Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

A network of chaperones and ubiquitin ligases sustain intracellular proteostasis, and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases: Spinocerebellar ataxia Type 3 (SCA3) and Huntington's disease (HD), we aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. We report here that Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain is diminished when polyQ repeat proteins (Ataxin-3/Huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in transgenic Drosophila model of SCA3. Thus, our findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyglutamine aggregates.

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Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

10.1101/2020.12.13.417964 ◽

2020 ◽

Author(s):

Atin Kumar Mandal ◽

Baijayanti Ghosh ◽

Susnata Karmakar ◽

Mohit Prasad

Keyword(s):

Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

Spinocerebellar Ataxia Type ◽

Spinocerebellar Ataxia Type 3 ◽

Protein Levels ◽

Drosophila Model ◽

Polyq Protein ◽

Cytotoxic Stress ◽

Polyq Diseases ◽

Type 3

A network of chaperones and ubiquitin ligases sustain intracellular proteostasis, and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases: Spinocerebellar ataxia Type 3 (SCA3) and Huntingtons disease (HD), we aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. We report here that Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain is diminished when polyQ repeat proteins (Ataxin-3/Huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in transgenic Drosophila model of SCA3. Thus, our findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyglutamine aggregates.

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A fine balance between Prpf19 and Exoc7 in achieving degradation of aggregated protein and suppression of cell death in spinocerebellar ataxia type 3

Cell Death and Disease ◽

10.1038/s41419-021-03444-x ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Zhefan Stephen Chen ◽

Xiaoying Huang ◽

Kevin Talbot ◽

Ho Yin Edwin Chan

Keyword(s):

Spinocerebellar Ataxia ◽

E3 Ligase ◽

Spinocerebellar Ataxia Type ◽

Disease Genes ◽

Spinocerebellar Ataxia Type 3 ◽

E3 Ligases ◽

Protein Ubiquitination ◽

Polyq Protein ◽

Polyq Diseases ◽

Type 3

AbstractPolyglutamine (polyQ) diseases comprise Huntington’s disease and several subtypes of spinocerebellar ataxia, including spinocerebellar ataxia type 3 (SCA3). The genomic expansion of coding CAG trinucleotide sequence in disease genes leads to the production and accumulation of misfolded polyQ domain-containing disease proteins, which cause cellular dysfunction and neuronal death. As one of the principal cellular protein clearance pathways, the activity of the ubiquitin–proteasome system (UPS) is tightly regulated to ensure efficient clearance of damaged and toxic proteins. Emerging evidence demonstrates that UPS plays a crucial role in the pathogenesis of polyQ diseases. Ubiquitin (Ub) E3 ligases catalyze the transfer of a Ub tag to label proteins destined for proteasomal clearance. In this study, we identified an E3 ligase, pre-mRNA processing factor 19 (Prpf19/prp19), that modulates expanded ataxin-3 (ATXN3-polyQ), disease protein of SCA3, induced neurodegeneration in both mammalian and Drosophila disease models. We further showed that Prpf19/prp19 promotes poly-ubiquitination and degradation of mutant ATXN3-polyQ protein. Our data further demonstrated the nuclear localization of Prpf19/prp19 is essential for eliciting its modulatory function towards toxic ATXN3-polyQ protein. Intriguingly, we found that exocyst complex component 7 (Exoc7/exo70), a Prpf19/prp19 interacting partner, modulates expanded ATXN3-polyQ protein levels and toxicity in an opposite manner to Prpf19/prp19. Our data suggest that Exoc7/exo70 exerts its ATXN3-polyQ-modifying effect through regulating the E3 ligase function of Prpf19/prp19. In summary, this study allows us to better define the mechanistic role of Exoc7/exo70-regulated Prpf19/prp19-associated protein ubiquitination pathway in SCA3 pathogenesis.

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Silencing of genes responsible for polyQ diseases using chemically modified single-stranded siRNAs

Acta Biochimica Polonica ◽

10.18388/abp.2016_1336 ◽

2017 ◽

Vol 63 (4) ◽

Cited By ~ 4

Author(s):

Agnieszka Fiszer ◽

Marianna E Ellison-Klimontowicz ◽

Wlodzimierz J Krzyzosiak

Keyword(s):

Genetic Disorders ◽

Mutant Gene ◽

Cag Repeat ◽

Spinocerebellar Ataxia Type ◽

Spinocerebellar Ataxia Type 3 ◽

Mutation Site ◽

Protein Levels ◽

Cellular Models ◽

Polyq Diseases ◽

Type 3

Polyglutamine (polyQ) diseases comprise a group of nine genetic disorders that are caused by the expansion of the CAG triplet repeat, which encodes glutamine, in unrelated single genes. The pathogenesis is caused by the disruption of cellular pathways by the expression products of the mutant gene, i.e., proteins containing polyQ tracts and mutant transcripts. In considering oligonucleotide (ON)-based therapeutic approaches for polyQ diseases, the very attractive CAG repeat-targeting strategy offers selective silencing of the mutant allele by directly targeting the mutation site. CAG repeat-targeting miRNA-like siRNAs have been shown to specifically inhibit mutant gene expression, and their characteristic feature is the formation of mismatches in their interactions with the target site. Here, we designed novel single-stranded siRNAs that contain base substitutions and chemical modifications and tested these oligonucleotides in cellular models of Huntington’s disease (HD), spinocerebellar ataxia type 3 (SCA3) and dentatorubral-pallidoluysian atrophy (DRPLA), including HD mouse striatal cells. Selected siRNAs caused the efficient and selective downregulation of the mutant protein levels.

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DNAzyme Cleavage of CAG Repeat RNA in Polyglutamine Diseases

Neurotherapeutics ◽

10.1007/s13311-021-01075-w ◽

2021 ◽

Author(s):

Nan Zhang ◽

Brittani Bewick ◽

Jason Schultz ◽

Anjana Tiwari ◽

Robert Krencik ◽

...

Keyword(s):

Spinocerebellar Ataxia ◽

Cag Repeat ◽

Spinocerebellar Ataxia Type ◽

Spinocerebellar Ataxia Type 3 ◽

Therapeutic Value ◽

Expanded Allele ◽

Allele Specific ◽

Aggresome Formation ◽

Polyq Diseases ◽

Type 3

AbstractCAG repeat expansion is the genetic cause of nine incurable polyglutamine (polyQ) diseases with neurodegenerative features. Silencing repeat RNA holds great therapeutic value. Here, we developed a repeat-based RNA-cleaving DNAzyme that catalyzes the destruction of expanded CAG repeat RNA of six polyQ diseases with high potency. DNAzyme preferentially cleaved the expanded allele in spinocerebellar ataxia type 1 (SCA1) cells. While cleavage was non-allele-specific for spinocerebellar ataxia type 3 (SCA3) cells, treatment of DNAzyme leads to improved cell viability without affecting mitochondrial metabolism or p62-dependent aggresome formation. DNAzyme appears to be stable in mouse brain for at least 1 month, and an intermediate dosage of DNAzyme in a SCA3 mouse model leads to a significant reduction of high molecular weight ATXN3 proteins. Our data suggest that DNAzyme is an effective RNA silencing molecule for potential treatment of multiple polyQ diseases.

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Inhalational Aesthetic Sevoflurane Exacerbates Eye Phenotype of SCA3 Transgenic Drosophila Model

10.21203/rs.2.14482/v1 ◽

2019 ◽

Author(s):

Chia-Wen Chen ◽

Wei-Yong Lin ◽

Jack Cheng ◽

Kuen-Bao Chen ◽

Yu-Cheng Kuo ◽

...

Keyword(s):

Motor Function ◽

Cell Number ◽

Retinal Cell ◽

Spinocerebellar Ataxia Type ◽

Potential Hazard ◽

Spinocerebellar Ataxia Type 3 ◽

Drosophila Model ◽

Type 3 ◽

Transgenic Drosophila ◽

Transgenic Flies

Abstract Background: Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant inherited neurodegenerative disease. The features of SCA3 include extremely short life expectancies, motor functions, and eye phenotypes. Sevoflurane is one of the most frequently used inhalational anesthetics and shows both neuroprotective and neurotoxic effects. Previous studies showed neurotoxicity of sevoflurane exposure to Alzheimer’s disease models. However, the effect of sevoflurane inhalation on SCA3 is not clear. Materials and Methods: Here, we exposed sevoflurane to SCA3-transgenic Drosophila model with clinically relevant concentrations and observed the consequent change of survival, motor function, and eye phenotype of the flies. Results: We found that sevoflurane exposure exacerbated eye phenotype but not survival or motor function of male SCA3-transgenic flies. The percentage of ommatidium retinal cell number of male SCA3-transgenic flies with 0%, 2.1%, or 3% of sevoflurane exposure was 70.2 ± 4.8%, 64.8 ± 4.5%, or 46.8 ± 2.9% respectively (ANOVA F = 27.86, total df = 10, p = 0.0002), while sevoflurane exposure did not show any harm to the control flies. Conclusions: Our results may acknowledge the need for caution of the potential hazard of sevoflurane application on patients with SCA3 or other poly-Q related neurodegenerative diseases.

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CRISPR/Cas9 mediated gene correction ameliorates abnormal phenotypes in spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cells

Translational Psychiatry ◽

10.1038/s41398-021-01605-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lang He ◽

Shang Wang ◽

Linliu Peng ◽

Huifang Zhao ◽

Shuai Li ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Spinocerebellar Ataxia Type ◽

Spinocerebellar Ataxia Type 3 ◽

Induced Pluripotent ◽

Polyq Diseases ◽

Type 3 ◽

Hr Strategy

AbstractSpinocerebellar ataxia type 3/Machado–Joseph disease (SCA3/MJD) is a progressive autosomal dominant neurodegenerative disease caused by abnormal CAG repeats in the exon 10 of ATXN3. The accumulation of the mutant ataxin-3 proteins carrying expanded polyglutamine (polyQ) leads to selective degeneration of neurons. Since the pathogenesis of SCA3 has not been fully elucidated, and no effective therapies have been identified, it is crucial to investigate the pathogenesis and seek new therapeutic strategies of SCA3. Induced pluripotent stem cells (iPSCs) can be used as the ideal cell model for the molecular pathogenesis of polyQ diseases. Abnormal CAG expansions mediated by CRISPR/Cas9 genome engineering technologies have shown promising potential for the treatment of polyQ diseases, including SCA3. In this study, SCA3-iPSCs can be corrected by the replacement of the abnormal CAG expansions (74 CAG) with normal repeats (17 CAG) using CRISPR/Cas9-mediated homologous recombination (HR) strategy. Besides, corrected SCA3-iPSCs retained pluripotent and normal karyotype, which can be differentiated into a neural stem cell (NSCs) and neuronal cells, and maintained electrophysiological characteristics. The expression of differentiation markers and electrophysiological characteristics were similar among the neuronal differentiation from normal control iPSCs (Ctrl-iPSCs), SCA3-iPSCs, and isogenic control SCA3-iPSCs. Furthermore, this study proved that the phenotypic abnormalities in SCA3 neurons, including aggregated IC2-polyQ protein, decreased mitochondrial membrane potential (MMP) and glutathione expressions, increased reactive oxygen species (ROS), intracellular Ca2+ concentrations, and lipid peroxidase malondialdehyde (MDA) levels, all were rescued in the corrected SCA3-NCs. For the first time, this study demonstrated the feasibility of CRISPR/Cas9-mediated HR strategy to precisely repair SCA3-iPSCs, and reverse the corresponding abnormal disease phenotypes. In addition, the importance of genetic control using CRISPR/Cas9-mediated iPSCs for disease modeling. Our work may contribute to providing a potential ideal model for molecular mechanism research and autologous stem cell therapy of SCA3 or other polyQ diseases, and offer a good gene therapy strategy for future treatment.

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