RESUMO
Fragile X-associated tremor and ataxia syndrome (FXTAS) is a late-onset, progressive neurodegenerative disorder characterized by tremors, ataxia and neuropsychological problems. This disease is quite common in the general population with approximately 20 million carriers worldwide. The risk of developing FXTAS increases dramatically with age, with about 45% of male carriers over the age of 50 being affected. FXTAS is caused by a CGG-repeat expansion (CGGexp) in the fragile X mental retardation 1 (FMR1) gene. CGGexp RNA is translated into the FMRpolyG protein by a mechanism called RAN translation. Although both gene and pathogenic trigger are known, no therapeutic interventions are available at this moment. Here, we present, for the first time, primary hippocampal neurons derived from the ubiquitous inducible mouse model which is used as a screening tool for targeted interventions. A promising candidate is the repeat binding, RAN translation blocking, small molecule 1a. Small molecule 1a shields the disease-causing CGGexp from being translated into the toxic FMRpolyG protein. Primary hippocampal neurons formed FMRpolyG-positive inclusions, and upon treatment with 1a, the numbers of FMRpolyG-positive inclusions are reduced. We also describe for the first time the formation of FMRpolyG-positive inclusions in the liver of this mouse model. Treatment with 1a reduced the insoluble FMRpolyG protein fraction in the liver but not the number of inclusions. Moreover, 1a treatment had a reducing effect on the number of Rad23b-positive inclusions and insoluble Rad23b protein levels. These data suggest that targeted small molecule therapy is effective in an FXTAS mouse model and has the potential to treat CGGexp-mediated diseases, including FXTAS.
Assuntos
Ataxia/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Tremor/genética , Animais , Ataxia/fisiopatologia , Comunicação Celular , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Tremor/fisiopatologia , Expansão das Repetições de TrinucleotídeosRESUMO
Fragile X syndrome (FXS) is the most common monogenetic cause of intellectual disability and autism spectrum disorders. Mostly, FXS is caused by transcriptional silencing of the FMR1 gene due to a repeat expansion in the 5' UTR, and consequently lack of the protein product FMRP. However, in rare cases FXS is caused by other types of variants in the FMR1 gene. We describe a missense variant in the FMR1 gene, identified through whole-exome sequencing, in a boy with intellectual disability and behavioral problems. The variant is located in the FMRP's nuclear export signal (NES). We performed expression and localization studies of the variant in hair roots and HEK293 cells. Our results show normal expression but significant retention of the FMRP in the cells' nucleus. This finding suggests a possible FMRP reduction at its essential functional sites in the dendrites and the synaptic compartments and possible interference of other cellular processes in the nucleus. Together, this might lead to a FXS phenotype in the boy.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Sinais de Exportação Nuclear/genética , Regiões 5' não Traduzidas/genética , Linhagem Celular , Feminino , Síndrome do Cromossomo X Frágil/genética , Células HEK293 , Humanos , Masculino , Fenótipo , Sequenciamento do Exoma/métodosRESUMO
Neuronal intranuclear inclusion disease (NIID) is a rare heterogeneous progressive neurodegenerative disease characterized by the presence of eosinophilic hyaline intranuclear inclusions in neuronal and glial cells of the CNS, peripheral cells of the autonomic nervous system, visceral organs and skin. The clinical presentation is broadly heterogeneous and includes limb weakness, dementia, seizures, ataxia, and parkinsonism. High-intensity signal in the corticomedullary junction on brain MRI is a characteristic finding in NIID. We describe a 65-year-old patient presenting with mild cognitive impairment, evolving in dementia with behavioral disturbances and parkinsonism. Brain MRI showed mild global cortical atrophy, more pronounced in the cingulate and temporal cortex and mild leukoaraiosis, but no high-intensity signal in corticomedullary junction on diffusion weighted imaging. Neuropathological examination showed p62- and optineurin-positive neuronal intranuclear inclusions in the hippocampus and in some subcortical structures. Glial cells did not present any intranuclear inclusions, and no spongiotic changes proximal to the U-fibers or diffuse myelin pallor were disclosed in the white matter. We report on a case with pathological features of NIID showing different neuroimaging and pathological findings. We noted an absence of typical MRI abnormalities, lack of intranuclear inclusions in glial cells, and prominent involvement of hippocampal neurons, refining the clinico-pathological spectrum of the disease.
Assuntos
Doenças Neurodegenerativas/patologia , Idoso , Atrofia , Autopsia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Córtex Cerebral/patologia , Disfunção Cognitiva/psicologia , Imagem de Difusão por Ressonância Magnética , Feminino , Humanos , Corpos de Inclusão Intranuclear/patologia , Leucoaraiose/patologia , Imageamento por Ressonância Magnética , Debilidade Muscular/patologia , Bainha de Mielina/patologia , Fibras Nervosas/patologia , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/psicologia , Neuroglia/patologiaRESUMO
Human homologue of yeast UV excision repair protein Rad23b (HR23B) inclusions are found in a number of neurodegenerative diseases, including frontotemporal dementia (FTD), Huntington's disease (HD), spinocerebellar ataxia type 3 and 7 (SCA3/7), fragile X associated tremor/ataxia syndrome (FXTAS) and Parkinson's disease (PD). Here, we describe HR23B pathology in C9ORF72 linked FTD and amyotrophic lateral sclerosis (ALS) cases. HR23B presented in neuropils, intranuclear inclusions and cytoplasmic and perinuclear inclusions and was predominantly found in cortices (frontal, temporal and motor), spinal cord and hippocampal dentate gyrus. HR23B co-localized with poly-GA-, pTDP-43- and p62-positive inclusions in frontal cortex and in hippocampal dentate gyrus, the latter showing higher co-localization percentages. HR23B binding partners XPC, 20S and ataxin-3, which are involved in nucleotide excision repair (NER) and the ubiquitin-proteasome system (UPS), did not show an aberrant distribution. However, C9ORF72 fibroblasts were more sensitive for UV-C damage than healthy control fibroblasts, even though all factors involved in NER localized normally to DNA damage and the efficiency of DNA repair was not reduced. HR23Bs other binding partner NGly1/PNGase, involved in ER-associated degradation (ERAD) of misfolded proteins, was not expressed in the majority of neurons in C9FTD/ALS brain sections compared to non-demented controls. Our results suggest a difference in HR23B aggregation and co-localization pattern with DPRs, pTDP-43 and p62 between different brain areas from C9FTD/ALS cases. We hypothesize that HR23B may play a role in C9ORF72 pathogenesis, possibly by aberrant ERAD functioning.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Idoso , Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/análise , Enzimas Reparadoras do DNA/análise , Proteínas de Ligação a DNA/análise , Feminino , Demência Frontotemporal/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Agregados Proteicos/fisiologia , Proteínas/análise , Proteínas de Ligação a RNA/análiseRESUMO
The fragile X premutation is a CGG trinucleotide repeat expansion between 55 and 200 repeats in the 5'-untranslated region of the fragile X mental retardation 1 (FMR1) gene. Human carriers of the premutation allele are at risk of developing the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Characteristic neuropathology associated with FXTAS includes intranuclear inclusions in neurons and astroglia. Previous studies recapitulated these histopathological features in neurons in a knock-in mouse model, but without significant astroglial pathology. To determine the role of astroglia in FXTAS, we generated a transgenic mouse line (Gfa2-CGG99-eGFP) that selectively expresses a 99-CGG repeat expansion linked to an enhanced green fluorescent protein (eGFP) reporter in astroglia throughout the brain, including cerebellar Bergmann glia. Behaviorally these mice displayed impaired motor performance on the ladder-rung test, but paradoxically better performance on the rotarod. Immunocytochemical analysis revealed that CGG99-eGFP co-localized with GFAP and S-100ß, but not with NeuN, Iba1, or MBP, indicating that CGG99-eGFP expression is specific to astroglia. Ubiquitin-positive intranuclear inclusions were found in eGFP-expressing glia throughout the brain. In addition, intracytoplasmic ubiquitin-positive inclusions were found outside the nucleus in distal astrocyte processes. Intriguingly, intranuclear inclusions, in the absence of eGFP mRNA and eGFP fluorescence, were present in neurons of the hypothalamus and neocortex. Furthermore, intranuclear inclusions in both neurons and astrocytes displayed immunofluorescent labeling for the polyglycine peptide FMRpolyG, implicating FMRpolyG in the pathology found in Gfa2-CGG99 mice. Considered together, these results show that Gfa2-CGG99 expression in mice is sufficient to induce key features of FXTAS pathology, including formation of intranuclear inclusions, translation of FMRpolyG, and deficits in motor function.
Assuntos
Astrócitos/fisiologia , Ataxia/genética , Comunicação Celular/fisiologia , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Transtornos das Habilidades Motoras/genética , Tremor/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Ataxia/metabolismo , Ataxia/patologia , Sequência de Bases , Proteína do X Frágil da Deficiência Intelectual/biossíntese , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos das Habilidades Motoras/metabolismo , Transtornos das Habilidades Motoras/patologia , Tremor/metabolismo , Tremor/patologiaRESUMO
Recent evidence indicates that specific RNAs promote the formation of ribonucleoprotein condensates by acting as scaffolds for RNA-binding proteins (RBPs). We systematically investigated RNA-RBP interaction networks to understand ribonucleoprotein assembly. We found that highly contacted RNAs are structured, have long UTRs, and contain nucleotide repeat expansions. Among the RNAs with such properties, we identified the FMR1 3' UTR that harbors CGG expansions implicated in fragile X-associated tremor/ataxia syndrome (FXTAS). We studied FMR1 binding partners in silico and in vitro and prioritized the splicing regulator TRA2A for further characterization. In a FXTAS cellular model, we validated the TRA2A-FMR1 interaction and investigated implications of its sequestration at both transcriptomic and post-transcriptomic levels. We found that TRA2A co-aggregates with FMR1 in a FXTAS mouse model and in post-mortem human samples. Our integrative study identifies key components of ribonucleoprotein aggregates, providing links to neurodegenerative disease and allowing the discovery of therapeutic targets.
Assuntos
Ataxia/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Tremor/metabolismo , Animais , Encéfalo/patologia , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Simulação por Computador , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Camundongos , Mapas de Interação de Proteínas , Splicing de RNA/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
Intellectual disability (ID) comprises a large group of heterogeneous disorders, often without a known molecular cause. X-linked ID accounts for 5-10% of male ID cases. We investigated a large, three-generation family with mild ID and behavior problems in five males and one female, with a segregation suggestive for X-linked inheritance. Linkage analysis mapped a disease locus to a 7.6 Mb candidate region on the X-chromosome (LOD score 3.3). Whole-genome sequencing identified a 2 bp insertion in exon 2 of the chromosome X open reading frame 56 gene (CXorf56), resulting in a premature stop codon. This insertion was present in all intellectually impaired individuals and carrier females. Additionally, X-inactivation status showed skewed methylation patterns favoring the inactivation of the mutated allele in the unaffected carrier females. We demonstrate that the insertion leads to nonsense-mediated decay and that CXorf56 mRNA expression is reduced in the impaired males and female. In murine brain slices and primary hippocampal neuronal cultures, CXorf56 protein was present and localized in the nucleus, cell soma, dendrites, and dendritic spines. Although no other families have been identified with pathogenic variants in CXorf56, these results suggest that CXorf56 is the causative gene in this family, and thus a novel candidate gene for X-linked ID with behavior problems.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Deficiência Intelectual/genética , Proteínas do Tecido Nervoso/genética , Adolescente , Adulto , Animais , Células Cultivadas , Códon de Terminação/genética , Metilação de DNA , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Deficiência Intelectual/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neurônios/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas NuclearesRESUMO
A CGG-repeat expansion in the premutation range in the Fragile X mental retardation 1 gene (FMR1) has been identified as the genetic cause of Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder that manifests with action tremor, gait ataxia and cognitive impairments. In this study, we used a bigenic mouse model, in which expression of a 90CGG premutation tract is activated in neural cells upon doxycycline administration-P90CGG mouse model. We, here, demonstrate the behavioural manifestation of clinically relevant features of FXTAS patients and premutation carrier individuals in this inducible mouse model. P90CGG mice display heightened anxiety, deficits in motor coordination and impaired gait and represent the first FXTAS model that exhibits an ataxia phenotype as observed in patients. The behavioural phenotype is accompanied by the formation of ubiquitin/FMRpolyglycine-positive intranuclear inclusions, as another hallmark of FXTAS, in the cerebellum, hippocampus and amygdala. Strikingly, upon cessation of transgene induction the anxiety phenotype of mice recovers along with a reduction of intranuclear inclusions in dentate gyrus and amygdala. In contrast, motor function deteriorates further and no reduction in intranuclear inclusions can be observed in the cerebellum. Our data thus demonstrate that expression of a 90CGG premutation expansion outside of the FMR1 context is sufficient to evoke an FXTAS-like behavioural phenotype. Brain region-specific neuropathology and (partial) behavioural reversibility make the inducible P90CGG a valuable mouse model for testing pathogenic mechanisms and therapeutic intervention methods.
Assuntos
Ataxia/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Tremor/genética , Animais , Ansiedade/genética , Ansiedade/metabolismo , Ataxia/metabolismo , Encéfalo/patologia , Ataxia Cerebelar/genética , Transtornos Cognitivos/genética , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Marcha , Marcha Atáxica/genética , Marcha Atáxica/metabolismo , Corpos de Inclusão Intranuclear/genética , Camundongos , Transtornos dos Movimentos/genética , Neurônios/patologia , Tremor/metabolismo , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of the fragile X-premutation, who have an expanded CGG repeat in the 5'-UTR of the FMR1 gene. FXTAS is characterized by progressive development of intention tremor, ataxia, parkinsonism and neuropsychological problems. The disease is thought to be caused by a toxic RNA gain-of-function mechanism, and the major hallmark of the disease is ubiquitin-positive intranuclear inclusions in neurons and astrocytes. We have developed a new transgenic mouse model in which we can induce expression of an expanded repeat in the brain upon doxycycline (dox) exposure (i.e. Tet-On mice). This Tet-On model makes use of the PrP-rtTA driver and allows us to study disease progression and possibilities of reversibility. In these mice, 8 weeks of dox exposure was sufficient to induce the formation of ubiquitin-positive intranuclear inclusions, which also stain positive for the RAN translation product FMRpolyG. Formation of these inclusions is reversible after stopping expression of the expanded CGG RNA at an early developmental stage. Furthermore, we observed a deficit in the compensatory eye movements of mice with inclusions, a functional phenotype that could be reduced by stopping expression of the expanded CGG RNA early in the disease development. Taken together, this study shows, for the first time, the potential of disease reversibility and suggests that early intervention might be beneficial for FXTAS patients.
Assuntos
Ataxia/genética , Ataxia/fisiopatologia , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Tremor/genética , Tremor/fisiopatologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Movimentos Oculares/genética , Expressão Gênica , Genes Reporter , Humanos , Corpos de Inclusão Intranuclear/patologia , Camundongos , Camundongos Transgênicos , Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico , Expansão das Repetições de Trinucleotídeos , Ubiquitina/metabolismoAssuntos
Esclerose Lateral Amiotrófica/metabolismo , Expansão das Repetições de DNA , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Camundongos Transgênicos , Proteínas/genética , Esclerose Lateral Amiotrófica/genética , Animais , Encéfalo/metabolismo , Proteína C9orf72 , Demência Frontotemporal/genética , Humanos , Corpos de Inclusão/metabolismo , Proteínas/metabolismo , Ubiquitina/metabolismoAssuntos
Ataxia/metabolismo , Ataxia/patologia , Proteína do X Frágil da Deficiência Intelectual/análise , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Tremor/metabolismo , Tremor/patologia , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Ataxia/genética , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Proteína do X Frágil da Deficiência Intelectual/química , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Heterozigoto , Humanos , Corpos de Inclusão Intranuclear , Rim/metabolismo , Rim/patologia , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Tremor/genética , Ubiquitina/metabolismoRESUMO
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of premutation forms of the FMR1 gene, resulting in a progressive development of tremor, ataxia and neuropsychological problems. The disease is caused by an expanded CGG repeat in the FMR1 gene, leading to an RNA gain-of-function toxicity mechanism. In order to study the pathogenesis of FXTAS, new inducible transgenic mouse models have been developed that expresses either 11CGGs or 90CGGs at the RNA level under control of a Tet-On promoter. When bred to an hnRNP-rtTA driver line, doxycycline (dox) induced expression of the transgene could be found in almost all tissues. Dox exposure resulted in loss of weight and death within 5 d for the 90CGG RNA expressing mice. Immunohistochemical examination of tissues of these mice revealed steatosis and apoptosis in the liver. Decreased expression of GPX1 and increased expression of cytochrome C is found. These effects were not seen in mice expressing a normal sized 11CGG repeat. In conclusion, we were able to show in vivo that expression of an expanded CGG-repeat rather than overexpression of a normal CGG-repeat causes pathology. In addition, we have shown that expanded CGG RNA expression can cause mitochondrial dysfunction by regulating expression levels of several markers. Although FTXAS patients do not display liver abnormalities, our findings contribute to understanding of the molecular mechanisms underlying toxicity of CGG repeat RNA expression in an animal model. In addition, the dox inducible mouse lines offer new opportunities to study therapeutic interventions for FXTAS.
Assuntos
Ataxia/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitocôndrias/metabolismo , RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Tremor/metabolismo , Animais , Antibacterianos/metabolismo , Apoptose/efeitos dos fármacos , Ataxia/genética , Modelos Animais de Doenças , Doxiciclina/metabolismo , Fígado Gorduroso/patologia , Síndrome do Cromossomo X Frágil/genética , Fígado/metabolismo , Fígado/ultraestrutura , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Regiões Promotoras Genéticas , RNA/genética , Espécies Reativas de Oxigênio , Tremor/genéticaRESUMO
Pathological accumulation of intermediate filaments can be observed in neurodegenerative disorders, such as Alzheimer's disease, frontotemporal dementia and Parkinson's disease, and is also characteristic of neuronal intermediate filament inclusion disease. Intermediate filaments type IV include three neurofilament proteins (light, medium and heavy molecular weight neurofilament subunits) and α-internexin. The phosphorylation of intermediate filament proteins contributes to axonal growth, and is regulated by protein kinase A. Here we describe a family with a novel late-onset neurodegenerative disorder presenting with dementia and/or parkinsonism in 12 affected individuals. The disorder is characterized by a unique neuropathological phenotype displaying abundant neuronal inclusions by haematoxylin and eosin staining throughout the brain with immunoreactivity for intermediate filaments. Combining linkage analysis, exome sequencing and proteomics analysis, we identified a heterozygous c.149T>G (p.Leu50Arg) missense mutation in the gene encoding the protein kinase A type I-beta regulatory subunit (PRKAR1B). The pathogenicity of the mutation is supported by segregation in the family, absence in variant databases, and the specific accumulation of PRKAR1B in the inclusions in our cases associated with a specific biochemical pattern of PRKAR1B. Screening of PRKAR1B in 138 patients with Parkinson's disease and 56 patients with frontotemporal dementia did not identify additional novel pathogenic mutations. Our findings link a pathogenic PRKAR1B mutation to a novel hereditary neurodegenerative disorder and suggest an altered protein kinase A function through a reduced binding of the regulatory subunit to the A-kinase anchoring protein and the catalytic subunit of protein kinase A, which might result in subcellular dislocalization of the catalytic subunit and hyperphosphorylation of intermediate filaments.
Assuntos
Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Polimorfismo de Nucleotídeo Único/genética , Idoso , Peptídeos beta-Amiloides/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Tomografia com Microscopia Eletrônica , Saúde da Família , Feminino , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Lobo Frontal/ultraestrutura , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMO
Primary open-angle glaucoma (POAG) is a hereditary neurodegenerative disease, characterized by optic nerve changes including increased excavation, notching and optic disc hemorrhages. The excavation can be described by the vertical cup-disc ratio (VCDR). Previously, genome-wide significant evidence for the association of rs10483727 in SIX1-SIX6 locus with VCDR and subsequent POAG was found. Using 1000 genomes-based imputation of four independent population-based cohorts in the Netherlands, we identified a missense variant rs33912345 (His141Asn) in SIX6 associated with VCDR (Pmeta = 7.74 × 10(-7), n = 11 473) and POAG (Pmeta = 6.09 × 10(-3), n = 292). Exome sequencing analysis revealed another missense variant rs146737847 (Glu129Lys) also in SIX6 associated with VCDR (P = 5.09 × 10(-3), n = 1208). These two findings point to SIX6 as the responsible gene for the previously reported association signal. Functional characterization of SIX6 in zebrafish revealed that knockdown of six6b led to a small eye phenotype. Histological analysis showed retinal lamination, implying an apparent normal development of the eye, but an underdeveloped lens, and reduced optic nerve diameter. Expression analysis of morphants at 3 dpf showed a 5.5-fold up-regulation of cdkn2b, a cyclin-dependent kinase inhibitor, involved in cell cycle regulation and previously associated with VCDR and POAG in genome-wide association studies (GWASs). Since both six6b and cdkn2b play a key role in cell proliferation, we assessed the proliferative activity in the eye of morphants and found an alteration in the proliferative pattern of retinal cells. Our findings in humans and zebrafish suggest a functional involvement of six6b in early eye development, and open new insights into the genetic architecture of POAG.
Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Degeneração Neural/genética , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Transativadores/genética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Animais , Proliferação de Células , Mapeamento Cromossômico , Exoma , Olho/embriologia , Olho/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Humanos , Pessoa de Meia-Idade , Modelos Biológicos , Organogênese/genética , Fenótipo , Locos de Características Quantitativas , Transativadores/metabolismo , Adulto Jovem , Peixe-ZebraRESUMO
Mutations in the gene encoding the F-box only protein 7 (FBXO7) cause PARK15, an autosomal recessive form of juvenile parkinsonism. Although the brain pathology in PARK15 patients remains unexplored, in vivo imaging displays severe loss of nigrostriatal dopaminergic terminals. Understanding the pathogenesis of PARK15 might therefore illuminate the mechanisms of the selective dopaminergic neuronal degeneration, which could also be important for understanding idiopathic Parkinson disease (PD). The expression of FBXO7 in the human brain remains poorly characterized, and its expression in idiopathic PD and different neurodegenerative diseases has not been investigated. Here, we studied FBXO7 protein expression in brain samples of normal controls (n = 9) and from patients with PD (n = 13), multiple system atrophy (MSA) (n = 5), Alzheimer disease (AD) (n = 5), and progressive supranuclear palsy (PSP) (n = 5) using immunohistochemistry with 2 anti-FBXO7 antibodies. We detected widespread brain FBXO7 immunoreactivity, with the highest levels in neurons of the cerebral cortex, putamen, and cerebellum. There were no major differences between normal and PD brains overall, but FBXO7 immunoreactivity was detected in large proportions of α-synuclein-positive inclusions (Lewy bodies, Lewy neurites, glial cytoplasmic inclusions), where it colocalized with α-synuclein in PD and MSA cases. By contrast, weak FBXO7 immunoreactivity was occasionally detected in tau-positive inclusions in AD and PSP. These findings suggest a role for FBXO7 in the pathogenesis of the synucleinopathies.
Assuntos
Proteínas F-Box/metabolismo , Corpos de Inclusão/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas F-Box/imunologia , Feminino , Células HEK293 , Humanos , Corpos de Inclusão/imunologia , Corpos de Inclusão/patologia , Masculino , Pessoa de Meia-Idade , Atrofia de Múltiplos Sistemas/imunologia , Atrofia de Múltiplos Sistemas/patologia , Doença de Parkinson/imunologia , Doença de Parkinson/patologia , alfa-Sinucleína/imunologiaRESUMO
Recessive mutations in the F-box only protein 7 gene (FBXO7) cause PARK15, a mendelian form of early-onset, levodopa-responsive parkinsonism with severe loss of nigrostriatal dopaminergic neurons. However, the function of the protein encoded by FBXO7, and the pathogenesis of PARK15 remain unknown. No animal models of this disease exist. Here, we report the generation of a vertebrate model of PARK15 in zebrafish. We first show that the zebrafish Fbxo7 homolog protein (zFbxo7) is expressed abundantly in the normal zebrafish brain. Next, we used two zFbxo7-specific morpholinos (targeting protein translation and mRNA splicing, respectively), to knock down the zFbxo7 expression. The injection of either of these zFbxo7-specific morpholinos in the fish embryos induced a marked decrease in the zFbxo7 protein expression, and a range of developmental defects. Furthermore, whole-mount in situ mRNA hybridization showed abnormal patterning and significant decrease in the number of diencephalic tyrosine hydroxylase-expressing neurons, corresponding to the human nigrostriatal or ventral tegmental dopaminergic neurons. Of note, the number of the dopamine transporter-expressing neurons was much more severely depleted, suggesting dopaminergic dysfunctions earlier and larger than those due to neuronal loss. Last, the zFbxo7 morphants displayed severe locomotor disturbances (bradykinesia), which were dramatically improved by the dopaminergic agonist apomorphine. The severity of these morphological and behavioral abnormalities correlated with the severity of zFbxo7 protein deficiency. Moreover, the effects of the co-injection of zFbxo7- and p53-specific morpholinos were similar to those obtained with zFbxo7-specific morpholinos alone, supporting further the contention that the observed phenotypes were specifically due to the knock down of zFbxo7. In conclusion, this novel vertebrate model reproduces pathologic and behavioral hallmarks of human parkinsonism (dopaminergic neuronal loss and dopamine-dependent bradykinesia), representing therefore a valid tool for investigating the mechanisms of selective dopaminergic neuronal death, and screening for modifier genes and therapeutic compounds.
Assuntos
Dopamina/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/fisiologia , Neurônios/metabolismo , Animais , Apomorfina/farmacologia , Padronização Corporal , Encéfalo/metabolismo , Modelos Animais de Doenças , Domperidona/farmacologia , Antagonistas de Dopamina/farmacologia , Éxons , Etiquetas de Sequências Expressas , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ , Íntrons , Locomoção , Transtornos Parkinsonianos/metabolismo , Fenótipo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Peixe-ZebraRESUMO
RATIONALE: Congenital heart malformations are a major cause of morbidity and mortality, especially in young children. Failure to establish normal left-right (L-R) asymmetry often results in cardiovascular malformations and other laterality defects of visceral organs. OBJECTIVE: To identify genetic mutations causing cardiac laterality defects. METHODS AND RESULTS: We performed a genome-wide linkage analysis in patients with cardiac laterality defects from a consanguineous family. The patients had combinations of defects that included dextrocardia, transposition of great arteries, double-outlet right ventricle, atrioventricular septal defects, and caval vein abnormalities. Sequencing of positional candidate genes identified mutations in NPHP4. We performed mutation analysis of NPHP4 in 146 unrelated patients with similar cardiac laterality defects. Forty-one percent of these patients also had laterality defects of the abdominal organs. We identified 8 additional missense variants that were absent or very rare in control subjects. To study the role of nphp4 in establishing L-R asymmetry, we used antisense morpholinos to knockdown nphp4 expression in zebrafish. Depletion of nphp4 disrupted L-R patterning as well as cardiac and gut laterality. Cardiac laterality defects were partially rescued by human NPHP4 mRNA, whereas mutant NPHP4 containing genetic variants found in patients failed to rescue. We show that nphp4 is involved in the formation of motile cilia in Kupffer's vesicle, which generate asymmetrical fluid flow necessary for normal L-R asymmetry. CONCLUSIONS: NPHP4 mutations are associated with cardiac laterality defects and heterotaxy. In zebrafish, nphp4 is essential for the development and function of Kupffer's vesicle cilia and is required for global L-R patterning.
Assuntos
Pleiotropia Genética/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Cardiopatias Congênitas/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Estudos de Coortes , Feminino , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/patologia , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Peixe-ZebraRESUMO
Manganese is essential for several metabolic pathways but becomes toxic in excessive amounts. Manganese levels in the body are therefore tightly regulated, but the responsible protein(s) remain incompletely known. We studied two consanguineous families with neurologic disorders including juvenile-onset dystonia, adult-onset parkinsonism, severe hypermanganesemia, polycythemia, and chronic hepatic disease, including steatosis and cirrhosis. We localized the genetic defect by homozygosity mapping and then identified two different homozygous frameshift SLC30A10 mutations, segregating with disease. SLC30A10 is highly expressed in the liver and brain, including in the basal ganglia. Its encoded protein belongs to a large family of membrane transporters, mediating the efflux of divalent cations from the cytosol. We show the localization of SLC30A10 in normal human liver and nervous system, and its depletion in liver from one affected individual. Our in silico analyses suggest that SLC30A10 possesses substrate specificity different from its closest (zinc-transporting) homologs. We also show that the expression of SLC30A10 and the levels of the encoded protein are markedly induced by manganese in vitro. The phenotype associated with SLC30A10 mutations is broad, including neurologic, hepatic, and hematologic disturbances. Intrafamilial phenotypic variability is also present. Chelation therapy can normalize the manganesemia, leading to marked clinical improvements. In conclusion, we show that SLC30A10 mutations cause a treatable recessive disease with pleomorphic phenotype, and provide compelling evidence that SLC30A10 plays a pivotal role in manganese transport. This work has broad implications for understanding of the manganese biology and pathophysiology in multiple human organs.
Assuntos
Proteínas de Transporte de Cátions/genética , Intoxicação por Manganês/genética , Proteínas de Membrana Transportadoras/genética , Doenças Metabólicas/genética , Transtornos Parkinsonianos/genética , Idoso , Sequência de Aminoácidos , Encéfalo/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Mapeamento Cromossômico/métodos , Feminino , Mutação da Fase de Leitura/genética , Genes Recessivos , Predisposição Genética para Doença , Células Hep G2 , Homozigoto , Humanos , Imuno-Histoquímica/métodos , Fígado/metabolismo , Masculino , Manganês/metabolismo , Intoxicação por Manganês/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Doenças Metabólicas/metabolismo , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fenótipo , Alinhamento de Sequência/métodos , Células Tumorais Cultivadas , Transportador 8 de ZincoRESUMO
Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder generally presenting with intention tremor and gait ataxia, but with a growing list of co-morbid medical conditions including hypothyroidism, hypertension, peripheral neuropathy, and cognitive decline. The pathological hallmark of FXTAS is the presence of intranuclear inclusions in both neurons and astroglia. However, it is unknown to what extent such inclusions are present outside the central nervous system (CNS). To address this issue, we surveyed non-CNS organs in ten human cases with FXTAS and in a CGG repeat knock-in (CGG KI) mouse model known to possess neuronal and astroglial inclusions. We find inclusions in multiple tissues from FXTAS cases and CGG KI mice, including pancreas, thyroid, adrenal gland, gastrointestinal, pituitary gland, pineal gland, heart, and mitral valve, as well as throughout the associated autonomic ganglia. Inclusions were observed in the testes, epididymis, and kidney of FXTAS cases, but were not observed in mice. These observations demonstrate extensive involvement of the peripheral nervous system and systemic organs. The finding of intranuclear inclusions in non-CNS somatic organ systems, throughout the PNS, and in the enteric nervous system of both FXTAS cases as well as CGG KI mice suggests that these tissues may serve as potential sites to evaluate early intervention strategies or be used as diagnostic factors.