ABSTRACT
Molecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids (ROs) provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-seq and spatial transcriptomics we present for the first-time a single cell spatiotemporal transcriptome of RO development. Our data demonstrate that ROs recapitulate key events of retinogenesis including optic vesicle/cup formation, presence of a putative ciliary margin zone, emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA- with scATAC-seq data, we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development, and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal neurons.
ABSTRACT
Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) mutations cause severe, progressive, and often lethal diseases with highly heterogeneous and tissue-specific clinical manifestations. This study investigates the molecular mechanisms triggered by three different mt-ARS defects caused by biallelic mutations in AARS2, EARS2, and RARS2, using an in vitro model of human neuronal cells. We report distinct molecular mechanisms of mitochondrial dysfunction among the mt-ARS defects studied. Our findings highlight the ability of proliferating neuronal progenitor cells (iNPCs) to compensate for mitochondrial translation defects and maintain balanced levels of oxidative phosphorylation (OXPHOS) components, which becomes more challenging in mature neurons. Mutant iNPCs exhibit unique compensatory mechanisms, involving specific branches of the integrated stress response, which may be gene-specific or related to the severity of the mitochondrial translation defect. RNA sequencing revealed distinct transcriptomic profiles showing dysregulation of neuronal differentiation and protein translation. This study provides valuable insights into the tissue-specific compensatory mechanisms potentially underlying the phenotypes of patients with mt-ARS defects. Our novel in vitro model may more accurately represent the neurological presentation of patients and offer an improved platform for future investigations and therapeutic development.
Subject(s)
Amino Acyl-tRNA Synthetases , Humans , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mutation , Neurons/metabolism , RNA, Transfer/metabolismABSTRACT
Abnormalities of the arterial valves, including bicuspid aortic valve (BAV) are amongst the most common congenital defects and are a significant cause of morbidity as well as predisposition to disease in later life. Despite this, and compounded by their small size and relative inaccessibility, there is still much to understand about how the arterial valves form and remodel during embryogenesis, both at the morphological and genetic level. Here we set out to address this in human embryos, using Spatial Transcriptomics (ST). We show that ST can be used to investigate the transcriptome of the developing arterial valves, circumventing the problems of accurately dissecting out these tiny structures from the developing embryo. We show that the transcriptome of CS16 and CS19 arterial valves overlap considerably, despite being several days apart in terms of human gestation, and that expression data confirm that the great majority of the most differentially expressed genes are valve-specific. Moreover, we show that the transcriptome of the human arterial valves overlaps with that of mouse atrioventricular valves from a range of gestations, validating our dataset but also highlighting novel genes, including four that are not found in the mouse genome and have not previously been linked to valve development. Importantly, our data suggests that valve transcriptomes are under-represented when using commonly used databases to filter for genes important in cardiac development; this means that causative variants in valve-related genes may be excluded during filtering for genomic data analyses for, for example, BAV. Finally, we highlight "novel" pathways that likely play important roles in arterial valve development, showing that mouse knockouts of RBP1 have arterial valve defects. Thus, this study has confirmed the utility of ST for studies of the developing heart valves and broadens our knowledge of the genes and signalling pathways important in human valve development.
Subject(s)
Bicuspid Aortic Valve Disease , Heart Valve Diseases , Humans , Mice , Animals , Heart Valve Diseases/genetics , Aortic Valve/abnormalities , Bicuspid Aortic Valve Disease/metabolism , Gene Expression Profiling , Transcriptome/geneticsABSTRACT
Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.
Subject(s)
Extracellular Vesicles , Macular Degeneration , Humans , Retinal Pigment Epithelium/metabolism , Extracellular Vesicles/metabolism , Retina/metabolism , Retina/pathology , Macular Degeneration/metabolism , PhenotypeABSTRACT
Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066-1315 (emGFP-1066:TetC), 1093-1315 (emGFP-1093:TetC) and 1109-1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.
Subject(s)
Microscopy, Confocal , Neuromuscular Junction/diagnostic imaging , Tetanus Toxin/toxicity , Animals , Animals, Newborn , Axons/drug effects , Axons/metabolism , Binding Sites , Fluorescence , Green Fluorescent Proteins/metabolism , Humans , Mice, Inbred C57BL , Motor Neurons/drug effects , Motor Neurons/metabolism , Nerve Tissue/drug effects , Nerve Tissue/metabolism , Neuromuscular Junction/drug effects , Neuromuscular Junction/pathology , Synapses/drug effects , Synapses/metabolism , Synaptic Transmission/drug effectsABSTRACT
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.
Subject(s)
Mitochondrial Diseases/genetics , Mitochondrial Diseases/metabolism , Mitochondrial Myopathies/genetics , Mitochondrial Myopathies/metabolism , Adolescent , Cell Line , DNA, Mitochondrial/genetics , Female , Gene Expression , Humans , Infant , Male , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Mutation , Pedigree , Proteomics , Quadriceps Muscle/metabolism , tRNA Methyltransferases/genetics , tRNA Methyltransferases/metabolismABSTRACT
The ß-adrenergic agonists salbutamol and ephedrine have proven to be effective as therapies for human disorders of the neuromuscular junction, in particular many subsets of congenital myasthenic syndromes. However, the mechanisms underlying this clinical benefit are unknown and improved understanding of the effect of adrenergic signalling on the neuromuscular junction is essential to facilitate the development of more targeted therapies. Here, we investigated the effect of salbutamol treatment on the neuromuscular junction in the ColQ deficient mouse, a model of end-plate acetylcholinesterase deficiency. ColQ-/- mice received 7 weeks of daily salbutamol injection, and the effect on muscle strength and neuromuscular junction morphology was analysed. We show that salbutamol leads to a gradual improvement in muscle strength in ColQ-/- mice. In addition, the neuromuscular junctions of salbutamol treated mice showed significant improvements in several postsynaptic morphological defects, including increased synaptic area, acetylcholine receptor area and density, and extent of postjunctional folds. These changes occurred without alterations in skeletal muscle fibre size or type. These findings suggest that ß-adrenergic agonists lead to functional benefit in the ColQ-/- mouse and to long-term structural changes at the neuromuscular junction. These effects are primarily at the postsynaptic membrane and may lead to enhanced neuromuscular transmission.
Subject(s)
Acetylcholinesterase/genetics , Adrenergic beta-Agonists/therapeutic use , Albuterol/therapeutic use , Collagen/genetics , Muscle Fibers, Skeletal/metabolism , Muscle Proteins/genetics , Myasthenic Syndromes, Congenital/genetics , Neuromuscular Junction/drug effects , Acetylcholinesterase/metabolism , Agrin/metabolism , Animals , Collagen/metabolism , Disease Models, Animal , Dystroglycans/metabolism , Mice , Mice, Knockout , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/ultrastructure , Muscle Proteins/metabolism , Muscle Weakness/therapy , Myasthenic Syndromes, Congenital/drug therapy , Neuromuscular Junction/diagnostic imaging , Neuromuscular Junction/metabolism , Receptors, Cholinergic , Signal Transduction , Synaptic Transmission/physiologyABSTRACT
Diagnosing primary mitochondrial diseases is challenging in clinical practice. Although, defective oxidative phosphorylation (OXPHOS) is the common final pathway, it is unknown why different mtDNA or nuclear mutations result in largely heterogeneous and often tissue -specific clinical presentations. Mitochondrial tRNA (mt-tRNA) mutations are frequent causes of mitochondrial diseases both in children and adults. However numerous nuclear mutations involved in mitochondrial protein synthesis affecting ubiquitously expressed genes have been reported in association with very tissue specific clinical manifestations suggesting that there are so far unknown factors determining the tissue specificity in mitochondrial translation. Most of these gene defects result in histological abnormalities and multiple respiratory chain defects in the affected organs. The clinical phenotypes are usually early-onset, severe, and often fatal, implying the importance of mitochondrial translation from birth. However, some rare, reversible infantile mitochondrial diseases are caused by very specific defects of mitochondrial translation. An unbiased genetic approach (whole exome sequencing, RNA sequencing) combined with proteomics and functional studies revealed novel factors involved in mitochondrial translation which contribute to the clinical manifestation and recovery in these rare reversible mitochondrial conditions.
Subject(s)
DNA, Mitochondrial/genetics , Mitochondrial Diseases/genetics , Mitochondrial Proteins/genetics , Protein Biosynthesis , Transcription, Genetic , Electron Transport/genetics , Humans , Mitochondrial Diseases/diagnosis , Mutation , Oxidative Phosphorylation , Phenotype , RNA Precursors/genetics , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Transfer/genetics , SyndromeABSTRACT
The nuclear-encoded glycyl-tRNA synthetase gene (GARS) is essential for protein translation in both cytoplasm and mitochondria. In contrast, different genes encode the mitochondrial and cytosolic forms of most other tRNA synthetases. Dominant GARS mutations were described in inherited neuropathies, while recessive mutations cause severe childhood-onset disorders affecting skeletal muscle and heart. The downstream events explaining tissue-specific phenotype-genotype relations remained unclear. We investigated the mitochondrial function of GARS in human cell lines and in the GarsC210R mouse model. Human-induced neuronal progenitor cells (iNPCs) carrying dominant and recessive GARS mutations showed alterations of mitochondrial proteins, which were more prominent in iNPCs with dominant, neuropathy-causing mutations. Although comparative proteomic analysis of iNPCs showed significant changes in mitochondrial respiratory chain complex subunits, assembly genes, Krebs cycle enzymes and transport proteins in both recessive and dominant mutations, proteins involved in fatty acid oxidation were only altered by recessive mutations causing mitochondrial cardiomyopathy. In contrast, significant alterations of the vesicle-associated membrane protein-associated protein B (VAPB) and its downstream pathways such as mitochondrial calcium uptake and autophagy were detected in dominant GARS mutations. The role of VAPB has been supported by similar results in the GarsC210R mice. Our data suggest that altered mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) may be important disease mechanisms leading to neuropathy in this condition.
Subject(s)
Endoplasmic Reticulum/genetics , Glycine-tRNA Ligase/genetics , Mitochondria/genetics , Vesicular Transport Proteins/genetics , Animals , Humans , Mice , Mitochondria/metabolism , Mutation , Neurons/metabolism , Neurons/pathology , Signal Transduction , Stem Cells/metabolismABSTRACT
PURPOSE: To understand the role of the mitochondrial oxodicarboxylate carrier (SLC25A21) in the development of spinal muscular atrophy-like disease. METHODS: We identified a novel pathogenic variant in a patient by whole-exome sequencing. The pathogenicity of the mutation was studied by transport assays, computer modeling, followed by targeted metabolic testing and in vitro studies in human fibroblasts and neurons. RESULTS: The patient carries a homozygous pathogenic variant c.695A>G; p.(Lys232Arg) in the SLC25A21 gene, encoding the mitochondrial oxodicarboxylate carrier, and developed spinal muscular atrophy and mitochondrial myopathy. Transport assays show that the mutation renders SLC25A21 dysfunctional and 2-oxoadipate cannot be imported into the mitochondrial matrix. Computer models of central metabolism predicted that impaired transport of oxodicarboxylate disrupts the pathways of lysine and tryptophan degradation, and causes accumulation of 2-oxoadipate, pipecolic acid, and quinolinic acid, which was confirmed in the patient's urine by targeted metabolomics. Exposure to 2-oxoadipate and quinolinic acid decreased the level of mitochondrial complexes in neuronal cells (SH-SY5Y) and induced apoptosis. CONCLUSION: Mitochondrial oxodicarboxylate carrier deficiency leads to mitochondrial dysfunction and the accumulation of oxoadipate and quinolinic acid, which in turn cause toxicity in spinal motor neurons leading to spinal muscular atrophy-like disease.
Subject(s)
Adipates/metabolism , DNA, Mitochondrial/genetics , Dicarboxylic Acid Transporters/genetics , Mitochondrial Membrane Transport Proteins/genetics , Muscular Atrophy, Spinal/genetics , Adipates/pharmacology , Apoptosis/drug effects , Cell Line , DNA, Mitochondrial/metabolism , Dicarboxylic Acid Transporters/metabolism , Fibroblasts/drug effects , Homozygote , Humans , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Motor Neurons/drug effects , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/physiopathology , Mutation , Pipecolic Acids/metabolism , Quinolinic Acid/metabolismABSTRACT
Mitochondrial dynamics play an important role in cellular homeostasis and a variety of human diseases are linked to its dysregulated function. Here, we describe a 15-year-old boy with a novel disease caused by altered mitochondrial dynamics. The patient was the second child of consanguineous Jewish parents. He developed progressive muscle weakness and exercise intolerance at 6 years of age. His muscle biopsy revealed mitochondrial myopathy with numerous ragged red and cytochrome c oxidase (COX) negative fibers and combined respiratory chain complex I and IV deficiency. MtDNA copy number was elevated and no deletions of the mtDNA were detected in muscle DNA. Whole exome sequencing identified a homozygous nonsense mutation (p.Q92*) in the MIEF2 gene encoding the mitochondrial dynamics protein of 49 kDa (MID49). Immunoblotting revealed increased levels of proteins promoting mitochondrial fusion (MFN2, OPA1) and decreased levels of the fission protein DRP1. Fibroblasts of the patient showed elongated mitochondria, and significantly higher frequency of fusion events, mtDNA abundance and aberrant mitochondrial cristae ultrastructure, compared with controls. Thus, our data suggest that mutations in MIEF2 result in imbalanced mitochondrial dynamics and a combined respiratory chain enzyme defect in skeletal muscle, leading to mitochondrial myopathy.
Subject(s)
Fibroblasts/metabolism , Mitochondrial Dynamics/genetics , Mitochondrial Proteins , Muscular Diseases , Mutation, Missense , Peptide Elongation Factors , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Female , Fibroblasts/pathology , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Humans , Male , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Muscular Diseases/genetics , Muscular Diseases/metabolism , Muscular Diseases/pathology , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Primary Cell CultureABSTRACT
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for charging tRNAs with their cognate amino acids, therefore essential for the first step in protein synthesis. Although the majority of protein synthesis happens in the cytosol, an additional translation apparatus is required to translate the 13 mitochondrial DNA-encoded proteins important for oxidative phosphorylation. Most ARS genes in these cellular compartments are distinct, but two genes are common, encoding aminoacyl-tRNA synthetases of glycine (GARS) and lysine (KARS) in both mitochondria and the cytosol. Mutations in the majority of the 37 nuclear-encoded human ARS genes have been linked to a variety of recessive and dominant tissue-specific disorders. Current data indicate that impaired enzyme function could explain the pathogenicity, however not all pathogenic ARSs mutations result in deficient catalytic function; thus, the consequences of mutations may arise from other molecular mechanisms. The peripheral nerves are frequently affected, as illustrated by the high number of mutations in cytosolic and bifunctional tRNA synthetases causing Charcot-Marie-Tooth disease (CMT). Here we provide insights on the pathomechanisms of CMT-causing tRNA synthetases with specific focus on the two bifunctional tRNA synthetases (GARS, KARS).
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Charcot-Marie-Tooth Disease , Cytosol , Mitochondria , Mitochondrial Proteins , Neuromuscular Diseases , Amino Acyl-tRNA Synthetases/genetics , Animals , Charcot-Marie-Tooth Disease/enzymology , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/pathology , Cytosol/enzymology , Cytosol/pathology , Humans , Mitochondria/enzymology , Mitochondria/genetics , Mitochondria/pathology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Neuromuscular Diseases/enzymology , Neuromuscular Diseases/genetics , Neuromuscular Diseases/pathology , Oxidative PhosphorylationABSTRACT
OBJECTIVE: To study the prevalence, molecular cause, and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the North of England. METHODS: Detailed neurologic and electrophysiologic assessments and next-generation panel testing or whole exome sequencing were performed in 105 patients with clinical symptoms of distal hereditary motor neuropathy (dHMN, 64 patients), axonal motor neuropathy (motor Charcot-Marie-Tooth disease [CMT2], 16 patients), or complex neurologic disease predominantly affecting the motor nerves (hereditary motor neuropathy plus, 25 patients). RESULTS: The prevalence of dHMN is 2.14 affected individuals per 100,000 inhabitants (95% confidence interval 1.62-2.66) in the North of England. Causative mutations were identified in 26 out of 73 index patients (35.6%). The diagnostic rate in the dHMN subgroup was 32.5%, which is higher than previously reported (20%). We detected a significant defect of neuromuscular transmission in 7 cases and identified potentially causative mutations in 4 patients with multifocal demyelinating motor neuropathy. CONCLUSIONS: Many of the genes were shared between dHMN and motor CMT2, indicating identical disease mechanisms; therefore, we suggest changing the classification and including dHMN also as a subcategory of Charcot-Marie-Tooth disease. Abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies.
Subject(s)
Charcot-Marie-Tooth Disease/epidemiology , Genetic Heterogeneity , Hereditary Sensory and Motor Neuropathy/epidemiology , Hereditary Sensory and Motor Neuropathy/genetics , Mutation/genetics , Adolescent , Adult , Aged , Analysis of Variance , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/physiopathology , Cohort Studies , Connexins/genetics , DNA Mutational Analysis , Electromyography , England/epidemiology , Family Health , Female , GTP Phosphohydrolases/genetics , Hereditary Sensory and Motor Neuropathy/physiopathology , Humans , Male , Middle Aged , Mitochondrial Proteins/genetics , Myelin Proteins/genetics , Neural Conduction/genetics , Young Adult , Gap Junction beta-1 ProteinABSTRACT
BACKGROUND: Mitochondrial encephalomyopathies are severe, relentlessly progressive conditions and there are very few effective therapies available to date. We have previously suggested that in two rare forms of reversible mitochondrial disease (reversible infantile respiratory chain deficiency and reversible infantile hepatopathy) supplementation with L-cysteine can improve mitochondrial protein synthesis, since cysteine is required for the 2-thiomodification of mitochondrial tRNAs. OBJECTIVES: We studied whether supplementation with L-cysteine or N-acetyl-cysteine (NAC) results in any improvement of the mitochondrial function in vitro in fibroblasts of patients with different genetic forms of abnormal mitochondrial translation. METHODS: We studied in vitro in fibroblasts of patients carrying the common m.3243A>G and m.8344A>G mutations or autosomal recessive mutations in genes affecting mitochondrial translation, whether L-cysteine or N-acetyl-cysteine supplementation have an effect on mitochondrial respiratory chain function. RESULTS: Here we show that supplementation with L-cysteine, but not with N-acetyl-cysteine partially rescues the mitochondrial translation defect in vitro in fibroblasts of patients carrying the m.3243A>G and m.8344A>G mutations. In contrast, N-acetyl-cysteine had a beneficial effect on mitochondrial translation in TRMU and MTO1 deficient fibroblasts. CONCLUSIONS: Our results suggest that L-cysteine or N-acetyl-cysteine supplementation may be a potential treatment for selected subgroups of patients with mitochondrial translation deficiencies. Further studies are needed to explore the full potential of cysteine supplementation as a treatment for patients with mitochondrial disease.
Subject(s)
Acetylcysteine/pharmacology , Cysteine/pharmacology , Fibroblasts/drug effects , MELAS Syndrome/metabolism , MERRF Syndrome/metabolism , Mitochondria/drug effects , Mitochondrial Diseases/metabolism , Protein Biosynthesis/drug effects , Carrier Proteins/genetics , Cyclooxygenase 2/genetics , Dietary Supplements , Fibroblasts/metabolism , Humans , In Vitro Techniques , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mutation , Neoplasm Proteins/genetics , Oxygen Consumption/drug effects , RNA-Binding Proteins , tRNA Methyltransferases/geneticsABSTRACT
Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.
Subject(s)
Frameshift Mutation/genetics , Mitochondrial Diseases/genetics , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/genetics , Nucleotidyltransferases/genetics , Riboflavin/pharmacology , Vitamin B Complex/pharmacology , Adult , Blotting, Western , Case-Control Studies , Cells, Cultured , Electron Transport , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Flavin-Adenine Dinucleotide/metabolism , Gene Expression Profiling , Humans , Infant , Infant, Newborn , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , Mitochondrial Diseases/drug therapy , Mitochondrial Diseases/pathology , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/drug therapy , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/pathology , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Mutagenesis, Site-Directed , Protein Binding , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Skin/drug effects , Skin/metabolism , Skin/pathology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Young AdultABSTRACT
The exosome complex is the most important RNA processing machinery within the cell. Mutations in its subunits EXOSC8 and EXOSC3 cause pontocerebellar hypoplasia, spinal muscular atrophy (SMA) and central nervous system demyelination. We present a patient with SMA-like phenotype carrying a homozygous mutation in RBM7-a subunit of the nuclear exosome targeting (NEXT) complex-which is known to bind and carry specific subtypes of coding and non-coding RNAs to the exosome. The NEXT complex with other protein complexes is responsible for the substrate specificity of the exosome. We performed RNA-sequencing (RNA-seq) analysis on primary fibroblasts of patients with mutations in EXOSC8 and RBM7 and gene knock-down experiments using zebrafish as a model system. RNA-seq analysis identified significantly altered expression of 62 transcripts shared by the two patient cell lines. Knock-down of rbm7, exosc8 and exosc3 in zebrafish showed a common pattern of defects in motor neurons and cerebellum. Our data indicate that impaired RNA metabolism may underlie the clinical phenotype by fine tuning gene expression which is essential for correct neuronal differentiation.
Subject(s)
Exosome Multienzyme Ribonuclease Complex/genetics , Muscular Atrophy, Spinal/genetics , RNA-Binding Proteins/genetics , Animals , Cerebellum/metabolism , Cerebellum/pathology , Disease Models, Animal , Exosomes/genetics , Humans , Motor Neurons/metabolism , Motor Neurons/pathology , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/pathology , Mutation , Sequence Analysis, RNA , Zebrafish/metabolismABSTRACT
Liver failure is a heterogeneous condition which may be fatal and the primary cause is frequently unknown. We investigated mitochondrial oxidative phosphorylation in patients undergoing liver transplantation. We studied 45 patients who had liver transplantation due to a variety of clinical presentations. Blue native polyacrylamide gel electrophoresis with immunodetection of respiratory chain complexes I-V, biochemical activity of respiratory chain complexes II and IV and quantification of mitochondrial DNA (mtDNA) copy number were investigated in liver tissue collected from the explanted liver during transplantation. Abnormal mitochondrial function was frequently present in this cohort: ten of 40 patients (25 %) had a defect of one or more respiratory chain enzyme complexes on blue native gels, 20 patients (44 %) had low activity of complex II and/or IV and ten (22 %) had a reduced mtDNA copy number. Combined respiratory chain deficiency and reduced numbers of mitochondria were detected in all three patients with acute liver failure. Low complex IV activity in biliary atresia and complex II defects in cirrhosis were common findings. All six patients diagnosed with liver tumours showed variable alterations in mitochondrial function, probably due to the heterogeneity of the presenting tumour. In conclusion, mitochondrial dysfunction is common in severe liver failure in non-mitochondrial conditions. Therefore, in contrast to the common practice detection of respiratory chain abnormalities in liver should not restrict the inclusion of patients for liver transplantation. Furthermore, improving mitochondrial function may be targeted as part of a complex therapy approach in different forms of liver diseases.
Subject(s)
Liver Failure/pathology , Liver/pathology , Mitochondria/pathology , Mitochondrial Diseases/pathology , Adolescent , Adult , Biliary Atresia/metabolism , Biliary Atresia/pathology , Child , Child, Preschool , DNA, Mitochondrial/metabolism , Electron Transport/physiology , Electron Transport Complex IV/metabolism , Female , Humans , Infant , Liver/metabolism , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Failure/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Transplantation/methods , Male , Middle Aged , Mitochondria/metabolism , Mitochondrial Diseases/metabolism , Oxidative Phosphorylation , Young AdultABSTRACT
Envoplakin and periplakin are the two smallest plakin family cytoskeletal linker proteins that connect intermediate filaments to cellular junctions and other membrane locations. These two plakins have a structural role in the assembly of the cornified envelope (CE), the terminal stage of epidermal differentiation. Analysis of gene-targeted mice lacking both these plakins and the third initial CE scaffold protein, involucrin, demonstrate the importance of the structural integrity of CE for a proper epidermal barrier function. It has emerged that periplakin, which also has a wider tissue distribution than envoplakin, has additional, independent roles. Periplakin participates in the cytoskeletal organization also in other tissues and interacts with a wide range of membrane-associated proteins such as kazrin and butyrophilin BTN3A1. This review covers methods used to understand periplakin and envoplakin functions in cell culture models, including siRNA ablation of periplakin expression and the use of tagged protein domain constructs to study localization and interactions. In addition, assays that can be used to analyze CEs and epidermal barrier function in gene-targeted mice are described and discussed.
Subject(s)
Cornified Envelope Proline-Rich Proteins/physiology , Membrane Proteins/physiology , Plakins/physiology , Protein Precursors/physiology , Animals , Cell Fractionation , Cell Line, Tumor , Cornified Envelope Proline-Rich Proteins/isolation & purification , Gene Knockdown Techniques , Humans , Keratinocytes/metabolism , Membrane Proteins/isolation & purification , Plakins/isolation & purification , Protein Precursors/isolation & purification , Two-Hybrid System TechniquesABSTRACT
Reversible infantile respiratory chain deficiency is characterised by spontaneous recovery of mitochondrial myopathy in infants. We studied whether a physiological isoform switch of nuclear cytochrome c oxidase subunits contributes to the age-dependent manifestation and spontaneous recovery in reversible mitochondrial disease. Some nuclear-encoded subunits of cytochrome c oxidase are present as tissue-specific isoforms. Isoforms of subunits COX6A and COX7A expressed in heart and skeletal muscle are different from isoforms expressed in the liver, kidney and brain. Furthermore, in skeletal muscle both the heart and liver isoforms of subunit COX7A have been demonstrated with variable levels, indicating that the tissue-specific expression of nuclear-encoded subunits could provide a basis for the fine-tuning of cytochrome c oxidase activity to the specific metabolic needs of the different tissues. We demonstrate a developmental isoform switch of COX6A and COX7A subunits in human and mouse skeletal muscle. While the liver type isoforms are more present soon after birth, the heart/muscle isoforms gradually increase around 3 months of age in infants, 4 weeks of age in mice, and these isoforms persist in muscle throughout life. Our data in follow-up biopsies of patients with reversible infantile respiratory chain deficiency indicate that the physiological isoform switch does not contribute to the clinical manifestation and to the spontaneous recovery of this disease. However, understanding developmental changes of the different cytochrome c oxidase isoforms may have implications for other mitochondrial diseases. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.