RESUMO
Spinocerebellar ataxia type 3 (SCA3) is the most common type of disease related to poly-glutamine (polyQ) repeats. Its hallmark pathology is related to the abnormal accumulation of ataxin 3 with a longer polyQ tract (polyQ-ATXN3). However, there are other mechanisms related to SCA3 progression that require identifying trait and state biomarkers for a more accurate diagnosis and prognosis. Moreover, the identification of potential pharmacodynamic targets and assessment of therapeutic efficacy necessitates valid biomarker profiles. The aim of this review was to identify potential trait and state biomarkers and their potential value in clinical trials. Our results show that, in SCA3, there are different fluid biomarkers involved in neurodegeneration, oxidative stress, metabolism, miRNA and novel genes. However, neurofilament light chain NfL and polyQ-ATXN3 stand out as the most prevalent in body fluids and SCA3 stages. A heterogeneity analysis of NfL revealed that it may be a valuable state biomarker, particularly when measured in plasma. Nonetheless, since it could be a more beneficial approach to tracking SCA3 progression and clinical trial efficacy, it is more convenient to perform a biomarker profile evaluation than to rely on only one.
Assuntos
Biomarcadores , Doença de Machado-Joseph , Humanos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Ataxina-3/genética , Ataxina-3/metabolismo , Proteínas de Neurofilamentos/metabolismo , Peptídeos/metabolismo , Progressão da Doença , Estresse OxidativoRESUMO
We aimed to produce a mouse model of spinocerebellar ataxia type 3 (SCA3) using the mouse blood-brain barrier (BBB)-penetrating adeno-associated virus (AAV)-PHP.B. Four-to-five-week-old C57BL/6 mice received injections of high-dose (2.0 × 1011 vg/mouse) or low-dose (5.0 × 1010 vg/mouse) AAV-PHP.B encoding a SCA3 causative gene containing abnormally long 89 CAG repeats [ATXN3(Q89)] under the control of the ubiquitous chicken ß-actin hybrid (CBh) promoter. Control mice received high doses of AAV-PHP.B encoding ATXN3 with non-pathogenic 15 CAG repeats [ATXN3(Q15)] or phosphate-buffered saline (PBS) alone. More than half of the mice injected with high doses of AAV-PHP.B encoding ATXN3(Q89) died within 4 weeks after the injection. No mice in other groups died during the 12-week observation period. Mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89) exhibited progressive motor uncoordination starting 4 weeks and a shorter stride in footprint analysis performed at 12 weeks post-AAV injection. Immunohistochemistry showed thinning of the molecular layer and the formation of nuclear inclusions in Purkinje cells from mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89). Moreover, ATXN3(Q89) expression significantly reduced the number of large projection neurons in the cerebellar nuclei to one third of that observed in mice expressing ATXN3(Q15). This AAV-based approach is superior to conventional methods in that the required number of model mice can be created simply by injecting AAV, and the expression levels of the responsible gene can be adjusted by changing the amount of AAV injected. Moreover, this method may be applied to produce SCA3 models in non-human primates.
Assuntos
Ataxina-3 , Dependovirus , Modelos Animais de Doenças , Vetores Genéticos , Doença de Machado-Joseph , Camundongos Endogâmicos C57BL , Animais , Dependovirus/genética , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/terapia , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Camundongos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Ataxina-3/genética , Ataxina-3/metabolismo , Injeções Intravenosas , Barreira Hematoencefálica/metabolismo , Regiões Promotoras GenéticasRESUMO
Aggregation of aberrant proteins is a common pathological hallmark in neurodegeneration such as polyglutamine (polyQ) and other repeat-expansion diseases. Here through overexpression of ataxin3 C-terminal polyQ expansion in Drosophila gut enterocytes, we generated an intestinal obstruction model of spinocerebellar ataxia type3 (SCA3) and reported a new role of nuclear-associated endosomes (NAEs)-the delivery of polyQ to the nucleoplasm. In this model, accompanied by the prominently increased RAB5-positive NAEs are abundant nucleoplasmic reticulum enriched with polyQ, abnormal nuclear envelope invagination, significantly reduced endoplasmic reticulum, indicating dysfunctional nucleocytoplasmic trafficking and impaired endomembrane organization. Consistently, Rab5 but not Rab7 RNAi further decreased polyQ-related NAEs, inhibited endomembrane disorganization, and alleviated disease model. Interestingly, autophagic proteins were enriched in polyQ-related NAEs and played non-canonical autophagic roles as genetic manipulation of autophagic molecules exhibited differential impacts on NAEs and SCA3 toxicity. Namely, the down-regulation of Atg1 or Atg12 mitigated while Atg5 RNAi aggravated the disease phenotypes both in Drosophila intestines and compound eyes. Our findings, therefore, provide new mechanistic insights and underscore the fundamental roles of endosome-centered nucleocytoplasmic trafficking and homeostatic endomembrane allocation in the pathogenesis of polyQ diseases.
Assuntos
Autofagia , Endossomos , Peptídeos , Animais , Peptídeos/metabolismo , Endossomos/metabolismo , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Transporte Ativo do Núcleo Celular , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/patologia , Enterócitos/metabolismo , Modelos Animais de Doenças , Ataxina-3/metabolismo , Ataxina-3/genética , Drosophila/metabolismoRESUMO
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is reported to be the most common type of autosomal dominant cerebellar ataxia (ADCA). SCA3 patients suffer from a progressive decline in motor coordination and other disease-associated symptoms. Moreover, recent studies have reported that SCA3 patients also exhibit symptoms of cerebellar cognitive affective syndrome (CCAS). We previously observed signs of CCAS in mouse model of SCA3. Particularly, SCA3-84Q mice suffer from anxiety, recognition memory decline, and also exhibit signs of low mood and aversion to activity. Here we studied the effect of long-term injections of SK channels activator chlorzoxazone (CHZ) together and separately with the folic acid (FA) on the cerebellar Purkinje cell (PC) firing and histology, and also on the motor and cognitive functions as well as mood alterations in SCA3-84Q hemizygous transgenic mice. We realized that both CHZ and CHZ-FA combination had similar positive effect on pure cerebellum impairments including PC firing precision, PC histology, and motor performance in SCA3-84Q mice. However, only the CHZ-FA combination, but not CHZ, had significantly ameliorated the signs of anxiety and depression, and also noticeably improved recognition memory in SCA3-84Q mice. Our results suggest that the combination therapy for both ataxia and non-motor symptoms is required for the complex treatment of ADCA.
Assuntos
Ansiedade , Clorzoxazona , Depressão , Modelos Animais de Doenças , Ácido Fólico , Doença de Machado-Joseph , Camundongos Transgênicos , Animais , Camundongos , Ansiedade/tratamento farmacológico , Ansiedade/fisiopatologia , Depressão/tratamento farmacológico , Depressão/genética , Depressão/fisiopatologia , Ácido Fólico/farmacologia , Ácido Fólico/administração & dosagem , Doença de Machado-Joseph/tratamento farmacológico , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/fisiopatologia , Doença de Machado-Joseph/patologia , Clorzoxazona/farmacologia , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Memória/efeitos dos fármacos , Humanos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Masculino , Ataxina-3/genética , Ataxina-3/metabolismoRESUMO
While deubiquitinase ATXN3 has been implicated as a potential oncogene in various types of human cancers, its role in colon adenocarcinoma remains understudied. Surprisingly, our findings demonstrate that ATXN3 exerts an antitumor effect in human colon cancers through potentiating Galectin-9-induced apoptosis. CRISPR-mediated ATXN3 deletion unexpectedly intensified colon cancer growth both in vitro and in xenograft colon cancers. At the molecular level, we identified ATXN3 as a bona fide deubiquitinase specifically targeting Galectin-9, as ATXN3 interacted with and inhibited Galectin-9 ubiquitination. Consequently, targeted ATXN3 ablation resulted in reduced Galectin-9 protein expression, thereby diminishing Galectin-9-induced colon cancer apoptosis and cell growth arrest. The ectopic expression of Galectin-9 fully reversed the growth of ATXN3-null colon cancer in mice. Furthermore, immunohistochemistry staining revealed a significant reduction in both ATXN3 and Galectin-9 protein expression, along with a positive correlation between them in human colon cancer. Our study identifies the first Galectin-9-specific deubiquitinase and unveils a tumor-suppressive role of ATXN3 in human colon cancer.
Assuntos
Adenocarcinoma , Apoptose , Ataxina-3 , Neoplasias do Colo , Galectinas , Humanos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/genética , Galectinas/metabolismo , Galectinas/genética , Animais , Ataxina-3/metabolismo , Ataxina-3/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/genética , Camundongos , Linhagem Celular Tumoral , Ubiquitinação , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas RepressorasRESUMO
Ribosome biogenesis is initiated in the nucleolus, a multiphase biomolecular condensate formed by liquid-liquid phase separation. The nucleolus is a powerful disease biomarker and stress biosensor whose morphology reflects function. Here we have used digital holographic microscopy (DHM), a label-free quantitative phase contrast microscopy technique, to detect nucleoli in adherent and suspension human cells. We trained convolutional neural networks to detect and quantify nucleoli automatically on DHM images. Holograms containing cell optical thickness information allowed us to define a novel index which we used to distinguish nucleoli whose material state had been modulated optogenetically by blue-light-induced protein aggregation. Nucleoli whose function had been impacted by drug treatment or depletion of ribosomal proteins could also be distinguished. We explored the potential of the technology to detect other natural and pathological condensates, such as those formed upon overexpression of a mutant form of huntingtin, ataxin-3, or TDP-43, and also other cell assemblies (lipid droplets). We conclude that DHM is a powerful tool for quantitatively characterizing nucleoli and other cell assemblies, including their material state, without any staining.
Assuntos
Nucléolo Celular , Holografia , Humanos , Nucléolo Celular/metabolismo , Holografia/métodos , Redes Neurais de Computação , Microscopia/métodos , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Ataxina-3/metabolismo , Ataxina-3/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Microscopia de Contraste de Fase/métodos , Imageamento Quantitativo de FaseRESUMO
Machado-Joseph disease (MJD) is a devastating and incurable neurodegenerative disease characterised by progressive ataxia, difficulty speaking and swallowing. Consequently, affected individuals ultimately become wheelchair dependent, require constant care, and face a shortened life expectancy. The monogenic cause of MJD is expansion of a trinucleotide (CAG) repeat region within the ATXN3 gene, which results in polyglutamine (polyQ) expansion within the resultant ataxin-3 protein. While it is well established that the ataxin-3 protein functions as a deubiquitinating (DUB) enzyme and is therefore critically involved in proteostasis, several unanswered questions remain regarding the impact of polyQ expansion in ataxin-3 on its DUB function. Here we review the current literature surrounding ataxin-3's DUB function, its DUB targets, and what is known regarding the impact of polyQ expansion on ataxin-3's DUB function. We also consider the potential neuroprotective effects of ataxin-3's DUB function, and the intersection of ataxin-3's role as a DUB enzyme and regulator of gene transcription. Ataxin-3 is the principal pathogenic protein in MJD and also appears to be involved in cancer. As aberrant deubiquitination has been linked to both neurodegeneration and cancer, a comprehensive understanding of ataxin-3's DUB function is important for elucidating potential therapeutic targets in these complex conditions. In this review, we aim to consolidate knowledge of ataxin-3 as a DUB and unveil areas for future research to aid therapeutic targeting of ataxin-3's DUB function for the treatment of MJD and other diseases.
Assuntos
Doença de Machado-Joseph , Neoplasias , Doenças Neurodegenerativas , Humanos , Ataxina-3/genética , Ataxina-3/metabolismo , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Doenças Neurodegenerativas/genéticaRESUMO
Spinocerebellar ataxia type 3 (SCA3) is the most common dominantly inherited ataxia. Currently, no preventive or disease-modifying treatments exist for this progressive neurodegenerative disorder, although efforts using gene silencing approaches are under clinical trial investigation. The disease is caused by a CAG repeat expansion in the mutant gene, ATXN3, producing an enlarged polyglutamine tract in the mutant protein. Similar to other paradigmatic neurodegenerative diseases, studies evaluating the pathogenic mechanism focus primarily on neuronal implications. Consequently, therapeutic interventions often overlook non-neuronal contributions to disease. Our lab recently reported that oligodendrocytes display some of the earliest and most progressive dysfunction in SCA3 mice. Evidence of disease-associated oligodendrocyte signatures has also been reported in other neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease. Here, we assess the effects of anti-ATXN3 antisense oligonucleotide (ASO) treatment on oligodendrocyte dysfunction in premanifest and symptomatic SCA3 mice. We report a severe, but modifiable, deficit in oligodendrocyte maturation caused by the toxic gain-of-function of mutant ATXN3 early in SCA3 disease that is transcriptionally, biochemically, and functionally rescued with anti-ATXN3 ASO. Our results highlight the promising use of an ASO therapy across neurodegenerative diseases that requires glial targeting in addition to affected neuronal populations.
Assuntos
Ataxina-3 , Modelos Animais de Doenças , Doença de Machado-Joseph , Oligodendroglia , Oligonucleotídeos Antissenso , Animais , Oligodendroglia/metabolismo , Camundongos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/terapia , Doença de Machado-Joseph/patologia , Doença de Machado-Joseph/metabolismo , Ataxina-3/genética , Ataxina-3/metabolismo , Humanos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Camundongos TransgênicosRESUMO
Spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) is a heritable proteinopathy disorder, whose causative gene, ATXN3, undergoes alternative splicing. Ataxin-3 protein isoforms differ in their toxicity, suggesting that certain ATXN3 splice variants may be crucial in driving the selective toxicity in SCA3. Using RNA-seq datasets we identified and determined the abundance of annotated ATXN3 transcripts in blood (n = 60) and cerebellum (n = 12) of SCA3 subjects and controls. The reference transcript (ATXN3-251), translating into an ataxin-3 isoform harbouring three ubiquitin-interacting motifs (UIMs), showed the highest abundance in blood, while the most abundant transcript in the cerebellum (ATXN3-208) was of unclear function. Noteworthy, two of the four transcripts that encode full-length ataxin-3 isoforms but differ in the C-terminus were strongly related with tissue expression specificity: ATXN3-251 (3UIM) was expressed in blood 50-fold more than in the cerebellum, whereas ATXN3-214 (2UIM) was expressed in the cerebellum 20-fold more than in the blood. These findings shed light on ATXN3 alternative splicing, aiding in the comprehension of SCA3 pathogenesis and providing guidance in the design of future ATXN3 mRNA-lowering therapies.
Assuntos
Doença de Machado-Joseph , Humanos , Doença de Machado-Joseph/metabolismo , Ataxina-3/genética , Ataxina-3/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Cerebelo/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant hereditary disorder, caused by an expansion of polyglutamine in the ataxin-3 protein. SCA3 symptoms include progressive motor decline caused by an atrophy of the cerebellum and brainstem. However, it was recently reported that SCA3 patients also suffer from the cerebellar cognitive affective syndrome. The majority of SCA3 patients exhibit cognitive decline and approximately half of them suffer from depression and anxiety. The necessity to find a combined therapy for both motor and cognitive deficits in a SCA3 mouse model is required for the development of SCA3 treatment. Here, we demonstrated that the SCA3-84Q transgenic mice exhibited anxiety over the novel brightly illuminated environment in the open field, novelty suppressed feeding, and light-dark place preference tests. Moreover, SCA3-84Q mice also suffered from a decline in recognition memory during the novel object recognition test. SCA3-84Q mice also demonstrated floating behavior during the Morris water maze that can be interpreted as a sign of low mood and aversion to activity, i.e. depressive-like state. SCA3-84Q mice also spent more time immobile during the forced swimming and tail suspension tests which is also evidence for depressive-like behavior. Therefore, the SCA3-84Q mouse model may be used as a model system to test the possible treatments for both ataxia and non-motor symptoms including depression, anxiety, and memory loss.
Assuntos
Doença de Machado-Joseph , Humanos , Camundongos , Animais , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Depressão/genética , Cerebelo/metabolismo , Ataxina-3/genética , Ataxina-3/metabolismo , Camundongos Transgênicos , Ansiedade/genéticaRESUMO
Spinocerebellar ataxia type 1 is caused by an expansion of the polyglutamine tract in ATAXIN-1. Ataxin-1 is broadly expressed throughout the brain and is involved in regulating gene expression. However, it is not yet known if mutant ataxin-1 can impact the regulation of alternative splicing events. We performed RNA sequencing in mouse models of spinocerebellar ataxia type 1 and identified that mutant ataxin-1 expression abnormally leads to diverse splicing events in the mouse cerebellum of spinocerebellar ataxia type 1. We found that the diverse splicing events occurred in a predominantly cell autonomous manner. A majority of the transcripts with misregulated alternative splicing events were previously unknown, thus allowing us to identify overall new biological pathways that are distinctive to those affected by differential gene expression in spinocerebellar ataxia type 1. We also provide evidence that the splicing factor Rbfox1 mediates the effect of mutant ataxin-1 on misregulated alternative splicing and that genetic manipulation of Rbfox1 expression modifies neurodegenerative phenotypes in a Drosophila model of spinocerebellar ataxia type 1 in vivo. Together, this study provides novel molecular mechanistic insight into the pathogenesis of spinocerebellar ataxia type 1 and identifies potential therapeutic strategies for spinocerebellar ataxia type 1.
Assuntos
Processamento Alternativo , Ataxias Espinocerebelares , Camundongos , Animais , Ataxina-1/genética , Ataxina-1/metabolismo , Processamento Alternativo/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Encéfalo/metabolismo , Ataxina-3/metabolismoRESUMO
Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by ataxia and other neurological manifestations, with a poor prognosis and a lack of effective therapies. The amyloid aggregation of the ataxin-3 protein is a hallmark of SCA3 and one of the main biochemical events prompting its onset, making it a prominent target for the development of preventive and therapeutic interventions. Here, we tested the efficacy of an aqueous Lavado cocoa extract and its polyphenolic components against ataxin-3 aggregation and neurotoxicity. The combination of biochemical assays and atomic force microscopy morphological analysis provided clear evidence of cocoa flavanols' ability to hinder ATX3 amyloid aggregation through direct physical interaction, as assessed by NMR spectroscopy. The chemical identity of the flavanols was investigated by ultraperformance liquid chromatography-high-resolution mass spectrometry. The use of the preclinical model Caenorhabditis elegans allowed us to demonstrate cocoa flavanols' ability to ameliorate ataxic phenotypes in vivo. To the best of our knowledge, Lavado cocoa is the first natural source whose extract is able to directly interfere with ATX3 aggregation, leading to the formation of off-pathway species.
Assuntos
Doença de Machado-Joseph , Animais , Ataxina-3/genética , Ataxina-3/metabolismo , Doença de Machado-Joseph/tratamento farmacológico , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Proteínas Amiloidogênicas/metabolismo , Amiloide/metabolismo , Caenorhabditis elegans , Polifenóis/uso terapêutico , Extratos Vegetais/farmacologiaRESUMO
Spinocerebellar ataxia type 3 (SCA3) is a genetic degeneration disease of the nervous system with ataxia as the main clinical manifestation, and the most frequent subtype of SCA3 is known to be caused by CAG repeat expansions of more than 55 units in ATXN3. In this study, we used peripheral blood mononuclear cells (PBMCs) from a volunteer with 14/63 CAG repeats in ATXN3 to generate induced pluripotent stem cells (iPSCs), which will be a good model for studying SCA3.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Machado-Joseph , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Ataxina-3/genética , Ataxina-3/metabolismo , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Mutação/genética , Voluntários , Proteínas Repressoras/genéticaRESUMO
BACKGROUND: Pancreatic cancer is a common digestive system cancer and one of the most lethal malignancies worldwide. Ataxin-3 (ATXN3) protein is a deubiquitinating enzyme implicated in the occurrence of diverse human cancers. The potential role of ATXN3 in pancreatic cancer still remains unclear. METHODS: ATXN3 was screened from differentially-upregulated genes of GSE71989, GSE27890 and GSE40098 datasets. The mRNA and protein levels of ATXN3 was evaluated in pancreatic cancer samples and cell lines. Through the gain- and loss-of-function experiments, the effects of ATXN3 on cell proliferation, migration and invasion were evaluated using cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) staining, wound healing and Transwell assays. Subsequently, the interaction between ATXN3 and HDAC6 was confirmed using double immunofluorescence staining, co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). The underlying mechanism of ATXN3 was determined by knockdown of HDAC6 in ATXN3-upregulated pancreatic cancer cells. The function of ATXN3 in vivo was verified through xenograft assay. RESULTS: High expression of ATXN3 was found in pancreatic cancer tissues. Increased ATXN3 expression dramatically promoted cell proliferation, migration, and invasion. The malignant phenotypes were suppressed in ATXN3-silenced pancreatic cancer cells. ATXN3 was proved to interact with HDAC6 and regulate its degradation through deubiquitination. Downregulation of HDAC6 inhibited ATXN3-induced development of pancreatic cancer cells through regulating the expression of PCNA, vimentin and E-cadherin. ATXN3 facilitated tumor growth of pancreatic cancer and increased HDAC6 expression in vivo. CONCLUSIONS: This study confirmed that ATXN3 facilitated malignant phenotypes of pancreatic cancer via reducing the ubiquitination of HDAC6.
Assuntos
Ataxina-3 , Desacetilase 6 de Histona , Neoplasias Pancreáticas , Humanos , Ataxina-3/genética , Ataxina-3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Repressoras/genética , Neoplasias PancreáticasRESUMO
Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by an abnormal polyglutamine expansion within the ataxin-3 protein (ATXN3). This leads to neurodegeneration of specific brain and spinal cord regions, resulting in a progressive loss of motor function. Despite neuronal death, non-neuronal cells, including astrocytes, are also involved in SCA3 pathogenesis. Astrogliosis is a common pathological feature in SCA3 patients and animal models of the disease. However, the contribution of astrocytes to SCA3 is not clearly defined. Inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is the predominant IP3R in mediating astrocyte somatic calcium signals, and genetically ablation of IP3R2 has been widely used to study astrocyte function. Here, we aimed to investigate the relevance of IP3R2 in the onset and progression of SCA3. For this, we tested whether IP3R2 depletion and the consecutive suppression of global astrocytic calcium signalling would lead to marked changes in the behavioral phenotype of a SCA3 mouse model, the CMVMJD135 transgenic line. This was achieved by crossing IP3R2 null mice with the CMVMJD135 mouse model and performing a longitudinal behavioral characterization of these mice using well-established motor-related function tests. Our results demonstrate that IP3R2 deletion in astrocytes does not modify SCA3 progression.
Assuntos
Doença de Machado-Joseph , Doenças Neurodegenerativas , Camundongos , Animais , Doença de Machado-Joseph/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Camundongos Transgênicos , Cálcio/metabolismo , Ataxina-3/genética , Ataxina-3/metabolismo , Camundongos Knockout , Modelos Animais de Doenças , Progressão da DoençaRESUMO
BACKGROUND: Prostate cancer (PC) is the most common neoplasm and is the second leading cause of cancer-related deaths in men worldwide. The Hippo tumor suppressor pathway is highly conserved in mammals and plays an important role in carcinogenesis. YAP is one of major key effectors of the Hippo pathway. However, the mechanism supporting abnormal YAP expression in PC remains to be characterized. METHODS: Western blot was used to measure the protein expression of ATXN3 and YAP, while the YAP target genes were measured by real-time PCR. CCK8 assay was used to detect cell viability; transwell invasion assay was used to measure the invasion ability of PC. The xeno-graft tumor model was used for in vivo study. Protein stability assay was used to detect YAP protein degradation. Immuno-precipitation assay was used to detect the interaction domain between YAP and ATXN3. The ubiquitin-based Immuno-precipitation assays were used to detect the specific ubiquitination manner happened on YAP. RESULTS: In the present study, we identified ATXN3, a DUB enzyme in the ubiquitin-specific proteases family, as a bona fide deubiquitylase of YAP in PC. ATXN3 was shown to interact with, deubiquitylate, and stabilize YAP in a deubiquitylation activity-dependent manner. Depletion of ATXN3 decreased the YAP protein level and the expression of YAP/TEAD target genes in PC, including CTGF, ANKRD1 and CYR61. Further mechanistic study revealed that the Josephin domain of ATXN3 interacted with the WW domain of YAP. ATXN3 stabilized YAP protein via inhibiting K48-specific poly-ubiquitination process on YAP protein. In addition, ATXN3 depletion significantly decreased PC cell proliferation, invasion and stem-like properties. The effects induced by ATXN3 depletion could be rescued by further YAP overexpression. CONCLUSIONS: In general, our findings establish a previously undocumented catalytic role for ATXN3 as a deubiquitinating enzyme of YAP and provides a possible target for the therapy of PC. Video Abstract.
Assuntos
Neoplasias da Próstata , Transdução de Sinais , Masculino , Animais , Humanos , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Neoplasias da Próstata/patologia , Via de Sinalização Hippo , Proliferação de Células , Mamíferos/metabolismo , Ataxina-3/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Nine polyglutamine (polyQ) proteins have already been identified that are considered to be associated with the pathologies of neurodegenerative disorders called polyQ diseases, but whether these polyQ proteins mutually interact and synergize in proteinopathies remains to be elucidated. In this study, 4 polyQ-containing proteins, androgen receptor (AR), ataxin-7 (Atx7), huntingtin (Htt) and ataxin-3 (Atx3), are used as model molecules to investigate their heterologous coaggregation and consequent impact on cellular proteostasis. Our data indicate that the N-terminal fragment of polyQ-expanded (PQE) Atx7 or Htt can coaggregate with and sequester AR and Atx3 into insoluble aggregates or inclusions through their respective polyQ tracts. In vitro coprecipitation and NMR titration experiments suggest that this specific coaggregation depends on polyQ lengths and is probably mediated by polyQ-tract interactions. Luciferase reporter assay shows that these coaggregation and sequestration effects can deplete the cellular availability of AR and consequently impair its transactivation function. This study provides valid evidence supporting the viewpoint that coaggregation of polyQ proteins is mediated by polyQ-tract interactions and benefits our understanding of the molecular mechanism underlying the accumulation of different polyQ proteins in inclusions and their copathological causes of polyQ diseases.
Assuntos
Doenças Neurodegenerativas , Proteostase , Humanos , Peptídeos/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Ataxina-3/genética , Ataxina-3/metabolismoRESUMO
Liquid-liquid phase separation is a phenomenon that features the formation of liquid droplets containing concentrated solutes. The droplets of neurodegeneration-associated proteins are prone to generate aggregates and cause diseases. To uncover the aggregation process from the droplets, it is necessary to analyze the protein structure with keeping the droplet state in a label-free manner, but there was no suitable method. In this study, we observed the structural changes of ataxin-3, a protein associated with Machado-Joseph disease, inside the droplets, using autofluorescence lifetime microscopy. Each droplet showed autofluorescence due to tryptophan (Trp) residues, and its lifetime increased with time, reflecting structural changes toward aggregation. We used Trp mutants to reveal the structural changes around each Trp and showed that the structural change consists of several steps on different timescales. We demonstrated that the present method visualizes the protein dynamics inside a droplet in a label-free manner. Further investigations revealed that the aggregate structure formed in the droplets differs from that formed in dispersed solutions and that a polyglutamine repeat extension in ataxin-3 hardly modulates the aggregation dynamics in the droplets. These findings highlight that the droplet environment facilitates unique protein dynamics different from those in solutions.
Assuntos
Doença de Machado-Joseph , Humanos , Ataxina-3/metabolismo , Doença de Machado-Joseph/metabolismoRESUMO
Machado-Joseph disease (MJD) or spinocerebellar ataxia 3 (SCA3) is a rare, inherited, monogenic, neurodegenerative disease, and the most common SCA worldwide. MJD/SCA3 causative mutation is an abnormal expansion of the triplet CAG at exon 10 within the ATXN3 gene. The gene encodes for ataxin-3, which is a deubiquitinating protein that is also involved in transcriptional regulation. In normal conditions, the ataxin-3 protein polyglutamine stretch has between 13 and 49 glutamines. However, in MJD/SCA3 patients, the size of the stretch increases from 55 to 87, contributing to abnormal protein conformation, insolubility, and aggregation. The formation of aggregates, which is a hallmark of MJD/SCA3, compromises different cell pathways, leading to an impairment of cell clearance mechanisms, such as autophagy. MJD/SCA3 patients display several signals and symptoms in which the most prominent is ataxia. Neuropathologically, the regions most affected are the cerebellum and the pons. Currently, there are no disease-modifying therapies, and patients rely only on supportive and symptomatic treatments. Due to these facts, there is a huge research effort to develop therapeutic strategies for this incurable disease. This review aims to bring together current state-of-the-art strategies regarding the autophagy pathway in MJD/SCA3, focusing on evidence for its impairment in the disease context and, importantly, its targeting for the development of pharmacological and gene-based therapies.
Assuntos
Doença de Machado-Joseph , Doenças Neurodegenerativas , Humanos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/terapia , Doença de Machado-Joseph/metabolismo , Ataxina-3/genética , Ataxina-3/metabolismo , Proteínas , Autofagia/genéticaRESUMO
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited neurodegenerative disease, which belongs to the trinucleotide repeat disease group with a CAG repeat expansion in exon 1 of the ATXN2 gene resulting in an ataxin-2 protein with an expanded polyglutamine (polyQ)-stretch. The disease is late manifesting leading to early death. Today, therapeutic interventions to cure the disease or even to decelerate disease progression are not available yet. Furthermore, primary readout parameter for disease progression and therapeutic intervention studies are limited. Thus, there is an urgent need for quantifiable molecular biomarkers such as ataxin-2 becoming even more important due to numerous potential protein-lowering therapeutic intervention strategies. The aim of this study was to establish a sensitive technique to measure the amount of soluble polyQ-expanded ataxin-2 in human biofluids to evaluate ataxin-2 protein levels as prognostic and/or therapeutic biomarker in SCA2. Time-resolved fluorescence energy transfer (TR-FRET) was used to establish a polyQ-expanded ataxin-2-specific immunoassay. Two different ataxin-2 antibodies and two different polyQ-binding antibodies were validated in three different concentrations and tested in cellular and animal tissue as well as in human cell lines, comparing different buffer conditions to evaluate the best assay conditions. We established a TR-FRET-based immunoassay for soluble polyQ-expanded ataxin-2 and validated measurements in human cell lines including iPSC-derived cortical neurons. Additionally, our immunoassay was sensitive enough to monitor small ataxin-2 expression changes by siRNA or starvation treatment. We successfully established the first sensitive ataxin-2 immunoassay to measure specifically soluble polyQ-expanded ataxin-2 in human biomaterials.