C9orf72 Poly(PR) Dipeptide Repeats Disturb Biomolecular Phase Separation and Disrupt Nucleolar Function

Abstract

Highlights d R-rich DPRs sequester NPM1 into large, soluble phaseseparation-inhibited complexes d NPM1 sequestration dissolves droplets in vitro and delocalizes nucleolar NPM1 in cells d poly(PR) entraps rRNA in static puncta in vitro and in nucleoli d poly(PR) interactions disrupt nucleolar organization in a length-dependent manner

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

“…This outcome is partially consistent with multiple previous reports that arginine-rich DPRs are cytotoxic when delivered either by way of intracellular expression or through exogenous incubation [15,[21][22][23][24][25][26][29][30][31][32]38]. In particular, these results echo findings that have shown that exogenous delivery of PR dipeptide repeat proteins to cells results in cytotoxicity [29][30][31][32]. Furthermore, these results are similar to previous findings suggesting that exogenous polyPR was toxic to primary rat neurons, but not to astrocytes [31].…”

“…First, different cell lines and incubation times used might account for different degrees of sensitivity; indeed, the different sensitivity between CHO cells and NSC-34 cells is one of our key observations. Secondly, various groups have studied DRPs of different lengths [15,[21][22][23][24][25][26][29][30][31][32][33]38] with one group providing clear evidence for changes in toxicity as a function of DRP size [30]. It is possible that results observed in our experiments with 15-mer DRP treatment on cells may not be representative of DRP modes of function at other dipeptide repeat lengths.…”

“…Uncovering the mechanisms of action of C9-DRPs and developing assay systems to test possible anti-DRP therapeutics remains imperative to understanding, and perhaps treating, C9ALS/FTD. Recently, multiple groups have conducted studies exploring the effects of cell-line incubation in the presence of synthesized C9-DRPs, typically between 10 and 20 repeats, and revealed signs of cytotoxicity and implicated various impaired cellular processes driving cell death [29][30][31][32][33].In this study, we developed multiple cell-based assay systems to assess changes in cellular function and health caused by exogenous treatment with C9-DRPs. Using these assay systems, we reveal increased DRP toxicity in a neuron-like cell line, as well as in primary neurons, when compared to non-neuronal cells.…”

Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3

“…Lopez-Gonzalez et al reported that poly-GR binds to mitochondrial ribosomal proteins and leads to mitochondrial dysfunction, oxidative stress, and DNA damage [32]. Others reported that poly-GR and poly-PR change the intranuclear distribution of NPM1, which is protective against DNA damage [15,54,62]. Thus, each neurotoxic DPR may affect DNA damage in an individual way.…”

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

“…Second, sense G4C2 and antisense C4G2 expanded repeats are repeat‐associated non‐AUG (RAN) translated into dipeptide repeat (DPR)‐containing proteins, which form inclusions throughout the brain of patients with C9‐ALS/FTD (Ash et al , ; Gendron et al , ; Mori et al , ; Zu et al , ), as well as in mice expressing expanded G4C2 repeats (Chew et al , ; O'Rourke et al , ; Peters et al , ; Jiang et al , ; Liu et al , ). Overexpression of DPR proteins using artificial ATG codons for their translation initiation leads to neurodegeneration in cell and animal models, notably through alteration of mitochondria (Dafinca et al , ; Lopez‐Gonzalez et al , ; Choi et al , ), DNA repair (Walker et al , ; Lopez‐Gonzalez et al , ), nuclear and nucleolar organization (White et al , ; Zhang et al , ), and/or nucleocytoplasmic transport (Kwon et al , ; May et al , ; Mizielinska et al , ; Wen et al , ; Zhang et al , , ; Freibaum et al , ; Jovičić et al , ; Tao et al , ; Boeynaems et al , ; Khosravi et al , ). Third, expanded G4C2 repeats promote DNA epigenetic changes that lead to decreased expression of C9ORF72 mRNA and protein levels in C9‐ALS/FTD individuals (DeJesus‐Hernandez et al , ; Gijselinck et al , ; Almeida et al , ; Waite et al , ; van Blitterswijk et al , ; Xiao et al , ; Saberi et al , ; Frick et al , ; Viodé et al , ).…”