Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

Determining minimal clinically important differences in the Hammersmith Functional Motor Scale Expanded for untreated spinal muscular atrophy patients: An international study.

BACKGROUND AND PURPOSE: Spinal muscular atrophy (SMA) is a rare and progressive neuromuscular disorder with varying severity levels. The aim of the study was to calculate minimal clinically important difference (MCID), minimal detectable change (MDC), and values for the Hammersmith Functional Motor Scale Expanded (HFMSE) in an untreated international SMA cohort. METHODS: The study employed two distinct methods. MDC was calculated using distribution-based approaches to consider standard error of measurement and effect size change in a population of 321 patients (176 SMA II and 145 SMA III), allowing for stratification based on age and function. MCID was assessed using anchor-based methods (receiver operating characteristic [ROC] curve analysis and standard error) on 76 patients (52 SMA II and 24 SMA III) for whom the 12-month HFMSE could be anchored to a caregiver-reported clinical perception questionnaire. RESULTS: With both approaches, SMA type II and type III patients had different profiles. The MCID, using ROC analysis, identified optimal cutoff points of -2 for type II and -4 for type III patients, whereas using the standard error we found the optimal cutoff points to be 1.5 for improvement and -3.2 for deterioration. Furthermore, distribution-based methods uncovered varying values across age and functional status subgroups within each SMA type. CONCLUSIONS: These results emphasize that the interpretation of a single MCID or MDC value obtained in large cohorts with different functional status needs to be made with caution, especially when these may be used to assess possible responses to new therapies.

VMA21 deficiency: a case of myocyte indigestion.

The Vma21p protein in yeast is an essential assembly chaperone for the vacuolar ATPase, the major proton pump of cellular membranes. In this issue, Ramachandran et al. (2009) report that mutations in the gene encoding the human homolog VMA21 cause the disease X-linked myopathy with excessive autophagy through an unexpected mechanism.

Diagnosis and management of glycogen storage disease type IV, including adult polyglucosan body disease: A clinical practice resource.

Glycogen storage disease type IV (GSD IV) is an ultra-rare autosomal recessive disorder caused by pathogenic variants in GBE1 which results in reduced or deficient glycogen branching enzyme activity. Consequently, glycogen synthesis is impaired and leads to accumulation of poorly branched glycogen known as polyglucosan. GSD IV is characterized by a remarkable degree of phenotypic heterogeneity with presentations in utero, during infancy, early childhood, adolescence, or middle to late adulthood. The clinical continuum encompasses hepatic, cardiac, muscular, and neurologic manifestations that range in severity. The adult-onset form of GSD IV, referred to as adult polyglucosan body disease (APBD), is a neurodegenerative disease characterized by neurogenic bladder, spastic paraparesis, and peripheral neuropathy. There are currently no consensus guidelines for the diagnosis and management of these patients, resulting in high rates of misdiagnosis, delayed diagnosis, and lack of standardized clinical care. To address this, a group of experts from the United States developed a set of recommendations for the diagnosis and management of all clinical phenotypes of GSD IV, including APBD, to support clinicians and caregivers who provide long-term care for individuals with GSD IV. The educational resource includes practical steps to confirm a GSD IV diagnosis and best practices for medical management, including (a) imaging of the liver, heart, skeletal muscle, brain, and spine, (b) functional and neuromusculoskeletal assessments, (c) laboratory investigations, (d) liver and heart transplantation, and (e) long-term follow-up care. Remaining knowledge gaps are detailed to emphasize areas for improvement and future research.

Two cases of MT-ND5-related mitochondrial disorder misdiagnosed as seronegative neuromyelitis optica spectrum disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease primarily affecting the optic nerves and spinal cord, which is usually associated with anti-aquaporin-4 antibodies. Here, we present two individuals who were negative for anti-aquaporin-4 antibodies and were initially diagnosed with seronegative NMOSD. Each patient's clinical course and radiographic features raised suspicion for an alternative disease process. Both individuals were found to have pathogenic variants of MT-ND5, encoding subunit 5 of mitochondrial complex I, ultimately leading to a revised diagnosis of a primary mitochondrial disorder. These cases illustrate the importance of biochemical and genetic testing in atypical cases of NMOSD.

Time to harmonize mitochondrial syndrome nomenclature and classification: A consensus from the North American Mitochondrial Disease Consortium (NAMDC).

OBJECTIVE: To harmonize terminology in mitochondrial medicine, we propose revised clinical criteria for primary mitochondrial syndromes. METHODS: The North American Mitochondrial Disease Consortium (NAMDC) established a Diagnostic Criteria Committee comprised of members with diverse expertise. It included clinicians, researchers, diagnostic laboratory directors, statisticians, and data managers. The Committee conducted a comprehensive literature review, an evaluation of current clinical practices and diagnostic modalities, surveys, and teleconferences to reach consensus on syndrome definitions for mitochondrial diseases. The criteria were refined after manual application to patients enrolled in the NAMDC Registry. RESULTS: By building upon published diagnostic criteria and integrating recent advances, NAMDC has generated updated consensus criteria for the clinical definition of classical mitochondrial syndromes. CONCLUSIONS: Mitochondrial diseases are clinically, biochemically, and genetically heterogeneous and therefore challenging to classify and diagnose. To harmonize terminology, we propose revised criteria for the clinical definition of mitochondrial disorders. These criteria are expected to standardize the diagnosis and categorization of mitochondrial diseases, which will facilitate future natural history studies and clinical trials.

Synergistic Deoxynucleoside and Gene Therapies for Thymidine Kinase 2 Deficiency.

OBJECTIVE: Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with pyrimidine deoxynucleosides deoxycytidine and thymidine ameliorates mitochondrial defects and extends the lifespan of Tk2 knock-in mouse (Tk2KI ) and compassionate use deoxynucleoside therapy in TK2 deficient patients have shown promising indications of efficacy. To augment therapy for Tk2 deficiency, we assessed gene therapy alone and in combination with deoxynucleoside therapy in Tk2KI mice. METHODS: We generated pAAVsc CB6 PI vectors containing human TK2 cDNA (TK2). Adeno-associated virus (AAV)-TK2 was administered to Tk2KI , which were serially assessed for weight, motor functions, and survival as well as biochemical functions in tissues. AAV-TK2 treated mice were further treated with deoxynucleosides. RESULTS: AAV9 delivery of human TK2 cDNA to Tk2KI mice efficiently rescued Tk2 activity in all the tissues tested except the kidneys, delayed disease onset, and increased lifespan. Sequential treatment of Tk2KI mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2KI mice dramatically improved mtDNA copy numbers in the liver and kidneys, animal growth, and lifespan. INTERPRETATION: Our data indicate that AAV-TK2 gene therapy as well as combination deoxynucleoside and gene therapies is more effective in Tk2KI mice than pharmacological alone. Thus, combination of gene therapy with substrate enhancement is a promising therapeutic approach for TK2 deficiency and potentially other metabolic disorders. ANN NEUROL 2021;90:640-652.

Cardiomyopathy and altered integrin-actin signaling in Fhl1 mutant female mice.

X-linked scapuloperoneal myopathy (X-SM), one of Four-and-a-half LIM 1 (FHL1) related diseases, is an adult-onset slowly progressive myopathy, often associated with cardiomyopathy. We previously generated a knock-in mouse model that has the same mutation (c.365 G > C, p.W122S) as human X-SM patients. The mutant male mouse developed late-onset slowly progressive myopathy without cardiomyopathy. In this study, we observed that heterozygous (Het) and homozygous (Homo) female mice did not show alterations of skeletal muscle function or histology. In contrast, 20-month-old mutant female mice showed signs of cardiomyopathy on echocardiograms with increased systolic diameter [wild-type (WT): 2.74 ± 0.22 mm, mean ± standard deviation (SD); Het: 3.13 ± 0.11 mm, P < 0.01; Homo: 3.08 ± 0.37 mm, P < 0.05) and lower fractional shortening (WT: 31.1 ± 4.4%, mean ± SD; Het: 22.7 ± 2.5%, P < 0.01; Homo: 22.4 ± 6.9%, P < 0.01]. Histological analysis of cardiac muscle revealed frequent extraordinarily large rectangular nuclei in mutant female mice that were also observed in human cardiac muscle from X-SM patients. Western blot demonstrated decreased Fhl1 protein levels in cardiac muscle, but not in skeletal muscle, of Homo mutant female mice. Proteomic analysis of cardiac muscle from 20-month-old Homo mutant female mice indicated abnormalities of the integrin signaling pathway (ISP) in association with cardiac dysfunction. The ISP dysregulation was further supported by altered levels of a subunit of the ISP downstream effectors Arpc1a in Fhl1 mutant mice and ARPC1A in X-SM patient muscles. This study reveals the first mouse model of FHL1-related cardiomyopathy and implicates ISP dysregulation in the pathogenesis of FHL1 myopathy.

GGPS1 Mutations Cause Muscular Dystrophy/Hearing Loss/Ovarian Insufficiency Syndrome.

OBJECTIVE: A hitherto undescribed phenotype of early onset muscular dystrophy associated with sensorineural hearing loss and primary ovarian insufficiency was initially identified in 2 siblings and in subsequent patients with a similar constellation of findings. The goal of this study was to understand the genetic and molecular etiology of this condition. METHODS: We applied whole exome sequencing (WES) superimposed on shared haplotype regions to identify the initial biallelic variants in GGPS1 followed by GGPS1 Sanger sequencing or WES in 5 additional families with the same phenotype. Molecular modeling, biochemical analysis, laser membrane injury assay, and the generation of a Y259C knock-in mouse were done. RESULTS: A total of 11 patients in 6 families carrying 5 different biallelic pathogenic variants in specific domains of GGPS1 were identified. GGPS1 encodes geranylgeranyl diphosphate synthase in the mevalonate/isoprenoid pathway, which catalyzes the synthesis of geranylgeranyl pyrophosphate, the lipid precursor of geranylgeranylated proteins including small guanosine triphosphatases. In addition to proximal weakness, all but one patient presented with congenital sensorineural hearing loss, and all postpubertal females had primary ovarian insufficiency. Muscle histology was dystrophic, with ultrastructural evidence of autophagic material and large mitochondria in the most severe cases. There was delayed membrane healing after laser injury in patient-derived myogenic cells, and a knock-in mouse of one of the mutations (Y259C) resulted in prenatal lethality. INTERPRETATION: The identification of specific GGPS1 mutations defines the cause of a unique form of muscular dystrophy with hearing loss and ovarian insufficiency and points to a novel pathway for this clinical constellation. ANN NEUROL 2020;88:332-347.

Regulatory environment for novel therapeutic development in mitochondrial diseases.

At present, there is just one approved therapy for patients with mitochondrial diseases in Europe, another in Japan, and none in the United States. These facts reveal an important and significant unmet need for approved therapies for these debilitating and often fatal disorders. To fill this need, it is critical for clinicians and drug developers to work closely with regulatory agencies. In the United States, mitochondrial disease patients and clinicians, the United Mitochondrial Disease Foundation, and pharmaceutical industry members have engaged with the Food and Drug Administration to educate each other about these complex and heterogeneous diseases and about regulatory requirements to obtain approvals for novel therapies. Clinical development of therapies for rare diseases has been facilitated by the 1983 US Orphan Drug Act (ODA) and similar legislation in Japan and the European Union. Further legislation and regulatory guidance have expanded and refined regulatory flexibility. While regulatory and financial incentives of the ODA have augmented involvement of pharmaceutical companies, clinicians, with patient advocacy groups and industry, need to conduct natural history studies, develop clinical outcome measures, and identify potential supportive surrogate endpoints predictive of clinical benefit, which together are critical foundations for clinical trials. Thus, the regulatory environment for novel therapeutic development is conducive and offers flexibility for mitochondrial diseases. Nevertheless, flexibility does not mean lower standards, as well-controlled rigorous clinical trials of high quality are still required to establish the efficacy of potential therapies and to obtain regulatory agency approvals for their commercial use. This process is illustrated through the authors' ongoing efforts to develop therapy for thymidine kinase 2 deficiency.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): Position paper on diagnosis, prognosis, and treatment by the MNGIE International Network.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disease caused by TYMP mutations and thymidine phosphorylase (TP) deficiency. Thymidine and deoxyuridine accumulate impairing the mitochondrial DNA maintenance and integrity. Clinically, patients show severe and progressive gastrointestinal and neurological manifestations. The onset typically occurs in the second decade of life and mean age at death is 37 years. Signs and symptoms of MNGIE are heterogeneous and confirmatory diagnostic tests are not routinely performed by most laboratories, accounting for common misdiagnosis. Factors predictive of progression and appropriate tests for monitoring are still undefined. Several treatment options showed promising results in restoring the biochemical imbalance of MNGIE. The lack of controlled studies with appropriate follow-up accounts for the limited evidence informing diagnostic and therapeutic choices. The International Consensus Conference (ICC) on MNGIE, held in Bologna, Italy, on 30 March to 31 March 2019, aimed at an evidence-based consensus on diagnosis, prognosis, and treatment of MNGIE among experts, patients, caregivers and other stakeholders involved in caring the condition. The conference was conducted according to the National Institute of Health Consensus Conference methodology. A consensus development panel formulated a set of statements and proposed a research agenda. Specifically, the ICC produced recommendations on: (a) diagnostic pathway; (b) prognosis and the main predictors of disease progression; (c) efficacy and safety of treatments; and (f) research priorities on diagnosis, prognosis, and treatment. The Bologna ICC on diagnosis, management and treatment of MNGIE provided evidence-based guidance for clinicians incorporating patients' values and preferences.

USMG5 Ashkenazi Jewish founder mutation impairs mitochondrial complex V dimerization and ATP synthesis.

Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of five protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, USMG5. The Ashkenazi population allele frequency is 0.57%. This mutation produces two USMG5 transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type USMG5 mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type USMG5 cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent USMG5 splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.

Natural underlying mtDNA heteroplasmy as a potential source of intra-person hiPSC variability.

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability, the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.

Successful liver transplantation in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a fatal disorder characterized by progressive gastrointestinal dysmotility, peripheral neuropathy, leukoencephalopathy, skeletal myopathy, ophthalmoparesis, and ptosis. MNGIE stems from deficient thymidine phosphorylase activity (TP) leading to toxic elevations of plasma thymidine. Hematopoietic stem cell transplant (HSCT) restores TP activity and halts disease progression but has high transplant-related morbidity and mortality. Liver transplant (LT) was reported to restore TP activity in two adult MNGIE patients. We report successful LT in four additional MNGIE patients, including a pediatric patient. Our patients were diagnosed between ages 14 months and 36 years with elevated thymidine levels and biallelic pathogenic variants in TYMP. Two patients presented with progressive gastrointestinal dysmotility, and three demonstrated progressive peripheral neuropathy with two suffering limitations in ambulation. Two patients, including the child, had liver dysfunction and cirrhosis. Following LT, thymidine levels nearly normalized in all four patients and remained low for the duration of follow-up. Disease symptoms stabilized in all patients, with some manifesting improvements, including intestinal function. No patient died, and LT appeared to have a more favorable safety profile than HSCT, especially when liver disease is present. Follow-up studies will need to document the long-term impact of this new approach on disease outcome. Take Home Message: Liver transplantation is effective in stabilizing symptoms and nearly normalizing thymidine levels in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and may have an improved safety profile over hematopoietic stem cell transplant.

Deoxynucleoside Therapy for Thymidine Kinase 2-Deficient Myopathy.

OBJECTIVE: Thymidine kinase 2, encoded by the nuclear gene TK2, is required for mitochondrial DNA maintenance. Autosomal recessive TK2 mutations cause depletion and multiple deletions of mtDNA that manifest predominantly as a myopathy usually beginning in childhood and progressing relentlessly. We investigated the safety and efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies. METHODS: We administered deoxynucleoside monophosphates and deoxynucleoside to 16 TK2-deficient patients under a compassionate use program. RESULTS: In 5 patients with early onset and severe disease, survival and motor functions were better than historically untreated patients. In 11 childhood and adult onset patients, clinical measures stabilized or improved. Three of 8 patients who were nonambulatory at baseline gained the ability to walk on therapy; 4 of 5 patients who required enteric nutrition were able to discontinue feeding tube use; and 1 of 9 patients who required mechanical ventilation became able to breathe independently. In motor functional scales, improvements were observed in the 6-minute walk test performance in 7 of 8 subjects, Egen Klassifikation in 2 of 3, and North Star Ambulatory Assessment in all 5 tested. Baseline elevated serum growth differentiation factor 15 levels decreased with treatment in all 7 patients tested. A side effect observed in 8 of the 16 patients was dose-dependent diarrhea, which did not require withdrawal of treatment. Among 12 other TK2 patients treated with deoxynucleoside, 2 adults developed elevated liver enzymes that normalized following discontinuation of therapy. INTERPRETATION: This open-label study indicates favorable side effect profiles and clinical efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies for TK2 deficiency. ANN NEUROL 2019;86:293-303.

Intracellular calcium leak as a therapeutic target for RYR1-related myopathies.

RYR1 encodes the type 1 ryanodine receptor, an intracellular calcium release channel (RyR1) on the skeletal muscle sarcoplasmic reticulum (SR). Pathogenic RYR1 variations can destabilize RyR1 leading to calcium leak causing oxidative overload and myopathy. However, the effect of RyR1 leak has not been established in individuals with RYR1-related myopathies (RYR1-RM), a broad spectrum of rare neuromuscular disorders. We sought to determine whether RYR1-RM affected individuals exhibit pathologic, leaky RyR1 and whether variant location in the channel structure can predict pathogenicity. Skeletal muscle biopsies were obtained from 17 individuals with RYR1-RM. Mutant RyR1 from these individuals exhibited pathologic SR calcium leak and increased activity of calcium-activated proteases. The increased calcium leak and protease activity were normalized by ex-vivo treatment with S107, a RyR stabilizing Rycal molecule. Using the cryo-EM structure of RyR1 and a new dataset of > 2200 suspected RYR1-RM affected individuals we developed a method for assigning pathogenicity probabilities to RYR1 variants based on 3D co-localization of known pathogenic variants. This study provides the rationale for a clinical trial testing Rycals in RYR1-RM affected individuals and introduces a predictive tool for investigating the pathogenicity of RYR1 variants of uncertain significance.

The clinical spectrum of the congenital myasthenic syndrome resulting from COL13A1 mutations.

Next generation sequencing techniques were recently used to show mutations in COL13A1 cause synaptic basal lamina-associated congenital myasthenic syndrome type 19. Animal studies showed COL13A1, a synaptic extracellular-matrix protein, is involved in the formation and maintenance of the neuromuscular synapse that appears independent of the Agrin-LRP4-MuSK-DOK7 acetylcholine receptor clustering pathway. Here, we report the phenotypic spectrum of 16 patients from 11 kinships harbouring homozygous or heteroallelic mutations in COL13A1. Clinical presentation was mostly at birth with hypotonia and breathing and feeding difficulties often requiring ventilation and artificial feeding. Respiratory crisis related to recurrent apnoeas, sometimes triggered by chest infections, were common early in life but resolved over time. The predominant pattern of muscle weakness included bilateral ptosis (non-fatigable in adulthood), myopathic facies and marked axial weakness, especially of neck flexion, while limb muscles were less involved. Other features included facial dysmorphism, skeletal abnormalities and mild learning difficulties. All patients tested had results consistent with abnormal neuromuscular transmission. Muscle biopsies were within normal limits or showed non-specific changes. Muscle MRI and serum creatine kinase levels were normal. In keeping with COL13A1 mutations affecting both synaptic structure and presynaptic function, treatment with 3,4-diaminopyridine and salbutamol resulted in motor and respiratory function improvement. In non-treated cases, disease severity and muscle strength improved gradually over time and several adults recovered normal muscle strength in the limbs. In summary, patients with COL13A1 mutations present mostly with severe early-onset myasthenic syndrome with feeding and breathing difficulties. Axial weakness is greater than limb weakness. Disease course improves gradually over time, which could be consistent with the less prominent role of COL13A1 once the neuromuscular junction is mature. This report emphasizes the role of collagens at the human muscle endplate and should facilitate the recognition of this disorder, which can benefit from pharmacological treatment.

Low-dose rapamycin extends lifespan in a mouse model of mtDNA depletion syndrome.

Mitochondrial disorders affecting oxidative phosphorylation (OxPhos) are caused by mutations in both the nuclear and mitochondrial genomes. One promising candidate for treatment is the drug rapamycin, which has been shown to extend lifespan in multiple animal models, and which was previously shown to ameliorate mitochondrial disease in a knock-out mouse model lacking a nuclear-encoded gene specifying an OxPhos structural subunit (Ndufs4). In that model, relatively high-dose intraperitoneal rapamycin extended lifespan and improved markers of neurological disease, via an unknown mechanism. Here, we administered low-dose oral rapamycin to a knock-in (KI) mouse model of authentic mtDNA disease, specifically, progressive mtDNA depletion syndrome, resulting from a mutation in the mitochondrial nucleotide salvage enzyme thymidine kinase 2 (TK2). Importantly, low-dose oral rapamycin was sufficient to extend Tk2KI/KI mouse lifespan significantly, and did so in the absence of detectable improvements in mitochondrial dysfunction. We found no evidence that rapamycin increased survival by acting through canonical pathways, including mitochondrial autophagy. However, transcriptomics and metabolomics analyses uncovered systemic metabolic changes pointing to a potential 'rapamycin metabolic signature.' These changes also implied that rapamycin may have enabled the Tk2KI/KI mice to utilize alternative energy reserves, and possibly triggered indirect signaling events that modified mortality through developmental reprogramming. From a therapeutic standpoint, our results support the possibility that low-dose rapamycin, while not targeting the underlying mtDNA defect, could represent a crucial therapy for the treatment of mtDNA-driven, and some nuclear DNA-driven, mitochondrial diseases.

Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes.

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein implicated in mitochondrial dynamics, nucleoid organization, protein translation, cell growth, and cholesterol metabolism. We identified a recurrent de novo ATAD3A c.1582C>T (p.Arg528Trp) variant by whole-exome sequencing (WES) in five unrelated individuals with a core phenotype of global developmental delay, hypotonia, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. We also describe two families with biallelic variants in ATAD3A, including a homozygous variant in two siblings, and biallelic ATAD3A deletions mediated by nonallelic homologous recombination (NAHR) between ATAD3A and gene family members ATAD3B and ATAD3C. Tissue-specific overexpression of borR534W, the Drosophila mutation homologous to the human c.1582C>T (p.Arg528Trp) variant, resulted in a dramatic decrease in mitochondrial content, aberrant mitochondrial morphology, and increased autophagy. Homozygous null bor larvae showed a significant decrease of mitochondria, while overexpression of borWT resulted in larger, elongated mitochondria. Finally, fibroblasts of an affected individual exhibited increased mitophagy. We conclude that the p.Arg528Trp variant functions through a dominant-negative mechanism that results in small mitochondria that trigger mitophagy, resulting in a reduction in mitochondrial content. ATAD3A variation represents an additional link between mitochondrial dynamics and recognizable neurological syndromes, as seen with MFN2, OPA1, DNM1L, and STAT2 mutations.