Specific Biomarkers in Spinocerebellar Ataxia Type 3: A Systematic Review of Their Potential Uses in Disease Staging and Treatment Assessment.

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.

Production of Spinocerebellar Ataxia Type 3 Model Mice by Intravenous Injection of AAV-PHP.B Vectors.

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.

Trehalose prevents the formation of aggregates of mutant ataxin-3 and reduces soluble ataxin-3 protein levels in an SCA3 cell model.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by mutant ataxin-3 with an abnormally expanded polyQ tract and is the most common dominantly inherited ataxia worldwide. There are no suitable therapeutic options for this disease. Autophagy, a defense mechanism against the toxic effects of aggregation-prone misfolded proteins, has been shown to have beneficial effects on neurodegenerative diseases. Thus, trehalose, which is an autophagy inducer, may have beneficial effects on SCA3. In the present study, we examined the effects of trehalose on an SCA3 cell model. After trehalose treatment, aggregate formation, soluble ataxin-3 protein levels and cell viability were evaluated in HEK293T cells overexpressing ataxin-3-15Q or ataxin-3-77Q. We also explored the mechanism by which trehalose affects autophagy and stress pathways. A filter trap assay showed that trehalose decreased the number of aggregates formed by mutant ataxin-3 containing an expanded polyQ tract. Western blot and Cell Counting Kit-8 (CCK-8) results demonstrated that trehalose also reduced the ataxin-3 protein levels and was safe for ataxin-3-expressing cells, respectively. Western blot and total antioxidant capacity assays suggested that trehalose had great therapeutic potential for treating SCA3, likely through its antioxidant activity. Our data indicate that trehalose plays a neuroprotective role in SCA3 by inhibiting the aggregation and reducing the protein level of ataxin-3, which is also known to protect against oxidative stress. These findings provide a new insight into the possibility of treating SCA3 with trehalose and highlight the importance of inducing autophagy in SCA3.

Global DNA methylation is not elevated in blood samples from Machado-Joseph disease mutation carriers.

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia (SCA) caused by a polyglutamine expansion in the ataxin-3 protein, which initiates a cascade of pathogenic events, including transcriptional dysregulation. Genotype-phenotype correlations in MJD are incomplete, suggesting an influence of additional factors, such as epigenetic modifications, underlying the MJD pathogenesis. DNA methylation is known to impact the pathophysiology of neurodegenerative disorders through gene expression regulation and increased methylation has been reported for other SCAs. In this work we aimed to analyse global methylation in MJD carriers. Global 5-mC levels were quantified in blood samples of 33 MJD mutation carriers (patients and preclinical subjects) and 33 healthy controls, matched by age, sex, and smoking status. For a subset of 16 MJD subjects, a pilot follow-up analysis with two time points was also conducted. No differences were found in median global 5-mC levels between MJD mutation carriers and controls and no correlations between methylation levels and clinical or genetic variables were detected. Also, no alterations in global 5-mC levels were observed over time. Our findings do not support an increase in global blood methylation levels associated with MJD.

Endosome mediated nucleocytoplasmic trafficking and endomembrane allocation is crucial to polyglutamine toxicity.

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.

ATXN3 functions as a tumor suppressor through potentiating galectin-9-mediated apoptosis in human colon adenocarcinoma.

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.

VCP/p97 UFMylation stabilizes BECN1 and facilitates the initiation of autophagy.

Macroautophagy/autophagy is essential for the degradation and recycling of cytoplasmic materials. The initiation of this process is determined by phosphatidylinositol-3-kinase (PtdIns3K) complex, which is regulated by factor BECN1 (beclin 1). UFMylation is a novel ubiquitin-like modification that has been demonstrated to modulate several cellular activities. However, the role of UFMylation in regulating autophagy has not been fully elucidated. Here, we found that VCP/p97 is UFMylated on K109 by the E3 UFL1 (UFM1 specific ligase 1) and this modification promotes BECN1 stabilization and assembly of the PtdIns3K complex, suggesting a role for VCP/p97 UFMylation in autophagy initiation. Mechanistically, VCP/p97 UFMylation stabilizes BECN1 through ATXN3 (ataxin 3)-mediated deubiquitination. As a key component of the PtdIns3K complex, stabilized BECN1 facilitates assembly of this complex. Re-expression of VCP/p97, but not the UFMylation-defective mutant, rescued the VCP/p97 depletion-induced increase in MAP1LC3B/LC3B protein expression. We also showed that several pathogenic VCP/p97 mutations identified in a variety of neurological disorders and cancers were associated with reduced UFMylation, thus implicating VCP/p97 UFMylation as a potential therapeutic target for these diseases. Abbreviation: ATG14:autophagy related 14; Baf A1:bafilomycin A1;CMT2Y: Charcot-Marie-Toothdisease, axonal, 2Y; CYB5R3: cytochromeb5 reductase 3; DDRGK1: DDRGK domain containing 1; DMEM:Dulbecco'smodified Eagle's medium;ER:endoplasmic reticulum; FBS:fetalbovine serum;FTDALS6:frontotemporaldementia and/or amyotrophic lateral sclerosis 6; IBMPFD1:inclusion bodymyopathy with early-onset Paget disease with or withoutfrontotemporal dementia 1; LC-MS/MS:liquid chromatography tandem mass spectrometry; MAP1LC3B/LC3B:microtubule associated protein 1 light chain 3 beta; MS: massspectrometry; NPLOC4: NPL4 homolog, ubiquitin recognition factor;PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3;PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K:phosphatidylinositol 3-kinase; RPL26: ribosomal protein L26; RPN1:ribophorin I; SQSTM1/p62: sequestosome 1; UBA5: ubiquitin likemodifier activating enzyme 5; UFC1: ubiquitin-fold modifierconjugating enzyme 1; UFD1: ubiquitin recognition factor in ERassociated degradation 1; UFL1: UFM1 specific ligase 1; UFM1:ubiquitin fold modifier 1; UFSP2: UFM1 specific peptidase 2; UVRAG:UV radiation resistance associated; VCP/p97: valosin containingprotein; WT: wild-type.

Gene editing as a therapeutic strategy for spinocerebellar ataxia type-3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a neurodegenerative disease caused by expanded polyglutamine repeats in exon 10 of the ataxin-3 gene, ATXN3. The accumulation of mutant ATXN3 protein leads to severe clinical manifestations and premature death. Clinically, SCA3 pathology is characterized by progressive ataxia leading to motor incoordination that may affect balance, gait and speech, and neuropathologically by a progressive degeneration of the spinal cord and cerebellum, as well as the cerebral cortex and basal ganglia. Although SCA3 is a rare disease, it is the most common autosomal dominant spinocerebellar ataxia worldwide. Its geographical distribution varies worldwide, with peak prevalence in certain regions of Brazil, Portugal and China. In 1994, the identification of the polyglutamine expansion in the ATXN3 gene made it possible not only to diagnose this pathology but also to dissect the mechanisms leading to cellular degeneration. As a monogenic disease for which only symptomatic treatment is available, the ATXN3 gene represents an attractive therapeutic target for gene editing strategies.

The deubiquitinase function of ataxin-3 and its role in the pathogenesis of Machado-Joseph disease and other diseases.

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.

5-methylcytosine-mediated upregulation of circular RNA 0102913 augments malignant properties of colorectal cancer cells through a microRNA-571/Rac family small GTPase 2 axis.

Circular RNAs (circRNAs) are a class of stable non-coding RNAs that have emerged as key regulators in human diseases including cancer. This study investigates the role of circRNA_0102913 (circ_0102913) in malignant behavior of colorectal cancer (CRC) cells and the underpinning mechanisms. By analyzing CRC-related GSE197991, GSE159669, and GSE223001 datasets, we obtained circ_0102913 as an aberrantly upregulated circRNA in CRC. Increased circ_0102913 expression was detected in CRC tissues and cells. By querying multiple bioinformatics systems (circBank, Circular RNA Interactome, TargetScan, miRDIP, miRwalk, and miRDB), we identified microRNA-571 (miR-571) as a target of circ_0102913 and Rac family small GTPase 2 (RAC2) mRNA as a target of miR-571. Biotinylated-RNA pull-down and/or luciferase assays showed that circ_0102913 bound to miR-571 to restore the expression of RAC2 mRNA. Circ_0102913 silencing or miR-571 overexpression repressed proliferation, migration and invasion, and in vivo tumorigenesis abilities of CRC cells. However, the malignant properties of cells were restored by RAC2 overexpression. The increased circ_0102913 expression in CRC cells was attributed to increased 5-methylcytosine (m5C) modification levels. Silencing of NOP2/Sun RNA methyltransferase 5 reduced the m5C level and therefore reduced stability and expression of circ_0102913 expression in CRC cells. In conclusion, this study demonstrates that m5C-mediated upregulation of circ_0102913 augments malignant properties of CRC cells through a miR-571/RAC2 axis.

Treatment with sodium butyrate induces autophagy resulting in therapeutic benefits for spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. Mutation of ATXN3 causes formation of ataxin-3 protein aggregates, neurodegeneration, and motor deficits. Here we investigated the therapeutic potential and mechanistic activity of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model SCA3. SCA3 SH-SY5Y cells were found to contain high molecular weight ataxin-3 species and detergent-insoluble protein aggregates. Treatment with SB increased the activity of the autophagy protein quality control pathway in the SCA3 cells, decreased the presence of ataxin-3 aggregates and presence of high molecular weight ataxin-3 in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo, improving swimming performance, increasing activity of the autophagy pathway, and decreasing the presence of insoluble ataxin-3 protein species in the transgenic SCA3 zebrafish. Co-treating the SCA3 zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB on zebrafish swimming, indicating that the improved swimming performance was autophagy-dependent. To understand the mechanism by which SB induces autophagy we performed proteomic analysis of protein lysates from the SB-treated and untreated SCA3 SH-SY5Y cells. We found that SB treatment had increased activity of Protein Kinase A and AMPK signaling, with immunoblot analysis confirming that SB treatment had increased levels of AMPK protein and its substrates. Together our findings indicate that treatment with SB can increase activity of the autophagy pathway process and that this has beneficial effects in vitro and in vivo. While our results suggested that this activity may involve activity of a PKA/AMPK-dependent process, this requires further confirmation. We propose that treatment with sodium butyrate warrants further investigation as a potential treatment for neurodegenerative diseases underpinned by mechanisms relating to protein aggregation including SCA3.

Cell-Free and In Vivo Characterization of the Inhibitory Activity of Lavado Cocoa Flavanols on the Amyloid Protein Ataxin-3: Toward New Approaches against Spinocerebellar Ataxia Type 3.

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.

CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1-positive regulator for tumor immune evasion.

Regulation of tumoral PD-L1 expression is critical to advancing our understanding of tumor immune evasion and the improvement of existing antitumor immunotherapies. Herein, we describe a CRISPR-based screening platform and identified ATXN3 as a positive regulator for PD-L1 transcription. TCGA database analysis revealed a positive correlation between ATXN3 and CD274 in more than 80% of human cancers. ATXN3-induced Pd-l1 transcription was promoted by tumor microenvironmental factors, including the inflammatory cytokine IFN-γ and hypoxia, through protection of their downstream transcription factors IRF1, STAT3, and HIF-2α. Moreover, ATXN3 functioned as a deubiquitinase of the AP-1 transcription factor JunB, indicating that ATNX3 promotes PD-L1 expression through multiple pathways. Targeted deletion of ATXN3 in cancer cells largely abolished IFN-γ- and hypoxia-induced PD-L1 expression and consequently enhanced antitumor immunity in mice, and these effects were partially reversed by PD-L1 reconstitution. Furthermore, tumoral ATXN3 suppression improved the preclinical efficacy of checkpoint blockade antitumor immunotherapy. Importantly, ATXN3 expression was increased in human lung adenocarcinoma and melanoma, and its levels were positively correlated with PD-L1 as well as its transcription factors IRF1 and HIF-2α. Collectively, our study identifies what we believe to be a previously unknown deubiquitinase, ATXN3, as a positive regulator for PD-L1 transcription and provides a rationale for targeting ATXN3 to sensitize checkpoint blockade antitumor immunotherapy.

ATXN3 promotes proliferation, stemness and motility of clear cell renal cell carcinoma cells by regulating S100A8 ubiquitination.

Background: Clear cell renal cell carcinoma (ccRCC) is a dominant subtype of kidney cancer with a dismal outcome at advanced stages. Ataxin 3 (ATXN3) has been proven to play a cancer-promoting role in several tumors and is upregulated in the patients with renal cell carcinoma. Thus, the objective of this research is to examine the biological roles and underlying mechanisms of ATXN3 in ccRCC. Methods: Bioinformatics analysis was carried out to analyze ATXN3 expression in ccRCC tissues and patient survival. Gain- and loss-of-function assays were applied to explore the effect of ATXN3 on ccRCC cell malignant behavior in vitro. The effect of ATXN3 on the NF-κB pathway was assessed by Western blot and immunofluorescence staining. The binding between ATXN3 and S100A8 and the effect of ATXN3 on S100A8 ubiquitination were verified using coimmunoprecipitation. Results: ATXN3 was upregulated in ccRCC tissues and correlated with adverse patient outcome. ATXN3 overexpression facilitated the proliferation, stemness, invasion and migratory capacity of ccRCC cells, whereas silencing had the opposite effect. ATXN3 enhanced the activity of the NF-κB pathway. Silencing ATXN3 facilitated S100A8 ubiquitination. Rescue experiments demonstrated that S100A8 downregulation reversed the promoting effect of ATXN3 on malignant behavior and NF-κB pathway activation in ccRCC cells. Conclusion: ATXN3 exerts a cancer-promoting effect in ccRCC by regulating S100A8 ubiquitination. Therefore, targeting the ATXN3/S100A8/NF-κB axis may provide a novel underlying therapeutic strategy for ccRCC.

Involvement of Ataxin-3 (ATXN3) in the malignant progression of pancreatic cancer via deubiquitinating HDAC6.

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.

ATXN3 promotes prostate cancer progression by stabilizing YAP.

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.

Coaggregation of polyglutamine (polyQ) proteins is mediated by polyQ-tract interactions and impairs cellular proteostasis.

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.

Extracellular vesicle-based delivery of silencing sequences for the treatment of Machado-Joseph disease/spinocerebellar ataxia type 3.

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominantly inherited ataxia worldwide. It is caused by an over-repetition of the trinucleotide CAG within the ATXN3 gene, which confers toxic properties to ataxin-3 (ATXN3) species. RNA interference technology has shown promising therapeutic outcomes but still lacks a non-invasive delivery method to the brain. Extracellular vesicles (EVs) emerged as promising delivery vehicles due to their capacity to deliver small nucleic acids, such as microRNAs (miRNAs). miRNAs were found to be enriched into EVs due to specific signal motifs designated as ExoMotifs. In this study, we aimed at investigating whether ExoMotifs would promote the packaging of artificial miRNAs into EVs to be used as non-invasive therapeutic delivery vehicles to treat MJD/SCA3. We found that miRNA-based silencing sequences, associated with ExoMotif GGAG and ribonucleoprotein A2B1 (hnRNPA2B1), retained the capacity to silence mutant ATXN3 (mutATXN3) and were 3-fold enriched into EVs. Bioengineered EVs containing the neuronal targeting peptide RVG on the surface significantly decreased mutATXN3 mRNA in primary cerebellar neurons from MJD YAC 84.2 and in a novel dual-luciferase MJD mouse model upon daily intranasal administration. Altogether, these findings indicate that bioengineered EVs carrying miRNA-based silencing sequences are a promising delivery vehicle for brain therapy.

Autophagy Function and Benefits of Autophagy Induction in Models of Spinocerebellar Ataxia Type 3.

BACKGROUND: Spinocerebellar ataxia 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. The presence of this genetic expansion results in an ataxin-3 protein containing a polyglutamine repeat region, which renders the ataxin-3 protein aggregation prone. Formation of ataxin-3 protein aggregates is linked with neuronal loss and, therefore, the development of motor deficits. METHODS: Here, we investigated whether the autophagy protein quality control pathway, which is important in the process of protein aggregate removal, is impaired in a cell culture and zebrafish model of SCA3. RESULTS: We found that SH-SY5Y cells expressing human ataxin-3 containing polyglutamine expansion exhibited aberrant levels of autophagy substrates, including increased p62 and decreased LC3II (following bafilomycin treatment), compared to the controls. Similarly, transgenic SCA3 zebrafish showed signs of autophagy impairment at early disease stages (larval), as well as p62 accumulation at advanced age stages (18 months old). We then examined whether treating with compounds known to induce autophagy activity, would aid removal of human ataxin-3 84Q and improve the swimming of the SCA3 zebrafish larvae. We found that treatment with loperamide, trehalose, rapamycin, and MG132 each improved the swimming of the SCA3 zebrafish compared to the vehicle-treated controls. CONCLUSION: We propose that signs of autophagy impairment occur in the SH-SY5Y model of SCA3 and SCA3 zebrafish at larval and advanced age stages. Treatment of the larval SCA3 zebrafish with various compounds with autophagy induction capacity was able to produce the improved swimming of the zebrafish, suggesting the potential benefit of autophagy-inducing compounds for the treatment of SCA3.

Integrative analysis of small non-coding RNAs predicts a piRNA/miRNA-CCND1/BRAF/HRH1/ATXN3 regulatory circuit that drives oncogenesis in glioblastoma.

The high-grade astrocytoma, glioblastoma multiforme (GBM), is the most common primary tumour of the brain, known for being aggressive and developing drug resistance. The non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), have critical functions in tumorigenesis and cancer drug resistance. Hence, we profiled miRNAs, piRNAs, and genes in U-87 MG GBM cells by next-generation sequencing and performed target prediction, pathway enrichment, protein-protein interaction, co-expression studies, and qRT-PCR validations to predict their possible roles in the malignancy. The study identified 335 miRNAs, 665 piRNAs, and 4286 genes differentially expressed (DE) in GBM. Among them 128 DE genes (DEGs) were targeted by both miRNAs and piRNAs, while 1817 and 192 were targeted solely by miRNAs or piRNAs, respectively. Interestingly, all the DEG targets enriched in cancer processes were overexpressed in GBM. Among these, BRAF was solely targeted by two piRNAs and this was found to be co-expressed with 19 sole targets of 5 miRNAs, including CCND1, and both were found to regulate cell proliferation in cancer. We conjectured that upregulated HRH1 and ATXN3 were targeted by both piRNAs and miRNAs, and along with BRAF and CCND1 might induce cell proliferation in GBM through G-protein-coupled receptor or Akt signalling pathways due to downregulation of the respective targeting small RNAs. These targets were also linked to the progression and overall survival of GBM patients, suggesting that they could be used as biomarkers. Overall, this study has identified a few novel ncRNA targets, which might aid in a better understanding of GBM pathogenesis.