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PURPOSE: Interneuronopathies are a group of neurodevelopmental disorders characterized by deficient migration and differentiation of gamma-aminobutyric acidergic interneurons resulting in a broad clinical spectrum, including autism spectrum disorders, early-onset epileptic encephalopathy, intellectual disability, and schizophrenic disorders. SP9 is a transcription factor belonging to the Krüppel-like factor and specificity protein family, the members of which harbor highly conserved DNA-binding domains. SP9 plays a central role in interneuron development and tangential migration, but it has not yet been implicated in a human neurodevelopmental disorder. METHODS: Cases with SP9 variants were collected through international data-sharing networks. To address the specific impact of SP9 variants, in silico and in vitro assays were carried out. RESULTS: De novo heterozygous variants in SP9 cause a novel form of interneuronopathy. SP9 missense variants affecting the glutamate 378 amino acid result in severe epileptic encephalopathy because of hypomorphic and neomorphic DNA-binding effects, whereas SP9 loss-of-function variants result in a milder phenotype with epilepsy, developmental delay, and autism spectrum disorder. CONCLUSION: De novo heterozygous SP9 variants are responsible for a neurodevelopmental disease. Interestingly, variants located in conserved DNA-binding domains of KLF/SP family transcription factors may lead to neomorphic DNA-binding functions resulting in a combination of loss- and gain-of-function effects.
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Trastorno del Espectro Autista , Epilepsia , Discapacidad Intelectual , Interneuronas , Factores de Transcripción Sp , Factores de Transcripción , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/patología , Epilepsia/genética , Epilepsia/patología , Heterocigoto , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Interneuronas/metabolismo , Interneuronas/patología , Mutación Missense/genética , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Fenotipo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción Sp/genéticaRESUMEN
Introduction: Inherited mitochondrial diseases are the most common group of metabolic disorders caused by a defect in oxidative phosphorylation. They are characterized by a wide clinical and genetic spectrum and can manifest at any age. In this study, we established novel phenotype-genotype correlations between the clinical and molecular features of a cohort of Tunisian patients with mitochondrial diseases. Materials and methods: Whole-exome sequencing was performed on five Tunisian patients with suspected mitochondrial diseases. Then, a combination of filtering and bioinformatics prediction tools was utilized to assess the pathogenicity of genetic variations. Sanger sequencing was subsequently performed to confirm the presence of potential deleterious variants in the patients and verify their segregation within families. Structural modeling was conducted to study the effect of novel variants on the protein structure. Results: We identified two novel homozygous variants in NDUFAF5 (c.827G>C; p.Arg276Pro) and FASTKD2 (c.496_497del; p.Leu166GlufsTer2) associated with a severe clinical form of Leigh and Leigh-like syndromes, respectively. Our results further disclosed two variants unreported in North Africa, in GFM2 (c.569G>A; p.Arg190Gln) and FOXRED1 (c.1261G>A; p.Val421Met) genes, and we described the first case of fumaric aciduria in a Tunisian patient harboring the c.1358T>C; p.Leu453Pro FH variant. Conclusion: Our study expands the mutational and phenotypic spectrum of mitochondrial diseases in Tunisia and highlights the importance of next-generation sequencing to decipher the pathomolecular mechanisms responsible for these disorders in an admixed population.
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Deoxyhypusine hydroxylase (DOHH) is the enzyme catalyzing the second step in the post-translational synthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] in the eukaryotic initiation factor 5A (eIF5A). Hypusine is formed exclusively in eIF5A by two sequential enzymatic steps catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusinated eIF5A is essential for translation and cell proliferation in eukaryotes, and all three genes encoding eIF5A, DHPS, and DOHH are highly conserved throughout eukaryotes. Pathogenic variants affecting either DHPS or EIF5A have been previously associated with neurodevelopmental disorders. Using trio exome sequencing, we identified rare bi-allelic pathogenic missense and truncating DOHH variants segregating with disease in five affected individuals from four unrelated families. The DOHH variants are associated with a neurodevelopmental phenotype that is similar to phenotypes caused by DHPS or EIF5A variants and includes global developmental delay, intellectual disability, facial dysmorphism, and microcephaly. A two-dimensional gel analyses revealed the accumulation of deoxyhypusine-containing eIF5A [eIF5A(Dhp)] and a reduction in the hypusinated eIF5A in fibroblasts derived from affected individuals, providing biochemical evidence for deficiency of DOHH activity in cells carrying the bi-allelic DOHH variants. Our data suggest that rare bi-allelic variants in DOHH result in reduced enzyme activity, limit the hypusination of eIF5A, and thereby lead to a neurodevelopmental disorder.
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Lisina , Oxigenasas de Función Mixta , Trastornos del Neurodesarrollo , Alelos , Expresión Génica , Humanos , Lisina/análogos & derivados , Oxigenasas de Función Mixta/genética , Trastornos del Neurodesarrollo/genéticaRESUMEN
Lactic acidosis, the extracellular accumulation of lactate and protons, is a consequence of increased glycolysis triggered by insufficient oxygen supply to tissues. Macrophages are able to differentiate from monocytes under such acidotic conditions, and remain active in order to resolve the underlying injury. Here we show that, in lactic acidosis, human monocytes differentiating into macrophages are characterized by depolarized mitochondria, transient reduction of mitochondrial mass due to mitophagy, and a significant decrease in nutrient absorption. These metabolic changes, resembling pseudostarvation, result from the low extracellular pH rather than from the lactosis component, and render these cells dependent on autophagy for survival. Meanwhile, acetoacetate, a natural metabolite produced by the liver, is utilized by monocytes/macrophages as an alternative fuel to mitigate lactic acidosis-induced pseudostarvation, as evidenced by retained mitochondrial integrity and function, retained nutrient uptake, and survival without the need of autophagy. Our results thus show that acetoacetate may increase tissue tolerance to sustained lactic acidosis.
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Acetoacetatos/farmacología , Acidosis Láctica/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Mitocondrias/metabolismo , Sustancias Protectoras/farmacología , Reprogramación Celular , Metabolismo Energético , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo , Macrófagos/metabolismo , Ingeniería Metabólica , Mitofagia , Microambiente TumoralRESUMEN
Mandibuloacral dysplasia syndromes are mainly due to recessive LMNA or ZMPSTE24 mutations, with cardinal nuclear morphological abnormalities and dysfunction. We report five homozygous null mutations in MTX2, encoding Metaxin-2 (MTX2), an outer mitochondrial membrane protein, in patients presenting with a severe laminopathy-like mandibuloacral dysplasia characterized by growth retardation, bone resorption, arterial calcification, renal glomerulosclerosis and severe hypertension. Loss of MTX2 in patients' primary fibroblasts leads to loss of Metaxin-1 (MTX1) and mitochondrial dysfunction, including network fragmentation and oxidative phosphorylation impairment. Furthermore, patients' fibroblasts are resistant to induced apoptosis, leading to increased cell senescence and mitophagy and reduced proliferation. Interestingly, secondary nuclear morphological defects are observed in both MTX2-mutant fibroblasts and mtx-2-depleted C. elegans. We thus report the identification of a severe premature aging syndrome revealing an unsuspected link between mitochondrial composition and function and nuclear morphology, establishing a pathophysiological link with premature aging laminopathies and likely explaining common clinical features.
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Acroosteólisis/metabolismo , Predisposición Genética a la Enfermedad/genética , Lipodistrofia/metabolismo , Mandíbula/anomalías , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Acroosteólisis/diagnóstico por imagen , Acroosteólisis/genética , Acroosteólisis/patología , Envejecimiento Prematuro/genética , Envejecimiento Prematuro/metabolismo , Animales , Apoptosis , Caenorhabditis elegans , Proliferación Celular , Niño , Regulación hacia Abajo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Genotipo , Homocigoto , Humanos , Lipodistrofia/diagnóstico por imagen , Lipodistrofia/genética , Lipodistrofia/patología , Masculino , Mandíbula/diagnóstico por imagen , Proteínas de la Membrana/genética , Metaloendopeptidasas , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas Mitocondriales/genética , Mutación , Fenotipo , Piel , Secuenciación Completa del GenomaRESUMEN
Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency.
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Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Ciclo del Ácido Cítrico , Fibroblastos/citología , Fibroblastos/metabolismo , Glucólisis , Humanos , Ingeniería Metabólica , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , NADH Deshidrogenasa/antagonistas & inhibidores , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Análisis de Componente Principal , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Angiogenesis is a complex process leading to the growth of new blood vessels from existing vasculature, triggered by local proangiogenic factors such as VEGF. An excess of angiogenesis is a recurrent feature of various pathologic conditions such as tumor growth. Phostines are a family of synthetic glycomimetic compounds that exhibit anticancer properties, and the lead compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST 3.1a) shows antiglioblastoma properties both in vitro and in vivo. In the present study, we assessed the effect of PST 3.1a on angiogenesis and endothelial metabolism. In vitro, PST 3.1a (10 µM) inhibited all steps that regulate angiogenesis, including migration, proliferation, adhesion, and tube formation. In vivo, PST 3.1a reduced intersegmental vessel formation and vascularization of the subintestinal plexus in zebrafish embryos and also altered pathologic angiogenesis and glioblastoma progression in vivo. Mechanistically, PST 3.1a altered interaction of VEGF receptor 2 and glycosylation-regulating protein galectin-1, a key component regulating angiogenesis associated with tumor resistance. Thus, these data show that use of PST 3.1a is an innovative approach to target angiogenesis.-Bousseau, S., Marchand, M., Soleti, R., Vergori, L., Hilairet, G., Recoquillon, S., Le Mao, M., Gueguen, N., Khiati, S., Clarion, L., Bakalara, N., Martinez, M. C., Germain, S., Lenaers, G., Andriantsitohaina, R. Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.
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Inhibidores de la Angiogénesis/farmacología , Neovascularización Patológica/tratamiento farmacológico , Fosfinas/farmacología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Galectina 1/metabolismo , Glioblastoma/metabolismo , Glicosilación , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Pez CebraRESUMEN
Thirty percent of medullary thyroid carcinomas (MTCs) are related to dominant germline pathogenic variants in the RET proto-oncogene. According to their aggressiveness, these pathogenic variants are classified in three risk levels: 'moderate', 'high' and 'highest'. The present study compares the metabolomics profiles of five pathogenic variants, whether already classified or not. We have generated six stable murine fibroblast cell lines (NIH3T3) expressing the WT allele or variants of the human RET gene, with different levels of pathogenicity, including the M918V variant that is yet to be accurately classified. We carried out a targeted metabolomics study of the cell extracts with a QTRAP mass spectrometer, using the Biocrates Absolute IDQ p180 kit, which allows the quantification of 188 endogenous molecules. The data were then subjected to multivariate statistical analysis. One hundred seventy three metabolites were accurately measured. The metabolic profiles of the cells expressing the RET variants were found to be correlated with their oncogenic risk. In addition, the statistical model we constructed for predicting the oncogenic risk attributed a moderate risk to the M918V variant. Our results indicate that metabolomics may be useful for characterizing the pathogenicity of the RET gene variants and their levels of aggressiveness.
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Carcinoma Neuroendocrino/genética , Carcinoma Neuroendocrino/metabolismo , Metaboloma/genética , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Animales , Carcinoma Neuroendocrino/clasificación , Carcinoma Neuroendocrino/patología , Proliferación Celular , Variación Genética , Humanos , Metabolómica , Ratones , Modelos Estadísticos , Mutación , Células 3T3 NIH , Fosforilación , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret/metabolismo , Riesgo , Transducción de Señal , Neoplasias de la Tiroides/clasificación , Neoplasias de la Tiroides/patologíaRESUMEN
OPA1 (Optic Atrophy 1) is a multi-isoform dynamin GTPase involved in the regulation of mitochondrial fusion and organization of the cristae structure of the mitochondrial inner membrane. Pathogenic OPA1 variants lead to a large spectrum of disorders associated with visual impairment due to optic nerve neuropathy. The aim of this study was to investigate the metabolomic consequences of complete OPA1 disruption in Opa1-/- mouse embryonic fibroblasts (MEFs) compared to their Opa1+/+ counterparts. Our non-targeted metabolomics approach revealed significant modifications of the concentration of several mitochondrial substrates, i.e. a decrease of aspartate, glutamate and α-ketoglutaric acid, and an increase of asparagine, glutamine and adenosine-5'-monophosphate, all related to aspartate metabolism. The signature further highlighted the altered metabolism of nucleotides and NAD together with deficient mitochondrial bioenergetics, reflected by the decrease of creatine/creatine phosphate and pantothenic acid, and the increase in pyruvate and glutathione. Interestingly, we recently reported significant variations of five of these molecules, including aspartate and glutamate, in the plasma of individuals carrying pathogenic OPA1 variants. Our findings show that the disruption of OPA1 leads to a remodelling of bioenergetic pathways with the central role being played by aspartate and related metabolites.
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Metabolismo Energético , Fibroblastos/química , Fibroblastos/metabolismo , GTP Fosfohidrolasas/deficiencia , Metaboloma , Animales , Ratones , Ratones Noqueados , Mitocondrias/metabolismoRESUMEN
Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status.