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

Abstract

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...

Hypothesis and Theory: Roles of Arginine Methylation in C9orf72-Mediated ALS and FTD

“…Toxicity of polyGR/PR has been observed in cell culture systems (Mori et al, 2013 ; Kwon et al, 2014 ; Kramer et al, 2018 ), in Drosophila models (Mizielinska et al, 2014 ; Wen et al, 2014 ), and in mouse models (Zhang et al, 2018 ; 2019 ; Choi et al, 2019 ; Cook et al, 2020 ). We also demonstrated consistent and significant cell type and differentiation-specific toxicity of polyGR/PR in cell culture models (Gill et al, 2019 ).…”

Type I PRMT Inhibition Protects Against C9ORF72 Arginine-Rich Dipeptide Repeat Toxicity

“…Our lab has reported that exogenous application of GR 15 and PR 15 to NSC-34 cells induces cellular toxicity, as measured by WST-1 metabolism, LDH release, BrdU labeling, and Caspase-3 activity (Gill et al, 2019). In the present study, we use the WST-1 metabolism and LDH release assays to evaluate changes in DRP toxicity in the presence of a PRMT inhibitor.…”

“…For example, when administered exogenously to U2OS cells, synthetic GR 20 and PR 20 are shown to bind to nucleoli, disrupt RNA splicing and processing, and decrease cell viability (Mori et al, 2013). Our lab has previously demonstrated that exogenous application of synthetic GR 15 and PR 15 to mouse spinal cord neuroblastoma hybrid cells (NSC-34) induces cellular toxicity, as measured by various cell health and function assays and that this toxic effect becomes more severe as the cells are further differentiated toward neurons, with primary neurons exhibiting the greatest toxicity (Gill et al, 2019). In addition, a series of studies involving the expression of the repeat expansion in Drosophila have demonstrated polyGR and polyPR related toxicity (Mizielinska et al, 2014;Freibaum et al, 2015;Lee et al, 2016), with one study revealing the disruption of stress granule assembly due to the presence of polyGR and polyPR (Lee et al, 2016).…”

QTL mapping of adult plant and seedling resistance to leaf rust (Puccinia triticina Eriks.) in a multiparent advanced generation intercross (MAGIC) wheat population

“…Quantitative resistances are effective at later growth stages and are therefore referred to as field resistance or adult plant resistance (APR, Krattinger and Keller 2016 ). To date, more than 80 resistance genes to leaf rust ( Lr genes) have been identified in bread wheat, durum wheat, and diploid wheat species (Gill et al 2019 ). While most of them show race-specific resistance at the seedling stage, genes like Lr12 , Lr13 , Lr22a/b , Lr34 , Lr35 , Lr37 , Lr46 , Lr67 , Lr68 , and Lr77 confer resistance at the adult plant stage (Dakouri et al 2013 ; McIntosh et al 2013 , 2017 ).…”