Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders

AbstractHexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). The nucleotide repeat expansions are translated into dipeptide repeat (DPR) proteins, which are aggregation-prone and may contribute to neurodegeneration. Studies in model organisms, including yeast and flies have converged upon nucleocytoplasmic transport as one underlying pathogenic mechanism, but a comprehensive understanding of the molecular and cellular underpinnings of DPR toxicity in human cells is still lacking. We used the bacteria-derived clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to perform genome-wide gene knockout screens for suppressors and enhancers of C9orf72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. Our screens revealed genes involved in nucleocytoplasmic transport, reinforcing the previous findings from model systems. We also uncovered new potent modifiers of DPR toxicity whose gene products function in the endoplasmic reticulum (ER), proteasome, RNA processing pathways, and in chromatin modification. Since regulators of ER stress emerged prominently from the screens, we further investigated one such modifier, TMX2, which we identified as a modulator of the ER-stress signature elicited by C9orf72 DPRs in neurons. Together, this work identifies novel suppressors of DPR toxicity that represent potential therapeutic targets and demonstrates the promise of CRISPR-Cas9 screens to define mechanisms of neurodegenerative diseases.One Sentence SummaryGenome-wide CRISPR-Cas9 screens in human cells reveal mechanisms and targets for ALS-associated C9orf72 dipeptide repeat protein toxicity.

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Circadian sleep/wake-associated cells show dipeptide repeat protein aggregates in C9orf72-related ALS and FTLD cases

Acta Neuropathologica Communications ◽

10.1186/s40478-019-0845-9 ◽

2019 ◽

Vol 7 (1) ◽

Cited By ~ 4

Author(s):

Lieselot Dedeene ◽

Evelien Van Schoor ◽

Rik Vandenberghe ◽

Philip Van Damme ◽

Koen Poesen ◽

...

Keyword(s):

Frontotemporal Lobar Degeneration ◽

Sleep Disturbances ◽

Motor Behavior ◽

Repeat Expansion ◽

Repeat Protein ◽

Hexanucleotide Repeat ◽

Gland Tissue ◽

Hexanucleotide Repeat Expansion ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractMotor-, behavior- and/or cognition-related symptoms are key hallmarks in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with TDP-43 pathology (FTLD-TDP), respectively. It has been reported that these patients also experience sleep disturbances, which might implicate a disturbed circadian rhythm of the sleep/wake cycle. It remains unknown, however, whether cells involved in the circadian sleep/wake cycle are affected by ALS- and FTLD-related neuropathological changes including phosphorylated TDP-43 (pTDP-43) aggregates and dipeptide repeat protein (DPR) inclusions resulting from the C9orf72 hexanucleotide repeat expansion. Immunohistochemistry for DPR and pTDP-43 pathology was performed in post-mortem hypothalamus and pineal gland tissue of patients with ALS and/or FTLD-TDP with and without the C9orf72 repeat expansion and healthy controls. Circadian sleep/wake-associated cells, including pinealocytes and hypothalamic neurons related to the suprachiasmatic nucleus (SCN), were microscopically assessed. We observed numerous DPR inclusions (poly(GA), poly(GP), poly(GR) and poly(PR)) in the pinealocytes and few poly(GA) inclusions in the SCN-related neurons in C9orf72-related ALS and/or FTLD-TDP cases. These circadian sleep/wake-associated cells, however, were devoid of pTDP-43 pathology both in C9orf72- and nonC9orf72-related ALS and/or FTLD-TDP cases. Our neuropathological findings show that pinealocytes and, to a lesser extent, SCN-related neurons are affected by DPR pathology. This may reflect an involvement of these cells in sleep/wake disturbances observed in ALS and/or FTLD-TDP patients.

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CRISPR–Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity

Nature Genetics ◽

10.1038/s41588-018-0070-7 ◽

2018 ◽

Vol 50 (4) ◽

pp. 603-612 ◽

Cited By ~ 81

Author(s):

Nicholas J. Kramer ◽

Michael S. Haney ◽

David W. Morgens ◽

Ana Jovičić ◽

Julien Couthouis ◽

...

Keyword(s):

Human Cells ◽

Primary Neurons ◽

Repeat Protein ◽

Protein Toxicity ◽

Dipeptide Repeat

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Dipeptide repeat protein toxicity in frontotemporal lobar degeneration and in motor neurone disease associated with expansions in C9ORF72—a cautionary note

Neurobiology of Aging ◽

10.1016/j.neurobiolaging.2014.10.011 ◽

2015 ◽

Vol 36 (2) ◽

pp. 1224-1226 ◽

Cited By ~ 8

Author(s):

David M.A. Mann

Keyword(s):

Frontotemporal Lobar Degeneration ◽

Motor Neurone ◽

Motor Neurone Disease ◽

Cautionary Note ◽

Repeat Protein ◽

Protein Toxicity ◽

Dipeptide Repeat

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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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Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20246238 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6238 ◽

Cited By ~ 1

Author(s):

Anna L. Gill ◽

Monica Z. Wang ◽

Beth Levine ◽

Alan Premasiri ◽

Fernando G. Vieira

Keyword(s):

Motor Neurons ◽

Laser Scanning ◽

Neuronal Cell ◽

Fluorescein Isothiocyanate ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Primary Neurons ◽

Repeat Protein ◽

Neuronal Cell Lines ◽

Dipeptide Repeat

A repeat expansion mutation in the C9orf72 gene is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In this study, using multiple cell-based assay systems, we reveal both increased dipeptide repeat protein (DRP) toxicity in primary neurons and in differentiated neuronal cell lines. Using flow cytometry and confocal laser scanning microscopy of cells treated with fluorescein isothiocyanate (FITC)-labeled DRPs, we confirm that poly-glycine-arginine (GR) and poly-proline-arginine (PR) DRPs entered cells more readily than poly-glycine-proline (GP) and poly-proline-alanine (PA) DRPs. Our findings suggest that the toxicity of C9-DRPs may be influenced by properties associated with differentiated and aging motor neurons. Further, our findings provide sensitive cell-based assay systems to test phenotypic rescue ability of potential interventions.

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