Repeat expansion mutations in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeatassociated non-AUG translation of this expansion produces dipeptide repeat proteins (DRPs). The arginine containing DRPs, polyGR and polyPR, are consistently reported to be the most toxic. Here we demonstrated that small molecule inhibition of type I protein arginine methyltransferases (PRMT) protects against polyGR and polyPR toxicity. Furthermore, our findings suggest that asymmetric dimethylation of polyGR and polyPR by Type I PRMTs plays important roles in their cytotoxicity.

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. Int. J. Mol. Sci. 2019, 20, 6238 2 of 20 suggest that it may act as a guanine nucleotide exchange factor for small GTPases, as it has been shown to regulate endosomal trafficking and autophagy in neurons [13]. Another hypothesis suggests RNA gain-of-function neurotoxicity, resulting from RNA binding protein-sequestering RNA foci accumulating in neurons following expression of repeat expanded, intronic, C9orf72 transcripts [14][15][16][17][18].A third suggests that dipeptide repeat proteins (DRPs) derived from RAN translation of C9HRE RNA transcripts constitute a toxic gain-of-function mutation. These DRPs: poly-glycine-arginine (GR), poly-proline-arginine (PR), poly-glycine-proline (GP), poly-proline-alanine (PA), and poly-glycine-alanine (GA) have been shown to cause toxicity as well as interfere with vital cellular processes, including RNA biogenesis, endoplasmic reticulum function, the Notch signaling pathway, and nucleocytoplasmic transport [15,[19][20][21][22][23][24][25][26]. Testing of post-mortem tissues from ALS/FTD patients has not shown a correlation between the amount or localization of C9-DRPs and neurodegenerative phenotype, which contributes to skepticism that C9-DRPs are the major contributor to C9-ALS/FTD pathogenesis [27]. However, polyGP has been detected in cerebral spinal fluid of people carrying C9HRE mutations both before and during ALS or FTD disease progression and is being explored as a biomarker of therapeutic effects in people carrying this mutation [28]. 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. Usin...

IntroductionIntronic repeat expansions in the C9orf72 gene are the most frequent known single genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These repeat expansions are believed to result in both loss-of-function and toxic gain-of-function. Gain-of-function results in the production of toxic arginine-rich dipeptide repeat proteins (DPRs), namely polyGR and polyPR. Small-molecule inhibition of Type I protein arginine methyltransferases (PRMTs) has been shown to protect against toxicity resulting from polyGR and polyPR challenge in NSC-34 cells and primary mouse-derived spinal neurons, but the effect in human motor neurons (MNs) has not yet been explored.MethodsTo study this, we generated a panel of C9orf72 homozygous and hemizygous knockout iPSCs to examine the contribution of C9orf72 loss-of-function toward disease pathogenesis. We differentiated these iPSCs into spinal motor neurons (sMNs).ResultsWe found that reduced levels of C9orf72 exacerbate polyGR15 toxicity in a dose-dependent manner. Type I PRMT inhibition was able to partially rescue polyGR15 toxicity in both wild-type and C9orf72-expanded sMNs.DiscussionThis study explores the interplay of loss-of-function and gain-of-function toxicity in C9orf72 ALS. It also implicates type I PRMT inhibitors as a possible modulator of polyGR toxicity.

Hexanucleotide repeat expansion (G4C2n) mutations in the gene C9ORF72 account for approximately 30% of familial cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as well as approximately 7% of sporadic cases of ALS. G4C2n mutations are known to result in the production of five species of dipeptide repeat proteins (DRPs) through non-canonical translation processes. Arginine-enriched dipeptide repeat proteins, glycine-arginine (polyGR), and proline-arginine (polyPR) have been demonstrated to be cytotoxic and deleterious in multiple experimental systems. Recently, we and others have implicated methylation of polyGR/polyPR arginine residues in disease processes related to G4C2n mutation-mediated neurodegeneration. We previously reported that inhibition of asymmetric dimethylation (ADMe) of arginine residues is protective in cell-based models of polyGR/polyPR cytotoxicity. These results are consistent with the idea that PRMT-mediated arginine methylation in the context of polyGR/polyPR exposure is harmful. However, it remains unclear why. Here we discuss the influence of arginine methylation on diverse cellular processes including liquid-liquid phase separation, chromatin remodeling, transcription, RNA processing, and RNA-binding protein localization, and we consider how methylation of polyGR/polyPR may disrupt processes essential for normal cellular function and survival.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a repeat expansion mutation in the C9orf72 gene. Repeat-associated non-AUG (RAN) translation of this expansion produces five species of dipeptide repeat proteins (DRPs). The arginine containing DRPs, polyGR and polyPR, are consistently reported to be the most toxic. Here, we uncover Type I protein arginine methyltransferase (PRMT) inhibitors as possible therapeutics for polyGR- and polyPR- related toxicity. Furthermore, we reveal data that suggest that asymmetric dimethylation (ADMe) of polyGR is a determining factor in its pathogenesis.