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1.
Neuron ; 105(4): 630-644.e9, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31859031

RESUMO

Sirtuin 1 (Sirt1) is a NAD+-dependent deacetylase capable of countering age-related neurodegeneration, but the basis of Sirt1 neuroprotection remains elusive. Spinocerebellar ataxia type 7 (SCA7) is an inherited CAG-polyglutamine repeat disorder. Transcriptome analysis of SCA7 mice revealed downregulation of calcium flux genes accompanied by abnormal calcium-dependent cerebellar membrane excitability. Transcription-factor binding-site analysis of downregulated genes yielded Sirt1 target sites, and we observed reduced Sirt1 activity in the SCA7 mouse cerebellum with NAD+ depletion. SCA7 patients displayed increased poly(ADP-ribose) in cerebellar neurons, supporting poly(ADP-ribose) polymerase-1 upregulation. We crossed Sirt1-overexpressing mice with SCA7 mice and noted rescue of neurodegeneration and calcium flux defects. NAD+ repletion via nicotinamide riboside ameliorated disease phenotypes in SCA7 mice and patient stem cell-derived neurons. Sirt1 thus achieves neuroprotection by promoting calcium regulation, and NAD+ dysregulation underlies Sirt1 dysfunction in SCA7, indicating that cerebellar ataxias exhibit altered calcium homeostasis because of metabolic dysregulation, suggesting shared therapy targets.


Assuntos
Cálcio/fisiologia , Homeostase/fisiologia , Neuroproteção/fisiologia , Niacinamida/metabolismo , Sirtuína 1/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Linhagem Celular , Cerebelo/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Transdução de Sinais/fisiologia , Sirtuína 1/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/prevenção & controle
2.
Curr Med Sci ; 39(5): 734-740, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31612390

RESUMO

In polyglutamine (PolyQ) diseases, mutant proteins cause not only neurological problems but also peripheral tissue abnormalities. Among all systemic damages, skeletal muscle dystrophy is the severest. Previously by studying knock-in (KI) mouse models of spinal cerebellar ataxia 17 (SCA17), it was found that mutant TATA box binding protein (TBP) decreases its interaction with myogenic differentiation antigen, thus reducing the expression of skeletal muscle structural proteins and resulting in muscle degeneration. In this paper, the role of mutant TBP in myogenesis was investigated. Single myofibers were isolated from tibialis anterior muscles of wild type (WT) and SCA17KI mice. The 1TBP18 staining confirmed the expression of mutant TBP in muscle satellite cells in SCA17KI mice. In the BaCl2-induced TA muscle injury, H&E cross-section staining showed no significant change in myofibril size before and after BaCl2 treatment, and there was no significant difference in centralized nuclei between WT and SCA17KI mice, suggesting that mutant TBP had no significant effect on muscle regeneration. In the cultured primary myoblasts from WT and SCA17KI mice in vitro, representative BrdU immunostaining showed no significant difference in proliferation of muscle satellite cells. The primary myoblasts were then induced to differentiate and immunostained for eMyHC, and the staining showed there was no significant difference in differentiation of primary myoblasts between WT and SCA1KI mice. Our findings confirmed that mutant TBP had no significant effect on myogenesis.


Assuntos
Desenvolvimento Muscular/genética , Proteína MyoD/genética , Mioblastos/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Ataxias Espinocerebelares/genética , Proteína de Ligação a TATA-Box/genética , Animais , Compostos de Bário/farmacologia , Diferenciação Celular , Cloretos/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação , Proteína MyoD/metabolismo , Mioblastos/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Cultura Primária de Células , Células Satélites de Músculo Esquelético/patologia , Transdução de Sinais , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia , Proteína de Ligação a TATA-Box/metabolismo
3.
Biochimie ; 163: 21-32, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31075282

RESUMO

Huntington's diseases (HD) is a very devastating disease caused by r(CAG) expansion in HTT gene, encoding the huntingtin protein. r(CAG) expansion causes disease via multiple pathways including, 1) loss of normal protein function like sequestration of RNA binding protein such as Muscleblind-like (MBNL) and nucleolin, 2) Gain of function for mutant proteins and 3) repeat-associated non-ATG (RAN) translation; in which expanded r(CAG) translates into toxic poly glu, poly ser, or poly ala without the use of any canonical start codon. Herein, we have rationally designed and synthesized a unique class of pyridocoumarin derivatives that target the r(CAG)exp involved in HD and spinocerebellar ataxia (SCA) pathogenesis. Notably, compounds 3 and 15 showed higher affinity (nanomolar Kd) and selectivity for diseased r(CAG)exp RNA compared to regular duplex AU-paired RNA. Interestingly, both scaffolds are cell permeable, exhibit low toxicity to healthy fibroblast cells and are also capable of reducing the level of poly Q aggregation in cellular models. Indeed, our current study offers promising facet for selectively targeting repeats containing RNAs that cause severe diseases like HD and SCAs.


Assuntos
Cumarínicos/química , Doença de Huntington/tratamento farmacológico , Proteínas Mutantes/genética , RNA Mensageiro/química , Ataxias Espinocerebelares/tratamento farmacológico , Células Cultivadas , Cumarínicos/farmacologia , Cumarínicos/uso terapêutico , Desenho de Fármacos , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Cinética , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/efeitos dos fármacos , Ataxias Espinocerebelares/metabolismo , Expansão das Repetições de Trinucleotídeos
4.
Nucleic Acids Res ; 47(8): 4086-4110, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30986824

RESUMO

Ataxia with oculomotor apraxia type 1 (AOA1) is an early onset progressive spinocerebellar ataxia caused by mutation in aprataxin (APTX). APTX removes 5'-AMP groups from DNA, a product of abortive ligation during DNA repair and replication. APTX deficiency has been suggested to compromise mitochondrial function; however, a detailed characterization of mitochondrial homeostasis in APTX-deficient cells is not available. Here, we show that cells lacking APTX undergo mitochondrial stress and display significant changes in the expression of the mitochondrial inner membrane fusion protein optic atrophy type 1, and components of the oxidative phosphorylation complexes. At the cellular level, APTX deficiency impairs mitochondrial morphology and network formation, and autophagic removal of damaged mitochondria by mitophagy. Thus, our results show that aberrant mitochondrial function is a key component of AOA1 pathology. This work corroborates the emerging evidence that impaired mitochondrial function is a characteristic of an increasing number of genetically diverse neurodegenerative disorders.


Assuntos
Proteínas de Ligação a DNA/genética , GTP Fosfo-Hidrolases/genética , Mitocôndrias/genética , Proteínas Nucleares/genética , Ataxias Espinocerebelares/congênito , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , GTP Fosfo-Hidrolases/deficiência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Nucleares/deficiência , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação Oxidativa , Transdução de Sinais , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia
5.
Neuroscience ; 400: 72-84, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30625334

RESUMO

Spino-cerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by neurodegeneration of the brain, cerebellum, and retina caused by a polyglutamine expansion in ataxin7. The presence of an expanded polyQ tract in a mutant protein is known to induce protein aggregation, cellular stress, toxicity, and finally cell death. However, the consequences of the presence of mutant ataxin7 in the retina and the mechanisms underlying photoreceptor degeneration remain poorly understood. In this study, we show that in a retinal SCA7 mouse model, polyQ ataxin7 induces stress within the retina and activates Muller cells. Moreover, unfolded protein response and autophagy are activated in SCA7 photoreceptors. We have also shown that the photoreceptor death does not involve a caspase-dependent apoptosis but instead involves apoptosis inducing factor (AIF) and Leukocyte Elastase Inhibitor (LEI/L-DNase II). When these two cell death effectors are downregulated by their siRNA, a significant reduction in photoreceptor death is observed. These results highlight the consequences of polyQ protein expression in the retina and the role of caspase-independent pathways involved in photoreceptor cell death.


Assuntos
Ataxina-7/metabolismo , Morte Celular , Peptídeos/metabolismo , Degeneração Retiniana/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Fator de Indução de Apoptose/metabolismo , Ataxina-7/genética , Calpaína/metabolismo , Caspases/metabolismo , Catepsinas/metabolismo , Modelos Animais de Doenças , Endodesoxirribonucleases/metabolismo , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/etiologia , Transdução de Sinais , Ataxias Espinocerebelares/complicações , Estresse Fisiológico
6.
Neurobiol Dis ; 124: 14-28, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30389403

RESUMO

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.


Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Ataxias Espinocerebelares/congênito , Animais , Feminino , Técnicas de Introdução de Genes , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Células de Purkinje/fisiologia , Células de Purkinje/ultraestrutura , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia
7.
FASEB J ; 33(2): 2982-2994, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30332300

RESUMO

Loss of sacsin, a large 520 kDa multidomain protein, causes autosomal recessive spastic ataxia of the Charlevoix-Saguenay, one of the most common childhood-onset recessive ataxias. A prominent feature is abnormal bundling of neurofilaments in many neuronal populations. This study shows the direct involvement of sacsin domains in regulating intermediate filament assembly and dynamics and identifies important domains for alleviating neurofilament bundles in neurons lacking sacsin. Peptides encoding sacsin internal repeat (SIRPT) 1, J-domains, and ubiquitin-like domain modified neurofilament assembly in vivo. The domains with chaperone homology, the SIRPT and the J-domain, had opposite effects, promoting and preventing filament assembly, respectively. In cultured Sacs-/- motor neurons, both the SIRPT1 and J-domain resolved preexisting neurofilament bundles. Increasing expression of heat shock proteins also resolved neurofilament bundles, indicating that this endogenous chaperone system can compensate to some extent for sacsin deficiency.-Gentil, B. J., Lai, G.-T., Menade, M., Larivière, R., Minotti, S., Gehring, K., Chapple, J.-P., Brais, B., Durham, H. D. Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics.


Assuntos
Fibroblastos/patologia , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/fisiologia , Filamentos Intermediários/patologia , Neurônios Motores/patologia , Espasticidade Muscular/patologia , Mutação , Ataxias Espinocerebelares/congênito , Animais , Células Cultivadas , Fibroblastos/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Filamentos Intermediários/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/metabolismo , Espasticidade Muscular/metabolismo , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia
8.
J Neurol ; 266(2): 533-544, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30284037

RESUMO

Spinocerebellar ataxia (SCA) is a heterogeneous group of neurodegenerative ataxic disorders with autosomal dominant inheritance. We aim to provide an update on the recent clinical and scientific progresses in SCA where numerous novel genes have been identified with next-generation sequencing techniques. The main disease mechanisms of these SCAs include toxic RNA gain-of-function, mitochondrial dysfunction, channelopathies, autophagy and transcription dysregulation. Recent studies have also demonstrated the importance of DNA repair pathways in modifying SCA with CAG expansions. In addition, we summarise the latest technological advances in detecting known and novel repeat expansion in SCA. Finally, we discuss the roles of antisense oligonucleotides and RNA-based therapy as potential treatments.


Assuntos
Ataxias Espinocerebelares/diagnóstico , Humanos , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/terapia
9.
IEEE J Biomed Health Inform ; 23(1): 26-37, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30176611

RESUMO

The advancement of scientific and medical research over the past years has generated a wealth of experimental data from multiple technologies, including genomics, transcriptomics, proteomics, and other forms of -omics data, which are available for a number of diseases. The integration of such multisource data is a key component toward the success of precision medicine. In this paper, we are investigating a multisource data integration method developed by our group, regarding its ability to drive to clusters of connected pathways under two different approaches: first, a disease-centric approach, where we integrate data around a disease, and second, a gene-centric approach, where we integrate data around a gene. We have used as a paradigm for the first approach Huntington's disease (HD), a disease with a plethora of available data, whereas for the second approach the GBA2, a gene that is related to spastic ataxia (SA), a phenotype with sparse availability of data. Our paper shows that valuable information at the level of disease-related pathway clusters can be obtained for both HD and SA. New pathways that classical pathway analysis methods were unable to reveal, emerged as necessary "connectors" to build connected pathway stories formed as pathway clusters. The capability to integrate multisource molecular data, concluding to something more than the sum of the existing information, empowers precision and personalized medicine approaches.


Assuntos
Biologia Computacional/métodos , Doença de Huntington , Deficiência Intelectual , Espasticidade Muscular , Atrofia Óptica , Mapas de Interação de Proteínas , Transdução de Sinais , Ataxias Espinocerebelares , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/fisiopatologia , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/fisiopatologia , Informática Médica , Espasticidade Muscular/genética , Espasticidade Muscular/metabolismo , Espasticidade Muscular/fisiopatologia , Atrofia Óptica/genética , Atrofia Óptica/metabolismo , Atrofia Óptica/fisiopatologia , Medicina de Precisão , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/fisiopatologia , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
10.
Dis Model Mech ; 12(1)2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30559154

RESUMO

Perturbation of protein homeostasis and aggregation of misfolded proteins is a major cause of many human diseases. A hallmark of the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7) is the intranuclear accumulation of mutant, misfolded ataxin-7 (polyQ-ATXN7). Here, we show that endogenous ATXN7 is modified by SUMO proteins, thus also suggesting a physiological role for this modification under conditions of proteotoxic stress caused by the accumulation of polyQ-ATXN7. Co-immunoprecipitation experiments, immunofluorescence microscopy and proximity ligation assays confirmed the colocalization and interaction of polyQ-ATXN7 with SUMO2 in cells. Moreover, upon inhibition of the proteasome, both endogenous SUMO2/3 and the RNF4 ubiquitin ligase surround large polyQ-ATXN7 intranuclear inclusions. Overexpression of RNF4 and/or SUMO2 significantly decreased levels of polyQ-ATXN7 and, upon proteasomal inhibition, led to a marked increase in the polyubiquitination of polyQ-ATXN7. This provides a mechanism for the clearance of polyQ-ATXN7 from affected cells that involves the recruitment of RNF4 by SUMO2/3-modified polyQ-ATXN7, thus leading to its ubiquitination and proteasomal degradation. In a SCA7 knock-in mouse model, we similarly observed colocalization of SUMO2/3 with polyQ-ATXN7 inclusions in the cerebellum and retina. Furthermore, we detected accumulation of SUMO2/3 high-molecular-mass species in the cerebellum of SCA7 knock-in mice, compared with their wild-type littermates, and changes in SUMO-related transcripts. Immunohistochemical analysis showed the accumulation of SUMO proteins and RNF4 in the cerebellum of SCA7 patients. Taken together, our results show that the SUMO pathway contributes to the clearance of aggregated ATXN7 and suggest that its deregulation might be associated with SCA7 disease progression.


Assuntos
Ataxina-7/metabolismo , Proteínas Nucleares/metabolismo , Dobramento de Proteína , Proteólise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ataxias Espinocerebelares/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Animais , Cerebelo/metabolismo , Criança , Modelos Animais de Doenças , Células HEK293 , Células HeLa , Humanos , Corpos de Inclusão/metabolismo , Células MCF-7 , Camundongos , Pessoa de Meia-Idade , Mutação/genética , Proteína da Leucemia Promielocítica/metabolismo , Inibidores de Proteassoma/farmacologia , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Ataxias Espinocerebelares/patologia , Sumoilação/efeitos dos fármacos , Ubiquitina/metabolismo
11.
Int J Mol Sci ; 21(1)2019 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-31892274

RESUMO

Spinocerebellar ataxias (SCAs) constitute a heterogeneous group of more than 40 autosomal-dominant genetic and neurodegenerative diseases characterized by loss of balance and motor coordination due to dysfunction of the cerebellum and its efferent connections. Despite a well-described clinical and pathological phenotype, the molecular and cellular events that underlie neurodegeneration are still poorly undaerstood. Emerging research suggests that mutations in SCA genes cause disruptions in multiple cellular pathways but the characteristic SCA pathogenesis does not begin until calcium signaling pathways are disrupted in cerebellar Purkinje cells. Ca2+ signaling in Purkinje cells is important for normal cellular function as these neurons express a variety of Ca2+ channels, Ca2+-dependent kinases and phosphatases, and Ca2+-binding proteins to tightly maintain Ca2+ homeostasis and regulate physiological Ca2+-dependent processes. Abnormal Ca2+ levels can activate toxic cascades leading to characteristic death of Purkinje cells, cerebellar atrophy, and ataxia that occur in many SCAs. The output of the cerebellar cortex is conveyed to the deep cerebellar nuclei (DCN) by Purkinje cells via inhibitory signals; thus, Purkinje cell dysfunction or degeneration would partially or completely impair the cerebellar output in SCAs. In the absence of the inhibitory signal emanating from Purkinje cells, DCN will become more excitable, thereby affecting the motor areas receiving DCN input and resulting in uncoordinated movements. An outstanding advantage in studying the pathogenesis of SCAs is represented by the availability of a large number of animal models which mimic the phenotype observed in humans. By mainly focusing on mouse models displaying mutations or deletions in genes which encode for Ca2+ signaling-related proteins, in this review we will discuss the several pathogenic mechanisms related to deranged Ca2+ homeostasis that leads to significant Purkinje cell degeneration and dysfunction.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Cerebelo/metabolismo , Homeostase/fisiologia , Humanos , Modelos Animais
12.
Elife ; 72018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30507379

RESUMO

The neurodegenerative disorder spinocerebellar ataxia type 1 (SCA1) affects the cerebellum and inferior olive, though previous research has focused primarily on the cerebellum. As a result, it is unknown what molecular alterations are present in the inferior olive, and whether these changes are found in other affected tissues. This study addresses these questions for the first time using two different SCA1 mouse models. We found that differentially regulated genes in the inferior olive segregated into several biological pathways. Comparison of the inferior olive and cerebellum demonstrates that vulnerable tissues in SCA1 are not uniform in their gene expression changes, and express largely discrete but some commonly enriched biological pathways. Importantly, we also found that brain-region-specific differences occur early in disease initiation and progression, and they are shared across the two mouse models of SCA1. This suggests different mechanisms of degeneration at work in the inferior olive and cerebellum.


Assuntos
Ataxina-1/genética , Cerebelo/metabolismo , Proteínas Nucleares/genética , Ataxias Espinocerebelares/genética , Animais , Cerebelo/fisiopatologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Transdução de Sinais/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/fisiopatologia
13.
Proc Natl Acad Sci U S A ; 115(52): E12407-E12416, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30530649

RESUMO

The genetically heterogeneous spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of nonreceptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-metastasis (MTSS1) is an ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically approved Src inhibitor corrects Purkinje neuron basal firing and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/fisiopatologia , Animais , Ataxia/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/genética , Proteínas/metabolismo , Células de Purkinje/fisiologia , Ataxias Espinocerebelares/metabolismo , Degenerações Espinocerebelares/metabolismo , Degenerações Espinocerebelares/fisiopatologia , Quinases da Família src/metabolismo
14.
Proc Natl Acad Sci U S A ; 115(45): E10748-E10757, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30348793

RESUMO

Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. Recently, coiled-coil structures in polyQ regions of such proteins were shown to facilitate aggregate formation and ultimately lead to cell death. However, the molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. Here, we demonstrate that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in Drosophila neurons. To functionally characterize coiled-coil structures in the Q repeat regions, we generated three structural variants of SCA3 polyQ proteins: (i) MJDtr-76Q, containing both α-helical coiled-coil and ß-sheet hairpin structures in the Q repeat region; (ii) MJDtr-70Q_cc0, possessing only α-helical coiled-coil structures due to the incorporation of ß-sheet-breaking residues (Q-to-N or Q-to-E mutations); and (iii) MJDtr-70Q_pQp, with no secondary structure due to the introduced proline residues (Q-to-P mutations). Through comparative analysis of these variants, we found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in Drosophila dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in Drosophila These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.


Assuntos
Ataxina-3/química , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Fatores de Transcrição Forkhead/química , Neurônios/metabolismo , Peptídeos/química , Ataxias Espinocerebelares/genética , Sequência de Aminoácidos , Animais , Ataxina-3/genética , Ataxina-3/metabolismo , Comportamento Animal , Sítios de Ligação , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Mutação , Neurônios/ultraestrutura , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia
15.
Int J Mol Sci ; 19(10)2018 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-30308956

RESUMO

The GBA2 gene encodes the non-lysosomal glucosylceramidase (NLGase), an enzyme that catalyzes the conversion of glucosylceramide (GlcCer) to ceramide and glucose. Mutations in GBA2 have been associated with the development of neurological disorders such as autosomal recessive cerebellar ataxia, hereditary spastic paraplegia, and Marinesco-Sjogren-Like Syndrome. Our group has previously identified the GBA2 c.1780G>C [p.Asp594His] missense mutation, in a Cypriot consanguineous family with spastic ataxia. In this study, we carried out a biochemical characterization of lymphoblastoid cell lines (LCLs) derived from three patients of this family. We found that the mutation strongly reduce NLGase activity both intracellularly and at the plasma membrane level. Additionally, we observed a two-fold increase of GlcCer content in LCLs derived from patients compared to controls, with the C16 lipid being the most abundant GlcCer species. Moreover, we showed that there is an apparent compensatory effect between NLGase and the lysosomal glucosylceramidase (GCase), since we found that the activity of GCase was three-fold higher in LCLs derived from patients compared to controls. We conclude that the c.1780G>C mutation results in NLGase loss of function with abolishment of the enzymatic activity and accumulation of GlcCer accompanied by a compensatory increase in GCase.


Assuntos
Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Linfócitos/metabolismo , Espasticidade Muscular/genética , Espasticidade Muscular/metabolismo , Mutação de Sentido Incorreto , Atrofia Óptica/genética , Atrofia Óptica/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , beta-Glucosidase/genética , Alelos , Biomarcadores , Linhagem Celular , Ativação Enzimática , Glucosilceramidase/metabolismo , Glucosilceramidas/metabolismo , Humanos , beta-Glucosidase/metabolismo
17.
Nat Commun ; 9(1): 3648, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194296

RESUMO

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of polyglutamine tract in the ATXN2 protein. We identified Staufen1 (STAU1) as an interactor of ATXN2, and showed elevation in cells from SCA2 patients, amyotrophic lateral sclerosis (ALS) patients, and in SCA2 mouse models. We demonstrated recruitment of STAU1 to mutant ATXN2 aggregates in brain tissue from patients with SCA2 human brain and in an SCA2 mouse model, and association of STAU1 elevation with dysregulation of SCA2-related transcript abundances. Targeting STAU1 in vitro by RNAi restored PCP2 transcript levels and lowering mutant ATXN2 also normalized STAU1 levels. Reduction of Stau1 in vivo improved motor behavior in an SCA2 mouse model, normalized the levels of several SCA2-related proteins, and reduced aggregation of polyglutamine-expanded ATXN2. These findings suggest a function for STAU1 in aberrant RNA metabolism associated with ATXN2 mutation, suggesting STAU1 is a possible novel therapeutic target for SCA2.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Ataxina-2/metabolismo , Encéfalo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos Knockout , Cultura Primária de Células
18.
J Neurosci Res ; 96(9): 1576-1585, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30113722

RESUMO

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of cerebellar degenerative disorders, characterized by progressive gait unsteadiness, hand incoordination, and dysarthria. Ataxia type 1 (SCA1) is caused by the expansion of a CAG trinucleotide repeat in the SCA1 gene resulting in the atypical extension of a polyglutamine (polyQ) tract within the ataxin-1 protein. Our main objective was to investigate the mitochondrial oxidative metabolism in the cerebellum of transgenic SCA1 mice. SCA1 transgenic mice develop clinical features in the early life stages (around 5 weeks of age) presenting pathological cerebellar signs with concomitant progressive Purkinje neuron atrophy and relatively little cell loss; this evidence suggests that the SCA1 phenotype is not the result of cell death per se, but a possible effect of cellular dysfunction that occurs before neuronal demise. We studied the mitochondrial oxidative metabolism in cerebellar cells from both homozygous and heterozygous transgenic SCA1 mice, aged 2 and 6 months. Histochemical examination showed a cytochrome-c-oxidase (COX) deficiency in the Purkinje cells (PCs) of both heterozygous and homozygous mice, the oxidative defect being more prominent in older mice, in which the percentage of COX-deficient PC was up to 30%. Using a laser-microdissector, we evaluated the mitochondrial DNA (mtDNA) content on selectively isolated COX-competent and COX-deficient PC by quantitative Polymerase Chain Reaction and we found mtDNA depletion in those with oxidative dysfunction. In conclusion, the selective oxidative metabolism defect observed in neuronal PC expressing mutant ataxin occurs as early as 8 weeks of age thus representing an early step in the PC degeneration process in SCA1 disease.


Assuntos
Deficiência de Citocromo-c Oxidase/metabolismo , DNA Mitocondrial/genética , Células de Purkinje/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Animais , Ataxina-1/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos Transgênicos , Células de Purkinje/ultraestrutura
19.
J Biol Chem ; 293(33): 12832-12842, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29945973

RESUMO

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease that is caused by mutations in the SACS gene. The product of this gene is a very large 520-kDa cytoplasmic protein, sacsin, with a ubiquitin-like (Ubl) domain at the N terminus followed by three large sacsin internal repeat (SIRPT) supradomains and C-terminal J and HEPN domains. The SIRPTs are predicted to contain Hsp90-like domains, suggesting a potential chaperone activity. In this work, we report the structures of the Hsp90-like Sr1 domain of SIRPT1 and the N-terminal Ubl domain determined at 1.55- and 2.1-Å resolutions, respectively. The Ubl domain crystallized as a swapped dimer that could be relevant in the context of full-length protein. The Sr1 domain displays the Bergerat protein fold with a characteristic nucleotide-binding pocket, although it binds nucleotides with very low affinity. The Sr1 structure reveals that ARSACS-causing missense mutations (R272H, R272C, and T201K) disrupt protein folding, most likely leading to sacsin degradation. This work lends structural support to the view of sacsin as a molecular chaperone and provides a framework for future studies of this protein.


Assuntos
Proteínas de Choque Térmico/química , Mutação de Sentido Incorreto , Dobramento de Proteína , Substituição de Aminoácidos , Cristalografia por Raios X , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Espasticidade Muscular/genética , Espasticidade Muscular/metabolismo , Domínios Proteicos , Ataxias Espinocerebelares/congênito , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo
20.
Biochim Biophys Acta Mol Cell Res ; 1865(11 Pt B): 1733-1744, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29777722

RESUMO

Spinocerebellar ataxia (SCA) is a neural disorder, which is caused by degenerative changes in the cerebellum. SCA is primarily characterized by gait ataxia, and additional clinical features include nystagmus, dysarthria, tremors and cerebellar atrophy. Forty-four hereditary SCAs have been identified to date, along with >35 SCA-associated genes. Despite the great diversity and distinct functionalities of the SCA-related genes, accumulating evidence supports the occurrence of a common pathophysiological event among several hereditary SCAs. Altered calcium (Ca2+) homeostasis in the Purkinje cells (PCs) of the cerebellum has been proposed as a possible pathological SCA trigger. In support of this, signaling events that are initiated from or lead to aberrant Ca2+ release from the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), which is highly expressed in cerebellar PCs, seem to be closely associated with the pathogenesis of several SCA types. In this review, we summarize the current research on pathological hereditary SCA events, which involve altered Ca2+ homeostasis in PCs, through IP3R1 signaling.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Suscetibilidade a Doenças , Ataxias Espinocerebelares/etiologia , Ataxias Espinocerebelares/metabolismo , Animais , Cerebelo/metabolismo , Cerebelo/fisiopatologia , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mutação , Células de Purkinje/metabolismo
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