis. Collectively, our data highlight that, by inhibiting ANT1 and mitochondrial dysfunction, SHP2 orchestrates an intrinsic regulatory loop to limit excessive NLRP3 inflammasome activation.

Dominance of yeast aac2R96H and aac2R252G mutations, equivalent to pathological mutations in ant1, is due to gain of function.

The mitochondrial ADP/ATP carrier is a nuclear encoded protein, which catalyzes the exchange of ATP generated in mitochondria with ADP produced in the cytosol. In humans, mutations in the major ADP/ATP carrier gene, ANT1, are involved in several degenerative mitochondrial pathologies, leading to instability of mitochondrial DNA. Recessive mutations have been associated with mitochondrial myopathy and cardiomyopathy whereas dominant mutations have been associated with autosomal dominant Progressive External Ophtalmoplegia (adPEO). Recently, two de novo dominant mutations, R80H and R235G, leading to extremely severe symptoms, have been identified. In order to evaluate if the dominance is due to haploinsufficiency or to a gain of function, the two mutations have been introduced in the equivalent positions of the AAC2 gene, the yeast orthologue of human ANT1, and their dominant effect has been studied in heteroallelic strains, containing both one copy of wild type AAC2 and one copy of mutant aac2 allele. Through phenotypic characterization of these yeast models we showed that the OXPHOS phenotypes in the heteroallelic strains were more affected than in the hemiallelic strain indicating that the dominant trait of the two mutations is due to gain of function.

Mitochondrial ANT-1 related adPEO leading to cognitive impairment: is there a link?

ANT1 is one of the nuclear genes responsible of autosomal dominant progressive external ophthalmoplegia (adPEO) with mitochondrial DNA multiple deletions. The course of ANT1- related adPEO is relatively benign, symptoms being generally restricted to skeletal muscle. Here we report the case of an Italian 74 years old woman with ANT1-related adPEO and dementia. Further studies are needed to assess the prevalence of central neurological manifestations in ANT1 mitochondrial disease.

A Mechanism Study Underlying the Protective Effects of Cyclosporine-A on Lung Ischemia-Reperfusion Injury.

AIM: This study is aimed at validating the hypothesis that administration of cyclosporine-A (CsA) would be protective in lung ischemia-reperfusion (I/R) injury and in exploring the underlying mechanism. METHODS: Rabbits were divided into 4 groups: the control, sham operation, I/R, and I/R with CsA treatment. Flow cytometry was used to measure the mitochondrial membrane potential. Laser scanning confocal microscope was used to analyze mitochondrion permeability transition pore (MPTP). The apoptotic cell was detected by the TUNEL staining. Western blot was performed to analyze the protein expression levels. RESULTS: CsA not only attenuated the histopathologic alterations in lung and mitochondria after I/R injury, but also attenuated I/R injury through increasing MPP and inhibiting MPTP opening. Besides, CsA attenuated I/R injury through suppressing the release of cytochrome-c (CytC), inhibiting cell apoptosis and decreasing the expression levels of cyclophilin-D (Cyp-D), adenine nucleotide translocase 1 (ANT1) and voltage-dependent anion channel 1 (VDAC1). Finally, we found that Cyp-D knockdown inhibits I/R injury-induced MPTP opening and cell apoptosis. CONCLUSION: Our study found that the protective role of CsA on lung I/R injury depends on the inhibition of MPTP and CytC release, suppression of the activation of mitochondrial apoptosis pathway and the expressions of apoptotic-related proteins, as well as the decreased expression levels of ANT1 and VDAC1.