RESUMEN
Amyotrophic Lateral Sclerosis (ALS) is the most common neurodegenerative disease affecting motor neurons. The pathophysiology of ALS is not well understood but TDP-43 proteinopathy (aggregation and mislocalization) is one of the major phenomena described. Several factors can influence TDP-43 behavior such as mild pH alterations that can induce conformational changes in recombinant TDP-43, increasing its propensity to aggregate. However to our knowledge, no studies have been conducted yet in a cellular setting, in the context of ALS. We therefore tested the effect of cellular pH alterations on the localization, aggregation, and phosphorylation of TDP-43. HEK293T cells overexpressing wildtype TDP-43 were incubated for 1 h with solutions of different pH (6.4, 7.2, and 8). Incubation of cells for 1 h in solutions of pH 6.4 and 8 led to an increase in TDP-43-positive puncta. This was accompanied by the mislocalization of TDP-43 from the nucleus to the cytoplasm. Our results suggest that small alterations in cellular pH affect TDP-43 and increase its mislocalization into cytoplasmic TDP-43-positive puncta, which might suggest a role of TDP-43 in the response of cells to pH alterations.
RESUMEN
The ionotropic glutamate delta receptor GluD1, encoded by the GRID1 gene, is involved in synapse formation, function, and plasticity. GluD1 does not bind glutamate, but instead cerebellin and D-serine, which allow the formation of trans-synaptic bridges, and trigger transmembrane signaling. Despite wide expression in the nervous system, pathogenic GRID1 variants have not been characterized in humans so far. We report homozygous missense GRID1 variants in five individuals from two unrelated consanguineous families presenting with intellectual disability and spastic paraplegia, without (p.Thr752Met) or with (p.Arg161His) diagnosis of glaucoma, a threefold phenotypic association whose genetic bases had not been elucidated previously. Molecular modeling and electrophysiological recordings indicated that Arg161His and Thr752Met mutations alter the hinge between GluD1 cerebellin and D-serine binding domains and the function of this latter domain, respectively. Expression, trafficking, physical interaction with metabotropic glutamate receptor mGlu1, and cerebellin binding of GluD1 mutants were not conspicuously altered. Conversely, upon expression in neurons of dissociated or organotypic slice cultures, we found that both GluD1 mutants hampered metabotropic glutamate receptor mGlu1/5 signaling via Ca2+ and the ERK pathway and impaired dendrite morphology and excitatory synapse density. These results show that the clinical phenotypes are distinct entities segregating in the families as an autosomal recessive trait, and caused by pathophysiological effects of GluD1 mutants involving metabotropic glutamate receptor signaling and neuronal connectivity. Our findings unravel the importance of GluD1 receptor signaling in sensory, cognitive and motor functions of the human nervous system.
Asunto(s)
Discapacidad Intelectual , Receptores de Glutamato Metabotrópico , Transducción de Señal , Sinapsis , Humanos , Discapacidad Intelectual/genética , Masculino , Sinapsis/metabolismo , Sinapsis/genética , Femenino , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal/genética , Homocigoto , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/genética , Linaje , Adulto , Paraplejía/genética , Paraplejía/metabolismo , Animales , Niño , Neuronas/metabolismo , Adolescente , Células HEK293 , Mutación/genéticaRESUMEN
The ubiquitin pathway, one of the main actors regulating cell signaling processes and cellular protein homeostasis, is directly involved in the pathophysiology of amyotrophic lateral sclerosis (ALS). We first analyzed, by a next-generation sequencing (NGS) strategy, a series of genes of the ubiquitin pathway in two cohorts of familial and sporadic ALS patients comprising 176 ALS patients. We identified several pathogenic variants in different genes of this ubiquitin pathway already described in ALS, such as FUS, CCNF and UBQLN2. Other variants of interest were discovered in new genes studied in this disease, in particular in the HECW1 gene. We have shown that the HECT E3 ligase called NEDL1, encoded by the HECW1 gene, is expressed in neurons, mainly in their somas. Its overexpression is associated with increased cell death in vitro and, very interestingly, with the cytoplasmic mislocalization of TDP-43, a major protein involved in ALS. These results give new support for the role of the ubiquitin pathway in ALS, and suggest further studies of the HECW1 gene and its protein NEDL1 in the pathophysiology of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Neuronas/metabolismo , Transducción de Señal/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Recent technological developments in ultrasound (US) imaging and ultrasound contrast agents (UCAs) have improved diagnostic confidence in echography. In the clinical management of melanoma, contrast-enhanced ultrasound (CEUS) imaging complements conventional US imaging (i.e., high-resolution US and Doppler imaging) for clinical examination and therapeutic follow-up. These developments have set into motion the combined use of ultrasound and UCAs as a new modality for drug delivery. This modality, called sonoporation, has emerged as a non-invasive, targeted and safe method for the delivery of therapeutic drugs into melanoma. This review focuses on the results and prospects of using US and UCAs as dual modalities for CEUS imaging and melanoma treatment.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Medios de Contraste , Humanos , Melanoma/diagnóstico por imagen , Melanoma/terapia , Microburbujas , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/terapia , Ultrasonografía/métodos , Melanoma Cutáneo MalignoRESUMEN
The ubiquitin pathway regulates the function of many proteins and controls cellular protein homeostasis. In recent years, it has attracted great interest in neurodevelopmental and neurodegenerative diseases. Here, we have presented the first review on the roles of the 9 proteins of the HECT E3 ligase NEDD4 subfamily in the development and function of neurons in the central nervous system (CNS). We discussed their regulation and their direct or indirect involvement in neurodevelopmental diseases, such as intellectual disability, and neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease or Amyotrophic Lateral Sclerosis. Further studies on the roles of these proteins, their regulation and their targets in neurons will certainly contribute to a better understanding of neuronal function and dysfunction, and will also provide interesting information for the development of therapeutics targeting them.
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Enfermedad de Parkinson , Ubiquitina-Proteína Ligasas , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Ubiquitina-Proteína Ligasas Nedd4/genética , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
In our previous study, in which array CGH was used on 19 Lebanese ASD subjects and their parents, we identified rare copy number variants (CNVs) in 14 subjects. The five remaining subjects did not show any CNVs related to autism spectrum disorders (ASD). In the present complementary study, we applied whole-exome sequencing (WES), which allows the identification of rare genetic variations such as single nucleotide variations and small insertions/deletions, to the five negative CNV subjects. After stringent filtering of initial data on the five families, three novel genes potentially related to neurodevelopment were identified, including a de novo mutation in the MIS18BP1 gene. In addition, genes already known to be related to ASD contained sequence variations. Our findings outline the potential involvement of the novel de novo mutation in the MIS18BP1 gene in the genetic etiology and pathophysiology of ASD and highlights the genetic complexity of these disorders. Further studies with larger cohorts of subjects are needed to confirm these observations, and functional analyses need to be performed to understand the precise pathophysiology in these cases.
Asunto(s)
Trastorno del Espectro Autista , Trastorno del Espectro Autista/genética , Variaciones en el Número de Copia de ADN , Exoma/genética , Humanos , Secuenciación del ExomaRESUMEN
The collapsin response mediator protein (CRMP) family proteins are intracellular mediators of neurotrophic factors regulating neurite structure/spine formation and are essential for dendrite patterning and directional axonal pathfinding during brain developmental processes. Among this family, CRMP5/DPYSL5 plays a significant role in neuronal migration, axonal guidance, dendrite outgrowth, and synapse formation by interacting with microtubules. Here, we report the identification of missense mutations in DPYSL5 in nine individuals with brain malformations, including corpus callosum agenesis and/or posterior fossa abnormalities, associated with variable degrees of intellectual disability. A recurrent de novo p.Glu41Lys variant was found in eight unrelated patients, and a p.Gly47Arg variant was identified in one individual from the first family reported with Ritscher-Schinzel syndrome. Functional analyses of the two missense mutations revealed impaired dendritic outgrowth processes in young developing hippocampal primary neuronal cultures. We further demonstrated that these mutations, both located in the same loop on the surface of DPYSL5 monomers and oligomers, reduced the interaction of DPYSL5 with neuronal cytoskeleton-associated proteins MAP2 and ßIII-tubulin. Our findings collectively indicate that the p.Glu41Lys and p.Gly47Arg variants impair DPYSL5 function on dendritic outgrowth regulation by preventing the formation of the ternary complex with MAP2 and ßIII-tubulin, ultimately leading to abnormal brain development. This study adds DPYSL5 to the list of genes implicated in brain malformation and in neurodevelopmental disorders.
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Agenesia del Cuerpo Calloso/genética , Cerebelo/anomalías , Mutación Missense/genética , Trastornos del Neurodesarrollo/genética , Adulto , Agenesia del Cuerpo Calloso/diagnóstico por imagen , Cerebelo/diagnóstico por imagen , Niño , Preescolar , Femenino , Humanos , Hidrolasas/química , Hidrolasas/genética , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/genética , Masculino , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Trastornos del Neurodesarrollo/diagnóstico por imagen , Tubulina (Proteína)/metabolismo , Adulto JovenRESUMEN
The X-linked PTCHD1 gene, encoding a synaptic membrane protein, has been involved in neurodevelopmental disorders with the description of deleterious genomic microdeletions or truncating coding mutations. Missense variants were also identified, however, without any functional evidence supporting their pathogenicity level. We investigated 13 missense variants of PTCHD1, including eight previously described (c.152G>A,p.(Ser51Asn); c.217C>T,p.(Leu73Phe); c.517A>G,p.(Ile173Val); c.542A>C,p.(Lys181Thr); c.583G>A,p.(Val195Ile); c.1076A>G,p.(His359Arg); c.1409C>A,p.(Ala470Asp); c.1436A>G,p.(Glu479Gly)), and five novel ones (c.95C>T,p.(Pro32Leu); c.95C>G,p.(Pro32Arg); c.638A>G,p.(Tyr213Cys); c.898G>C,p.(Gly300Arg); c.928G>C,p.(Ala310Pro)) identified in male patients with intellectual disability (ID) and/or autism spectrum disorder (ASD). Interestingly, several of these variants involve amino acids localized in structural domains such as transmembrane segments. To evaluate their potentially deleterious impact on PTCHD1 protein function, we performed in vitro overexpression experiments of the wild-type and mutated forms of PTCHD1-GFP in HEK 293T and in Neuro-2a cell lines as well as in mouse hippocampal primary neuronal cultures. We found that six variants impaired the expression level of the PTCHD1 protein, and were retained in the endoplasmic reticulum suggesting abnormal protein folding. Our functional analyses thus provided evidence of the pathogenic impact of missense variants in PTCHD1, which reinforces the involvement of the PTCHD1 gene in ID and in ASD.
Asunto(s)
Trastorno del Espectro Autista , Discapacidad Intelectual , Proteínas de la Membrana , Animales , Trastorno del Espectro Autista/genética , Membrana Celular/metabolismo , Humanos , Discapacidad Intelectual/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Mutación MissenseRESUMEN
Protein aggregates in affected motor neurons are a hallmark of amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to their formation remain incompletely understood. Oxidative stress associated with age, the major risk factor in ALS, contributes to this neurodegeneration in ALS. We show that several genes coding for enzymes of the ubiquitin and small ubiquitin-related modifier (SUMO) pathways exhibit altered expression in motor neuronal cells exposed to oxidative stress, such as the CCNF gene mutated in ALS patients. Eleven of these genes were further studied in conditions combining oxidative stress and the expression of an ALS related mutant of the superoxide dismutase 1 (SOD1) gene. We observed a combined effect of these two environmental and genetic factors on the expression of genes, such as Uhrf2, Rbx1, Kdm2b, Ube2d2, Xaf1, and Senp1. Overall, we identified dysregulations in the expression of enzymes of the ubiquitin and SUMO pathways that may be of interest to better understand the pathophysiology of ALS and to protect motor neurons from oxidative stress and genetic alterations.
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Esclerosis Amiotrófica Lateral/metabolismo , Regulación de la Expresión Génica , Modelos Neurológicos , Neuronas Motoras/metabolismo , Estrés Oxidativo , Proteína SUMO-1/biosíntesis , Superóxido Dismutasa-1/metabolismo , Ubiquitina/biosíntesis , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Línea Celular , Humanos , Neuronas Motoras/patología , Mutación , Proteína SUMO-1/genética , Superóxido Dismutasa-1/genética , Ubiquitina/genéticaRESUMEN
OBJECTIVES: To determine whether the familial clustering of amyotrophic lateral sclerosis (ALS) cases and the phenotype of the disease may help identify the pathogenic genes involved. METHODS: We conducted a targeted next-generation sequencing analysis on 235 French familial ALS (FALS), unrelated probands to identify mutations in 30 genes linked to the disease. The genealogy, that is, number of cases and generations with ALS, gender, age, site of onset and the duration of the disease were analysed. RESULTS: Regarding the number of generations, 49 pedigrees had only one affected generation, 152 had two affected generations and 34 had at least three affected generations. Among the 149 pedigrees (63.4%) for which a deleterious variant was found, an abnormal G4C2 expansion in C9orf72 was found in 98 cases as well as SOD1, TARBP or FUS mutations in 30, 9 and 7 cases, respectively. Considering pedigrees from the number of generations, abnormal G4C2 expansion in C9orf72 was more frequent in pedigrees with pairs of affected ALS cases, which represented 65.2% of our cohort. SOD1 mutation involved all types of pedigrees. No TARDBP nor FUS mutation was present in monogenerational pedigrees. TARDBP mutation predominated in bigenerational pedigrees with at least three cases and FUS mutation in multigenerational pedigrees with more than seven cases, on average, and with an age of onset younger than 45 years. CONCLUSION: Our results suggest that familial clustering, phenotypes and genotypes are interconnected in FALS, and thus it might be possible to target the genetic screening from the familial architecture and the phenotype of ALS cases.
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Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Mutación , Anciano , Análisis por Conglomerados , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Femenino , Pruebas Genéticas , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Proteína FUS de Unión a ARN/genética , Superóxido Dismutasa-1/genéticaRESUMEN
The 22q11.2 deletion syndrome (22q11DS) is associated with a wide spectrum of cognitive and psychiatric symptoms. Despite the considerable work performed over the past 20 years, the genetic etiology of the neurodevelopmental phenotype remains speculative. Here, we report de novo heterozygous truncating variants in the HIRA (Histone cell cycle regulation defective, S. Cerevisiae, homolog of, A) gene associated with a neurodevelopmental disorder in two unrelated patients. HIRA is located within the commonly deleted region of the 22q11DS and encodes a histone chaperone that regulates neural progenitor proliferation and neurogenesis, and that belongs to the WD40 Repeat (WDR) protein family involved in brain development and neuronal connectivity. To address the specific impact of HIRA haploinsufficiency in the neurodevelopmental phenotype of 22q11DS, we combined Hira knock-down strategies in developing mouse primary hippocampal neurons, and the direct study of brains from heterozygous Hira+/- mice. Our in vitro analyses revealed that Hira gene is mostly expressed during neuritogenesis and early dendritogenesis stages in mouse total brain and in developing primary hippocampal neurons. Moreover, shRNA knock-down experiments showed that a twofold decrease of endogenous Hira expression level resulted in an impaired dendritic growth and branching in primary developing hippocampal neuronal cultures. In parallel, in vivo analyses demonstrated that Hira+/- mice displayed subtle neuroanatomical defects including a reduced size of the hippocampus, the fornix and the corpus callosum. Our results suggest that HIRA haploinsufficiency would likely contribute to the complex pathophysiology of the neurodevelopmental phenotype of 22q11DS by impairing key processes in neurogenesis and by causing neuroanatomical defects during cerebral development.
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Proteínas de Ciclo Celular/genética , Síndrome de DiGeorge/genética , Haploinsuficiencia , Chaperonas de Histonas/genética , Trastornos del Neurodesarrollo/genética , Plasticidad Neuronal/genética , Neuronas/metabolismo , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Niño , Preescolar , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Síndrome de DiGeorge/metabolismo , Síndrome de DiGeorge/patología , Femenino , Fórnix/metabolismo , Fórnix/patología , Expresión Génica , Heterocigoto , Hipocampo/metabolismo , Hipocampo/patología , Chaperonas de Histonas/antagonistas & inhibidores , Chaperonas de Histonas/deficiencia , Chaperonas de Histonas/metabolismo , Humanos , Ratones , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Neurogénesis/genética , Neuronas/patología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismoRESUMEN
In amyotrophic lateral sclerosis, motor neurons undergoing degeneration are characterized by the presence of cytoplasmic aggregates containing TDP-43 protein. SUMOylation, a posttranslational modification of proteins, has been previously implicated in the formation of aggregates positives for SOD1, another protein enriched in a subset of ALS patients. We show in this study that TDP-43 is also a target of SUMOylation. The inhibition of the first step of the SUMOylation process by anacardic acid significantly reduces the presence of TDP-43 aggregates and improves neuritogenesis and cell viability in vitro. Interestingly, the mutation of the unique SUMOylation site on TDP-43, using site-directed mutagenesis, modifies the intracellular localization of TDP-43 aggregates. Instead of being cytoplasmic where they are associated with toxic effects, they are located inside the nucleus. This change of localization results in improvement in cell viability and in global cellular functions. Our results implicate the SUMOylation site of TDP-43 in the formation of cytoplasmic TDP-43 aggregates, a hallmark of ALS, and thus identifies this region as a new target for novel therapeutic strategies.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/metabolismo , Neuronas Motoras/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Ratones , Mutación/genética , Sumoilación/genéticaRESUMEN
BACKGROUND: There is a strong evidence for genetic factors as the main causes of Autism Spectrum Disorders (ASD). To date, hundreds of genes have been identified either by copy number variations (CNVs) and/or single nucleotide variations. However, despite all the findings, the genetics of these disorders have not been totally explored. METHODS: Thus, the aim of our work was to identify rare CNVs and genes present in these regions in ASD children, using a high-resolution comparative genomic hybridization technique and quantitative PCR (qPCR) approach. RESULTS: Our results have shown 60-70 chromosomal aberrations per patient. We have initially selected 66 CNVs that have been further assessed using qPCR. Finally, we have validated 22 CNVs including 11 deletions and 11 duplications. Ten CNVs are de novo, 11 are inherited and one of unknown origin of transmission. Among the CNVs detected, novel ASD candidate genes PJA2, SYNPO, APCS, and TAC1 have been identified in our group of Lebanese patients. In addition, previously described CNVs have been identified containing genes such as SHANK3, MBP, CHL1, and others. CONCLUSION: Our study broadens the population spectrum of studied ASD patients and adds new candidates at the list of genes contributing to these disorders.
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Trastorno del Espectro Autista/genética , Predisposición Genética a la Enfermedad , Adulto , Anciano , Trastorno del Espectro Autista/sangre , Trastorno del Espectro Autista/diagnóstico , Niño , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Femenino , Humanos , Líbano , Masculino , Proteínas de Microfilamentos/genética , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Reacción en Cadena en Tiempo Real de la Polimerasa , Componente Amiloide P Sérico/genética , Taquicininas/genética , Ubiquitina-Proteína Ligasas/genética , Adulto JovenRESUMEN
LIMK2 is involved in neuronal functions by regulating actin dynamics. Different isoforms of LIMK2 are described in databanks. LIMK2a and LIMK2b are the most characterized. A few pieces of evidence suggest that LIMK2 isoforms might not have overlapping functions. In this study, we focused our attention on a less studied human LIMK2 isoform, LIMK2-1. Compared to the other LIMK2 isoforms, LIMK2-1 contains a supplementary C-terminal phosphatase 1 inhibitory domain (PP1i). We found out that this isoform was hominidae-specific and showed that it was expressed in human fetal brain and faintly in adult brain. Its coding sequence was sequenced in 173 patients with sporadic non-syndromic intellectual disability (ID), and we observed an association of a rare missense variant in the PP1i domain (rs151191437, p.S668P) with ID. Our results also suggest an implication of LIMK2-1 in neurite outgrowth and neurons arborization which appears to be affected by the p.S668P variation. Therefore our results suggest that LIMK2-1 plays a role in the developing brain, and that a rare variation of this isoform is a susceptibility factor in ID.
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Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Discapacidad Intelectual/metabolismo , Quinasas Lim/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Sistema Nervioso Central/citología , Predisposición Genética a la Enfermedad , Hominidae , Discapacidad Intelectual/genética , Quinasas Lim/genética , Ratones , Modelos Moleculares , Mutación Missense , Proyección Neuronal/fisiología , Neuronas/citología , Neuronas/metabolismo , Isoformas de Proteínas , Ratas , Homología de SecuenciaRESUMEN
Mutations in the TAR-DNA Binding Protein-43 (TDP-43) encoding the TARDBP gene are present in amyotrophic lateral sclerosis (ALS). TDP-43 is the major component of ubiquitin-positive inclusions in motor neurons in ALS patients. We report here a novel heterozygous missense mutation in TARDBP in an ALS patient presenting a rapid form of ALS. This mutation p.N259S is located within the RNA recognition motif 2 (RRM2) in very close proximity with nucleotides in RNA. It is the first time a mutation was reported in this RRM2 domain of TDP-43. Expression of TDP-43N259S in neuronal cells NSC-34 and in primary cultures of motor neurons was associated with cytoplasmic TDP-43/ubiquitin positive inclusions. Our findings identified for the first time a mutation in ALS in the RRM2 domain of TDP-43, reinforcing the link between this RNA-binding protein, perturbations in RNA metabolism, disruption in protein homeostasis and ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Neuronas Motoras/metabolismo , Mutación Missense , Esclerosis Amiotrófica Lateral/diagnóstico , Células Cultivadas , Progresión de la Enfermedad , Resultado Fatal , Humanos , Cuerpos de Inclusión/metabolismo , Masculino , Persona de Mediana Edad , Ubiquitina/metabolismoRESUMEN
The selective degeneration of motoneuron that typifies amyotrophic lateral sclerosis (ALS) implicates non-cell-autonomous effects of astrocytes. However, mechanisms underlying astrocyte-mediated neurotoxicity remain largely unknown. According to the determinant role of astrocyte metabolism in supporting neuronal function, we propose to explore the metabolic status of astrocytes exposed to ALS-associated conditions. We found a significant metabolic dysregulation including purine, pyrimidine, lysine, and glycerophospholipid metabolism pathways in astrocytes expressing an ALS-causing mutated superoxide dismutase-1 (SOD1) when co-cultured with motoneurons. SOD1 astrocytes exposed to glutamate revealed a significant modification of the astrocyte metabolic fingerprint. More importantly, we observed that SOD1 mutation and glutamate impact the cellular shuttling of lactate between astrocytes and motoneurons with a decreased in extra- and intra-cellular lactate levels in astrocytes. Based on the emergent strategy of metabolomics, this work provides novel insight for understanding metabolic dysfunction of astrocytes in ALS conditions and opens the perspective of therapeutics targets through focusing on these metabolic pathways. GLIA 2017 GLIA 2017;65:592-605.
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Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Ácido Glutámico/farmacología , Ácido Láctico/metabolismo , Neuronas Motoras/metabolismo , Superóxido Dismutasa/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Técnicas de Cocultivo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Imagen por Resonancia Magnética , Redes y Vías Metabólicas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Análisis de Componente Principal , Médula Espinal/citología , Superóxido Dismutasa/metabolismo , Tritio/metabolismoRESUMEN
In amyotrophic lateral sclerosis (ALS), motor neuron degeneration is associated with systemic metabolic impairment. However, the evolution of metabolism alteration is partially unknown and its link with disease progression has never been described. For the first time, we ran a study focused on (1) the evolution of metabolism disturbance during disease progression through omics approaches and (2) the relation between metabolome profile and clinical evolution. SOD1-G93A (mSOD1) transgenic mice (n = 11) and wild-type (WT) littermates (n = 17) were studied during 20 weeks. Metabolomic profile of muscle and cerebral cortex was analysed at week 20, and plasma samples were assessed at four time points over 20 weeks. The relevant metabolic pathways highlighted by metabolomic analysis were explored by a targeted transcriptomic approach in mice. Plasma metabolomics were also performed in 24 ALS patients and 24 gender- and age-matched controls. Metabolomic analysis of muscle and cerebral cortex enabled an excellent discrimination between mSOD1 and WT mice (p < 0.001). These alterations included especially tryptophan, arginine, and proline metabolism pathways (including polyamines) as also revealed by transcriptomic analysis and findings in ALS patients. Multivariate models performed to explain clinical findings in ALS mice, and patients were excellent (p < 0.01) and highlighted three main metabolic pathways: arginine and proline, tryptophan, and branched amino acid metabolism. This work is the first longitudinal study that evaluates metabolism alteration in ALS, including the analysis of different tissues and using a combination of omics methods. We particularly identified arginine and proline metabolism. This pathway is also associated with disease progression and may open new perspectives of therapeutic targets.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Arginina/metabolismo , Neuronas Motoras/metabolismo , Prolina/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Estudios Longitudinales , Masculino , Metabolómica/métodos , Ratones Transgénicos , Superóxido Dismutasa/genética , Triptófano/metabolismoRESUMEN
In amyotrophic lateral sclerosis (ALS), motor neuron degeneration occurs simultaneously with systemic metabolic impairment and neuroinflammation. Playing an important role in the regulation of both phenomena, interleukin (IL)-6, a major cytokine of the inflammatory response has been proposed as a target for management of ALS. Although a pilot clinical trial provided promising results in humans, another recent preclinical study showed that knocking out the IL-6 gene in mice carrying ALS did not improve clinical outcome. In this study, we aimed to determine the relevance of the IL-6 pathway blockade in a mouse model of ALS by using a pharmacological antagonist of IL-6, a murine surrogate of tocilizumab, namely MR16-1. We analyzed the immunological and metabolic effects of IL-6 blockade by cytokine measurement, blood cell immunophenotyping, targeted metabolomics, and transcriptomics. A deleterious clinical effect of MR16-1 was revealed, with a speeding up of weight loss (p = 0.0041) and decreasing body weight (p < 0.05). A significant increase in regulatory T-cell count (p = 0.0268) and a decrease in C-X-C ligand-1 concentrations in plasma (p = 0.0479) were observed. Metabolomic and transcriptomic analyses revealed that MR16-1 mainly affected branched-chain amino acid, lipid, arginine, and proline metabolism. IL-6 blockade negatively affected body weight, despite a moderated anti-inflammatory effect. Metabolic effects of IL-6 were mild compared with metabolic disturbances observed in ALS, but a modification of lipid metabolism by therapy was identified. These results indicate that IL-6 blockade did not improve clinical outcome of a mutant superoxide dismutase 1 mouse model of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Interleucina-6/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Esclerosis Amiotrófica Lateral/genética , Animales , Peso Corporal/genética , Citocinas/sangre , Evaluación de la Discapacidad , Modelos Animales de Enfermedad , Estudios de Seguimiento , Redes Reguladoras de Genes , Factores Inmunológicos/farmacología , Factores Inmunológicos/uso terapéutico , Interleucina-6/genética , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons. Mutations in the SOD1 gene encoding the superoxide dismutase 1 are present in 15% of familial ALS cases and in 2% of sporadic cases. These mutations are associated with the formation of SOD1-positive aggregates. The mechanisms of aggregation remain unknown, but posttranslational modifications of SOD1 may be involved. Here, we report that NSC-34 motor neuronal cells expressing mutant SOD1 contained aggregates positive for small ubiquitin modifier-1 (SUMO-1), and in parallel a reduced level of free SUMO-1. CLEM (correlative light and electron microscopy) analysis showed nonorganized cytosolic aggregates for all mutations tested (SOD1A4V, SOD1V31A, and SOD1G93C). We next show that preventing the SUMOylation of mutant SOD1 by the substitution of lysine 75, the SUMOylation site of SOD1, significantly reduces the number of motor neuronal cells with aggregates. These results support the need for further research on the SUMOylation pathways, which may be a potential therapeutic target in ALS.
Asunto(s)
Lisina/metabolismo , Neuronas Motoras/metabolismo , Agregación Patológica de Proteínas/metabolismo , Proteína SUMO-1/metabolismo , Sumoilación/fisiología , Superóxido Dismutasa-1/metabolismo , Animales , Células CHO , Línea Celular , Supervivencia Celular/fisiología , Cricetulus , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Neuronas Motoras/ultraestructura , Mutación , Estructura Secundaria de Proteína , Superóxido Dismutasa-1/genéticaRESUMEN
Many genes are now thought to confer susceptibility to autism. Despite the fact that this neuropsychiatric disease appears to be related to several different causes, common cellular and molecular pathways have emerged and point to synaptic dysfunction or cellular growth. Several studies have indicated the importance of the ubiquitin pathway in synaptic function and the aetiology of autism. Here, we focused on the ring finger protein 135 (RNF135) gene, encoding an E3 ubiquitin ligase expressed in the cortex and cerebellum, and located in the NF1 gene locus in 17q11.2, a region linked to autism. We carried out a genetic analysis of the coding sequence of RFN135 in a French cohort of patients with autism and observed a significantly increased frequency of genotypes carrying the rare allele of the rs111902263 (p.R115K) missense variant in patients (P=0.0019, odds ratio: 4.23, 95% confidence interval: 1.87-9.57). Particularly, three unrelated patients showed a homozygous genotype for K115, a situation not observed in the 1812 control individuals. Further cellular and molecular studies are required to elucidate the role of this gene and the variant K115 in brain development and neuronal function.