TYMP Variants Result in Late-Onset Mitochondrial Myopathy With Altered Muscle Mitochondrial DNA Homeostasis.

Biallelic TYMP variants result in the mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a juvenile-onset disorder with progressive course and fatal outcome. Milder late-onset (>40 years) form has been rarely described. Gene panel sequencing in a cohort of 60 patients featuring muscle accumulation of mitochondrial DNA (mtDNA) deletions detected TYMP defects in three subjects (5%), two of them with symptom onset in the fifth decade. One of the patients only displayed ptosis and ophthalmoparesis. Biochemical and molecular studies supported the diagnosis. Screening of TYMP is recommended in adult patients with muscle mtDNA instability, even in the absence of cardinal MNGIE features.

Dystonia-ataxia syndrome with permanent torsional nystagmus caused by ECHS1 deficiency.

Biallelic mutations in ECHS1, encoding the mitochondrial enoyl-CoA hydratase, have been associated with mitochondrial encephalopathies with basal ganglia involvement. Here, we describe a novel clinical presentation consisting of dystonia-ataxia syndrome with hearing loss and a peculiar torsional nystagmus observed in two adult siblings. The presence of a 0.9-ppm peak at MR spectroscopy analysis suggested the accumulation of branched-chain amino acids. Exome sequencing in index probands identified two ECHS1 mutations, one of which was novel (p.V82L). ECHS1 protein levels and residual activities were reduced in patients' fibroblasts. This paper expands the phenotypic spectrum observed in patients with impaired valine catabolism.

Loss of the nucleoporin Aladin in central nervous system and fibroblasts of Allgrove Syndrome.

Allgrove syndrome (AS) is a rare disease with broad neurological involvement. Neurodegeneration can affect spinal motor neurons, Purkinje cells, striatal neurons and the autonomic system. The mechanisms that lead to neuronal loss are still unclear. Recessive mutations in the AAAS gene affect the encoded protein Aladin, which would normally localize to the cytoplasmic face of the nuclear membrane as part of the nuclear pore complex (NPC). While the NPC is known to be a key factor for nucleocytoplasmic transport, the precise role of Aladin has not been elucidated yet. Here, we explored the consequences of the homozygous AAAS mutation c.464G>A (p.R155H) in central nervous system tissues and fibroblasts of a novel AS patient presenting motor neuron disease, cerebellar ataxia and autonomic dysfunction. Neuropathological analyses showed severe loss of motor neurons and Purkinje cells, with significant reduction in the perinuclear expression of Aladin. A reduced amount of protein was detected in the nuclear membrane fraction of the patient's brain. RNA analysis revealed a significant reduction of the transcript AAAS-1, while the AAAS-2 transcript was upregulated in fibroblasts. To our knowledge, this is the first study to demonstrate the effects of AAAS mutations in the human central nervous system.

Neurofascin (NFASC) gene mutation causes autosomal recessive ataxia with demyelinating neuropathy.

INTRODUCTION: Neurofascin, encoded by NFASC, is a transmembrane protein that plays an essential role in nervous system development and node of Ranvier function. Anti-Neurofascin autoantibodies cause a specific type of chronic inflammatory demyelinating polyneuropathy (CIDP) often characterized by cerebellar ataxia and tremor. Recently, homozygous NFASC mutations were recently associated with a neurodevelopmental disorder in two families. METHODS: A combined approach of linkage analysis and whole-exome sequencing was performed to find the genetic cause of early-onset cerebellar ataxia and demyelinating neuropathy in two siblings from a consanguineous Italian family. Functional studies were conducted on neurons from induced pluripotent stem cells (iPSCs) generated from the patients. RESULTS: Genetic analysis revealed a homozygous p.V1122E mutation in NFASC. This mutation, affecting a highly conserved hydrophobic transmembrane domain residue, led to significant loss of Neurofascin protein in the iPSC-derived neurons of affected siblings. CONCLUSIONS: The identification of NFASC mutations paves the way for genetic research in the developing field of nodopathies, an emerging pathological entity involving the nodes of Ranvier, which are associated for the first time with a hereditary ataxia syndrome with neuropathy.

Key role of SMN/SYNCRIP and RNA-Motif 7 in spinal muscular atrophy: RNA-Seq and motif analysis of human motor neurons.

Spinal muscular atrophy is a motor neuron disorder caused by mutations in SMN1. The reasons for the selective vulnerability of motor neurons linked to SMN (encoded by SMN1) reduction remain unclear. Therefore, we performed deep RNA sequencing on human spinal muscular atrophy motor neurons to detect specific altered gene splicing/expression and to identify the presence of a common sequence motif in these genes. Many deregulated genes, such as the neurexin and synaptotagmin families, are implicated in critical motor neuron functions. Motif-enrichment analyses of differentially expressed/spliced genes, including neurexin2 (NRXN2), revealed a common motif, motif 7, which is a target of SYNCRIP. Interestingly, SYNCRIP interacts only with full-length SMN, binding and modulating several motor neuron transcripts, including SMN itself. SYNCRIP overexpression rescued spinal muscular atrophy motor neurons, due to the subsequent increase in SMN and their downstream target NRXN2 through a positive loop mechanism and ameliorated SMN-loss-related pathological phenotypes in Caenorhabditis elegans and mouse models. SMN/SYNCRIP complex through motif 7 may account for selective motor neuron degeneration and represent a potential therapeutic target.

Mitochondrial Dysregulation and Impaired Autophagy in iPSC-Derived Dopaminergic Neurons of Multiple System Atrophy.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets.

Mitochondrial dysfunction in fibroblasts of Multiple System Atrophy.

Multiple System Atrophy is a severe neurodegenerative disorder which is characterized by a variable clinical presentation and a broad neuropathological spectrum. The pathogenic mechanisms are almost completely unknown. In the present study, we established a cellular model of MSA by using fibroblasts' primary cultures and performed several experiments to investigate the causative mechanisms of the disease, with a particular focus on mitochondrial functioning. Fibroblasts' analyses (7 MSA-P, 7 MSA-C and 6 healthy controls) displayed several anomalies in patients: an impairment of respiratory chain activity, in particular for succinate Coenzyme Q reductase (p < 0.05), and a reduction of complex II steady-state level (p < 0.01); a reduction of Coenzyme Q10 level (p < 0.001) and an up-regulation of some CoQ10 biosynthesis enzymes, namely COQ5 and COQ7; an impairment of mitophagy, demonstrated by a decreased reduction of mitochondrial markers after mitochondrial inner membrane depolarization (p < 0.05); a reduced basal autophagic activity, shown by a decreased level of LC3 II (p < 0.05); an increased mitochondrial mass in MSA-C, demonstrated by higher TOMM20 levels (p < 0.05) and suggested by a wide analysis of mitochondrial DNA content in blood of large cohorts of patients. The present study contributes to understand the causative mechanisms of Multiple System Atrophy. In particular, the observed impairment of respiratory chain activity, mitophagy and Coenzyme Q10 biosynthesis suggests that mitochondrial dysfunction plays a crucial role in the pathogenesis of the disease. Furthermore, these findings will hopefully contribute to identify novel therapeutic targets for this still incurable disorder.

Glucose-free/high-protein diet improves hepatomegaly and exercise intolerance in glycogen storage disease type III mice.

Glycogen disease type III (GSDIII), a rare incurable autosomal recessive disorder due to glycogen debranching enzyme deficiency, presents with liver, heart and skeletal muscle impairment, hepatomegaly and ketotic hypoglycemia. Muscle weakness usually worsens to fixed myopathy and cardiac involvement may present in about half of the patients during disease. Management relies on careful follow-up of symptoms and diet. No common agreement was reached on sugar restriction and treatment in adulthood. We administered two dietary regimens differing in their protein and carbohydrate content, high-protein (HPD) and high-protein/glucose-free (GFD), to our mouse model of GSDIII, starting at one month of age. Mice were monitored, either by histological, biochemical and molecular analysis and motor functional tests, until 10 months of age. GFD ameliorated muscle performance up to 10 months of age, while HPD showed little improvement only in young mice. In GFD mice, a decreased muscle glycogen content and fiber vacuolization was observed, even in aged animals indicating a protective role of proteins against skeletal muscle degeneration, at least in some districts. Hepatomegaly was reduced by about 20%. Moreover, the long-term administration of GFD did not worsen serum parameters even after eight months of high-protein diet. A decreased phosphofructokinase and pyruvate kinase activities and an increased expression of Krebs cycle and gluconeogenesis genes were seen in the liver of GFD fed mice. Our data show that the concurrent use of proteins and a strictly controlled glucose supply could reduce muscle wasting, and indicate a better metabolic control in mice with a glucose-free/high-protein diet.

Purkinje cell COX deficiency and mtDNA depletion in an animal model of spinocerebellar ataxia type 1.

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of cerebellar degenerative disorders, characterized by progressive gait unsteadiness, hand incoordination, and dysarthria. Ataxia type 1 (SCA1) is caused by the expansion of a CAG trinucleotide repeat in the SCA1 gene resulting in the atypical extension of a polyglutamine (polyQ) tract within the ataxin-1 protein. Our main objective was to investigate the mitochondrial oxidative metabolism in the cerebellum of transgenic SCA1 mice. SCA1 transgenic mice develop clinical features in the early life stages (around 5 weeks of age) presenting pathological cerebellar signs with concomitant progressive Purkinje neuron atrophy and relatively little cell loss; this evidence suggests that the SCA1 phenotype is not the result of cell death per se, but a possible effect of cellular dysfunction that occurs before neuronal demise. We studied the mitochondrial oxidative metabolism in cerebellar cells from both homozygous and heterozygous transgenic SCA1 mice, aged 2 and 6 months. Histochemical examination showed a cytochrome-c-oxidase (COX) deficiency in the Purkinje cells (PCs) of both heterozygous and homozygous mice, the oxidative defect being more prominent in older mice, in which the percentage of COX-deficient PC was up to 30%. Using a laser-microdissector, we evaluated the mitochondrial DNA (mtDNA) content on selectively isolated COX-competent and COX-deficient PC by quantitative Polymerase Chain Reaction and we found mtDNA depletion in those with oxidative dysfunction. In conclusion, the selective oxidative metabolism defect observed in neuronal PC expressing mutant ataxin occurs as early as 8 weeks of age thus representing an early step in the PC degeneration process in SCA1 disease.

Genome-wide RNA-seq of iPSC-derived motor neurons indicates selective cytoskeletal perturbation in Brown-Vialetto disease that is partially rescued by riboflavin.

Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown-Vialetto-Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell-selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy-lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.

Selective mitochondrial depletion, apoptosis resistance, and increased mitophagy in human Charcot-Marie-Tooth 2A motor neurons.

Charcot-Marie-Tooth 2A (CMT2A) is an inherited peripheral neuropathy caused by mutations in MFN2, which encodes a mitochondrial membrane protein involved in mitochondrial network homeostasis. Because MFN2 is expressed ubiquitously, the reason for selective motor neuron (MN) involvement in CMT2A is unclear. To address this question, we generated MNs from induced pluripotent stem cells (iPSCs) obtained from the patients with CMT2A as an in vitro disease model. CMT2A iPSC-derived MNs (CMT2A-MNs) exhibited a global reduction in mitochondrial content and altered mitochondrial positioning without significant differences in survival and axon elongation. RNA sequencing profiles and protein studies of key components of the apoptotic executioner program (i.e. p53, BAX, caspase 8, cleaved caspase 3, and the anti-apoptotic marker Bcl2) demonstrated that CMT2A-MNs are more resistant to apoptosis than wild-type MNs. Exploring the balance between mitochondrial biogenesis and the regulation of autophagy-lysosome transcription, we observed an increased autophagic flux in CMT2A-MNs that was associated with increased expression of PINK1, PARK2, BNIP3, and a splice variant of BECN1 that was recently demonstrated to be a trigger for mitochondrial autophagic removal. Taken together, these data suggest that the striking reduction in mitochondria in MNs expressing mutant MFN2 is not the result of impaired biogenesis, but more likely the consequence of enhanced mitophagy. Thus, these pathways represent possible novel molecular therapeutic targets for the development of an effective cure for this disease.

Changes in Whole-Body Oxygen Consumption and Skeletal Muscle Mitochondria During Linezolid-Induced Lactic Acidosis.

OBJECTIVE: To better clarify the pathogenesis of linezolid-induced lactic acidosis. DESIGN: Case report. SETTING: ICU. PATIENT: A 64-year-old man who died with linezolid-induced lactic acidosis. INTERVENTIONS: Skeletal muscle was sampled at autopsy to study mitochondrial function. MEASUREMENTS AND MAIN RESULTS: Lactic acidosis developed during continuous infusion of linezolid while oxygen consumption and oxygen extraction were diminishing from 172 to 52 mL/min/m and from 0.27 to 0.10, respectively. Activities of skeletal muscle respiratory chain complexes I, III, and IV, encoded by nuclear and mitochondrial DNA, were abnormally low, whereas activity of complex II, entirely encoded by nuclear DNA, was not. Protein studies confirmed stoichiometric imbalance between mitochondrial (cytochrome c oxidase subunits 1 and 2) and nuclear (succinate dehydrogenase A) DNA-encoded respiratory chain subunits. These findings were not explained by defects in mitochondrial DNA or transcription. There were no compensatory mitochondrial biogenesis (no induction of nuclear respiratory factor 1 and mitochondrial transcript factor A) or adaptive unfolded protein response (reduced concentration of heat shock proteins 60 and 70). CONCLUSIONS: Linezolid-induced lactic acidosis is associated with diminished global oxygen consumption and extraction. These changes reflect selective inhibition of mitochondrial protein synthesis (probably translation) with secondary mitonuclear imbalance. One novel aspect of linezolid toxicity that needs to be confirmed is blunting of reactive mitochondrial biogenesis and unfolded protein response.

Impaired Muscle Mitochondrial Biogenesis and Myogenesis in Spinal Muscular Atrophy.

IMPORTANCE: The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively). OBJECTIVE: To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA. DESIGN, SETTING, AND PARTICIPANTS: We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca' Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015. EXPOSURES: We used histochemical, biochemical, and molecular techniques to examine the muscle samples. MAIN OUTCOMES AND MEASURES: Respiratory chain activity and mitochondrial content. RESULTS: Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P < .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P < .05). We linked these alterations to downregulation of peroxisome proliferator-activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors-muscle-specific myogenic factor 5, myoblast determination 1, and myogenin-were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05). CONCLUSIONS AND RELEVANCE: Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes.

Glycogen storage disease type III: A novel Agl knockout mouse model.

Glycogen storage disease type III is an autosomal recessive disease characterized by a deficiency in the glycogen debranching enzyme, encoded by AGL. Essential features of this disease are hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation. Progressive skeletal myopathy, neuropathy, and/or cardiomyopathy become prominent in adults. Currently, there is no available cure. We generated an Agl knockout mouse model by deletion of the carboxy terminus of the protein, including the carboxy end of the glucosidase domain and the glycogen-binding domain. Agl knockout mice presented serious hepatomegaly, but we did not observe signs of cirrhosis or adenomas. In affected tissues, glycogen storage was higher than in wild-type mice, even in the central nervous system which has never been tested in GSDIII patients. The biochemical findings were in accordance with histological data, which clearly documented tissue impairment due to glycogen accumulation. Indeed, electron microscopy revealed the disruption of contractile units due to glycogen infiltrations. Furthermore, adult Agl knockout animals appeared less prompt to move, and they exhibited kyphosis. Three-mo-old Agl knockout mice could not run, and adult mice showed exercise intolerance. In addition, older affected animals exhibited an accelerated respiratory rate even at basal conditions. This observation was correlated with severe glycogen accumulation in the diaphragm. Diffuse glycogen deposition was observed in the tongues of affected mice. Our results demonstrate that this Agl knockout mouse is a reliable model for human glycogenosis type III, as it recapitulates the essential phenotypic features of the disease.

Lower motor neuron disease with respiratory failure caused by a novel MAPT mutation.

OBJECTIVE: To investigate the molecular defect underlying a large Italian kindred with progressive adult-onset respiratory failure, proximal weakness of the upper limbs, and evidence of lower motor neuron degeneration. METHODS: We describe the clinical features of 5 patients presenting with prominent respiratory insufficiency, proximal weakness of the upper limbs, and no signs of frontotemporal lobar degeneration or semantic dementia. Molecular analysis was performed combining linkage and exome sequencing analyses. Further investigations included transcript analysis and immunocytochemical and protein studies on established cell models. RESULTS: Genome-wide linkage analysis showed an association with chromosome 17q21. Exome analysis disclosed a missense change in MAPT segregating dominantly with the disease and resulting in D348G-mutated tau protein. Motor neuron cell lines overexpressing mutated D348G tau isoforms displayed a consistent reduction in neurite length and arborization. The mutation does not seem to modify tau interactions with microtubules. Neuropathologic studies were performed in one affected subject, which exhibited α-motoneuron loss and atrophy of the spinal anterior horns with accumulation of phosphorylated tau within the surviving motor neurons. Staining for 3R- and 4R-tau revealed pathology similar to that observed in familial cases harboring MAPT mutations. CONCLUSION: Our study broadens the phenotype of tauopathies to include lower motor neuron disease and implicate tau degradation pathway defects in motor neuron degeneration.

Extended phenotype description and new molecular findings in late onset glycogen storage disease type II: a northern Italy population study and review of the literature.

Glycogen storage disease type II (GSDII) is a lysosomal storage disorder caused by acid alpha-1,4-glucosidase deficiency and associated with recessive mutations in its coding gene GAA. Few studies have provided so far a detailed phenotypical characterization in late onset GSDII (LO-GSDII) patients. Genotype-phenotype correlation has been previously attempted with controversial results. We aim to provide an in-depth description of a cohort (n = 36) of LO-GSDII patients coming from the north of Italy and compare our population's findings to the literature. We performed a clinical record-based retrospective and prospective study of our patients. LO-GSDII in our cohort covers a large variability of phenotype including subtle clinical presentation and did not differ significantly from previous data. In all patients, molecular analysis disclosed GAA mutations, five of them being novel. To assess potential genotype-phenotype correlations we divided IVS1-32-13T>G heterozygous patients into two groups following the severity of the mutations on the second allele. Our patients harbouring "severe" mutations (n = 21) presented a strong tendency to have more severe phenotypes and more disability, more severe phenotypes and more disability, higher prevalence of assisted ventilation and a shorter time of evolution to show it. The determination of prognostic factors is mandatory in order to refine the accuracy of prognostic information, to develop follow-up strategy and, more importantly, to improve the decision algorithm for enzyme replacement therapy administration. The demonstration of genotype-phenotype correlations could help to reach this objective. Clinical assessment homogeneity is required to overcome limitations due to the lack of power of most studies.

Postural effects on lung and chest wall volumes in late onset type II glycogenosis patients.

Respiratory failure associated with diaphragmatic weakness is the first cause of death in late-onset type II glycogenosis (LO-GSDII). We aim to identify predictive factors of diaphragmatic weakness and investigate the pathophysiology of respiratory muscles impairment. Pulmonary function and chest wall volumes were measured in ten patients and eight controls (supine and seated). According to the change in forced vital capacity in supine (ΔFVC) we considered patients with (DW, ΔFVC>25%) and without (noDW, ΔFVC<25%) diaphragmatic weakness. Postural change made the supine abdominal contribution to tidal volume (%VAB) of DW to fall and the ribcage to increase and good correlation was found between %VAB and ΔFVC (R=0.776). Patients showed reduced chest wall and abdominal inspiratory capacity (ICCW and ICAB) (p<0.001) and low abdominal expiratory reserve volume (p<0.01). Passing to supine DW did not increase ICCW and ICAB. ΔFVC occurs in LO-GSDII due to weakened diaphragm and abdominal muscles while intercostals are preserved. %VAB represents a new reliable index to detect diaphragmatic weakness.