Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS

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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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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The carboxyl-termini of RAN translated GGGGCC nucleotide repeat expansions modulate toxicity in models of ALS/FTD

10.1101/2020.05.27.119651 ◽

2020 ◽

Cited By ~ 1

Author(s):

Fang He ◽

Brittany N. Flores ◽

Amy Krans ◽

Michelle Frazer ◽

Sam Natla ◽

...

Keyword(s):

Nuclear Accumulation ◽

Carboxyl Terminus ◽

Sporadic Amyotrophic Lateral Sclerosis ◽

Reading Frame ◽

Dipeptide Repeat Proteins ◽

Hexanucleotide Repeat ◽

Repeat Proteins ◽

Differential Toxicity ◽

Reading Frames ◽

Dipeptide Repeat

AbstractAn intronic hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat is thought to elicit toxicity through RNA mediated protein sequestration and repeat-associated non-AUG (RAN) translation of dipeptide repeat proteins (DPRs). We generated a series of transgenic Drosophila models expressing GGGGCC (G4C2) repeats either inside of an artificial intron within a GFP reporter or within the 5’ UTR of GFP placed in different downstream reading frames. Expression of 484 intronic repeats elicited minimal alterations in eye morphology, viability, longevity, or larval crawling but did trigger RNA foci formation, consistent with prior reports. In contrast, insertion of repeats into the 5’ UTR elicited differential toxicity that was dependent on the reading frame of GFP relative to the repeat. Greater toxicity correlated with a short and unstructured carboxyl terminus in the glycine-arginine (GR) RAN protein reading frame. This change in C-terminal sequence triggered nuclear accumulation of all three RAN DPRs. A similar differential toxicity and dependence on the GR carboxyl terminus was observed when repeats were expressed in rodent neurons. The presence of the native carboxyl-termini across all three reading frames was partly protective. Taken together, these findings suggest that carboxyl terminal sequences outside of the repeat region may alter the behavior and toxicity of dipeptide repeat proteins derived from GGGGCC repeats.

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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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