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

AbstractDisruption of nucleocytoplasmic transport (NCT), including mislocalization of the importin β cargo, TDP-43, is a hallmark of amyotrophic lateral sclerosis (ALS), including ALS caused by a hexanucleotide repeat expansion in C9orf72. However, the mechanism(s) remain unclear. Importin β and its cargo adaptors have been shown to co-precipitate with the C9orf72-arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to NCT protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with nuclear transport receptors in the vicinity of the nuclear envelope.

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

Cell Death and Disease ◽

10.1038/s41419-019-1983-5 ◽

2019 ◽

Vol 10 (10) ◽

Cited By ~ 4

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.

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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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C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import

eLife ◽

10.7554/elife.51685 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 12

Author(s):

Lindsey R Hayes ◽

Lauren Duan ◽

Kelly Bowen ◽

Petr Kalab ◽

Jeffrey D Rothstein

Keyword(s):

Nuclear Import ◽

Genetic Modifier ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Dipeptide Repeat Proteins ◽

Hexanucleotide Repeat ◽

Repeat Proteins ◽

Importin Α ◽

Dipeptide Repeat

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases, including ALS caused by a C9orf72 hexanucleotide repeat expansion. However, the mechanism(s) remain unclear. Karyopherins, including importin β and its cargo adaptors, have been shown to co-precipitate with the C9orf72 arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import of importin β, importin α/β, and transportin cargoes in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to nucleocytoplasmic transport protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with cargo loading on karyopherins.

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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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Enhanced insulin signalling ameliorates C9orf72 hexanucleotide repeat expansion toxicity in Drosophila

eLife ◽

10.7554/elife.58565 ◽

2021 ◽

Vol 10 ◽

Author(s):

Magda L Atilano ◽

Sebastian Grönke ◽

Teresa Niccoli ◽

Liam Kempthorne ◽

Oliver Hahn ◽

...

Keyword(s):

Mammalian Cells ◽

Insulin Signalling ◽

Repeat Expansion ◽

Adult Brain ◽

Systemic Injection ◽

Hexanucleotide Repeat ◽

Repeat Expansions ◽

Hexanucleotide Repeat Expansion ◽

Lateral Sclerosis ◽

Dipeptide Repeat

G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/IGF signalling is reduced in fly models of C9orf72 repeat expansion using RNA sequencing of adult brain. We further demonstrate that activation of insulin/IGF signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/IGF signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/IGF signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.

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