RESUMO
Spinocerebellar ataxia type 7 (SCA7) is a progressive neurodegenerative disorder resulting from abnormal expansion of an uninterrupted polyglutamine (polyQ) repeat in its disease protein, ataxin-7 (ATXN7). ATXN7 is part of Spt-Ada-Gcn5 acetyltransferase (SAGA), an evolutionarily conserved transcriptional coactivation complex with critical roles in chromatin remodeling, cell signaling, neurodifferentiation, mitochondrial health and autophagy. SCA7 is dominantly inherited and characterized by genetic anticipation and high repeat-length instability. Patients with SCA7 experience progressive ataxia, atrophy, spasticity, and blindness. There is currently no cure for SCA7, and therapies are aimed at alleviating symptoms to increase quality of life. Here, we report novel Drosophila lines of SCA7 with polyQ repeats in wild-type and human disease patient range. We find that ATXN7 expression has age- and polyQ repeat length-dependent reduction in fruit fly survival and retinal instability, concomitant with increased ATXN7 protein aggregation. These new lines will provide important insight on disease progression that can be used in the future to identify therapeutic targets for SCA7 patients.
Assuntos
Ataxina-7 , Modelos Animais de Doenças , Peptídeos , Ataxias Espinocerebelares , Animais , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Ataxias Espinocerebelares/metabolismo , Ataxina-7/genética , Ataxina-7/metabolismo , Humanos , Peptídeos/metabolismo , Peptídeos/genética , Drosophila/genética , Animais Geneticamente Modificados , Progressão da Doença , Drosophila melanogaster/genética , Retina/metabolismo , Retina/patologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMO
Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs). PolyQ-expanded SCA proteins are toxic for cerebellar neurons, with Purkinje cells (PCs) being the most vulnerable. RNA interference (RNAi) reagents targeting transcripts with expanded CAG reduce the level of various mutant SCA proteins in an allele-selective manner in vitro and represent promising universal tools for treating multiple CAG/polyQ SCAs. However, it remains unclear whether the therapeutic targeting of CAG expansion can be achieved in vivo and if it can ameliorate cerebellar functions. Here, using a mouse model of SCA7 expressing a mutant Atxn7 allele with 140 CAGs, we examined the efficacy of short hairpin RNAs (shRNAs) targeting CAG repeats expressed from PHP.eB adeno-associated virus vectors (AAVs), which were introduced into the brain via intravascular injection. We demonstrated that shRNAs carrying various mismatches with the CAG target sequence reduced the level of polyQ-expanded ATXN7 in the cerebellum, albeit with varying degrees of allele selectivity and safety profile. An shRNA named A4 potently reduced the level of polyQ-expanded ATXN7, with no effect on normal ATXN7 levels and no adverse side effects. Furthermore, A4 shRNA treatment improved a range of motor and behavioral parameters 23 weeks after AAV injection and attenuated the disease burden of PCs by preventing the downregulation of several PC-type-specific genes. Our results show the feasibility of the selective targeting of CAG expansion in the cerebellum using a blood-brain barrier-permeable vector to attenuate the disease phenotype in an SCA mouse model. Our study represents a significant advancement in developing CAG-targeting strategies as a potential therapy for SCA7 and possibly other CAG/polyQ SCAs.
Assuntos
Ataxina-7 , Dependovirus , Modelos Animais de Doenças , Peptídeos , Fenótipo , RNA Interferente Pequeno , Ataxias Espinocerebelares , Expansão das Repetições de Trinucleotídeos , Animais , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/terapia , Ataxias Espinocerebelares/metabolismo , Peptídeos/genética , Dependovirus/genética , Camundongos , Ataxina-7/genética , Ataxina-7/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , RNA Interferente Pequeno/genética , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Camundongos Transgênicos , Cerebelo/metabolismo , Cerebelo/patologia , Humanos , Terapia Genética/métodos , AlelosRESUMO
Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant inherited disease characterized by progressive ataxia and retinal degeneration. SCA7 belongs to a group of neurodegenerative diseases caused by an expanded CAG repeat in the disease-causing gene, resulting in aberrant polyglutamine (polyQ) protein synthesis. PolyQ ataxin-7 is prone to aggregate in intracellular inclusions, perturbing cellular processes leading to neuronal death in specific regions of the central nervous system (CNS). Currently, there is no treatment for SCA7; however, a promising approach successfully applied to other polyQ diseases involves the clearance of polyQ protein aggregates through pharmacological activation of autophagy. Nonetheless, the blood-brain barrier (BBB) poses a challenge for delivering drugs to the CNS, limiting treatment effectiveness. This study aimed to develop a polymeric nanocarrier system to deliver therapeutic agents across the BBB into the CNS. We prepared poly(lactic-co-glycolic acid) nanoparticles (NPs) modified with Poloxamer188 and loaded with rapamycin to enable NPs to activate autophagy. We demonstrated that these rapamycin-loaded NPs were successfully taken up by neuronal and glial cells, demonstrating high biocompatibility without adverse effects. Remarkably, rapamycin-loaded NPs effectively cleared mutant ataxin-7 aggregates in a SCA7 glial cell model, highlighting their potential as a therapeutic approach to fight SCA7 and other polyQ diseases.
Assuntos
Ataxias Espinocerebelares , Humanos , Ataxina-7/genética , Ataxina-7/metabolismo , Ataxias Espinocerebelares/tratamento farmacológico , Ataxias Espinocerebelares/genética , Neurônios/metabolismo , Neuroglia/metabolismo , SirolimoRESUMO
Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7's yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation. Impairment of such regulation increases Sgf73's abundance, which enhances recruitment of TATA box-binding protein (TBP) (that nucleates PIC formation) to the promoter but impairs transcription elongation. Further, decreased Sgf73 level reduces PIC formation and transcription. Thus, Sgf73 is fine-tuned by ubiquitin-proteasome system (UPS) in orchestrating transcription. Likewise, ataxin-7 undergoes ubiquitylation and proteasomal degradation, alteration of which changes ataxin-7's abundance that is associated with altered transcription and cellular pathologies/diseases. Collectively, our results unveil a novel UPS regulation of Sgf73/ataxin-7 for normal cellular health and implicate alteration of such regulation in diseases.
Assuntos
Proteínas de Saccharomyces cerevisiae , Ubiquitina , Ataxina-7/genética , Ataxina-7/metabolismo , Ubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Histona Acetiltransferases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Spinocerebellar ataxia type 7 (SCA7) and other polyglutamine (polyQ) diseases are caused by expansions of polyQ repeats in disease-specific proteins. Aggregation of the polyQ proteins resulting in various forms of cellular stress, that could induce the stress granule (SG) response, is believed to be a common pathological mechanism in these disorders. SGs can contribute to cell survival but have also been suggested to exacerbate disease pathology by seeding protein aggregation. In this study, we show that two SG-related proteins, TDP-43 and TIA1, are sequestered into the aggregates formed by polyQ-expanded ATXN7 in SCA7 cells. Interestingly, mutant ATXN7 also localises to induced SGs, and this association altered the shape of the SGs. In spite of this, neither the ability to induce nor to disassemble SGs, in response to arsenite stress induction or relief, was affected in SCA7 cells. Moreover, we could not observe any change in the number of ATXN7 aggregates per cell following SG induction, although a small, non-significant, increase in total aggregated ATXN7 material could be detected using filter trap. However, mutant ATXN7 expression in itself increased the speckling of the SG-nucleating protein G3BP1 and the SG response. Taken together, our results indicate that the SG response is induced, and although some key modulators of SGs show altered behaviour, the dynamics of SGs appear normal in the presence of mutant ATXN7.
Assuntos
DNA Helicases , Ataxias Espinocerebelares , Ataxina-7/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ataxias Espinocerebelares/genética , Grânulos de Estresse , Antígeno-1 Intracelular de Células T/metabolismoRESUMO
BACKGROUND: Circular RNAs (circRNAs) participate in the occurrence and progression of many cancers. CircRNA ataxin 7 (circATXN7) (circBase ID: hsa_circ_0066436) plays a promoting influence on gastric cancer progression. However, the biological role of circATXN7 in non-small cell lung cancer (NSCLC) is indistinct. METHODS: Levels of circATXN7, microRNA (miR)-7-5p, and profilin 2 (PFN2) mRNA were detected using quantitative real-time polymerase chain reaction (RT-qPCR). Proliferation, apoptosis, metastasis, and invasion were analyzed using cell counting kit-8 (CCK-8), colony formation, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and transwell assays. Protein levels were analyzed using western blotting (WB) and immunohistochemistry (IHC). The relationship between circATXN7 or PFN2 and miR-7-5p was analyzed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The biological function of circATXN7 was verified by xenograft assay. RESULTS: CircATXN7 and PFN2 were highly expressed in NSCLC, whereas miR-7-5p expression had the opposite trend. CircATXN7 overexpression constrained apoptosis and promoted proliferation, metastasis, invasion, and epithelial-mesenchymal transition of NSCLC cells, but circATXN7 silencing played the opposing influence and repressed xenograft tumor growth in vivo. CircATXN7 served as a miR-7-5p sponge, and circATXN7 regulated malignant behaviors of NSCLC cells through sponging miR-7-5p. PFN2 acted as a miR-7-5p target. PFN2 silencing overturned the promoting effect of miR-7-5p inhibitor on NSCLC cell malignancy, while PFN2 overexpression reversed the inhibitory impact of miR-7-5p mimic on NSCLC cell malignancy. CONCLUSION: CircATXN7 accelerated the malignancy of NSCLC cells through adsorbing miR-7-5p and upregulating PFN2, offering evidence to support circATXN7 as a target for NSCLC treatment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Ataxina-7/genética , Ataxina-7/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/genética , Regulação para Baixo , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Profilinas/genética , Profilinas/metabolismo , RNA Circular/genéticaRESUMO
A common mechanism in inherited ataxia is a vulnerability of DNA damage. Spinocerebellar ataxia type 7 (SCA7) is a CAG-polyglutamine-repeat disorder characterized by cerebellar and retinal degeneration. Polyglutamine-expanded ataxin-7 protein incorporates into STAGA co-activator complex and interferes with transcription by altering histone acetylation. We performed chromatic immunoprecipitation sequencing ChIP-seq on cerebellum from SCA7 mice and observed increased H3K9-promoter acetylation in DNA repair genes, resulting in increased expression. After detecting increased DNA damage in SCA7 cells, mouse primary cerebellar neurons, and patient stem-cell-derived neurons, we documented reduced homology-directed repair (HDR) and single-strand annealing (SSA). To evaluate repair at endogenous DNA in native chromosome context, we modified linear amplification-mediated high-throughput genome-wide translocation sequencing and found that DNA translocations are less frequent in SCA7 models, consistent with decreased HDR and SSA. Altered DNA repair function in SCA7 may predispose the subject to excessive DNA damage, leading to neuron demise and highlights DNA repair as a therapy target.
Assuntos
Ataxina-7/metabolismo , Doenças Cerebelares/patologia , Reparo do DNA , Histonas/metabolismo , Neurônios/patologia , Peptídeos/genética , Ataxias Espinocerebelares/complicações , Acetilação , Animais , Ataxina-7/genética , Doenças Cerebelares/etiologia , Doenças Cerebelares/metabolismo , Feminino , Histonas/genética , Humanos , Masculino , Camundongos , Neurônios/metabolismoRESUMO
Polyglutamine (polyQ) diseases, such as Spinocerebellar ataxia type 7 (SCA7), are caused by expansions of polyQ repeats in disease specific proteins. The sequestration of vital proteins into aggregates formed by polyQ proteins is believed to be a common pathological mechanism in these disorders. The RNA-binding protein FUS has been observed in polyQ aggregates, though if disruption of this protein plays a role in the neuronal dysfunction in SCA7 or other polyQ diseases remains unclear. We therefore analysed FUS localisation and function in a stable inducible PC12 cell model expressing the SCA7 polyQ protein ATXN7. We found that there was a high degree of FUS sequestration, which was associated with a more cytoplasmic FUS localisation, as well as a decreased expression of FUS regulated mRNAs. In contrast, the role of FUS in the formation of γH2AX positive DNA damage foci was unaffected. In fact, a statistical increase in the number of γH2AX foci, as well as an increased trend of single and double strand DNA breaks, detected by comet assay, could be observed in mutant ATXN7 cells. These results were further corroborated by a clear trend towards increased DNA damage in SCA7 patient fibroblasts. Our findings suggest that both alterations in the RNA regulatory functions of FUS, and increased DNA damage, may contribute to the pathology of SCA7.
Assuntos
Ataxina-7/genética , Dano ao DNA , Proteína FUS de Ligação a RNA/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Ataxina-7/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Células PC12 , Peptídeos/química , Peptídeos/genética , Transporte Proteico , Ratos , Ataxias Espinocerebelares/genéticaRESUMO
Spinocerebellar ataxia type 7 (SCA7) is a rare autosomal dominant neurodegenerative disorder characterized by progressive neuronal loss in the cerebellum, brainstem, and retina, leading to cerebellar ataxia and blindness as major symptoms. SCA7 is due to the expansion of a CAG triplet repeat that is translated into a polyglutamine tract in ATXN7. Larger SCA7 expansions are associated with earlier onset of symptoms and more severe and rapid disease progression. Here, we summarize the pathological and genetic aspects of SCA7, compile the current knowledge about ATXN7 functions, and then focus on recent advances in understanding the pathogenesis and in developing biomarkers and therapeutic strategies. ATXN7 is a bona fide subunit of the multiprotein SAGA complex, a transcriptional coactivator harboring chromatin remodeling activities, and plays a role in the differentiation of photoreceptors and Purkinje neurons, two highly vulnerable neuronal cell types in SCA7. Polyglutamine expansion in ATXN7 causes its misfolding and intranuclear accumulation, leading to changes in interactions with native partners and/or partners sequestration in insoluble nuclear inclusions. Studies of cellular and animal models of SCA7 have been crucial to unveil pathomechanistic aspects of the disease, including gene deregulation, mitochondrial and metabolic dysfunctions, cell and non-cell autonomous protein toxicity, loss of neuronal identity, and cell death mechanisms. However, a better understanding of the principal molecular mechanisms by which mutant ATXN7 elicits neurotoxicity, and how interconnected pathogenic cascades lead to neurodegeneration is needed for the development of effective therapies. At present, therapeutic strategies using nucleic acid-based molecules to silence mutant ATXN7 gene expression are under development for SCA7.
Assuntos
Ataxina-7/genética , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos/tendências , Marcação de Genes/tendências , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/terapia , Animais , Ataxina-7/metabolismo , Autofagia/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Sistemas de Liberação de Medicamentos/métodos , Marcação de Genes/métodos , Terapia Genética/métodos , Terapia Genética/tendências , Humanos , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/genética , Peptídeos/metabolismo , Ataxias Espinocerebelares/metabolismoRESUMO
Atxn7, a subunit of SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the Atxn7 amino terminus anchors Non-stop, a deubiquitinase, to the complex. To understand the scope of Atxn7-dependent regulation of Non-stop, substrates of the deubiquitinase were sought. This revealed Non-stop, dissociated from Atxn7, interacts with Arp2/3 and WAVE regulatory complexes (WRC), which control actin cytoskeleton assembly. There, Non-stop countered polyubiquitination and proteasomal degradation of WRC subunit SCAR. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop augmentation increased protein levels, and directed subcellular localization, of SCAR, decreasing cell area and number of protrusions. In vivo, heterozygous mutation of SCAR did not significantly rescue knockdown of Atxn7, but heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR.
Assuntos
Citoesqueleto de Actina/metabolismo , Ataxina-7/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Endopeptidases/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Regulação da Expressão Gênica , Mapeamento de Interação de Proteínas , Mapas de Interação de ProteínasRESUMO
Ataxin-7 (Atx7) is a disease-related protein associated with the pathogenesis of spinocerebellar ataxia 7, while its polyglutamine (polyQ) tract in N-terminus is the causative source of aggregation and proteinopathy. We investigated the structure, dynamics and aggregation properties of the N-terminal 62-residue fragment of Atx7 (Atx7-N) by biochemical and biophysical approaches. The results showed that the normal Atx7-N with a tract of 10 glutamines (10Q) overall adopts a flexible and disordered structure, but it may contain a short or small population of helical structure in solution. PolyQ expansion increases the α-helical propensity of the polyQ tract and consequently enhances its transformation into ß-sheet structures during amyloid aggregation. An alanine-rich region (ARR) just ahead of the polyQ tract forms a local and relatively stable α-helix. The ARR α-helix can initiate and stabilize helical formation of the following polyQ tract, but it may suppress aggregation of the polyQ-expanded Atx7-N both in vitro and in cell. Thus, the preceding ARR segment in Atx7-N may influence the dynamic structure and aggregation property of the polyQ tract and even determine the threshold of the pathogenic polyQ lengths. This study may gain structural and dynamic insights into amyloid aggregation of Atx7 and help us further understand the Atx7 proteinopathy based on polyQ expansion.
Assuntos
Amiloide/química , Ataxina-7/química , Simulação de Dinâmica Molecular , Multimerização Proteica , Amiloide/metabolismo , Ataxina-7/metabolismo , Células HEK293 , Humanos , Peptídeos/química , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha betaRESUMO
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 FisiológicoRESUMO
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/metabolismoRESUMO
Attention-deficit/hyperactivity disorder (ADHD) is a heterogeneous neurodevelopmental disorder characterized by varying levels of hyperactivity, inattention, and impulsivity. Patients with ADHD are often classified as (1) predominantly hyperactive-impulsive, (2) predominantly inattentive, and (3) combined type. There is a growing interest in developing specific animal models that would recapitulate specific clinical forms of ADHD, with the goal of developing specific therapeutic strategies. In our previous study, we have identified Ataxin-7 (Atxn7) as a hyperactivity-associated gene. Here, we generated Atxn7 overexpressing (Atxn7 OE) mice to investigate whether the increased Atxn7 expression in the brain correlates with ADHD-like behaviors. Quantitative real-time polymerase chain reaction and immunofluorescence confirmed overexpression of the Atxn7 gene and protein in the prefrontal cortex (PFC) and striatum (STR) of the Atxn7 OE mice. The Atxn7 OE mice displayed hyperactivity and impulsivity, but not inattention. Interestingly, treatment with the ADHD drug, atomoxetine (3â¯mg/kg, intraperitoneal), attenuated ADHD-like behaviors and reduced Atxn7 gene expression in the PFC and STR of these mice. These findings suggest that Atxn7 plays a role in the pathophysiology of ADHD, and that the Atxn7 OE mice can be used as an animal model of the hyperactive-impulsive phenotype of this disorder. Although confirmatory studies are warranted, the present study provides valuable information regarding the potential genetic underpinnings of ADHD.
Assuntos
Inibidores da Captação Adrenérgica/uso terapêutico , Ataxina-7/metabolismo , Cloridrato de Atomoxetina/uso terapêutico , Hipercinese/tratamento farmacológico , Hipercinese/genética , Comportamento Impulsivo/efeitos dos fármacos , Animais , Ataxina-7/genética , Desvalorização pelo Atraso/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Comportamento Impulsivo/fisiologia , Locomoção/efeitos dos fármacos , Locomoção/genética , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Teste de Desempenho do Rota-Rod , Estatísticas não ParamétricasRESUMO
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder characterized by cerebellar and retinal degeneration, and is caused by a CAG-polyglutamine repeat expansion in the ATAXIN-7 gene. Patients with SCA7 develop progressive cone-rod dystrophy, typically resulting in blindness. Antisense oligonucleotides (ASOs) are single-stranded chemically modified nucleic acids designed to mediate the destruction, prevent the translation, or modify the processing of targeted RNAs. Here, we evaluated ASOs as treatments for SCA7 retinal degeneration in representative mouse models of the disease after injection into the vitreous humor of the eye. Using Ataxin-7 aggregation, visual function, retinal histopathology, gene expression, and epigenetic dysregulation as outcome measures, we found that ASO-mediated Ataxin-7 knockdown yielded improvements in treated SCA7 mice. In SCA7 mice with retinal disease, intravitreal injection of Ataxin-7 ASOs also improved visual function despite initiating treatment after symptom onset. Using color fundus photography and autofluorescence imaging, we also determined the nature of retinal degeneration in human SCA7 patients. We observed variable disease severity and cataloged rapidly progressive retinal degeneration. Given the accessibility of neural retina, availability of objective, quantitative readouts for monitoring therapeutic response, and the rapid disease progression in SCA7, ASOs targeting ATAXIN-7 might represent a viable treatment for SCA7 retinal degeneration.
Assuntos
Ataxina-7/metabolismo , Proteínas Mutantes/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ataxias Espinocerebelares/fisiopatologia , Visão Ocular/efeitos dos fármacos , Animais , Ataxina-7/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Injeções Intravítreas , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Peptídeos/metabolismo , Fenótipo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Agregados Proteicos/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/metabolismo , Degeneração Retiniana/complicações , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Ataxias Espinocerebelares/complicações , Ataxias Espinocerebelares/patologiaRESUMO
Myotonic dystrophy types 1 (DM1) and 2 (DM2) are dominantly inherited neuromuscular disorders caused by a toxic gain of function of expanded CUG and CCUG repeats, respectively. Although both disorders are clinically similar, congenital myotonic dystrophy (CDM), a severe DM form, is found only in DM1. CDM is also characterized by muscle fiber immaturity not observed in adult DM, suggesting specific pathological mechanisms. Here, we revealed upregulation of the interleukin-6 (IL-6) myokine signaling pathway in CDM muscles. We also found a correlation between muscle immaturity and not only IL-6 expression but also expanded CTG repeat length and CpG methylation status upstream of the repeats. Aberrant CpG methylation was associated with transcriptional dysregulation at the repeat locus, increasing the toxic RNA burden that upregulates IL-6. Because the IL-6 pathway is involved in myocyte maturation and muscle atrophy, our results indicate that enhanced RNA toxicity contributes to severe CDM phenotypes through aberrant IL-6 signaling.
Assuntos
Distrofia Miotônica/patologia , Transdução de Sinais/fisiologia , Processamento Alternativo , Ataxina-7/genética , Ataxina-7/metabolismo , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Ilhas de CpG , Metilação de DNA , Humanos , Lactente , Interleucina-6/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculos/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Miotonina Proteína Quinase/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica , Expansão das Repetições de Trinucleotídeos , Regulação para CimaRESUMO
Several studies suggest a strong genetic component of attention-deficit/hyperactivity disorder (ADHD), a complex neurodevelopmental disorder characterized by inappropriate levels of hyperactivity, impulsivity and inattention. Determining specific genetic risk variants for each symptom dimension of ADHD may aid in the identification of the biological risk factors of the disorder. In this study, we explored the potential genetic underpinnings of the hyperactive phenotype of ADHD. To this end, we examined differentially expressed genes (DEGs) in the prefrontal cortex (PFC) of SHR/NCrl, an animal model of ADHD, compared with its genetic control, the Wistar Kyoto (WKY/NCrl) rat and the Wistar rat, strain used to represent the 'normal' heterogeneous population. Relative to WKY/NCrl and Wistar controls, SHR/NCrl showed hyperactivity in the open-field test. Treatment with the ADHD drug, amphetamine (AMPH) reduced hyperactivity in SHR/NCrl. Meanwhile, AMPH increased locomotor activity in WKY/NCrl and Wistar rats. Gene expression analysis found 21 common upregulated and 36 downregulated genes in the PFC of drug-naive SHR/NCrl when compared with WKY/NCrl and Wistar rats. Of these DEGs, expression levels of two genes, Atxn7 and Per2, which are involved in transcription and circadian rhythm, respectively, were downregulated following AMPH treatment in SHR/NCrl. Quantitative real-time-polymerase chain reaction analyses verified expression patterns of these genes in the PFC of drug-naïve and AMPH-treated SHR/NCrl. The present findings indicate genetic risk variants that may be associated with the hyperactive phenotype in ADHD. Further studies are warranted to establish the roles of Atxn7 and Per2 in mediating hyperactivity.
Assuntos
Anfetamina/farmacologia , Transtorno do Deficit de Atenção com Hiperatividade/genética , Estimulantes do Sistema Nervoso Central/farmacologia , Córtex Pré-Frontal/metabolismo , Transcriptoma , Anfetamina/uso terapêutico , Animais , Ataxina-7/genética , Ataxina-7/metabolismo , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Estimulantes do Sistema Nervoso Central/uso terapêutico , Regulação para Baixo , Locomoção , Masculino , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos WistarRESUMO
BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a lethal disease with nearly equal morbidity and mortality. Thus, the discovery and application of more useful predictive biomarkers for improving therapeutic effects and prediction of clinical outcomes is of crucial significance. METHODS: A total of 475 HBV-related HCC patients were enrolled. Ataxin 7 (ATXN7) single nucleotide polymorphisms (SNPs) were genotyped by Sanger DNA sequencing after PCR amplification. The associations between ATXN7 SNPs and mRNA expression with the prognosis of HBV-related HCC were analyzed. RESULTS: In all, rs3774729 was significantly associated with overall survival (OS) of HBV-related HCC (P = 0.013, HR = 0.66, 95% CI: 0.48-0.94). And patients with the AA genotype and a high level of serum alpha fetoprotein (AFP) had significantly worse OS when compared to patients with AG/GG genotypes and a low level of AFP (adjusted P = 0.007, adjusted HR = 1.83, 95% CI = 1.18-2.82). Furthermore, low expression of ATXN7 was significantly associated with poor recurrence-free survival (RFS) and OS (P = 0.007, HR = 2.38, 95% CI = 1.27-4.45 and P = 0.025, HR = 1.75, 95% CI = 1.18-2.62). CONCLUSION: ATXN7 may be a potential predictor of post-operative prognosis of HBV-related HCC.
Assuntos
Ataxina-7/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Vírus da Hepatite B/fisiologia , Neoplasias Hepáticas/genética , Polimorfismo de Nucleotídeo Único/genética , Ataxina-7/metabolismo , Carcinoma Hepatocelular/sangue , Feminino , Estudos de Associação Genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resultado do Tratamento , alfa-Fetoproteínas/metabolismoRESUMO
BACKGROUND: We used lentiviral vectors (LVs) to generate a new SCA7 animal model overexpressing a truncated mutant ataxin-7 (MUT ATXN7) fragment in the mouse cerebellum, in order to characterize the specific neuropathological and behavioral consequences of the genetic defect in this brain structure. RESULTS: LV-mediated overexpression of MUT ATXN7 into the cerebellum of C57/BL6 adult mice induced neuropathological features similar to that observed in patients, such as intranuclear aggregates in Purkinje cells (PC), loss of synaptic markers, neuroinflammation, and neuronal death. No neuropathological changes were observed when truncated wild-type ataxin-7 (WT ATXN7) was injected. Interestingly, the local delivery of LV-expressing mutant ataxin-7 (LV-MUT-ATXN7) into the cerebellum of wild-type mice also mediated the development of an ataxic phenotype at 8 to 12 weeks post-injection. Importantly, our data revealed abnormal levels of the FUS/TLS, MBNL1, and TDP-43 RNA-binding proteins in the cerebellum of the LV-MUT-ATXN7 injected mice. MUT ATXN7 overexpression induced an increase in the levels of the pathological phosphorylated TDP-43, and a decrease in the levels of soluble FUS/TLS, with both proteins accumulating within ATXN7-positive intranuclear inclusions. MBNL1 also co-aggregated with MUT ATXN7 in most PC nuclear inclusions. Interestingly, no MBNL2 aggregation was observed in cerebellar MUT ATXN7 aggregates. Immunohistochemical studies in postmortem tissue from SCA7 patients and SCA7 knock-in mice confirmed SCA7-induced nuclear accumulation of FUS/TLS and MBNL1, strongly suggesting that these proteins play a physiopathological role in SCA7. CONCLUSIONS: This study validates a novel SCA7 mouse model based on lentiviral vectors, in which strong and sustained expression of MUT ATXN7 in the cerebellum was found sufficient to generate motor defects.
Assuntos
Ataxina-7/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ataxias Espinocerebelares/genética , Animais , Ataxina-7/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Humanos , Lentivirus/genética , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , FenótipoRESUMO
BACKGROUND: Fusion proteins have unique oncogenic properties and their identification can be useful either as diagnostic or therapeutic targets. Next generation sequencing data have previously shown a fusion gene formed between Rad51C and ATXN7 genes in the MCF7 breast cancer cell line. However, the existence of this fusion gene in colorectal patient tumor tissues is largely still unknown. METHODS: We evaluated for the presence of Rad51C-ATXN7 fusion gene in colorectal tumors and cells by RT-PCR, PCR, Topo TA cloning, Real time PCR, immunoprecipitation and immunoblotting techniques. RESULTS: We identified two forms of fusion mRNAs between Rad51C and ATXN7 in the colorectal tumors, including a Variant 1 (fusion transcript between Rad51C exons 1-7 and ATXN7 exons 6-13), and a Variant 2 (between Rad51C exons 1-6 and ATXN7 exons 6-13). In silico analysis showed that the Variant 1 produces a truncated protein, whereas the Variant 2 was predicted to produce a fusion protein with molecular weight of 110 KDa. Immunoprecipitation and Western blot analysis further showed a 110 KDa protein in colorectal tumors. 5-Azacytidine treatment of LS-174 T cells caused a 3.51-fold increase in expression of the fusion gene (Variant 2) as compared to no treatment controls evaluated by real time PCR. CONCLUSION: In conclusion we found a fusion gene between DNA repair gene Rad51C and neuro-cerebral ataxia Ataxin-7 gene in colorectal tumors. The in-frame fusion transcript of Variant 2 results in a fusion protein with molecular weight of 110 KDa. In addition, we found that expression of fusion gene is associated with functional impairment of Fanconi Anemia (FA) DNA repair pathway in colorectal tumors. The expression of Rad51C-ATXN7 in tumors warrants further investigation, as it suggests the potential of the fusion gene in treatment and predictive value in colorectal cancers.