scholarly journals The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity

2021 ◽  
Author(s):  
Carley Snoznik ◽  
Valentina Medvedeva ◽  
Jelena Mojsilovic-Petrovic ◽  
Paige Rudich ◽  
James Oosten ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Repeat Expansion ◽  
Dipeptide Repeat Proteins ◽  
C Elegans ◽  
Repeat Proteins ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

AbstractA hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs (PR)50 and (GR)50 are highly toxic when expressed in C. elegans and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNAi screen for suppressors of (PR)50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by (PR)50 expression. All of these genes have vertebrate homologs and 7/12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for (GR)50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that genetic inhibition of bet-1, the C. elegans homolog of the known SPOP ubiquitination targets BRD2/3/4, suppresses the protective effect of SPOP mutations. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent a novel entry point for therapeutic intervention to treat C9 FTD/ALS.Significance statementThe G4C2 repeat expansion in the C9orf72 gene is a major cause of Fronto-Temporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Unusual translation of the repeat sequence produces two highly toxic dipeptide repeat proteins, PRX and GRX, which accumulate in the brain tissue of individuals with these diseases. Here, we show that PR and GR toxicity in both C. elegans and mammalian neurons depends on the E3 ubiquitin ligase adaptor SPOP. SPOP acts through the bromodomain protein BET-1 to mediate dipeptide toxicity. SPOP inhibitors, which are currently being developed to treat SPOP-dependent renal cancer, also protect neurons against DPR toxicity. Our findings identify a highly conserved and ‘druggable’ pathway that may represent a new strategy for treating these currently incurable diseases.

scholarly journals Modeling C9orf72-Related Frontotemporal Dementia and Amyotrophic Lateral Sclerosis in Drosophila

2021 ◽  
Vol 15 ◽  
Author(s):  
Joanne L. Sharpe ◽  
Nikki S. Harper ◽  
Duncan R. Garner ◽  
Ryan J. H. West
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Frontotemporal Dementia ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Dipeptide Repeat Proteins ◽  
Repeat Proteins ◽  
Individual Contributions ◽  
The Individual ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

An intronic hexanucleotide (GGGGCC) expansion in the C9orf72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In the decade following its discovery, much progress has been made in enhancing our understanding of how it precipitates disease. Both loss of function caused by reduced C9orf72 transcript levels, and gain of function mechanisms, triggered by the production of repetitive sense and antisense RNA and dipeptide repeat proteins, are thought to contribute to the toxicity. Drosophila models, with their unrivaled genetic tractability and short lifespan, have played a key role in developing our understanding of C9orf72-related FTD/ALS. There is no C9orf72 homolog in fly, and although this precludes investigations into loss of function toxicity, it is useful for elucidating mechanisms underpinning gain of function toxicity. To date there are a range of Drosophila C9orf72 models, encompassing different aspects of gain of function toxicity. In addition to pure repeat transgenes, which produce both repeat RNA and dipeptide repeat proteins (DPRs), RNA only models and DPR models have been generated to unpick the individual contributions of RNA and each dipeptide repeat protein to C9orf72 toxicity. In this review, we discuss how Drosophila models have shaped our understanding of C9orf72 gain of function toxicity, and address opportunities to utilize these models for further research.

scholarly journals SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72

2021 ◽  
Vol 134 (4) ◽  
pp. jcs256602 ◽  
Author(s):  
Mirjana Malnar ◽  
Boris Rogelj
Keyword(s):  
Antisense Rna ◽  
Rna Binding ◽  
Dipeptide Repeat Proteins ◽  
Rna Transcripts ◽  
Repeat Proteins ◽  
Rna Foci ◽  
Hek Cells ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

ABSTRACTThe expanded GGGGCC repeat mutation in the C9orf72 gene is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion is transcribed to sense and antisense RNA, which form RNA foci and bind cellular proteins. This mechanism of action is considered cytotoxic. Translation of the expanded RNA transcripts also leads to the accumulation of toxic dipeptide repeat proteins (DPRs). The RNA-binding protein splicing factor proline and glutamine rich (SFPQ), which is being increasingly associated with ALS and FTD pathology, binds to sense RNA foci. Here, we show that SFPQ plays an important role in the C9orf72 mutation. Overexpression of SFPQ resulted in higher numbers of both sense and antisense RNA foci and DPRs in transfected human embryonic kidney (HEK) cells. Conversely, reduced SPFQ levels resulted in lower numbers of RNA foci and DPRs in both transfected HEK cells and C9orf72 mutation-positive patient-derived fibroblasts and lymphoblasts. Therefore, we have revealed a role of SFPQ in regulating the C9orf72 mutation that has implications for understanding and developing novel therapeutic targets for ALS and FTD.This article has an associated First Person interview with the first author of the paper.

scholarly journals Type I PRMT inhibition protects against C9ORF72 arginine-rich dipeptide repeat toxicity

2020 ◽  
Author(s):  
Alan S. Premasiri ◽  
Anna L. Gill ◽  
Fernando G. Vieira
Keyword(s):  
Repeat Expansion ◽  
Type I ◽  
Arginine Methyltransferase ◽  
Repeat Proteins ◽  
C9orf72 Gene ◽  
Expansion Mutation ◽  
Determining Factor ◽  
Lateral Sclerosis ◽  
Ran Translation ◽  
Dipeptide Repeat

ABSTRACTThe most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a repeat expansion mutation in the C9orf72 gene. Repeat-associated non-AUG (RAN) translation of this expansion produces five species of dipeptide repeat proteins (DRPs). The arginine containing DRPs, polyGR and polyPR, are consistently reported to be the most toxic. Here, we uncover Type I protein arginine methyltransferase (PRMT) inhibitors as possible therapeutics for polyGR- and polyPR- related toxicity. Furthermore, we reveal data that suggest that asymmetric dimethylation (ADMe) of polyGR is a determining factor in its pathogenesis.

Ribosome Profiling Reveals Novel Regulation of C9ORF72 GGGGCC Repeat-Containing RNA Translation

RNA ◽  
2021 ◽  
pp. rna.078963.121
Author(s):  
Heleen M van 't Spijker ◽  
Emily E Stackpole ◽  
Sandra Almeida ◽  
Olga Katsara ◽  
Botao Liu ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Translation Initiation ◽  
Repeat Expansion ◽  
Ribosome Profiling ◽  
Initiation Factor ◽  
Rna Translation ◽  
First Intron ◽  
Translational Mechanisms ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5’ cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

scholarly journals C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Hiroaki Suzuki ◽  
Yoshio Shibagaki ◽  
Seisuke Hattori ◽  
Masaaki Matsuoka
Keyword(s):  
Dna Binding Protein ◽  
Repeat Expansion ◽  
Hexanucleotide Repeat ◽  
Repeat Proteins ◽  
Non Coding Rna ◽  
Heterogeneous Nuclear Ribonucleoproteins ◽  
Hexanucleotide Repeat Expansion ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

Abstract A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce five dipeptide repeat proteins (DPRs). Although DPRs are thought to be neurotoxic, the molecular mechanism underlying the DPR-caused neurotoxicity has not been fully elucidated. The current study shows that poly-proline-arginine (poly-PR), the most toxic DPR in vitro, binds to and up-regulates nuclear paraspeckle assembly transcript 1 (NEAT1) that plays an essential role as a scaffold non-coding RNA during the paraspeckle formation. The CRISPR-assisted up-regulation of endogenous NEAT1 causes neurotoxicity. We also show that the poly-PR modulates the function of several paraspeckle-localizing heterogeneous nuclear ribonucleoproteins. Furthermore, dysregulated expression of TAR DNA-binding protein 43 (TDP-43) up-regulates NEAT1 expression and induces neurotoxicity. These results suggest that the increase in the paraspeckle formation may be involved in the poly-PR- and TDP-43-mediated neurotoxicity.

scholarly journals Development of a sensitive trial-ready poly(GP) CSF biomarker assay for C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis

2021 ◽  
Author(s):  
Katherine M Wilson ◽  
Eszter Katona ◽  
Idoia Glaria ◽  
Imogen J Swift ◽  
Aitana Sogorb-Esteve ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Amyotrophic Lateral Sclerosis ◽  
Frontotemporal Dementia ◽  
Single Molecule ◽  
Repeat Expansion ◽  
Target Engagement ◽  
Limit Of Quantification ◽  
Highly Sensitive ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. We utilised the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the repeat expansion in CSF of people with C9orf72-associated FTD/ALS. We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intra- and inter-plate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) DPRs in the CSF of samples collected through the Genetic FTD Initiative. We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had 8-fold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.

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