Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis

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.

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

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.770937 ◽

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.

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

Journal of Cell Science ◽

10.1242/jcs.256602 ◽

2021 ◽

Vol 134 (4) ◽

pp. jcs256602 ◽

Cited By ~ 1

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.

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Hippocampal protein aggregation signatures fully distinguish pathogenic and wildtype UBQLN2 in amyotrophic lateral sclerosis

10.1101/2022.01.12.475792 ◽

2022 ◽

Author(s):

Kyrah M Thumbadoo ◽

Birger V Dieriks ◽

Helen C Murray ◽

Molly EV Swanson ◽

Ji Hun Yoo ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Protein Aggregation ◽

Gene Mutations ◽

Repeat Proteins ◽

Familial Als ◽

Fluorescent Immunohistochemistry ◽

Self Aggregation ◽

Lateral Sclerosis ◽

Dipeptide Repeat

Mutations in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD) characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, cerebellum, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial ALS or FTD cases not caused by UBQLN2 mutations, particularly C9ORF72-linked cases. This makes the mechanistic role of ubiquilin 2 mutations and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 31 genotypically diverse ALS cases with or without FTD, including four cases with UBQLN2 mutations (resulting in p.P497H, p.P506S, and two cases with p.T487I). Using double-, triple-, and six-label fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43 (pTDP-43), dipeptide repeat aggregates, and p62, in the hippocampus of controls (n=5), or ALS with or without FTD in sporadic (n=19), unknown familial (n=3), SOD1-linked (n=1), C9ORF72-linked (n=4), and UBQLN2-linked (n=4) cases. We differentiate between i) ubiquilin 2 aggregation together with, or driven by, pTDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation driven by UBQLN2 gene mutations. Together we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in ALS with or without FTD, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene mutations and to understand the mechanisms of UBQLN2-linked disease.

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Proline/arginine dipeptide repeat polymers derail protein folding in amyotrophic lateral sclerosis

Nature Communications ◽

10.1038/s41467-021-23691-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Maria Babu ◽

Filippo Favretto ◽

Alain Ibáñez de Opakua ◽

Marija Rankovic ◽

Stefan Becker ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Protein Folding ◽

Neurodegenerative Diseases ◽

Catalyst Activity ◽

Coding Region ◽

Prolyl Isomerase ◽

X Ray Crystallography ◽

Hexanucleotide Repeat ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractAmyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with overlapping clinical features and the pathological hallmark of cytoplasmic deposits of misfolded proteins. The most frequent cause of familial forms of these diseases is a hexanucleotide repeat expansion in the non-coding region of the C9ORF72 gene that is translated into dipeptide repeat polymers. Here we show that proline/arginine repeat polymers derail protein folding by sequestering molecular chaperones. We demonstrate that proline/arginine repeat polymers inhibit the folding catalyst activity of PPIA, an abundant molecular chaperone and prolyl isomerase in the brain that is altered in amyotrophic lateral sclerosis. NMR spectroscopy reveals that proline/arginine repeat polymers bind to the active site of PPIA. X-ray crystallography determines the atomic structure of a proline/arginine repeat polymer in complex with the prolyl isomerase and defines the molecular basis for the specificity of disease-associated proline/arginine polymer interactions. The combined data establish a toxic mechanism that is specific for proline/arginine dipeptide repeat polymers and leads to derailed protein homeostasis in C9orf72-associated neurodegenerative diseases.

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Methylation of C9orf72 expansion reduces RNA foci formation and dipeptide-repeat proteins expression in cells

Neuroscience Letters ◽

10.1016/j.neulet.2015.12.018 ◽

2016 ◽

Vol 612 ◽

pp. 204-209 ◽

Cited By ~ 16

Author(s):

Peter O. Bauer

Keyword(s):

C9orf72 Expansion ◽

Dipeptide Repeat Proteins ◽

Repeat Proteins ◽

Rna Foci ◽

Dipeptide Repeat ◽

In Cells

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Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

10.1101/2020.08.30.274597 ◽

2020 ◽

Author(s):

Anna B. Loveland ◽

Egor Svidritskiy ◽

Denis Susorov ◽

Soojin Lee ◽

Alexander Park ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Specific Binding ◽

Structural Basis ◽

Transferase Activity ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

Ribosomal Tunnel ◽

Cellular Translation ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractToxic dipeptide repeat (DPR) proteins are produced from expanded G4C2 hexanucleotide repeats in the C9ORF72 gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥ 20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with the DPR proteins and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

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