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1.
Brain ; 147(1): 91-99, 2024 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-37804319

RESUMEN

Pathogenic variants in the MFN2 gene are commonly associated with autosomal dominant (CMT2A2A) or recessive (CMT2A2B) Charcot-Marie-Tooth disease, with possible involvement of the CNS. Here, we present a case of severe antenatal encephalopathy with lissencephaly, polymicrogyria and cerebellar atrophy. Whole genome analysis revealed a homozygous deletion c.1717-274_1734 del (NM_014874.4) in the MFN2 gene, leading to exon 16 skipping and in-frame loss of 50 amino acids (p.Gln574_Val624del), removing the proline-rich domain and the transmembrane domain 1 (TM1). MFN2 is a transmembrane GTPase located on the mitochondrial outer membrane that contributes to mitochondrial fusion, shaping large mitochondrial networks within cells. In silico modelling showed that the loss of the TM1 domain resulted in a drastically altered topological insertion of the protein in the mitochondrial outer membrane. Fetus fibroblasts, investigated by fluorescent cell imaging, electron microscopy and time-lapse recording, showed a sharp alteration of the mitochondrial network, with clumped mitochondria and clusters of tethered mitochondria unable to fuse. Multiple deficiencies of respiratory chain complexes with severe impairment of complex I were also evidenced in patient fibroblasts, without involvement of mitochondrial DNA instability. This is the first reported case of a severe developmental defect due to MFN2 deficiency with clumped mitochondria.


Asunto(s)
Encefalopatías , Enfermedad de Charcot-Marie-Tooth , Embarazo , Humanos , Femenino , Homocigoto , Mutación/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Eliminación de Secuencia , Mitocondrias/metabolismo , Encefalopatías/genética , Enfermedad de Charcot-Marie-Tooth/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo
2.
Am J Hum Genet ; 108(6): 1126-1137, 2021 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-34010604

RESUMEN

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.


Asunto(s)
Enfermedades Óseas/etiología , Enfermedades Cardiovasculares/etiología , Enfermedades del Tejido Conjuntivo/etiología , Inmunidad Celular/inmunología , Mutación con Pérdida de Función , Pérdida de Heterocigocidad , beta Carioferinas/genética , Adolescente , Adulto , Animales , Enfermedades Óseas/patología , Enfermedades Cardiovasculares/patología , Niño , Enfermedades del Tejido Conjuntivo/patología , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Transducción de Señal , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Adulto Joven , Pez Cebra , beta Carioferinas/metabolismo
3.
J Transl Med ; 22(1): 495, 2024 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-38796496

RESUMEN

BACKGROUND: The pathophysiology of toxico-nutritional optic neuropathies remains debated, with no clear understanding of the respective roles played by the direct alcohol toxicity, smoking and the often associated vitamin deficiencies, which are risk factors for optic neuropathy. Our aim was to investigate genetic susceptibility in patients with bilateral infraclinical optic neuropathy associated with chronic alcohol use disorder. METHODS: This retrospective cohort study included 102 visually asymptomatic patients with documented alcohol use disorder from a French reference center. Optic neuropathy was identified with optical coherence tomography (OCT), after which genetic susceptibility in the group of affected patients was investigated. Genetic testing was performed using panel sequencing of 87 nuclear genes and complete mitochondrial DNA sequencing. RESULTS: Optic neuropathy was detected in 36% (37/102) of the included patients. Genetic testing of affected patients disclosed two patients (2/30, 6.7%) with optic neuropathy associated with pathogenic variants affecting the SPG7 gene and five patients (5/30, 16.7%) who harbored variants of uncertain significance close to probable pathogenicity in the genes WFS1, LOXL1, MMP19, NR2F1 and PMPCA. No pathogenic mitochondrial DNA variants were found in this group. CONCLUSIONS: OCT can detect presence of asymptomatic optic neuropathy in patients with chronic alcohol use disorder. Furthermore, genetic susceptibility to optic neuropathy in this setting is found in almost a quarter of affected patients. Further studies may clarify the role of preventative measures in patients who might be predisposed to avoidable visual loss and blindness.


Asunto(s)
Predisposición Genética a la Enfermedad , Enfermedades del Nervio Óptico , Humanos , Masculino , Femenino , Enfermedades del Nervio Óptico/genética , Persona de Mediana Edad , Adulto , Alcoholismo/genética , Alcoholismo/complicaciones , Anciano , Estudios Retrospectivos
4.
J Med Virol ; 96(9): e29886, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39246064

RESUMEN

Mitochondria are vital for most cells' functions. Viruses hijack mitochondria machinery for misappropriation of energy supply or to bypass defense mechanisms. Many of these mitochondrial dysfunctions persist after recovery from treated or untreated viral infections, particularly when mitochondrial DNA is permanently damaged. Quantitative defects and structural rearrangements of mitochondrial DNA accumulate in post-mitotic tissues as recently reported long after SARS-CoV-2 or HIV infection, or following antiviral therapy. These observations are consistent with the "hit-and-run" concept proposed decades ago to explain viro-induced cell transformation and it could apply to delayed post-viral onsets of symptoms and advocate for complementary supportive care. Thus, according to this concept, following exposure to viruses or antiviral agents, mitochondrial damage could evolve into an autonomous clinical condition. It also establishes a pathogenic link between communicable and non-communicable chronic diseases.


Asunto(s)
Antivirales , COVID-19 , ADN Mitocondrial , Mitocondrias , Virosis , Humanos , Antivirales/uso terapéutico , Mitocondrias/efectos de los fármacos , ADN Mitocondrial/genética , COVID-19/virología , Virosis/tratamiento farmacológico , Virosis/virología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , SARS-CoV-2 , Tratamiento Farmacológico de COVID-19
5.
Brain ; 146(2): 455-460, 2023 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-36317462

RESUMEN

Hereditary optic neuropathies are caused by the degeneration of retinal ganglion cells whose axons form the optic nerves, with a consistent genetic heterogeneity. As part of our diagnostic activity, we retrospectively evaluated the combination of Leber hereditary optic neuropathy mutations testing with the exon sequencing of 87 nuclear genes on 2186 patients referred for suspected hereditary optic neuropathies. The positive diagnosis rate in individuals referred for Leber hereditary optic neuropathy testing was 18% (199/1126 index cases), with 92% (184/199) carrying one of the three main pathogenic variants of mitochondrial DNA (m.11778G>A, 66.5%; m.3460G>A, 15% and m.14484T>C, 11%). The positive diagnosis rate in individuals referred for autosomal dominant or recessive optic neuropathies was 27% (451/1680 index cases), with 10 genes accounting together for 96% of this cohort. This represents an overall positive diagnostic rate of 30%. The identified top 10 nuclear genes included OPA1, WFS1, ACO2, SPG7, MFN2, AFG3L2, RTN4IP1, TMEM126A, NR2F1 and FDXR. Eleven additional genes, each accounting for less than 1% of cases, were identified in 17 individuals. Our results show that 10 major genes account for more than 96% of the cases diagnosed with our nuclear gene panel.


Asunto(s)
Atrofia Óptica Autosómica Dominante , Atrofia Óptica Hereditaria de Leber , Enfermedades del Nervio Óptico , Humanos , Atrofia Óptica Hereditaria de Leber/genética , Estudios Retrospectivos , Atrofia Óptica Autosómica Dominante/genética , Atrofia Óptica Autosómica Dominante/patología , Enfermedades del Nervio Óptico/genética , Mutación/genética , ADN Mitocondrial/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , Proteasas ATP-Dependientes/genética , Proteínas Portadoras/genética , Proteínas Mitocondriales/genética , Proteínas de la Membrana/genética
6.
Int J Mol Sci ; 24(21)2023 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-37958880

RESUMEN

Long noncoding RNAs (lncRNAs) are a subclass of noncoding RNAs composed of more than 200 nucleotides without the ability to encode functional proteins. Given their involvement in critical cellular processes such as gene expression regulation, transcription, and translation, lncRNAs play a significant role in organism homeostasis. Breast cancer (BC) is the second most common cancer worldwide and evidence has shown a relationship between aberrant lncRNA expression and BC development. One of the main obstacles in BC control is multidrug chemoresistance, which is associated with the deregulation of multiple mechanisms such as efflux transporter activity, mitochondrial metabolism reprogramming, and epigenetic regulation as well as apoptosis and autophagy. Studies have shown the involvement of a large number of lncRNAs in the regulation of such pathways. However, the underlying mechanism is not clearly elucidated. In this review, we present the principal mechanisms associated with BC chemoresistance that can be directly or indirectly regulated by lncRNA, highlighting the importance of lncRNA in controlling BC chemoresistance. Understanding these mechanisms in deep detail may interest the clinical outcome of BC patients and could be used as therapeutic targets to overcome BC therapy resistance.


Asunto(s)
Neoplasias de la Mama , ARN Largo no Codificante , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica
7.
Hum Mutat ; 43(2): 128-142, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34837429

RESUMEN

Pathogenic variants of the nuclear receptor subfamily 2 group F member 1 gene (NR2F1) are responsible for Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), an autosomal dominant disorder characterized by optic atrophy associated with developmental delay and intellectual disability, but with a clinical presentation which appears to be multifaceted. We created the first public locus-specific database dedicated to NR2F1. All variants and clinical cases reported in the literature, as well as new unpublished cases, were integrated into the database using standard nomenclature to describe both molecular and phenotypic anomalies. We subsequently pursued a comprehensive approach based on computed representation and analysis suggesting a refinement of the BBSOAS clinical description with respect to neurological features and the inclusion of additional signs of hypotonia and feeding difficulties. This database is fully accessible for both clinician and molecular biologists and should prove useful in further refining the clinical synopsis of NR2F1 as new data is recorded.


Asunto(s)
Factor de Transcripción COUP I , Bases de Datos Genéticas , Discapacidad Intelectual , Atrofias Ópticas Hereditarias , Atrofia Óptica , Factor de Transcripción COUP I/genética , Humanos , Discapacidad Intelectual/genética , Hipotonía Muscular/genética , Atrofias Ópticas Hereditarias/genética , Atrofia Óptica/diagnóstico , Atrofia Óptica/genética
8.
Clin Genet ; 102(5): 438-443, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35861300

RESUMEN

Leigh syndrome (LS) is a progressive neurodegenerative disease, characterized by extensive clinical, biochemical, and genetic heterogeneity. Recently, biallelic variants in DNAJC30 gene, encoding a protein crucial for the repair of mitochondrial complex I subunits, have been associated with Leber hereditary optic neuropathy and LS. It was suggested that clinical heterogeneity of DNAJC30-associated mitochondrial disease may be attributed to digenic inheritance. We describe three Polish patients, a 9-year-old boy, and female and male siblings, aged 17 and 11 years, with clinical and biochemical manifestations of LS. Exome sequencing (ES) identified a homozygous pathogenic variant in DNAJC30 c.152A>G, p.(Tyr51Cys) in the 9-year-old boy. In the siblings, ES identified two DNAJC30 variants: c.152A>G, p.(Tyr51Cys) and c.130_131del, p.(Ser44ValfsTer8) in a compound heterozygous state. In addition, both siblings carried a novel heterozygous c.484G>T, p.(Val162Leu) variant in NDUFS8 gene. This report provides further evidence for the association of DNAJC30 variants with LS. DNAJC30-associated LS is characterized by variable age at onset, movement disorder phenotype and normal or moderately elevated blood lactate level. Identification of a candidate heterozygous variant in NDUFS8 supports the hypothesis of digenic inheritance. Importantly, DNAJC30 pathogenic variants should be suspected in patients with LS irrespective of optic nerve involvement.


Asunto(s)
Enfermedad de Leigh , Enfermedades Mitocondriales , Enfermedades Neurodegenerativas , Femenino , Humanos , Lactatos , Enfermedad de Leigh/genética , Enfermedad de Leigh/patología , Masculino , Enfermedades Mitocondriales/genética , Mutación , Fenotipo
9.
Genet Med ; 23(9): 1769-1778, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34040194

RESUMEN

PURPOSE: Diseases caused by defects in mitochondrial DNA (mtDNA) maintenance machinery, leading to mtDNA deletions, form a specific group of disorders. However, mtDNA deletions also appear during aging, interfering with those resulting from mitochondrial disorders. METHODS: Here, using next-generation sequencing (NGS) data processed by eKLIPse and data mining, we established criteria distinguishing age-related mtDNA rearrangements from those due to mtDNA maintenance defects. MtDNA deletion profiles from muscle and urine patient samples carrying pathogenic variants in nuclear genes involved in mtDNA maintenance (n = 40) were compared with age-matched controls (n = 90). Seventeen additional patient samples were used to validate the data mining model. RESULTS: Overall, deletion number, heteroplasmy level, deletion locations, and the presence of repeats at deletion breakpoints were significantly different between patients and controls, especially in muscle samples. The deletion number was significantly relevant in adults, while breakpoint repeat lengths surrounding deletions were discriminant in young subjects. CONCLUSION: Altogether, eKLIPse analysis is a powerful tool for measuring the accumulation of mtDNA deletions between patients of different ages, as well as in prioritizing novel variants in genes involved in mtDNA stability.


Asunto(s)
Genoma Mitocondrial , Enfermedades Mitocondriales , Adulto , ADN Mitocondrial/genética , Genoma Mitocondrial/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mitocondrias/genética , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/genética , Eliminación de Secuencia/genética
10.
Int J Mol Sci ; 22(17)2021 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-34502189

RESUMEN

Since its discovery, mitophagy has been viewed as a protective mechanism used by cancer cells to prevent the induction of mitochondrial apoptosis. Most cancer treatments directly or indirectly cause mitochondrial dysfunction in order to trigger signals for cell death. Elimination of these dysfunctional mitochondria by mitophagy could thus prevent the initiation of the apoptotic cascade. In breast cancer patients, resistance to doxorubicin (DOX), one of the most widely used cancer drugs, is an important cause of poor clinical outcomes. However, the role played by mitophagy in the context of DOX resistance in breast cancer cells is not well understood. We therefore tried to determine whether an increase in mitophagic flux was associated with the resistance of breast cancer cells to DOX. Our first objective was to explore whether DOX-resistant breast cancer cells were characterized by conditions that favor mitophagy induction. We next tried to determine whether mitophagic flux was increased in DOX-resistant cells in response to DOX treatment. For this purpose, the parental (MCF-7) and DOX-resistant (MCF-7dox) breast cancer cell lines were used. Our results show that mitochondrial reactive oxygen species (ROS) production and hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression are higher in MCF-7dox in a basal condition compared to MCF-7, suggesting DOX-resistant breast cancer cells are prone to stimuli to induce a mitophagy-related event. Our results also showed that, in response to DOX, autophagolysosome formation is induced in DOX-resistant breast cancer cells. This mitophagic step following DOX treatment seems to be partly due to mitochondrial ROS production as autophagolysosome formation is moderately decreased by the mitochondrial antioxidant mitoTEMPO.


Asunto(s)
Neoplasias de la Mama/fisiopatología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Lisosomas , Mitofagia , Especies Reactivas de Oxígeno/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Doxorrubicina/uso terapéutico , Femenino , Humanos , Células MCF-7 , Mitocondrias/metabolismo
11.
J Hum Genet ; 65(2): 91-98, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31645654

RESUMEN

Hereditary spastic paraplegias (HSPs) are characterized by lower extremity spasticity and weakness. HSP is often caused by mutations in SPG genes, but it may also be produced by inborn errors of metabolism. We performed next-generation sequencing of 4813 genes in one adult twin pair with HSP and severe muscular weakness occurring at the same age. We found two pathogenic compound heterozygous variants in MTHFR, including a variant not referenced in international databases, c.197C>T (p.Pro66Leu) and a known variant, c.470G>A (p.Arg157Gln), and two heterozygous pathogenic variants in POLG, c.1760C>T (p.Pro587Leu) and c.752C>T (p.Thr251Ile). MTHFR and POLG mutations were consistent with the severe muscle weakness and the metabolic changes, including hyperhomocysteinemia and decreased activity of both N(5,10)methylenetetrahydrofolate reductase (MTHFR) and complexes I and II of the mitochondrial respiratory chain. These data suggest the potential role of MTHFR and POLG mutations through consequences on mitochondrial dysfunction in the occurrence of spastic paraparesis phenotype with combined metabolic, muscular, and neurological components.


Asunto(s)
ADN Polimerasa gamma/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Enfermedades Mitocondriales/genética , Paraparesia Espástica/genética , Paraplejía Espástica Hereditaria/genética , Femenino , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Persona de Mediana Edad , Enfermedades Mitocondriales/diagnóstico , Mutación , Paraparesia Espástica/diagnóstico , Análisis de Secuencia de ADN , Paraplejía Espástica Hereditaria/diagnóstico , Gemelos Monocigóticos
12.
Mol Biol Rep ; 47(5): 3779-3787, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-32319008

RESUMEN

Mitochondrial diseases are a clinically heterogeneous group of multisystemic disorders that arise as a result of various mitochondrial dysfunctions. Autosomal recessive aARS deficiencies represent a rapidly growing group of severe rare inherited mitochondrial diseases, involving multiple organs, and currently without curative option. They might be related to defects of mitochondrial aminoacyl t-RNA synthetases (mtARS) that are ubiquitous enzymes involved in mitochondrial aminoacylation and the translation process. Here, using NGS analysis of 281 nuclear genes encoding mitochondrial proteins, we identified 4 variants in different mtARS in three patients from unrelated Tunisian families, with clinical features of mitochondrial disorders. Two homozygous variants were found in KARS (c.683C>T) and AARS2 (c.1150-4C>G), respectively in two patients, while two heterozygous variants in EARS2 (c.486-7C>G) and DARS2 (c.1456C>T) were concomitantly found in the third patient. Bio-informatics investigations predicted their pathogenicity and deleterious effects on pre-mRNA splicing and on protein stability. Thus, our results suggest that mtARS mutations are common in Tunisian patients with mitochondrial diseases.


Asunto(s)
Alanina-ARNt Ligasa/genética , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Alanina-ARNt Ligasa/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Aspartato-ARNt Ligasa/genética , Aspartato-ARNt Ligasa/metabolismo , Niño , Preescolar , Femenino , Estudios de Asociación Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Homocigoto , Humanos , Masculino , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Mutación/genética , Linaje
13.
Int J Mol Sci ; 21(8)2020 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-32344771

RESUMEN

Leber's hereditary optic neuropathy (LHON, MIM#535000) is the most common form of inherited optic neuropathies and mitochondrial DNA-related diseases. The pathogenicity of mutations in genes encoding components of mitochondrial Complex I is well established, but the underlying pathomechanisms of the disease are still unclear. Hypothesizing that oxidative stress related to Complex I deficiency may increase protein S-glutathionylation, we investigated the proteome-wide S-glutathionylation profiles in LHON (n = 11) and control (n = 7) fibroblasts, using the GluICAT platform that we recently developed. Glutathionylation was also studied in healthy fibroblasts (n = 6) after experimental Complex I inhibition. The significantly increased reactive oxygen species (ROS) production in the LHON group by Complex I was shown experimentally. Among the 540 proteins which were globally identified as glutathionylated, 79 showed a significantly increased glutathionylation (p < 0.05) in LHON and 94 in Complex I-inhibited fibroblasts. Approximately 42% (33/79) of the altered proteins were shared by the two groups, suggesting that Complex I deficiency was the main cause of increased glutathionylation. Among the 79 affected proteins in LHON fibroblasts, 23% (18/79) were involved in energetic metabolism, 31% (24/79) exhibited catalytic activity, 73% (58/79) showed various non-mitochondrial localizations, and 38% (30/79) affected the cell protein quality control. Integrated proteo-metabolomic analysis using our previous metabolomic study of LHON fibroblasts also revealed similar alterations of protein metabolism and, in particular, of aminoacyl-tRNA synthetases. S-glutathionylation is mainly known to be responsible for protein loss of function, and molecular dynamics simulations and 3D structure predictions confirmed such deleterious impacts on adenine nucleotide translocator 2 (ANT2), by weakening its affinity to ATP/ADP. Our study reveals a broad impact throughout the cell of Complex I-related LHON pathogenesis, involving a generalized protein stress response, and provides a therapeutic rationale for targeting S-glutathionylation by antioxidative strategies.


Asunto(s)
Atrofia Óptica Hereditaria de Leber/metabolismo , Proteína S/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Anciano , Susceptibilidad a Enfermedades , Complejo I de Transporte de Electrón/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Modelos Moleculares , Atrofia Óptica Hereditaria de Leber/tratamiento farmacológico , Atrofia Óptica Hereditaria de Leber/etiología , Conformación Proteica , Procesamiento Proteico-Postraduccional , Proteína S/química , Proteoma , Proteómica/métodos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Adulto Joven
14.
J Cell Sci ; 130(11): 1940-1951, 2017 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-28424233

RESUMEN

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.


Asunto(s)
Ciclo del Ácido Cítrico/genética , ADN Mitocondrial/genética , Mitocondrias/metabolismo , Dinámicas Mitocondriales/genética , Proteínas de Unión al ARN/metabolismo , Adenosina Trifosfato/biosíntesis , Sistemas CRISPR-Cas , Ciclo del Ácido Cítrico/efectos de los fármacos , Daño del ADN , ADN Mitocondrial/metabolismo , Etidio/toxicidad , Eliminación de Gen , Células HeLa , Humanos , Metabolómica , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/efectos de los fármacos , Imagen Óptica , Oxidación-Reducción , Fosforilación Oxidativa/efectos de los fármacos , Palmitoilcarnitina/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Unión al ARN/genética
15.
Genet Med ; 21(6): 1407-1416, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30393377

RESUMEN

PURPOSE: Accurate detection of mitochondrial DNA (mtDNA) alterations is essential for the diagnosis of mitochondrial diseases. The development of high-throughput sequencing technologies has enhanced the detection sensitivity of mtDNA pathogenic variants, but the detection of mtDNA rearrangements, especially multiple deletions, is still poorly processed. Here, we present eKLIPse, a sensitive and specific tool allowing the detection and quantification of large mtDNA rearrangements from single and paired-end sequencing data. METHODS: The methodology was first validated using a set of simulated data to assess the detection sensitivity and specificity, and second with a series of sequencing data from mitochondrial disease patients carrying either single or multiple deletions, related to pathogenic variants in nuclear genes involved in mtDNA maintenance. RESULTS: eKLIPse provides the precise breakpoint positions and the cumulated percentage of mtDNA rearrangements at a given gene location with a detection sensitivity lower than 0.5% mutant. eKLIPse software is available either as a script to be integrated in a bioinformatics pipeline, or as user-friendly graphical interface to visualize the results through a Circos representation ( https://github.com/dooguypapua/eKLIPse ). CONCLUSION: Thus, eKLIPse represents a useful resource to study the causes and consequences of mtDNA rearrangements, for further genotype/phenotype correlations in mitochondrial disorders.


Asunto(s)
ADN Mitocondrial/genética , Análisis de Secuencia de ADN/métodos , Eliminación de Secuencia/genética , Secuencia de Bases/genética , Estudios de Asociación Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mitocondrias/genética , Enfermedades Mitocondriales/diagnóstico , Programas Informáticos
16.
Biochim Biophys Acta Mol Basis Dis ; 1864(5 Pt A): 1596-1608, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29454073

RESUMEN

Ketogenic diet (KD) which combined carbohydrate restriction and the addition of ketone bodies has emerged as an alternative metabolic intervention used as an anticonvulsant therapy or to treat different types of neurological or mitochondrial disorders including MELAS syndrome. MELAS syndrome is a severe mitochondrial disease mainly due to the m.3243A > G mitochondrial DNA mutation. The broad success of KD is due to multiple beneficial mechanisms with distinct effects of very low carbohydrates and ketones. To evaluate the metabolic part of carbohydrate restriction, transmitochondrial neuronal-like cybrid cells carrying the m.3243A > G mutation, shown to be associated with a severe complex I deficiency was exposed during 3 weeks to glucose restriction. Mitochondrial enzyme defects were combined with an accumulation of complex I (CI) matrix intermediates in the untreated mutant cells, leading to a drastic reduction in CI driven respiration. The severe reduction of CI was also paralleled in post-mortem brain tissue of a MELAS patient carrying high mutant load. Importantly, lowering significantly glucose concentration in cell culture improved CI assembly with a significant reduction of matrix assembly intermediates and respiration capacities were restored in a sequential manner. In addition, OXPHOS protein expression and mitochondrial DNA copy number were significantly increased in mutant cells exposed to glucose restriction. The accumulation of CI matrix intermediates appeared as a hallmark of MELAS pathophysiology highlighting a critical pathophysiological mechanism involving CI disassembly, which can be alleviated by lowering glucose fuelling and the induction of mitochondrial biogenesis, emphasizing the usefulness of metabolic interventions in MELAS syndrome.


Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Glucosa/metabolismo , Síndrome MELAS/enzimología , Mitocondrias/enzimología , Neuronas/enzimología , Mutación Puntual , Línea Celular Tumoral , Complejo I de Transporte de Electrón/genética , Femenino , Humanos , Síndrome MELAS/genética , Síndrome MELAS/patología , Masculino , Mitocondrias/genética , Mitocondrias/patología , Neuronas/patología , Fosforilación Oxidativa
17.
J Cell Mol Med ; 21(10): 2284-2297, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28378518

RESUMEN

Optic Atrophy 1 (OPA1) gene mutations cause diseases ranging from isolated dominant optic atrophy (DOA) to various multisystemic disorders. OPA1, a large GTPase belonging to the dynamin family, is involved in mitochondrial network dynamics. The majority of OPA1 mutations encodes truncated forms of the protein and causes DOA through haploinsufficiency, whereas missense OPA1 mutations are predicted to cause disease through deleterious dominant-negative mechanisms. We used 3D imaging and biochemical analysis to explore autophagy and mitophagy in fibroblasts from seven patients harbouring OPA1 mutations. We report new genotype-phenotype correlations between various types of OPA1 mutation and mitophagy. Fibroblasts bearing dominant-negative OPA1 mutations showed increased autophagy and mitophagy in response to uncoupled oxidative phosphorylation. In contrast, OPA1 haploinsufficiency was correlated with a substantial reduction in mitochondrial turnover and autophagy, unless subjected to experimental mitochondrial injury. Our results indicate distinct alterations of mitochondrial physiology and turnover in cells with OPA1 mutations, suggesting that the level and profile of OPA1 may regulate the rate of mitophagy.


Asunto(s)
Autofagia/genética , GTP Fosfohidrolasas/genética , Mutación , Atrofia Óptica Autosómica Dominante/genética , Adolescente , Adulto , Células Cultivadas , Preescolar , Femenino , Fibroblastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Mitofagia/genética
18.
Hum Mol Genet ; 24(17): 5015-23, 2015 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-26071363

RESUMEN

Acute intermittent porphyria (AIP), an autosomal dominant metabolic disease (MIM #176000), is due to a deficiency of hydroxymethylbilane synthase (HMBS), which catalyzes the third step of the heme biosynthetic pathway. The clinical expression of the disease is mainly neurological, involving the autonomous, central and peripheral nervous systems. We explored mitochondrial oxidative phosphorylation (OXPHOS) in the brain and skeletal muscle of the Hmbs(-/-) mouse model first in the basal state (BS), and then after induction of the disease with phenobarbital and treatment with heme arginate (HA). The modification of the respiratory parameters, determined in mice in the BS, reflected a spontaneous metabolic energetic adaptation to HMBS deficiency. Phenobarbital induced a sharp alteration of the oxidative metabolism with a significant decrease of ATP production in skeletal muscle that was restored by treatment with HA. This OXPHOS defect was due to deficiencies in complexes I and II in the skeletal muscle whereas all four respiratory chain complexes were affected in the brain. To date, the pathogenesis of AIP has been mainly attributed to the neurotoxicity of aminolevulinic acid and heme deficiency. Our results show that mitochondrial energetic failure also plays an important role in the expression of the disease.


Asunto(s)
Encéfalo/metabolismo , Hidroximetilbilano Sintasa/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Músculos/metabolismo , Porfiria Intermitente Aguda/genética , Porfiria Intermitente Aguda/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Complejo I de Transporte de Electrón/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Ratones Noqueados , Modelos Biológicos , Músculos/efectos de los fármacos , Fenobarbital/farmacología
19.
Am J Hum Genet ; 95(6): 637-48, 2014 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-25466283

RESUMEN

Galloway-Mowat syndrome is a rare autosomal-recessive condition characterized by nephrotic syndrome associated with microcephaly and neurological impairment. Through a combination of autozygosity mapping and whole-exome sequencing, we identified WDR73 as a gene in which mutations cause Galloway-Mowat syndrome in two unrelated families. WDR73 encodes a WD40-repeat-containing protein of unknown function. Here, we show that WDR73 was present in the brain and kidney and was located diffusely in the cytoplasm during interphase but relocalized to spindle poles and astral microtubules during mitosis. Fibroblasts from one affected child and WDR73-depleted podocytes displayed abnormal nuclear morphology, low cell viability, and alterations of the microtubule network. These data suggest that WDR73 plays a crucial role in the maintenance of cell architecture and cell survival. Altogether, WDR73 mutations cause Galloway-Mowat syndrome in a particular subset of individuals presenting with late-onset nephrotic syndrome, postnatal microcephaly, severe intellectual disability, and homogenous brain MRI features. WDR73 is another example of a gene involved in a disease affecting both the kidney glomerulus and the CNS.


Asunto(s)
Hernia Hiatal/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Nefrosis/genética , Síndrome Nefrótico/genética , Proteínas/genética , Adolescente , Encéfalo/fisiopatología , Línea Celular , Supervivencia Celular , Niño , Preescolar , Citosol/metabolismo , Exoma/genética , Hernia Hiatal/fisiopatología , Homocigoto , Humanos , Glomérulos Renales/fisiopatología , Masculino , Microcefalia/fisiopatología , Microtúbulos/metabolismo , Mitosis , Modelos Moleculares , Mutación , Nefrosis/fisiopatología , Síndrome Nefrótico/fisiopatología , Podocitos , Transporte de Proteínas , Proteínas/metabolismo , Polos del Huso/metabolismo
20.
Biochim Biophys Acta Mol Basis Dis ; 1863(1): 284-291, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27815040

RESUMEN

Ketogenic Diet used to treat refractory epilepsy for almost a century may represent a treatment option for mitochondrial disorders for which effective treatments are still lacking. Mitochondrial complex I deficiencies are involved in a broad spectrum of inherited diseases including Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes syndrome leading to recurrent cerebral insults resembling strokes and associated with a severe complex I deficiency caused by mitochondrial DNA (mtDNA) mutations. The analysis of MELAS neuronal cybrid cells carrying the almost homoplasmic m.3243A>G mutation revealed a metabolic switch towards glycolysis with the production of lactic acid, severe defects in respiratory chain activity and complex I disassembly with an accumulation of assembly intermediates. Metabolites, NADH/NAD+ ratio, mitochondrial enzyme activities, oxygen consumption and BN-PAGE analysis were evaluated in mutant compared to control cells. A severe complex I enzymatic deficiency was identified associated with a major complex I disassembly with an accumulation of assembly intermediates of 400kDa. We showed that Ketone Bodies (KB) exposure for 4weeks associated with glucose deprivation significantly restored complex I stability and activity, increased ATP synthesis and reduced the NADH/NAD+ ratio, a key component of mitochondrial metabolism. In addition, without changing the mutant load, mtDNA copy number was significantly increased with KB, indicating that the absolute amount of wild type mtDNA copy number was higher in treated mutant cells. Therefore KB may constitute an alternative and promising therapy for MELAS syndrome, and could be beneficial for other mitochondrial diseases caused by complex I deficiency.


Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Cuerpos Cetónicos/farmacología , Síndrome MELAS/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Línea Celular , Respiración de la Célula/efectos de los fármacos , Variaciones en el Número de Copia de ADN/efectos de los fármacos , ADN Mitocondrial/genética , Dieta Cetogénica , Complejo I de Transporte de Electrón/deficiencia , Humanos , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Síndrome MELAS/patología , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/complicaciones , Neuronas/metabolismo , Neuronas/patología
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