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.

Anna L Gill,Monica Z Wang,Fernando G Vieira,Beth Levine,Alan Premasiri

doi:10.3390/ijms20246238

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.

Highlights

A hexanucleotide repeat expansion (HRE) mutation in a non-coding region of the C9orf72 gene is currently the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [1,2]
48-h incubation of Chinese hamster ovary (CHO) and NSC-34 cells with 30 and 3 μM doses of non-arginine-rich dipeptide repeat protein (DRP) did not produce decreases in cellular metabolism compared to Dimethyl Sulfoxide (DMSO) control with the exception of 30 μM GP15 in NSC-34
The results indicated that the neuron-like cell line NSC-34 was significantly more sensitive to arginine-rich DRP treatment when compared to the non-neuronal CHO cell line under the same experimental conditions

Summary

Introduction

A hexanucleotide repeat expansion (HRE) mutation in a non-coding region of the C9orf gene is currently the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [1,2]. In ALS and FTD, having 30 or more repeats of the intronic C9orf (GGGGCC)n hexanucleotide is associated with disease pathology [4] This repeat expansion of C-G-rich motifs lends itself to a phenomenon known as repeat-associated non-ATG (RAN) translation, a non-canonical form of translation associated with DNA and RNA R-loops, quadruplex structures, and hairpins [5,6,7]. In contrast to canonical translation, RAN translation occurs with no confirmed specific initiation signal or start codon and, in the case of C9HRE, does so in multiple reading frames in both sense and antisense directions [5]. Impacts of RAN translation have been examined in many neurodegenerative diseases with repeat expansion mutations, including Huntington’s disease, numerous forms of spinocerebellar ataxia, myotonic dystrophy types 1 and 2, and spinal bulbar muscular atrophy [9,10,11,12]

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Results

Conclusion