Mechanisms of toxicity in C9FTLD/ALS

Abstract

A hexanucleotide repeat expansion within a non-coding region of the C9ORF72 gene is the most common mutation causative of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Elucidating how this bidirectionally transcribed G4C2·C4G2 expanded repeat causes "C9FTLD/ALS" has since become an important goal of the field. Likely pathogenic mechanisms include toxicity induced by repeat-containing RNAs, and loss of C9orf72 function due to epigenetic changes resulting in decreased C9ORF72 mRNA expression. With regards to the former, sense and antisense transcripts of the expanded repeat aberrantly interact with various RNA-binding proteins and form discrete nuclear structures, termed RNA foci. These foci have the capacity to sequester select RNA-binding proteins, thereby impairing their function. (G4C2)exp and (C4G2)exp transcripts also succumb to an alternative fate: repeat-associated non-ATG (RAN) translation. This unconventional mode of translation, which occurs in the absence of an initiating codon, results in the abnormal production of poly(GA), poly(GP), poly(GR), poly(PR) and poly(PA) peptides, collectively referred to as C9RAN proteins. C9RAN proteins form neuronal inclusions throughout the central nervous system of C9FTLD/ALS patients and may contribute to disease pathogenesis. This review aims to summarize the important findings from studies examining mechanisms of disease in C9FTLD/ALS, and will also highlight some of the many questions in need of further investigation.

Figure 1. Potential mechanisms of disease in C9FTLD/ALS

Expansion of the G₄C₂ C₄G₂ repeat within intron 1 of the C9ORF72 gene may cause C9FTLD/ALS through various mechanisms. 1) Abnormal DNA and histone methylation leads to a decrease in C9ORF72 mRNA expression which may consequently result in C9orf2 protein loss of function; 2) (G₄C₂)_exp and (C₄G₂)_exp transcripts are bound by select RNA-binding proteins and this may impair the ability of such proteins to bind their actual RNA targets. In addition, because (G₄C₂)_exp and (C₄G₂)_exp transcripts form nuclear foci, RNA-binding proteins that interact with these transcripts may too be sequestered in foci, also resulting in their loss of function; 3) In the cytosol, (G₄C₂)_exp and (C₄G₂)_exp transcripts are susceptible to repeat-associated non-ATG translation, producing poly(GA), poly(GP), poly(GR), poly(PR) and poly(PA) C9RAN proteins. Each C9RAN protein may have a different toxicity profile, and may contribute to neurodegeneration through the formation of soluble oligomers or their aggregation into insoluble inclusions within neurons.

Figure 2. RNA foci and C9RAN protein pathology are present in various regions of the central nervous system in C9FTLD/ALS

Fluorescence in situ hybridization of C9FTLD/ALS tissue sections using a probe against sense G₄C₂ transcripts (spinal cord, hippocampus) or antisense C₄G₂ transcripts (cerebellum, frontal cortex) was followed by immunofluorescence staining to detect poly(GP) inclusions. Note that RNA foci (red) in the nucleus (stained with DAPI, blue), and star-shaped cytoplasmic poly-GP inclusions (green), seldom co-occur in the same cells. Scale bar = 10 μm.