Increased mitophagy in the skeletal muscle of spinal and bulbar muscular atrophy patients

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by polyglutamine expansion in the androgen receptor (AR) and characterized by the loss of lower motor neurons. Here we investigated pathological processes occurring in muscle biopsy specimens derived from SBMA patients and, as controls, age-matched healthy subjects and patients suffering from amyotrophic lateral sclerosis (ALS) and neurogenic atrophy. We detected atrophic fibers in the muscle of SBMA, ALS and neurogenic atrophy patients. In addition, SBMA muscle was characterized by the presence of a large number of hypertrophic fibers, with oxidative fibers having a larger size compared with glycolytic fibers. Polyglutamine-expanded AR expression was decreased in whole muscle, yet enriched in the nucleus, and localized to mitochondria. Ultrastructural analysis revealed myofibrillar disorganization and streaming in zones lacking mitochondria and degenerating mitochondria. Using molecular (mtDNA copy number), biochemical (citrate synthase and respiratory chain enzymes) and morphological (dark blue area in nicotinamide adenine dinucleotide-stained muscle cross-sections) analyses, we found a depletion of the mitochondria associated with enhanced mitophagy. Mass spectrometry analysis revealed an increase of phosphatidylethanolamines and phosphatidylserines in mitochondria isolated from SBMA muscles, as well as a 50% depletion of cardiolipin associated with decreased expression of the cardiolipin synthase gene. These observations suggest a causative link between nuclear polyglutamine-expanded AR accumulation, depletion of mitochondrial mass, increased mitophagy and altered mitochondrial membrane composition in SBMA muscle patients. Given the central role of mitochondria in cell bioenergetics, therapeutic approaches toward improving the mitochondrial network are worth considering to support SBMA patients

The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a \u3b2-thalassemia mouse model

Anemia in \u3b2-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired \u3b1-globin chains impose substantial oxidative stress on \u3b2-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for \u3b2-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in \u3b2-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2\u3b1 axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing \u3b2-thalassemia\u2013related liver iron overload. In ex vivo studies on erythroid precursors from patients with \u3b2-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for \u3b2-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials

Chronic kidney disease and coenzyme Q10 supplementation

Among the potential causes of chronic kidney disease (CKD), mitochondrial respiratory chain (MRC) dysfunction, oxidative stress and inflammation have been implicated as contributor factors to the pathogenesis of this disorder. It is thought that CoQ10 supplementation may offer some therapeutic potential in the treatment of patients with CKD, since CoQ10 has a key role in normal MRC function, as well as having antioxidant and anti-inflammatory action. This article will outline the current knowledge on the use of CoQ10 in the treatment of CK

The multiple roles of coenzyme Q in cellular homeostasis and their relevance for the pathogenesis of coenzyme Q deficiency

Coenzyme Q (CoQ) is a redox active lipid that plays a central role in cellular homeostasis. It was discovered more than 60 years ago because of its role as electron transporter in the mitochondrial respiratory chain. Since then it has become evident that CoQ has many other functions, not directly related to bioenergetics. It is a cofactor of several mitochondrial dehydrogenases involved in the metabolism of lipids, amino acids, and nucleotides, and in sulfide detoxification. It is a powerful antioxidant and it is involved in the control of programmed cell death by modulating both apoptosis and ferroptosis. CoQ deficiency is a clinically and genetically heterogeneous group of disorders characterized by the impairment of CoQ biosynthesis. CoQ deficient patients display defects in cellular bioenergetics, but also in the other pathways in which CoQ is involved. In this review we will focus on the functions of CoQ not directly related to the respiratory chain, and on how their impairment is relevant for the pathophysiology of CoQ deficiency. A better understanding of the complex set of events triggered by CoQ deficiency will allow to design novel approaches for the treatment of this condition

Molecular and Cellular Studies Reveal Folding Defects of Human Ornithine Aminotransferase Variants Associated With Gyrate Atrophy of the Choroid and Retina

The deficit of human ornithine aminotransferase (hOAT) is responsible for gyrate atrophy (GA), a rare recessive inherited disorder. Although more than 60 disease-associated mutations have been identified to date, the molecular mechanisms explaining how each mutation leads to the deficit of OAT are mostly unknown. To fill this gap, we considered six representative missense mutations present in homozygous patients concerning residues spread over the hOAT structure. E. coli expression, spectroscopic, kinetic and bioinformatic analyses, reveal that the R154L and G237D mutations induce a catalytic more than a folding defect, the Q90E and R271K mutations mainly impact folding efficiency, while the E318K and C394Y mutations give rise to both folding and catalytic defects. In a human cellular model of disease folding-defective variants, although at a different extent, display reduced protein levels and/or specific activity, due to increased aggregation and/or degradation propensity. The supplementation with Vitamin B6, to mimic a treatment strategy available for GA patients, does not significantly improve the expression/activity of folding-defective variants, in contrast with the clinical responsiveness of patients bearing the E318K mutation. Thus, we speculate that the action of vitamin B6 could be also independent of hOAT. Overall, these data represent a further effort toward a comprehensive analysis of GA pathogenesis at molecular and cellular level, with important relapses for the improvement of genotype/phenotype correlations and the development of novel treatments

Hybrid minigene assay: An efficient tool to characterize mrna splicing profiles of nf1 variants

Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the NF1 gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. NF1 displays allelic heterogeneity, with a high proportion of variants affecting splicing, including deep intronic alleles and changes outside the canonical splice sites, making validation problematic. Next Generation Sequencing (NGS) technologies integrated with multiplex ligation-dependent probe amplification (MLPA) have largely overcome RNA-based techniques but do not detect splicing defects. A rapid minigene-based system was set up to test the effects of NF1 variants on splicing. We investigated 29 intronic and exonic NF1 variants identified in patients during the diagnostic process. The minigene assay showed the coexistence of multiple mechanisms of splicing alterations for seven variants. A leaky effect on splicing was documented in one de novo substitution detected in a sporadic patient with a specific phenotype without neurofibromas. Our splicing assay proved to be a reliable and fast method to validate novel NF1 variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient\u2019s RNA

Functional Analysis of Missense Mutations of OAT, Causing Gyrate Atrophy of Choroid and Retina.

We studied eight kindreds with gyrate atrophy of choroid and retina (GA), a rare autosomal recessive disorder caused by mutations of the OAT gene, encoding the homoexameric enzyme ornithine-delta-aminotransferase. We identified four novel and five previously reported mutations. Missense alleles were expressed in yeast strain carrying a deletion of the orthologous of human OAT. All mutations markedly reduced enzymatic activity. However, the effect on the yeast growth was variable, suggesting that some mutations retain residual activity, below the threshold of the enzymatic assay. Mutant proteins were either highly unstable and rapidly degraded, or failed to assemble to form the active OAT hexamer. Where possible, fibroblast analysis confirmed these data. We found no correlation between the residual enzymatic activity and the age of onset, or the severity of symptoms. Moreover, the response to B6 was apparently not related to the specific mutations carried by patients. Overall these data suggest that other factors besides the specific OAT genotype modulate (GA) phenotype in patients. Finally, we found that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator known to increase mitochondrial biogenesis, markedly stimulates OAT expression, thus representing a possible treatment for a subset of GA patients with hypomorphic alleles

New pathogenic variants in COQ4 cause ataxia and neurodevelopmental disorder without detectable CoQ10 deficiency in muscle or skin fibroblasts

COQ4 is a component of an enzyme complex involved in the biosynthesis of coenzyme Q10 (CoQ10), a molecule with primary importance in cell metabolism. Mutations in the COQ4 gene are responsible for mitochondrial diseases showing heterogeneous age at onset, clinical presentations and association with CoQ10 deficiency. We herein expand the phenotypic and genetic spectrum of COQ4-related diseases, by reporting two patients harboring bi-allelic variants but not showing CoQ10 deficiency. One patient was found to harbor compound heterozygous mutations (specifically, c.577C>T/p.Pro193Ser and the previously reported c.718C>T/p.Arg240Cys) associated with progressive spasticity, while the other harbored two novel missense (c.284G>A/p.Gly95Asp and c.305G>A/p.Arg102His) associated with a neurodevelopmental disorder. Both patients presented motor impairment and ataxia. To further understand the role of COQ4, we performed functional studies in patient-derived fibroblasts, yeast and \u201ccrispant\u201d zebrafish larvae. Micro-oxygraphy showed impaired oxygen consumption rates in one patient, while yeast complementation assays showed that all the mutations were presumably disease related. Moreover, characterization of the coq4 F0 CRISPR zebrafish line showed motor defects and cell reduction in a specific area of the hindbrain, a region reminiscent of the human cerebellum. Our expanded phenotype associated with COQ4 mutations allowed us to investigate, for the first time, the role of COQ4 in brain development in vivo