Assuntos
CADASIL/diagnóstico por imagem , CADASIL/epidemiologia , Vasculite Retiniana/diagnóstico por imagem , Vasculite Retiniana/epidemiologia , Adulto , CADASIL/genética , Feminino , Humanos , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/epidemiologia , Leucoencefalopatias/genética , Masculino , Pessoa de Meia-Idade , Vasculite Retiniana/genéticaRESUMO
Facioscapulohumeral muscular dystrophy cases with facial weakness before the age of 5 and signs of shoulder weakness by the age of 10 are defined as early onset. Contraction of the D4Z4 repeat on chromosome 4q35 is causally related to facioscapulohumeral muscular dystrophy type 1, and the residual size of the D4Z4 repeat shows a roughly inverse correlation with the severity of the disease. Contraction of the D4Z4 repeat on chromosome 4q35 is believed to induce a local change in chromatin structure and consequent transcriptional deregulation of 4qter genes. We present early-onset cases in the Polish population that amounted to 21% of our total population with facioscapulohumeral muscular dystrophy. More than 27% of them presented with severe phenotypes (wheelchair dependency). The residual D4Z4 repeat sizes ranged from 1 to 4 units. In addition, even within early-onset facioscapulohumeral muscular dystrophy type 1 phenotypes, some cases had uncommon features (head drop, early disabling contractures, progressive ptosis, and respiratory insufficiency and cardiomyopathy).
Assuntos
Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/fisiopatologia , Idade de Início , Criança , Pré-Escolar , Cromossomos Humanos Par 4 , Análise Mutacional de DNA , Eletroforese em Gel de Campo Pulsado , Feminino , Seguimentos , Quadril/patologia , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Músculos/patologia , Distrofia Muscular Facioescapuloumeral/diagnóstico , Distrofia Muscular Facioescapuloumeral/patologia , Fenótipo , Polônia , Sequências Repetitivas de Ácido Nucleico , Índice de Gravidade de Doença , Cadeiras de RodasRESUMO
BACKGROUND: Various CACNA1A missense mutations cause familial hemiplegic migraine type 1 (FHM1), a rare monogenic subtype of migraine with aura. FHM1 mutation R192Q is associated with pure hemiplegic migraine, whereas the S218L mutation causes hemiplegic migraine, cerebellar ataxia, seizures, and mild head trauma-induced brain edema. Transgenic knock-in (KI) migraine mouse models were generated that carried either the FHM1 R192Q or the S218L mutation and were shown to exhibit increased CaV2.1 channel activity. Here we investigated their cerebellar and caudal cortical transcriptome. METHODS: Caudal cortical and cerebellar RNA expression profiles from mutant and wild-type mice were studied using microarrays. Respective brain regions were selected based on their relevance to migraine aura and ataxia. Relevant expression changes were further investigated at RNA and protein level by quantitative polymerase chain reaction (qPCR) and/or immunohistochemistry, respectively. RESULTS: Expression differences in the cerebellum were most pronounced in S218L mice. Particularly, tyrosine hydroxylase, a marker of delayed cerebellar maturation, appeared strongly upregulated in S218L cerebella. In contrast, only minimal expression differences were observed in the caudal cortex of either mutant mice strain. CONCLUSION: Despite pronounced consequences of migraine gene mutations at the neurobiological level, changes in cortical RNA expression in FHM1 migraine mice compared to wild-type are modest. In contrast, pronounced RNA expression changes are seen in the cerebellum of S218L mice and may explain their cerebellar ataxia phenotype.
Assuntos
Encéfalo/fisiopatologia , Canais de Cálcio Tipo N/genética , Ataxia Cerebelar/genética , Cerebelo/fisiopatologia , Córtex Cerebral/fisiopatologia , Transtornos de Enxaqueca/genética , Proteínas do Tecido Nervoso/genética , RNA/metabolismo , Animais , Ataxia Cerebelar/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transtornos de Enxaqueca/metabolismo , Mutação , RNA/genética , Distribuição Tecidual , Transcriptoma/genéticaRESUMO
Migraine is the most common brain disorder, affecting approximately 14% of the adult population, but its molecular mechanisms are poorly understood. We report the results of a meta-analysis across 29 genome-wide association studies, including a total of 23,285 individuals with migraine (cases) and 95,425 population-matched controls. We identified 12 loci associated with migraine susceptibility (P<5×10(-8)). Five loci are new: near AJAP1 at 1p36, near TSPAN2 at 1p13, within FHL5 at 6q16, within C7orf10 at 7p14 and near MMP16 at 8q21. Three of these loci were identified in disease subgroup analyses. Brain tissue expression quantitative trait locus analysis suggests potential functional candidate genes at four loci: APOA1BP, TBC1D7, FUT9, STAT6 and ATP5B.
Assuntos
Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Transtornos de Enxaqueca/genética , Cerebelo/metabolismo , Biologia Computacional , Lobo Frontal/metabolismo , Humanos , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Dysferlin is mutated in a group of muscular dystrophies commonly referred to as dysferlinopathies. It is highly expressed in skeletal muscle, where it is important for sarcolemmal maintenance. Recent studies show that dysferlin is also expressed in monocytes. Moreover, muscle of dysferlinopathy patients is characterized by massive immune cell infiltrates, and dysferlin-negative monocytes were shown to be more aggressive and phagocytose more particles. This suggests that dysferlin deregulation in monocytes might contribute to disease progression, but the molecular mechanism is unclear. Here we show that dysferlin expression is increased with differentiation in human monocytes and the THP1 monocyte cell model. Freshly isolated monocytes of dysferlinopathy patients show deregulated expression of fibronectin and fibronectin-binding integrins, which is recapitulated by transient knockdown of dysferlin in THP1 cells. Dysferlin forms a protein complex with these integrins at the cell membrane, and its depletion impairs cell adhesion. Moreover, patient macrophages show altered adhesion and motility. These findings suggest that dysferlin is involved in regulating cellular interactions and provide new insight into dysferlin function in inflammatory cells.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Monócitos/citologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Adesão Celular , Diferenciação Celular , Disferlina , Humanos , Sistema Imunitário , Inflamação , Integrina beta3/metabolismo , Macrófagos , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismoRESUMO
Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy caused by decreased epigenetic repression of the D4Z4 macrosatellite repeats and ectopic expression of DUX4, a retrogene encoding a germline transcription factor encoded in each repeat. Unaffected individuals generally have more than 10 repeats arrayed in the subtelomeric region of chromosome 4, whereas the most common form of FSHD (FSHD1) is caused by a contraction of the array to fewer than 10 repeats, associated with decreased epigenetic repression and variegated expression of DUX4 in skeletal muscle. We have generated transgenic mice carrying D4Z4 arrays from an FSHD1 allele and from a control allele. These mice recapitulate important epigenetic and DUX4 expression attributes seen in patients and controls, respectively, including high DUX4 expression levels in the germline, (incomplete) epigenetic repression in somatic tissue, and FSHD-specific variegated DUX4 expression in sporadic muscle nuclei associated with D4Z4 chromatin relaxation. In addition we show that DUX4 is able to activate similar functional gene groups in mouse muscle cells as it does in human muscle cells. These transgenic mice therefore represent a valuable animal model for FSHD and will be a useful resource to study the molecular mechanisms underlying FSHD and to test new therapeutic intervention strategies.
Assuntos
Epigênese Genética/genética , Proteínas de Homeodomínio , Distrofia Muscular Facioescapuloumeral/genética , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Células Cultivadas , Cromatina/genética , Metilação de DNA/genética , Modelos Animais de Doenças , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/metabolismoRESUMO
AIM: Migraine, in particular with aura, has been associated with an increased risk for ischemic stroke and coronary heart disease. The underlying mechanism is unknown. In a cross-sectional case control study we investigated whether an enhanced risk of atherosclerosis in migraineurs explains this increased cardiovascular risk. METHODS: Subjects were participants from the population-based Erasmus Rucphen Family study. Atherosclerosis was assessed in 360 migraineurs (209 without aura and 151 with aura) and 617 subjects without migraine or severe headache. Atherosclerosis was quantified by intima media thickness, pulse wave velocity and ankle-brachial index. RESULTS: Migraineurs, especially with aura, were found more likely to smoke, have diabetes or a modestly decreased HDL-cholesterol. No differences were found for the atherosclerosis parameters. CONCLUSION: In this large population-based study, migraineurs have no increased risk of atherosclerosis. Therefore, enhanced atherosclerosis is an unlikely explanation for the increased cardiovascular risk seen in migraineurs.
Assuntos
Aterosclerose/complicações , Aterosclerose/epidemiologia , Transtornos de Enxaqueca/complicações , Índice Tornozelo-Braço , Espessura Intima-Media Carotídea , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Análise de Onda de PulsoRESUMO
Facioscapulohumeral dystrophy (FSHD) is characterized by chromatin relaxation of the D4Z4 macrosatellite array on chromosome 4 and expression of the D4Z4-encoded DUX4 gene in skeletal muscle. The more common form, autosomal dominant FSHD1, is caused by contraction of the D4Z4 array, whereas the genetic determinants and inheritance of D4Z4 array contraction-independent FSHD2 are unclear. Here, we show that mutations in SMCHD1 (encoding structural maintenance of chromosomes flexible hinge domain containing 1) on chromosome 18 reduce SMCHD1 protein levels and segregate with genome-wide D4Z4 CpG hypomethylation in human kindreds. FSHD2 occurs in individuals who inherited both the SMCHD1 mutation and a normal-sized D4Z4 array on a chromosome 4 haplotype permissive for DUX4 expression. Reducing SMCHD1 levels in skeletal muscle results in D4Z4 contraction-independent DUX4 expression. Our study identifies SMCHD1 as an epigenetic modifier of the D4Z4 metastable epiallele and as a causal genetic determinant of FSHD2 and possibly other human diseases subject to epigenetic regulation.
Assuntos
Proteínas Cromossômicas não Histona/genética , Hereditariedade/genética , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Mutação , Adulto , Idoso , Cromossomos Humanos Par 18/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Epigênese Genética , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Migraine without aura is the most common form of migraine, characterized by recurrent disabling headache and associated autonomic symptoms. To identify common genetic variants associated with this migraine type, we analyzed genome-wide association data of 2,326 clinic-based German and Dutch individuals with migraine without aura and 4,580 population-matched controls. We selected SNPs from 12 loci with 2 or more SNPs associated with P values of <1 × 10(-5) for replication testing in 2,508 individuals with migraine without aura and 2,652 controls. SNPs at two of these loci showed convincing replication: at 1q22 (in MEF2D; replication P = 4.9 × 10(-4); combined P = 7.06 × 10(-11)) and at 3p24 (near TGFBR2; replication P = 1.0 × 10(-4); combined P = 1.17 × 10(-9)). In addition, SNPs at the PHACTR1 and ASTN2 loci showed suggestive evidence of replication (P = 0.01; combined P = 3.20 × 10(-8) and P = 0.02; combined P = 3.86 × 10(-8), respectively). We also replicated associations at two previously reported migraine loci in or near TRPM8 and LRP1. This study identifies the first susceptibility loci for migraine without aura, thereby expanding our knowledge of this debilitating neurological disorder.
Assuntos
Enxaqueca sem Aura/genética , Adulto , Estudos de Casos e Controles , Feminino , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Domínio MADS/genética , Fatores de Transcrição MEF2 , Masculino , Proteínas dos Microfilamentos/genética , Fatores de Regulação Miogênica/genética , Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Canais de Cátion TRPM/genéticaRESUMO
The Cacna1a gene encodes the α(1A) subunit of voltage-gated Ca(V)2.1 Ca(2+) channels that are involved in neurotransmission at central synapses. Ca(V)2.1-α(1)-knockout (α1KO) mice, which lack Ca(V)2.1 channels in all neurons, have a very severe phenotype of cerebellar ataxia and dystonia, and usually die around postnatal day 20. This early lethality, combined with the wide expression of Ca(V)2.1 channels throughout the cerebellar cortex and nuclei, prohibited determination of the contribution of particular cerebellar cell types to the development of the severe neurobiological phenotype in Cacna1a mutant mice. Here, we crossed conditional Cacna1a mice with transgenic mice expressing Cre recombinase, driven by the Purkinje cell-specific Pcp2 promoter, to specifically ablate the Ca(V)2.1-α(1A) subunit and thereby Ca(V)2.1 channels in Purkinje cells. Purkinje cell Ca(V)2.1-α(1A)-knockout (PCα1KO) mice aged without difficulties, rescuing the lethal phenotype seen in α1KO mice. PCα1KO mice exhibited cerebellar ataxia starting around P12, much earlier than the first signs of progressive Purkinje cell loss, which appears in these mice between P30 and P45. Secondary cell loss was observed in the granular and molecular layers of the cerebellum and the volume of all individual cerebellar nuclei was reduced. In this mouse model with a cell type-specific ablation of Ca(V)2.1 channels, we show that ablation of Ca(V)2.1 channels restricted to Purkinje cells is sufficient to cause cerebellar ataxia. We demonstrate that spatial ablation of Ca(V)2.1 channels may help in unraveling mechanisms of human disease.
Assuntos
Canais de Cálcio Tipo N/deficiência , Ataxia Cerebelar/genética , Ataxia Cerebelar/metabolismo , Córtex Cerebelar/patologia , Predisposição Genética para Doença/genética , Células de Purkinje/patologia , Animais , Canais de Cálcio Tipo N/genética , Ataxia Cerebelar/patologia , Córtex Cerebelar/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células de Purkinje/metabolismoRESUMO
AHNAK is a 700-kDa protein involved in cytoarchitecture and calcium signaling. It is secondarily reduced in muscle of dysferlinopathy patients and accumulates in muscle of calpainopathy patients, both affected by a muscular dystrophy. AHNAK directly interacts with dysferlin. This interaction is lost on cleavage of AHNAK by the protease calpain 3, explaining the molecular observations in patients. Currently, little is known of AHNAK regulation. We describe the self-regulation of multiple mRNA transcripts emanating from the AHNAK locus in muscle cells. We show that the AHNAK gene consists of a 17-kb exon flanked by multiple small exons. This genetic structure is shared by AHNAK2 and Periaxin, which share a common ancestor. Two major AHNAK transcripts are differentially expressed during muscle differentiation that encode for a small (17-kDa) and a large (700-kDa) protein isoform. These proteins interact in the cytoplasm, but the small AHNAK is also present in the nucleus. During muscle differentiation the small AHNAK is strongly increased, thereby establishing a positive feedback loop to regulate mRNA splicing of its own locus. A small 17-kDa isoform of Periaxin similarly traffics between the cytoplasm and the nucleus to regulate mRNA splicing. Thus, AHNAK constitutes a novel mechanism in post-transcriptional control of gene expression.
Assuntos
Processamento Alternativo/fisiologia , Sinalização do Cálcio/fisiologia , Proteínas de Membrana/genética , Mioblastos Esqueléticos/fisiologia , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Evolução Molecular , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica/genética , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Mioblastos Esqueléticos/citologia , Proteínas de Neoplasias/metabolismo , Filogenia , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/genéticaRESUMO
Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining patients carry unknown genetic defects (ICF2) but share with ICF1 patients the same immunological and epigenetic features, including hypomethylation of juxtacentromeric repeat sequences. We performed homozygosity mapping in five unrelated ICF2 patients with consanguineous parents and then performed whole-exome sequencing in one of these patients and Sanger sequencing in all to identify mutations in the zinc-finger- and BTB (bric-a-bric, tramtrack, broad complex)-domain-containing 24 (ZBTB24) gene in four consanguineously descended ICF2 patients. Additionally, we found ZBTB24 mutations in an affected sibling pair and in one patient for whom it was not known whether his parents were consanguineous. ZBTB24 belongs to a large family of transcriptional repressors that include members, such as BCL6 and PATZ1, with prominent regulatory roles in hematopoietic development and malignancy. These data thus indicate that ZBTB24 is involved in DNA methylation of juxtacentromeric DNA and in B cell development and/or B and T cell interactions. Because ZBTB24 is a putative DNA-binding protein highly expressed in the lymphoid lineage, we predict that by studying the molecular function of ZBTB24, we will improve our understanding of the molecular pathophysiology of ICF syndrome and of lymphocyte biology in general.
Assuntos
Centrômero/genética , Metilação de DNA/genética , Proteínas Repressoras/genética , Dedos de Zinco , Adolescente , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA , Epigenômica , Face/anormalidades , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Masculino , Mutação , Linhagem , Doenças da Imunodeficiência PrimáriaRESUMO
OBJECTIVE: Our objective was to study the long-term prognosis of sporadic hemiplegic migraine (SHM). METHODS: We performed a longitudinal follow-up study in 18 patients who were diagnosed with SHM between 1993 and 1996. Follow-up time between the first and second survey ranged from nine to 14 years. These patients were included as part of a genetic study in which we systematically analysed the role of the three known familial hemiplegic migraine (FHM) genes. RESULTS: In 12 out of 18 patients the clinical diagnosis was unchanged. In two of the six remaining patients the attacks were no longer associated with hemiplegia; one of them had an ATP1A2 gene mutation (E120A). In the four other patients, the diagnosis changed into FHM, because a family member had developed hemiplegic migraine since the initial diagnosis was made. In two of the four patients a mutation was demonstrated (CACNA1A [R583Q] and ATP1A2 [R834X]). CONCLUSION: This study shows that the diagnosis of SHM changes into FHM in a considerable percentage of patients (22% [4 of 18]), almost a decade after the initial diagnosis. This indicates that a careful follow-up of SHM patients and their families is advisable for optimal care and counseling. Diagnostic screening of FHM genes in SHM patients can be of value. Our genetic and clinical follow-up studies reinforce the evidence that FHM and SHM are part of the same spectrum of migraine.
Assuntos
Testes Genéticos , Hemiplegia , Transtornos de Enxaqueca , Adolescente , Adulto , Canais de Cálcio/genética , Criança , Progressão da Doença , Saúde da Família , Feminino , Seguimentos , Hemiplegia/diagnóstico , Hemiplegia/etiologia , Hemiplegia/genética , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Transtornos de Enxaqueca/complicações , Transtornos de Enxaqueca/diagnóstico , Transtornos de Enxaqueca/genética , Linhagem , Prognóstico , ATPase Trocadora de Sódio-Potássio/genética , Adulto JovemRESUMO
Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity.
Assuntos
Proteínas de Membrana/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Proteômica/métodos , Sarcolema/metabolismo , Animais , Western Blotting , Células Cultivadas , Análise por Conglomerados , Disferlina , Perfilação da Expressão Gênica , Imunoprecipitação , Proteínas de Membrana/genética , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Ligação Proteica , Proteoma/genética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Insight into the molecular mechanisms involved in primary headaches is important to identify drug targets for improving treatment of patients, but essentially lacking. Genetic research is increasingly successful in pinpointing these mechanisms. Most progress has been made for Familial Hemiplegic Migraine, a rare subtype of migraine with aura. Three genes (CACNA1A, ATP1A2 and SCN1A) have been identified that all encode ion transporters. Cellular and transgenic mouse studies suggest that neuronal hyperexcitability and increased susceptibility to cortical spreading depression, the correlate of migraine aura, are important molecular mechanisms in migraine. Investigating monogenic diseases in which migraine is a prominent feature such as CADASIL, which is caused by mutations in the NOTCH3 gene, can help understanding the pathology of migraine. Candidate gene association studies and linkage studies in the common forms of migraine were less successful. Except for the MTHFR gene no gene variant has been identified yet. Convincingly demonstrated genetic findings in other primary headaches such as cluster headache and tension-type headache are even rarer. However, with current technical possibilities of massive genotyping and international efforts to collect large well-phenotyped patient cohorts, the first gene variants for various primary headache types are likely to be discovered in the coming decade.
Assuntos
Genótipo , Cefaleia , Animais , Depressão Alastrante da Atividade Elétrica Cortical , Humanos , Transtornos de Enxaqueca , Enxaqueca com Aura , MutaçãoRESUMO
Calpain 3 (CAPN3) is a cysteine protease that when mutated causes Limb Girdle Muscular Dystrophy 2A. It is thereby the only described Calpain family member that genetically causes a disease. Due to its inherent instability little is known of its substrates or its mechanism of activity and pathogenicity. In this investigation we define a primary sequence motif underlying CAPN3 substrate cleavage. This motif can transform non-related proteins into substrates, and identifies >300 new putative CAPN3 targets. Bioinformatic analyses of these targets demonstrate a critical role in muscle cytoskeletal remodeling and identify novel CAPN3 functions. Among the new CAPN3 substrates are three E3 SUMO ligases of the Protein Inhibitor of Activated Stats (PIAS) family. CAPN3 can cleave PIAS proteins and negatively regulates PIAS3 sumoylase activity. Consequently, SUMO2 is deregulated in patient muscle tissue. Our study thus uncovers unexpected crosstalk between CAPN3 proteolysis and protein sumoylation, with strong implications for muscle remodeling.
Assuntos
Calpaína/metabolismo , Biologia Computacional , Músculos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Calpaína/química , Sequência Consenso , Citoesqueleto/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Músculos/citologiaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy in adults that is foremost characterized by progressive wasting of muscles in the upper body. FSHD is associated with contraction of D4Z4 macrosatellite repeats on chromosome 4q35, but this contraction is pathogenic only in certain "permissive" chromosomal backgrounds. Here, we show that FSHD patients carry specific single-nucleotide polymorphisms in the chromosomal region distal to the last D4Z4 repeat. This FSHD-predisposing configuration creates a canonical polyadenylation signal for transcripts derived from DUX4, a double homeobox gene of unknown function that straddles the last repeat unit and the adjacent sequence. Transfection studies revealed that DUX4 transcripts are efficiently polyadenylated and are more stable when expressed from permissive chromosomes. These findings suggest that FSHD arises through a toxic gain of function attributable to the stabilized distal DUX4 transcript.
Assuntos
Cromossomos Humanos Par 4/genética , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Sequências Repetitivas de Ácido Nucleico , Adolescente , Adulto , Idoso , Sequência de Bases , Pré-Escolar , Cromossomos Humanos Par 10/genética , Feminino , Predisposição Genética para Doença , Haplótipos , Proteínas de Homeodomínio/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Dados de Sequência Molecular , Poliadenilação , Polimorfismo de Nucleotídeo Único , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Transfecção , Adulto JovemRESUMO
Familial hemiplegic migraine type 1 (FHM1) is caused by mutations in the CACNA1A gene, encoding neuronal presynaptic Ca(V)2.1 (P/Q-type) Ca(2+) channels. These channels mediate neurotransmitter release at many central synapses and at the neuromuscular junction (NMJ). Mutation S218L causes a severe neurological phenotype of FHM and, additionally, ataxia and susceptibility to seizures, delayed brain edema, and fatal coma after minor head trauma. Recently, we generated a Cacna1a S218L knock-in mutant mouse, displaying these features and reduced survival. A first electrophysiological study showed high susceptibility for cortical spreading depression, enhanced neuronal soma Ca(2+) influx, and at diaphragm NMJs, a considerable increase of neurotransmitter release. We here assessed the function of S218L knock-in NMJs at several muscle types in great detail. Pharmacological analyses using specific Ca(V) subtype-blocking toxins excluded compensatory contribution of non-Ca(V)2.1 channels. Endplate potentials were considerably broadened at many NMJs. High rate (40 Hz)-evoked acetylcholine release was slightly reduced; however, it was not associated with block of neurotransmission causing weakness, as assessed with grip strength measurements and in vitro muscle contraction experiments. The synaptopathy clearly progressed with age, including development of an increased acetylcholine release at low-rate nerve stimulation at physiological extracellular Ca(2+) concentration and further endplate potential broadening. Our results suggest enhanced Ca(2+) influx into motor nerve terminals through S218L-mutated presynaptic Ca(V)2.1 channels, likely because of the earlier reported negative shift of activation potential and reduced inactivation. Similar severe aberrations at central synapses of S218L mutant mice and humans may underlie or contribute to the drastic neurological phenotype.
Assuntos
Acetilcolina/metabolismo , Canais de Cálcio Tipo P/genética , Canais de Cálcio Tipo Q/genética , Progressão da Doença , Técnicas de Introdução de Genes , Enxaqueca com Aura/genética , Índice de Gravidade de Doença , Animais , Canais de Cálcio Tipo N , Canais de Cálcio Tipo P/biossíntese , Canais de Cálcio Tipo Q/biossíntese , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Enxaqueca com Aura/metabolismo , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Transmissão Sináptica/genéticaRESUMO
Familial hemiplegic migraine type 1 (FHM1) is caused by missense mutations in the CACNA1A gene that encodes the alpha1A pore-forming subunit of Ca(V)2.1 Ca(2+) channels. Knock-in (KI) transgenic mice expressing Ca(V)2.1 Ca(2+) channels with a human pathogenic FHM1 mutation reveal enhanced glutamatergic neurotransmission in the cortex. In this study, we employed an iTRAQ-based LC-LC MS/MS approach to identify differentially expressed proteins in cortical synapse proteomes of Cacna1a R192Q KI and wild-type mice. All expression differences determined were subtle and in the range of 10-30%. Observed upregulated proteins in the mutant mice are involved in processes, such as neurite outgrowth and actin dynamics, vesicle turnover, and glutamate transporters. Our data support the view that in Cacna1a R192Q KI mice, several compensatory mechanisms counterbalancing a dysregulated glutamatergic signaling have come into effect. We propose that such adaptation mechanisms at the synapse level may play a role in the pathophysiology of FHM and possibly in the common forms of migraine.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Transtornos de Enxaqueca/metabolismo , Sinapses/química , Animais , Canais de Cálcio Tipo N/genética , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Mutação , Proteômica , Transdução de Sinais , Sinapses/metabolismoRESUMO
BACKGROUND: Relatively little is known about the role of specific saturated fatty acids in the development of high fat diet induced obesity and insulin resistance. Here, we have studied the effect of stearate in high fat diets (45% energy as fat) on whole body energy metabolism and tissue specific insulin sensitivity. METHODS: C57Bl/6 mice were fed a low stearate diet based on palm oil or one of two stearate rich diets, one diet based on lard and one diet based on palm oil supplemented with tristearin (to the stearate level of the lard based diet), for a period of 5 weeks. Ad libitum fed Oxidative metabolism was assessed by indirect calorimetry at week 5. Changes in body mass and composition was assessed by DEXA scan analysis. Tissue specific insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp analysis and Western blot at the end of week 5. RESULTS: Indirect calorimetry analysis revealed that high levels of dietary stearate resulted in lower caloric energy expenditure characterized by lower oxidation of fatty acids. In agreement with this metabolic phenotype, mice on the stearate rich diets gained more adipose tissue mass. Whole body and tissue specific insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp and analysis of insulin induced PKBser473 phosphorylation. Whole body insulin sensitivity was decreased by all high fat diets. However, while insulin-stimulated glucose uptake by peripheral tissues was impaired by all high fat diets, hepatic insulin sensitivity was affected only by the stearate rich diets. This tissue-specific pattern of reduced insulin sensitivity was confirmed by similar impairment in insulin-induced phosphorylation of PKBser473 in both liver and skeletal muscle. CONCLUSION: In C57Bl/6 mice, 5 weeks of a high fat diet rich in stearate induces a metabolic state favoring low oxidative metabolism, increased adiposity and whole body insulin resistance characterized by severe hepatic insulin resistance. These results indicate that dietary fatty acid composition per sé rather than dietary fat content determines insulin sensitivity in liver of high fat fed C57Bl/6 mice.