RESUMO
SQSTM1 (sequestosome 1; also known as p62) encodes a multidomain scaffolding protein involved in various key cellular processes, including the removal of damaged mitochondria by its function as a selective autophagy receptor. Heterozygous variants in SQSTM1 have been associated with Paget disease of the bone and might contribute to neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Using exome sequencing, we identified three different biallelic loss-of-function variants in SQSTM1 in nine affected individuals from four families with a childhood- or adolescence-onset neurodegenerative disorder characterized by gait abnormalities, ataxia, dysarthria, dystonia, vertical gaze palsy, and cognitive decline. We confirmed absence of the SQSTM1/p62 protein in affected individuals' fibroblasts and found evidence of a defect in the early response to mitochondrial depolarization and autophagosome formation. Our findings expand the SQSTM1-associated phenotypic spectrum and lend further support to the concept of disturbed selective autophagy pathways in neurodegenerative diseases.
Assuntos
Ataxia/genética , Autofagia/genética , Distonia/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Proteína Sequestossoma-1/deficiência , Paralisia Supranuclear Progressiva/genética , Adolescente , Adulto , Idade de Início , Ataxia/complicações , Autofagossomos/metabolismo , Autofagossomos/patologia , Criança , Transtornos Cognitivos/genética , Disartria/complicações , Disartria/genética , Distonia/complicações , Feminino , Fibroblastos/metabolismo , Marcha/genética , Humanos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Transtornos dos Movimentos/complicações , Transtornos dos Movimentos/genética , Doenças Neurodegenerativas/complicações , Linhagem , Fenótipo , RNA Mensageiro/análise , Proteína Sequestossoma-1/genética , Paralisia Supranuclear Progressiva/complicações , Adulto JovemRESUMO
Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA.
Assuntos
Encéfalo/efeitos dos fármacos , Exoma , Ferro/metabolismo , Degeneração Neural/patologia , Encéfalo/patologia , Clonagem Molecular , Coenzima A/metabolismo , Escherichia coli/genética , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mutação de Sentido Incorreto , Panteteína/análogos & derivados , Panteteína/metabolismo , Linhagem , Fosforilação , Saccharomyces cerevisiae/genética , Transferases/genética , Transferases/metabolismoRESUMO
NBIA (neurodegeneration with brain iron accumulation) comprises a heterogeneous group of neurodegenerative diseases having as a common denominator, iron overload in specific brain areas, mainly basal ganglia and globus pallidus. In the past decade a bunch of disease genes have been identified, but NBIA pathomechanisms are still not completely clear. PKAN (pantothenate kinase-associated neurodegeneration), an autosomal recessive disorder with progressive impairment of movement, vision and cognition, is the most common form of NBIA. It is caused by mutations in the PANK2 (pantothenate kinase 2) gene, coding for a mitochondrial enzyme that phosphorylates vitamin B5 in the first reaction of the CoA (coenzyme A) biosynthetic pathway. A distinct form of NBIA, denominated CoPAN (CoA synthase protein-associated neurodegeneration), is caused by mutations in the CoASY (CoA synthase) gene coding for a bifunctional mitochondrial enzyme, which catalyses the final steps of CoA biosynthesis. These two inborn errors of CoA metabolism further support the concept that dysfunctions in CoA synthesis may play a crucial role in the pathogenesis of NBIA.
Assuntos
Encéfalo/metabolismo , Coenzima A/metabolismo , Ferro/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Humanos , Erros Inatos do Metabolismo/metabolismo , Mitocôndrias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Pantothenate kinase-associated neurodegeneration (PKAN) is a rare, inborn error of metabolism characterized by iron accumulation in the basal ganglia and by the presence of dystonia, dysarthria, and retinal degeneration. Mutations in pantothenate kinase 2 (PANK2), the rate-limiting enzyme in mitochondrial coenzyme A biosynthesis, represent the most common genetic cause of this disorder. How mutations in this core metabolic enzyme give rise to such a broad clinical spectrum of pathology remains a mystery. To systematically explore its pathogenesis, we performed global metabolic profiling on plasma from a cohort of 14 genetically defined patients and 18 controls. Notably, lactate is elevated in PKAN patients, suggesting dysfunctional mitochondrial metabolism. As predicted, but never previously reported, pantothenate levels are higher in patients with premature stop mutations in PANK2. Global metabolic profiling and follow-up studies in patient-derived fibroblasts also reveal defects in bile acid conjugation and lipid metabolism, pathways that require coenzyme A. These findings raise a novel therapeutic hypothesis, namely, that dietary fats and bile acid supplements may hold potential as disease-modifying interventions. Our study illustrates the value of metabolic profiling as a tool for systematically exploring the biochemical basis of inherited metabolic diseases.
Assuntos
Coenzima A/deficiência , Mitocôndrias/enzimologia , Distrofias Neuroaxonais/metabolismo , Neurodegeneração Associada a Pantotenato-Quinase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Adolescente , Adulto , Ácidos e Sais Biliares/metabolismo , Criança , Pré-Escolar , Códon sem Sentido , Coenzima A/biossíntese , Coenzima A/genética , Estudos de Coortes , Feminino , Humanos , Distúrbios do Metabolismo do Ferro , Ácido Láctico/sangue , Metabolismo dos Lipídeos/genética , Transtornos do Metabolismo dos Lipídeos/genética , Transtornos do Metabolismo dos Lipídeos/metabolismo , Masculino , Metaboloma , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Distrofias Neuroaxonais/diagnóstico , Distrofias Neuroaxonais/enzimologia , Neurodegeneração Associada a Pantotenato-Quinase/enzimologia , Neurodegeneração Associada a Pantotenato-Quinase/genética , Ácido Pantotênico/sangue , Esfingomielinas/sangue , Adulto JovemRESUMO
Congenital Muscular Dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders presenting at birth with psychomotor delay, cognitive impairment, muscle weakness and hypotonia. Here we described an alteration of mitochondrial inner membrane potential and mitochondrial network in cells derived from Italian patients carrying three novel mutations in CHKB gene, recently associated with "megaconial CMD". On the bases of our findings, we hypothesize that the mitochondrial membrane potential alteration, presumably as a consequence of the altered biosynthesis of phosphatidylcholine, could be responsible for the peculiar morphological aspect of mitochondria in this disease and might be involved in the disease pathogenesis.
Assuntos
Colina Quinase , Potencial da Membrana Mitocondrial/genética , Mitocôndrias Musculares , Membranas Mitocondriais , Músculo Esquelético , Distrofias Musculares , Mutação , Criança , Pré-Escolar , Colina Quinase/genética , Colina Quinase/metabolismo , Feminino , Humanos , Itália , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologiaRESUMO
Pantothenate kinase-associated neurodegeneration (PKAN) is an early onset and severely disabling neurodegenerative disease for which no therapy is available. PKAN is caused by mutations in PANK2, which encodes for the mitochondrial enzyme pantothenate kinase 2. Its function is to catalyze the first limiting step of Coenzyme A (CoA) biosynthesis. We generated induced pluripotent stem cells from PKAN patients and showed that their derived neurons exhibited premature death, increased ROS production, mitochondrial dysfunctions-including impairment of mitochondrial iron-dependent biosynthesis-and major membrane excitability defects. CoA supplementation prevented neuronal death and ROS formation by restoring mitochondrial and neuronal functionality. Our findings provide direct evidence that PANK2 malfunctioning is responsible for abnormal phenotypes in human neuronal cells and indicate CoA treatment as a possible therapeutic intervention.
Assuntos
Coenzima A/metabolismo , Neurônios/patologia , Neurodegeneração Associada a Pantotenato-Quinase/fisiopatologia , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Morte Celular , Células Cultivadas , Humanos , Mitocôndrias/patologia , Células-Tronco Pluripotentes/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
AIMS: To assess and classify exposure to Polycyclic Aromatic Hydrocarbons (PAHs) in some specific working areas of a steel foundry operating with a continuous casting process and evaluate biomonitoring data in different job tasks. METHODS: Exposure to dusts and six PAHs classified as carcinogenic by EU directives was studied in a cohort of 35 male foundry workers (aged 41.1 +/- 6.9 years), who were examined both prior to and at the end of the work-shift (06:00 a.m.-02:00 p.m.) in two different periods. The urinary excretion of 1-hydroxypyrene (1-OH-P) was measured as a biomarker of exposure to pyrene. RESULTS: PAHs concentrations ranged from 461.8 to 935.6 ng/m3 near the continuous casting area, whereas lower values were measured near the ladle furnace. End of shift 1-OH-P values were higher in 11 non-smoking workers involved in continuous casting process as compared to those employed in mantenance and furnace areas (median of the second determination: 5.70 microg/g creatinine--range: 1.24-21.24 vs 1.17 microg/g creatinine--range: 0.23-4.49; p< 0.001). 1-OH-P excretion was significantly correlated with both the sum of six carcinogenic PAHs and pyrene airborne concentrations. In two biomonitoring sessions, 9.1% and 34.3% of the workers respectively showed end-of-shift 1-OH-P values exceeding the occupational exposure limit (OEL) (4.4 microg/g creatinine or 2.3 micromol/mol(-1) creatinine) recommended for coke-oven workers. CONCLUSIONS: 1-OH-P is a useful biomarker in assessing PAH exposure and is associated with job category at a Steelplant. Due to exposure variability, to assess risk associated with PAHs exposure, biological monitoring should be carried out periodically.
Assuntos
Poluentes Ocupacionais do Ar/análise , Carcinógenos Ambientais/análise , Monitoramento Ambiental , Metalurgia , Exposição Ocupacional , Hidrocarbonetos Policíclicos Aromáticos/análise , Pirenos/análise , Adulto , Poluentes Ocupacionais do Ar/efeitos adversos , Biomarcadores , Creatinina/urina , Poeira , Monitoramento Ambiental/estatística & dados numéricos , Humanos , Exposição por Inalação , Masculino , Concentração Máxima Permitida , Pessoa de Meia-Idade , Ocupações , Hidrocarbonetos Policíclicos Aromáticos/efeitos adversos , Medição de Risco , Fumaça , UrináliseRESUMO
Mutations in C19orf12 have been identified in patients affected by Neurodegeneration with Brain Iron Accumulation (NBIA), a clinical entity characterized by iron accumulation in the basal ganglia. By using western blot analysis with specific antibody and confocal studies, we showed that wild-type C19orf12 protein was not exclusively present in mitochondria, but also in the Endoplasmic Reticulum (ER) and MAM (Mitochondria Associated Membrane), while mutant C19orf12 variants presented a different localization. Moreover, after induction of oxidative stress, a GFP-tagged C19orf12 wild-type protein was able to relocate to the cytosol. On the contrary, mutant isoforms were not able to respond to oxidative stress. High mitochondrial calcium concentration and increased H2O2 induced apoptosis were found in fibroblasts derived from one patient as compared to controls. C19orf12 protein is a 17 kDa mitochondrial membrane-associated protein whose function is still unknown. Our in silico investigation suggests that, the glycine zipper motifs of C19orf12 form helical regions spanning the membrane. The N- and C-terminal regions with respect to the transmembrane portion, on the contrary, are predicted to rearrange in a structural domain, which is homologs to the N-terminal regulatory domain of the magnesium transporter MgtE, suggesting that C19orf12 may act as a regulatory protein for human MgtE transporters. The mutations here described affect respectively one glycine residue of the glycine zipper motifs, which are involved in dimerization of transmembrane helices and predicted to impair the correct localization of the protein into the membranes, and one residue present in the regulatory domain, which is important for protein-protein interaction.
RESUMO
Neurodegeneration with brain iron accumulation (NBIA) defines a wide spectrum of clinical entities characterized by iron accumulation in specific regions of the brain, predominantly in the basal ganglia. We evaluated the presence of FA2H and C19orf12 mutations in a cohort of 46 Italian patients with early onset NBIA, which were negative for mutations in the PANK2 and PLA2G6 genes. Follow-up molecular genetic and in vitro analyses were then performed. We did not find any mutations in the FA2H gene, although we identified 3 patients carrying novel mutations in the C19orf12 gene. The recent discovery of new genes responsible for NBIA extends the spectrum of the genetic investigation now available for these disorders and makes it possible to delineate a clearer clinical-genetic classification of different forms of this syndrome. A large fraction of patients still remain without a molecular genetics diagnosis, suggesting that additional NBIA genes are still to be discovered.