RESUMO
Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length protein and a shorter isoform lacking its N-terminal acidic domain. To investigate functions selectively associated with the full-length hnRNP R isoform, we generated a Hnrnpr knockout mouse (Hnrnprtm1a/tm1a) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnprtm1a/tm1a mice did not show any axonal growth defects but exhibited enhanced accumulation of double-strand breaks and an impaired DNA damage response upon exposure to genotoxic agents. Proteomic analysis of the hnRNP R interactome revealed the multifunctional protein Yb1 as a top interactor. Yb1-depleted motoneurons were defective in DNA damage repair. We show that Yb1 is recruited to chromatin upon DNA damage where it interacts with γ-H2AX, a mechanism that is dependent on full-length hnRNP R. Our findings thus suggest a novel role of hnRNP R in maintaining genomic integrity and highlight the function of its N-terminal acidic domain in this context.
Assuntos
Cromatina/genética , Dano ao DNA , Reparo do DNA/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Neurônios Motores/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Animais , Axônios/metabolismo , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Immunoblotting , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/citologia , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
Alterations in fear learning/generalization are considered to be relevant mechanisms engendering the development of anxiety disorders being the most prevalent mental disorders. Although anxiety disorders almost exclusively have their first onset in childhood and adolescence, etiological research focuses on adult individuals. In this study, we evaluated findings of a recent meta-analysis of genome-wide association studies in adult anxiety disorders with significant associations of four single nucleotide polymorphisms (SNPs) in a large cohort of 347 healthy children (8-12 years) characterized for dimensional anxiety. We investigated the modulation of anxiety parameters by these SNPs in a discriminative fear conditioning and generalization paradigm in the to-date largest sample of children. Results extended findings of the meta-analysis showing a genomic locus on 2p21 to modulate anxious personality traits and arousal ratings. These SNPs might, thus, serve as susceptibility markers for a shared risk across pathological anxiety, presumably mediated by alterations in arousal.
Assuntos
Transtornos de Ansiedade/psicologia , Cromossomos Humanos Par 2/genética , Medo/psicologia , Generalização Psicológica/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Criança , Feminino , Técnicas de Genotipagem , Humanos , MasculinoRESUMO
First line pain relief medication during pregnancy relies nearly entirely on the over-the-counter analgesic acetaminophen, which is generally considered safe to use during gestation. However, recent epidemiological studies suggest a risk of developing attention-deficit/hyperactivity disorder (ADHD)-like symptoms in children if mothers use acetaminophen during pregnancy. Currently, there are no experimental proofs that prenatal acetaminophen exposure causes developmental brain alterations of progeny. Exposure to high acetaminophen concentrations causes liver toxicity, which is well investigated in different model organisms. However, sub-liver-toxic concentrations have not been experimentally investigated with respect to ADHD endophenotypes such as hyperactivity. We used zebrafish to investigate the potential impact of acetaminophen exposure on locomotor activity levels, and compared it to the established zebrafish Latrophilin 3 (Lphn3) ADHD-model. We determined the sub-liver-toxic concentration of acetaminophen in zebrafish larvae and treated wild-type and lphn3.1 knockdown larvae with increasing concentrations of acetaminophen. We were able to confirm that lphn3.1 knockdown alone causes hyperactivity, strengthening the implication of Lphn3 dysfunction as an ADHD risk factor. Neither acute nor chronic exposure to acetaminophen at sub-liver-toxic concentrations in wild-type or lphn3.1 knock-downs increases locomotor activity levels. Together our findings show that embryonic to larval exposure to acetaminophen does not cause hyperactivity in zebrafish larvae. Furthermore, there are no additive and/or synergistic effects of acetaminophen exposure in a susceptible background induced by knock-down of lphn3.1. Our experimental study suggests that there is, at least in zebrafish larvae, no direct link between embryonic acetaminophen exposure and hyperactivity. Further work is necessary to clarify this issue in humans.
Assuntos
Acetaminofen/toxicidade , Analgésicos não Narcóticos/toxicidade , Transtorno do Deficit de Atenção com Hiperatividade/induzido quimicamente , Fatores Etários , Análise de Variância , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Larva/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Locomoção/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , RNA Mensageiro/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Peixe-ZebraRESUMO
The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Neurônios Motores/metabolismo , Proteoma/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Diferenciação Celular , Linhagem Celular , Citoesqueleto/genética , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Anotação de Sequência Molecular , Neurônios Motores/química , Neurônios Motores/patologia , Especificidade de Órgãos , Cultura Primária de Células , Proteoma/genética , Transdução de SinaisRESUMO
Diabetic neuropathy (DNP), afflicting sensory and motor nerve fibers, is a major complication in diabetes. The underlying cellular mechanisms of axon degeneration are poorly understood. IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP. We investigated the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP5 in motor axons and sensory nerve fibers. These mice develop motor axonopathy and sensory deficits similar to those seen in DNP. Motor axon degeneration was also observed in mice in which the IGF1 receptor (IGF1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF1 on IGF1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration. Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.
Assuntos
Axônios/fisiologia , Proteínas de Transporte/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas/fisiopatologia , Neurônios Motores/fisiologia , Degeneração Neural/fisiopatologia , Animais , Axônios/patologia , Crescimento Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Neuropatias Diabéticas/patologia , Humanos , Camundongos Transgênicos , Atividade Motora/fisiologia , Neurônios Motores/patologia , Degeneração Neural/patologia , Nervo Frênico/patologia , Nervo Frênico/fisiopatologia , Receptor IGF Tipo 1/metabolismo , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Sensação/fisiologiaRESUMO
Vacuolar-vesicular protein sorting (Vps) factors are involved in vesicular trafficking in eukaryotic cells. We identified the missense mutation L967Q in Vps54 in the wobbler mouse, an animal model of amyotrophic lateral sclerosis, and also characterized a lethal allele, Vps54(beta-geo). Motoneuron survival and spermiogenesis are severely compromised in the wobbler mouse, indicating that Vps54 has an essential role in these processes.
Assuntos
Doença dos Neurônios Motores/genética , Mutação de Sentido Incorreto/genética , Espermatogênese/genética , Proteínas de Transporte Vesicular/genética , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Animais , Cromossomos Artificiais Bacterianos , Clonagem Molecular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Dados de Sequência Molecular , Doença dos Neurônios Motores/patologia , Homologia de Sequência de AminoácidosRESUMO
A large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72, a gene located on chromosome 9p21, has been recently reported to be responsible for ~40% of familial amyotrophic lateral sclerosis cases of European ancestry. The aim of the current article was to describe the phenotype of amyotrophic lateral sclerosis cases carrying the expansion by providing a detailed clinical description of affected cases from representative multi-generational kindreds, and by analysing the age of onset, gender ratio and survival in a large cohort of patients with familial amyotrophic lateral sclerosis. We collected DNA and analysed phenotype data for 141 index Italian familial amyotrophic lateral sclerosis cases (21 of Sardinian ancestry) and 41 German index familial amyotrophic lateral sclerosis cases. Pathogenic repeat expansions were detected in 45 (37.5%) patients from mainland Italy, 12 (57.1%) patients of Sardinian ancestry and nine (22.0%) of the 41 German index familial amyotrophic lateral sclerosis cases. The disease was maternally transmitted in 27 (49.1%) pedigrees and paternally transmitted in 28 (50.9%) pedigrees (P = non-significant). On average, children developed disease 7.0 years earlier than their parents [children: 55.8 years (standard deviation 7.9), parents: 62.8 (standard deviation 10.9); P = 0.003]. Parental phenotype influenced the type of clinical symptoms manifested by the child: of the 13 cases where the affected parent had an amyotrophic lateral sclerosis-frontotemporal dementia or frontotemporal dementia, the affected child also developed amyotrophic lateral sclerosis-frontotemporal dementia in nine cases. When compared with patients carrying mutations of other amyotrophic lateral sclerosis-related genes, those with C9ORF72 expansion had commonly a bulbar onset (42.2% compared with 25.0% among non-C9ORF72 expansion cases, P = 0.03) and cognitive impairment (46.7% compared with 9.1% among non-C9ORF72 expansion cases, P = 0.0001). Median survival from symptom onset among cases carrying C9ORF72 repeat expansion was 3.2 years lower than that of patients carrying TARDBP mutations (5.0 years; 95% confidence interval: 3.6-7.2) and longer than those with FUS mutations (1.9 years; 95% confidence interval: 1.7-2.1). We conclude that C9ORF72 hexanucleotide repeat expansions were the most frequent mutation in our large cohort of patients with familial amyotrophic lateral sclerosis of Italian, Sardinian and German ancestry. Together with mutation of SOD1, TARDBP and FUS, mutations of C9ORF72 account for ~60% of familial amyotrophic lateral sclerosis in Italy. Patients with C9ORF72 hexanucleotide repeat expansions present some phenotypic differences compared with patients with mutations of other genes or with unknown mutations, namely a high incidence of bulbar-onset disease and comorbidity with frontotemporal dementia. Their pedigrees typically display a high frequency of cases with pure frontotemporal dementia, widening the concept of familial amyotrophic lateral sclerosis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteínas/genética , Adulto , Idade de Início , Idoso , Proteína C9orf72 , Estudos de Coortes , DNA/genética , Expansão das Repetições de DNA , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Mutação/genética , Pais , Linhagem , Fenótipo , Caracteres Sexuais , Análise de SobrevidaRESUMO
Phosphatase and tensin homolog (PTEN), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, is widely involved in the regulation of protein synthesis. Here we show that the PTEN protein is enriched in cell bodies and axon terminals of purified motor neurons. We explored the role of the PTEN pathway by manipulating PTEN expression in healthy and diseased motor neurons. PTEN depletion led to an increase in growth cone size, promotion of axonal elongation and increased survival of these cells. These changes were associated with alterations of downstream signaling pathways for local protein synthesis as revealed by an increase in pAKT and p70S6. Most notably, this treatment also restores beta-actin protein levels in axonal growth cones of SMN-deficient motor neurons. Furthermore, we report here that a single injection of adeno-associated virus serotype 6 (AAV6) expressing siPTEN into hind limb muscles at postnatal day 1 in SMNDelta7 mice leads to a significant PTEN depletion and robust improvement in motor neuron survival. Taken together, these data indicate that PTEN-mediated regulation of protein synthesis in motor neurons could represent a target for therapy in spinal muscular atrophy.
Assuntos
Axônios/fisiologia , Neurônios Motores/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Actinas/genética , Análise de Variância , Animais , Axônios/metabolismo , Western Blotting , Sobrevivência Celular , Células Cultivadas , Cones de Crescimento/metabolismo , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Neurônios Motores/citologia , PTEN Fosfo-Hidrolase/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Serina-Treonina Quinases TORRESUMO
Members of the family of metabotropic glutamate receptors are involved in the pathomechanism of several disorders of the nervous system. Besides the well-investigated function of dysfunctional glutamate receptor signaling in neurodegenerative diseases, neurodevelopmental disorders (NDD), like autism spectrum disorders (ASD) and attention-deficit and hyperactivity disorder (ADHD) might also be partly caused by disturbed glutamate signaling during development. However, the underlying mechanism of the type III metabotropic glutamate receptor 8 (mGluR8 or GRM8) involvement in neurodevelopment and disease mechanism is largely unknown. Here we show that the expression pattern of the two orthologs of human GRM8, grm8a and grm8b, have evolved partially distinct expression patterns in the brain of zebrafish (Danio rerio), especially at adult stages, suggesting sub-functionalization of these two genes during evolution. Using double in situ hybridization staining in the developing brain we demonstrate that grm8a is expressed in a subset of gad1a-positive cells, pointing towards glutamatergic modulation of GABAergic signaling. Building on this result we generated loss-of-function models of both genes using CRISPR/Cas9. Both mutant lines are viable and display no obvious gross morphological phenotypes making them suitable for further analysis. Initial behavioral characterization revealed distinct phenotypes in larvae. Whereas grm8a mutant animals display reduced swimming velocity, grm8b mutant animals show increased thigmotaxis behavior, suggesting an anxiety-like phenotype. We anticipate that our two novel metabotropic glutamate receptor 8 zebrafish models may contribute to a deeper understanding of its function in normal development and its role in the pathomechanism of disorders of the central nervous system.
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Recent advances in the genetics of neurodevelopmental disorders (NDDs) have identified the transcription factor FOXP2 as one of numerous risk genes, e.g. in autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD). FOXP2 function is suggested to be involved in GABAergic signalling and numerous studies demonstrate that GABAergic function is altered in NDDs, thus disrupting the excitation/inhibition balance. Interestingly, GABAergic signalling components, including glutamate-decarboxylase 1 (Gad1) and GABA receptors, are putative transcriptional targets of FOXP2. However, the specific role of FOXP2 in the pathomechanism of NDDs remains elusive. Here we test the hypothesis that Foxp2 affects behavioural dimensions via GABAergic signalling using zebrafish as model organism. We demonstrate that foxp2 is expressed by a subset of GABAergic neurons located in brain regions involved in motor functions, including the subpallium, posterior tuberculum, thalamus and medulla oblongata. Using CRISPR/Cas9 gene-editing we generated a novel foxp2 zebrafish loss-of-function mutant that exhibits increased locomotor activity. Further, genetic and/or pharmacological disruption of Gad1 or GABA-A receptors causes increased locomotor activity, resembling the phenotype of foxp2 mutants. Application of muscimol, a GABA-A receptor agonist, rescues the hyperactive phenotype induced by the foxp2 loss-of-function. By reverse translation of the therapeutic effect on hyperactive behaviour exerted by methylphenidate, we note that application of methylphenidate evokes different responses in wildtype compared to foxp2 or gad1b loss-of-function animals. Together, our findings support the hypothesis that foxp2 regulates locomotor activity via GABAergic signalling. This provides one targetable mechanism, which may contribute to behavioural phenotypes commonly observed in NDDs.
Assuntos
Transtornos do Neurodesenvolvimento , Peixe-Zebra , Animais , Neurônios GABAérgicos , Locomoção , Ácido gama-AminobutíricoRESUMO
The glucose-fructose oxidoreductase domain containing gene family (GFOD) is small and contains only two members in human (GFOD1 and GFOD2). Information about its function is scarce. As the name implies the proteins contain an enzyme-defining domain, however, if this is functional and has enzymatic activity remains to be shown. A single nucleotide polymorphism situated in an intron of GFOD1 was found to be associated with inattentive symptomology in patients with attention-deficit/hyperactivity disorder. Further, in a large schizophrenia genome-wide association study the GFOD2 locus was found to be associated with the psychiatric condition. Until now, however, it is unclear what specific functions are associated with the two GFOD-family members, if they might be involved in neurodevelopment and how this may relate to the development of psychiatric disorders. In order to gain first insights into the hypothesis that GFOD-family members are involved in brain development and/or function we performed RNA in situ hybridization on zebrafish (Danio rerio) tissues at different developmental stages. We found that both family members are expressed in the central nervous system at embryonic, larvae and adult stages. We were able to define subtle differences of expression of the two gfod genes and we showed that a subset of GABAergic neurons express gfod1. Taken together, we conclude that both gfod gene family members are expressed in overlapping as well as in distinct regions in the zebrafish central nervous system. Our data suggest that gfod1 and gfod2 are relevant both for the developing and adult zebrafish brain. This study paves the way for further functional analyses of this yet unexplored gene family.
Assuntos
Encéfalo/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Encéfalo/embriologia , Embrião não Mamífero/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , Humanos , Hibridização In SituRESUMO
The transport of glucose across the cell plasma membrane is vital to most mammalian cells. The glucose transporter (GLUT; also called SLC2A) family of transmembrane solute carriers is responsible for this function in vivo. GLUT proteins encompass 14 different isoforms in humans with different cell type-specific expression patterns and activities. Central to glucose utilization and delivery in the brain is the neuronally expressed GLUT3. Recent research has shown an involvement of GLUT3 genetic variation or altered expression in several different brain disorders, including Huntington's and Alzheimer's diseases. Furthermore, GLUT3 was identified as a potential risk gene for multiple psychiatric disorders. To study the role of GLUT3 in brain function and disease a more detailed knowledge of its expression in model organisms is needed. Zebrafish (Danio rerio) has in recent years gained popularity as a model organism for brain research and is now well-established for modeling psychiatric disorders. Here, we have analyzed the sequence of GLUT3 orthologs and identified two paralogous genes in the zebrafish, slc2a3a and slc2a3b. Interestingly, the Glut3b protein sequence contains a unique stretch of amino acids, which may be important for functional regulation. The slc2a3a transcript is detectable in the central nervous system including distinct cellular populations in telencephalon, diencephalon, mesencephalon and rhombencephalon at embryonic and larval stages. Conversely, the slc2a3b transcript shows a rather diffuse expression pattern at different embryonic stages and brain regions. Expression of slc2a3a is maintained in the adult brain and is found in the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The slc2a3b transcripts are present in overlapping as well as distinct regions compared to slc2a3a. Double in situ hybridizations were used to demonstrate that slc2a3a is expressed by some GABAergic neurons at embryonic stages. This detailed description of zebrafish slc2a3a and slc2a3b expression at developmental and adult stages paves the way for further investigations of normal GLUT3 function and its role in brain disorders.
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OBJECTIVES: During the last two decades transcranial sonography (TCS) of the brain parenchyma evolved from a pure research tool to a clinical relevant neuroimaging method especially in Parkinson's disease and related movement disorders. The aim of this systematic review is to update and summarise the published TCS findings in psychiatric disorders and critically address the question whether TCS may be a valuable tool for the diagnosis or differential diagnosis of psychiatric disorders similarly to the field of movement disorders. METHODS: This paper provides detailed information about the perspectives and limitations of TCS, including guidelines for the scanning procedures, assessment of midbrain structures and discusses the potential causes of the ultrasound abnormalities in psychiatric disorders. RESULTS: Changes in the echogenicity of subcortical brain structures were detected in different disorders, such as obsessive-compulsive disorder, autism spectrum disorder, schizophrenia, panic disorder, attention-deficit/hyperactivity (ADHD), bipolar disorder and depressive disorder. Although the physical properties of brain tissue underlying the echogenic features in TCS are largely unknown, no alternative technique provides the same insight into the specific central nervous structural characteristics. CONCLUSIONS: Urgent research questions to further clarify the underlying pathophysiological and structural alterations are further outlined to bring this promising technique to the clinic.
Assuntos
Transtornos Mentais/diagnóstico , Neuroimagem/métodos , Ultrassonografia Doppler Transcraniana , Pesquisa Biomédica , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Diagnóstico Diferencial , HumanosRESUMO
Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Proteínas/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Encéfalo/metabolismo , Proteína C9orf72 , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Humanos , Camundongos , Proteínas dos Microfilamentos/metabolismoRESUMO
MATR3 is an RNA- and DNA-binding protein that interacts with TDP-43, a disease protein linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Using exome sequencing, we identified mutations in MATR3 in ALS kindreds. We also observed MATR3 pathology in ALS-affected spinal cords with and without MATR3 mutations. Our data provide more evidence supporting the role of aberrant RNA processing in motor neuron degeneration.
Assuntos
Esclerose Lateral Amiotrófica/genética , Saúde da Família , Predisposição Genética para Doença/genética , Mutação/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteínas de Ligação a RNA/genética , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/patologia , Biologia Computacional , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Exame Neurológico , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
BACKGROUND: We aimed to accurately estimate the frequency of a hexanucleotide repeat expansion in C9orf72 that has been associated with a large proportion of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). METHODS: We screened 4448 patients diagnosed with ALS (El Escorial criteria) and 1425 patients with FTD (Lund-Manchester criteria) from 17 regions worldwide for the GGGGCC hexanucleotide expansion using a repeat-primed PCR assay. We assessed familial disease status on the basis of self-reported family history of similar neurodegenerative diseases at the time of sample collection. We compared haplotype data for 262 patients carrying the expansion with the known Finnish founder risk haplotype across the chromosomal locus. We calculated age-related penetrance using the Kaplan-Meier method with data for 603 individuals with the expansion. FINDINGS: In patients with sporadic ALS, we identified the repeat expansion in 236 (7·0%) of 3377 white individuals from the USA, Europe, and Australia, two (4·1%) of 49 black individuals from the USA, and six (8·3%) of 72 Hispanic individuals from the USA. The mutation was present in 217 (39·3%) of 552 white individuals with familial ALS from Europe and the USA. 59 (6·0%) of 981 white Europeans with sporadic FTD had the mutation, as did 99 (24·8%) of 400 white Europeans with familial FTD. Data for other ethnic groups were sparse, but we identified one Asian patient with familial ALS (from 20 assessed) and two with familial FTD (from three assessed) who carried the mutation. The mutation was not carried by the three Native Americans or 360 patients from Asia or the Pacific Islands with sporadic ALS who were tested, or by 41 Asian patients with sporadic FTD. All patients with the repeat expansion had (partly or fully) the founder haplotype, suggesting a one-off expansion occurring about 1500 years ago. The pathogenic expansion was non-penetrant in individuals younger than 35 years, 50% penetrant by 58 years, and almost fully penetrant by 80 years. INTERPRETATION: A common Mendelian genetic lesion in C9orf72 is implicated in many cases of sporadic and familial ALS and FTD. Testing for this pathogenic expansion should be considered in the management and genetic counselling of patients with these fatal neurodegenerative diseases. FUNDING: Full funding sources listed at end of paper (see Acknowledgments).
Assuntos
Esclerose Lateral Amiotrófica/genética , Cromossomos Humanos Par 9/genética , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Estudos de Coortes , Estudos Transversais , Feminino , Loci Gênicos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , Adulto JovemRESUMO
Amyotrophic lateral sclerosis (ALS), the major form of motor neuron disease in the adult occurs as a sporadic disease in more than 95% of all cases. Analysis of familial forms is considered as a key to understand the pathophysiology of the disease. It is expected that mutations responsible for familial forms are also found in sporadic ALS. During the past years, several loci and genes have been identified in which disease associated mutations have been discovered. We report here on the screening of 596 sporadic ALS patients, 41 familial ALS cases and other motor neuron disease patients from Germany for mutations in the FUS/TLS gene. Sequencing of the last two exons in all patients revealed the C1561T transversion, which leads to the amino acid substitution at R521C, in one familial and one sporadic ALS patient. In addition three patients with a synonymous mutation at codon 522 were identified. None of these variants were present in the control population. Our results indicate that mutations in FUS/TLS are not a major cause of sporadic ALS in the German population.
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Esclerose Lateral Amiotrófica/genética , Mutação/genética , Proteína FUS de Ligação a RNA/genética , Idoso , Esclerose Lateral Amiotrófica/classificação , Arginina/genética , Cisteína/genética , Análise Mutacional de DNA/métodos , Éxons/genética , Feminino , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Cultured spinal motoneurons are a valuable tool for studying the basic mechanisms of axon and dendrite growth and also for analyses of pathomechanisms underlying diseases like amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). As motoneurons in the developing spinal cord of mice constitute only a minor population of neurons, these cells need to be enriched in order to study them in the absence of contaminating neuronal and non-neuronal cells. Here, we describe a protocol for the isolation and in vitro cultivation of embryonic primary motoneurons from individual mouse embryos. Tissue dissection, cell isolation and a p75(NTR)-antibody-based panning technique, which highly enriches motoneurons within <8 h are described. This protocol is aimed to provide an alternative to the established FACS-based protocols describing p75(NTR)-based enrichments of neurons. This protocol will help in facilitating the research on molecular mechanisms underlying motoneuron development, survival and disease mechanisms.
Assuntos
Embrião de Mamíferos/citologia , Neurônios Motores/citologia , Medula Espinal/citologia , Medula Espinal/embriologia , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Meios de Cultura , Região Lombossacral , CamundongosRESUMO
The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (Galpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha(13) both suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Galpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.