Proteomics and <i>C9orf72</i> neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity

Hartmann, Hannelore; Hornburg, Daniel; Czuppa, Mareike; Bader, Jakob; Michaelsen, Meike; Farny, Daniel; Arzberger, Thomas; Mann, Matthias; Meissner, Felix; Edbauer, Dieter

doi:10.26508/lsa.201800070

Cited by 97 publications

(144 citation statements)

References 44 publications

Supporting

Mentioning

115

Contrasting

Order By: Relevance

“…One was that the Arg appears to mediate promiscuous binding to the proteome and the second was that these interactions are responsible for mitigating toxicity. The Arg-rich DPRs in particular enriched for proteins involved in ribosome biogenesis and RNA splicing machinery, which is consistent with prior findings (17,(34)(35)(36). However, we also found additional novel interactions with proteins involved in ribosome-translation, cytoskeleton and chromatin machineries (Fig 2B).…”

Section: Resultssupporting

confidence: 91%

Arginine valency inC9ORF72dipolypeptides mediates promiscuous proteome binding that stalls ribosomes, disable actin cytoskeleton assembly and impairs arginine methylation of endogenous proteins

Radwan

Ang

Ormsby

et al. 2019

Preprint

View full text Add to dashboard Cite

C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a Neuro2a cell model to demonstrate that the valency of Arg in the most toxic DPRS, PR and GR, drives promiscuous binding to the proteome, compared to a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats robustly stalled the ribosome suggesting high-valency Arg electrostatically jams the ribosome exit tunnel during synthesis. Poly-GR also bound to arginine methylases and induced hypomethylation of endogenous proteins, with a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation with concomitant downstream effects on widespread biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly. SIGNIFICANCE STATEMENT. The major genetic cause of MND are mutations in an intron of the C9ORF72 gene that lead to the expansion in the length of a hexanucleotide repeat sequence, and subsequent non-AUG mediated translation of the intron into 5 different DPRs. The two DPRs containing Arg are potently toxic in animal and cell models. Our research shows that the valency of Arg mediates widespread proteome binding especially affecting machinery involvedin Arg-methylation, cytoskeleton and translation. We suggest the mechanisms for toxicity are multipronged and involve electrostatic jamming of ribosomes during translation, acting as substrate mimetics for arginine methylase activity that renders the endogenous proteome hypomethylated and impairing actin cytoskeleton assembly. These mechanisms explain pathologic signatures previous reported in human brain pathology.

show abstract

Section: Resultssupporting

confidence: 91%

Arginine valency inC9ORF72dipolypeptides mediates promiscuous proteome binding that stalls ribosomes, disable actin cytoskeleton assembly and impairs arginine methylation of endogenous proteins

Radwan

Ang

Ormsby

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…We next measured the basal rate of protein synthesis in C9 patient and control iPSC-derived MNs and at the same time asked whether ETF1 could in fact regulate this process in human neurons. ALS-C9 patient MNs exhibited significantly reduced levels of de novo protein synthesis relative to control MNs in accordance with previous reports 17, [54][55][56] (day 50, n=2 patients vs 1 control, n=3 independent biological replicates; siSCR control vs siSCR C9: p < 0.0001, 44% decrease ± 4) ( Supplementary Fig. 6f).…”

Section: Etf1 Mediates a Shift From Protein Translation To Mrna Degrasupporting

confidence: 91%

Nucleocytoplasmic Proteomic Analysis Uncovers eRF1 and Nonsense Mediated Decay as Modifiers of ALS C9orf72 Toxicity

Ortega

Daley

Kour

et al. 2019

Preprint

View full text Add to dashboard Cite

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by the C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift towards a more cytosolic proteome in C9-HRE cells. Amongst these was ETF1, which regulates translation termination and nonsense-mediated decay (NMD). ETF1 accumulates within elaborate nuclear envelope invaginations in patient iPSC-neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of ETF1 and the NMD-driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegenerationassociated repeat expansion mutations and highlight ETF1 and NMD as therapeutic targets in C9orf72-associated ALS/FTD. KEYWORDSAmyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), C9orf72, ETF1, nonsense mediated decay (NMD), UPF1, nucleocytoplasmic proteome, iPSCs, motor neurons. through three non-exclusive mechanisms 5-8 . Several reports have shown that C9orf72 expression is decreased in patients suggesting that haploinsufficiency contributes to pathogenesis 9, 10 . Gain-of-function neurotoxic effects occur through the production of aberrant C9-HRE RNA molecules 11 , as well as through dipeptide repeat proteins (DPRs) translated by a non-ATG dependent mechanism from the HRE 12, 13 . The repeat is bi-directionally transcribed and corresponding RNA sense and antisense molecules are detected by fluorescence in situ hybridization (FISH) as predominantly nuclear RNA foci in patient cells and tissue 11,13,14 . Moreover, five C9-HRE DPRs (GA, GP, GR, PR, PA) have been detected with antigen-specific antibodies in patient samples where they accumulate in cytoplasmic and nuclear aggregates in the frontal and motor cortex as well as the spinal cord 15,16 .While the relative contribution of loss-of-function effects, as well as the dominant source of toxic gain-of-function effects (RNA or DPRs) is still an open-ended question, substantial and converging evidence suggests that the C9-HRE disrupts the balance of proteins that are transported between the nucleus and the cytoplasm. The long C9-RNA that is transcribed from the repeat expansion binds and sequesters RNA-binding proteins in the nucleus 11 . The argininerich GR and PR C9-DPRs bind and sequester a number of proteins with low complexity domains that assemble into membrane-less organelles through liquid-liquid phase separation 17, 18 . These include RNA granules, nucleoli and various components of the nuclear pore complex (NPC). Consequently, a series of groundbreaking genetic studies in C9-HRE Drosophila and yeast mod...

show abstract

“…We observed upregulation of many genes involved in ribosome biogenesis and assembly. This is of interest in view of recent interactome studies and yeast genetic modifier screens that show toxic di‐peptide repeat proteins play a role in ribosomal processing/biogenesis and reduce overall cell translation (Chai & Gitler, ; Hartmann et al, ). These studies thus provide indirect support for astrocyte DPRs having a role in the observed dysregulation of ribosomal processing genes.…”

Section: Discussionmentioning

confidence: 99%

Mutant C9orf72 human iPSC‐derived astrocytes cause non‐cell autonomous motor neuron pathophysiology

Zhao

Devlin

Chouhan

et al. 2019

Glia

View full text Add to dashboard Cite

Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non‐cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72‐mediated ALS. Here, we use a human iPSC‐based model to study the cell autonomous and non‐autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72‐related ALS pathology and, upon co‐culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage‐activated Na+ and K+ currents. Importantly, CRISPR/Cas‐9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell‐autonomous astrocyte pathology and non‐cell autonomous motor neuron pathophysiology.

show abstract

Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity

Abstract: Proteomics and neuropathological validation show that aberrant poly-GR/PR proteins in C9orf72 ALS/FTD bind STAU2 and ribosomes and inhibit translation.

Cited by 97 publications

References 44 publications

Arginine valency inC9ORF72dipolypeptides mediates promiscuous proteome binding that stalls ribosomes, disable actin cytoskeleton assembly and impairs arginine methylation of endogenous proteins

Arginine valency inC9ORF72dipolypeptides mediates promiscuous proteome binding that stalls ribosomes, disable actin cytoskeleton assembly and impairs arginine methylation of endogenous proteins

Nucleocytoplasmic Proteomic Analysis Uncovers eRF1 and Nonsense Mediated Decay as Modifiers of ALS C9orf72 Toxicity

Mutant C9orf72 human iPSC‐derived astrocytes cause non‐cell autonomous motor neuron pathophysiology

Contact Info

Product

Resources

About