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

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

10.1101/2021.03.09.434618 ◽

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.

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Order controls disordered droplets: structure-function relationships in C9orf72-derived poly(PR)

AJP Cell Physiology ◽

10.1152/ajpcell.00372.2021 ◽

2021 ◽

Author(s):

Kohsuke Kanekura ◽

Yuhei Hayamizu ◽

Masahiko Kuroda

Keyword(s):

Amino Acids ◽

Aromatic Amino Acids ◽

Polar Amino Acid ◽

Repeat Proteins ◽

Intron 1 ◽

Lateral Sclerosis ◽

Ran Translation ◽

Dipeptide Repeat ◽

Positively Charged

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have been thought as two distinct neurodegenerative diseases. However, recent genetic screening and careful investigations found the genetic and pathological overlap among these disorders. Hexanucleotide expansions in intron 1 of C9orf72 are a leading cause of familial ALS and familial FTD. These expansions facilitate the repeat-associated non-ATG initiated translation (RAN translation), producing five dipeptide repeat proteins (DRPs), including Arg-rich poly(PR: Pro-Arg) and poly-(GR: Gly-Arg) peptides. Arg is a positively charged, highly polar amino acid that facilitates interactions with anionic molecules such as nucleic acids and acidic amino acids via electrostatic forces and aromatic amino acids via cation-pi interaction, suggesting that Arg-rich DRPs underlie the pathophysiology of ALS via Arg-mediated molecular interactions. Arg-rich DRPs have also been reported to induce neurodegeneration in cellular and animal models via multiple mechanisms; however, it remains unclear why the Arg-rich DRPs exhibit such diverse toxic properties, because not all Arg-rich peptides are toxic. In this mini-review, we discuss the current understanding of the pathophysiology of Arg-rich C9orf72 DRPs and introduce recent findings on the role of Arg distribution as a determinant of the toxicity and its contribution to the pathogenesis of ALS.

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Soluble and insoluble dipeptide repeat protein measurements in C9orf72-frontotemporal dementia brains show regional differential solubility and correlation of poly-GR with clinical severity

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01036-y ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Annelies Quaegebeur ◽

Idoia Glaria ◽

Tammaryn Lashley ◽

Adrian M. Isaacs

Keyword(s):

Frontotemporal Dementia ◽

Potential Role ◽

Brain Regions ◽

Clinical Severity ◽

Repeat Proteins ◽

Differential Solubility ◽

Shed Light ◽

Lateral Sclerosis ◽

Dipeptide Repeat

Abstract A C9orf72 repeat expansion is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis. One of the suggested pathomechanisms is toxicity from dipeptide repeat proteins (DPRs), which are generated via unconventional translation of sense and antisense repeat transcripts with poly-GA, poly-GP and poly-GR being the most abundant dipeptide proteins. Animal and cellular studies highlight a neurotoxic role of poly-GR and poly-PR and to a lesser degree of poly-GA. Human post-mortem studies in contrast have been much less clear on a potential role of DPR toxicity but have largely focused on immunohistochemical methods to detect aggregated DPR inclusions. This study uses protein fractionation and sensitive immunoassays to quantify not only insoluble but also soluble poly-GA, poly-GP and poly-GR concentrations in brain homogenates of FTD patients with C9orf72 mutation across four brain regions. We show that soluble DPRs are less abundant in clinically affected areas (i.e. frontal and temporal cortices). In contrast, the cerebellum not only shows the largest DPR load but also the highest relative DPR solubility. Finally, poly-GR levels and poly-GP solubility correlate with clinical severity. These findings provide the first cross-comparison of soluble and insoluble forms of all sense DPRs and shed light on the distribution and role of soluble DPRs in the etiopathogenesis of human C9orf72-FTD.

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A 30-unit hexanucleotide repeat expansion in C9orf72 induces pathological lesions with dipeptide-repeat proteins and RNA foci, but not TDP-43 inclusions and clinical disease

Acta Neuropathologica ◽

10.1007/s00401-015-1473-5 ◽

2015 ◽

Vol 130 (4) ◽

pp. 599-601 ◽

Cited By ~ 25

Author(s):

Priya Gami ◽

Christina Murray ◽

Lucia Schottlaender ◽

Conceição Bettencourt ◽

Eduardo De Pablo Fernandez ◽

...

Keyword(s):

Repeat Expansion ◽

Clinical Disease ◽

Pathological Lesions ◽

Dipeptide Repeat Proteins ◽

Hexanucleotide Repeat ◽

Repeat Proteins ◽

Rna Foci ◽

Hexanucleotide Repeat Expansion ◽

Dipeptide Repeat

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Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice

Neuron ◽

10.1016/j.neuron.2015.11.018 ◽

2015 ◽

Vol 88 (5) ◽

pp. 902-909 ◽

Cited By ~ 141

Author(s):

Owen M. Peters ◽

Gabriela Toro Cabrera ◽

Helene Tran ◽

Tania F. Gendron ◽

Jeanne E. McKeon ◽

...

Keyword(s):

Transgenic Mice ◽

Dipeptide Repeat Proteins ◽

Repeat Proteins ◽

Rna Foci ◽

Dipeptide Repeat ◽

Bac Transgenic Mice

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Expression of C9orf72 hexanucleotide repeat expansion leads to formation of RNA foci and dipeptide repeat proteins but does not influence autophagy or proteasomal function in neuronal cells

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2021.119021 ◽

2021 ◽

pp. 119021

Author(s):

Stina Leskelä ◽

Nadine Huber ◽

Dorit Hoffmann ◽

Hannah Rostalski ◽

Anne M. Remes ◽

...

Keyword(s):

Neuronal Cells ◽

Repeat Expansion ◽

Dipeptide Repeat Proteins ◽

Hexanucleotide Repeat ◽

Repeat Proteins ◽

Rna Foci ◽

Hexanucleotide Repeat Expansion ◽

Dipeptide Repeat

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Unaffected mosaic C9orf72 case

Neurology ◽

10.1212/wnl.0000000000004865 ◽

2017 ◽

Vol 90 (4) ◽

pp. e323-e331 ◽

Cited By ~ 16

Author(s):

Philip McGoldrick ◽

Ming Zhang ◽

Marka van Blitterswijk ◽

Christine Sato ◽

Danielle Moreno ◽

...

Keyword(s):

Southern Blotting ◽

Mrna Levels ◽

Disease Manifestation ◽

Repeat Allele ◽

Rna Foci ◽

Disease Mechanisms ◽

Large Expansion ◽

Lateral Sclerosis ◽

Dipeptide Repeat

ObjectiveSuggested C9orf72 disease mechanisms for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration include C9orf72 haploinsufficiency, G4C2/C4G2 RNA foci, and dipeptide repeat (DPR) proteins translated from the G4C2 expansion; however, the role of small expansions (e.g., 30–90 repeats) is unknown and was investigated here.MethodsWe conducted a molecular and pathology study of a family in which the father (unaffected at age 90) carried a 70-repeat allele in blood DNA that expanded to ≈1,750 repeats in his children, causing ALS.ResultsSouthern blotting revealed different degrees of mosaicism of small and large expansions in the father's tissues from the CNS. Surprisingly, in each mosaic tissue, C9orf72 mRNA levels were significantly increased compared to an ALS-affected daughter with a large expansion. Increased expression correlated with higher levels of the 70-repeat allele (the upregulation was also evident at the protein level). Remarkably, RNA foci and DPR burdens were similar or even significantly increased (in cerebellum) in the unaffected father compared to the daughter with ALS. However, the father did not display TDP-43 pathology and signs of neurodegeneration.ConclusionThe presence of RNA foci and DPR pathology was insufficient for disease manifestation and TDP-43 pathology in the mosaic C9orf72 carrier with upregulated C9orf72 expression. It is important to conduct an investigation of similar cases, which could be found among unaffected parents of sporadic C9orf72 patients (e.g., 21% among Finnish patients with ALS). Caution should be taken when consulting carriers of small expansions because disease manifestation could be dependent on the extent of the somatic instability in disease-relevant tissues.

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Hypothesis and Theory: Roles of Arginine Methylation in C9orf72-Mediated ALS and FTD

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.633668 ◽

2021 ◽

Vol 15 ◽

Author(s):

Anna L. Gill ◽

Alan S. Premasiri ◽

Fernando G. Vieira

Keyword(s):

Rna Binding ◽

Protein Localization ◽

Arginine Methylation ◽

Dipeptide Repeat Proteins ◽

Cellular Processes ◽

Experimental Systems ◽

Hexanucleotide Repeat ◽

Repeat Proteins ◽

Arginine Residues ◽

Dipeptide Repeat

Hexanucleotide repeat expansion (G4C2n) mutations in the gene C9ORF72 account for approximately 30% of familial cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as well as approximately 7% of sporadic cases of ALS. G4C2n mutations are known to result in the production of five species of dipeptide repeat proteins (DRPs) through non-canonical translation processes. Arginine-enriched dipeptide repeat proteins, glycine-arginine (polyGR), and proline-arginine (polyPR) have been demonstrated to be cytotoxic and deleterious in multiple experimental systems. Recently, we and others have implicated methylation of polyGR/polyPR arginine residues in disease processes related to G4C2n mutation-mediated neurodegeneration. We previously reported that inhibition of asymmetric dimethylation (ADMe) of arginine residues is protective in cell-based models of polyGR/polyPR cytotoxicity. These results are consistent with the idea that PRMT-mediated arginine methylation in the context of polyGR/polyPR exposure is harmful. However, it remains unclear why. Here we discuss the influence of arginine methylation on diverse cellular processes including liquid-liquid phase separation, chromatin remodeling, transcription, RNA processing, and RNA-binding protein localization, and we consider how methylation of polyGR/polyPR may disrupt processes essential for normal cellular function and survival.

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