MECHANISMS OF TOXICITY AND THERAPEUTIC APPROACHES FOR C9FTD/ALS

Abstract

A significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two diseases commonly seen in comorbidity, carry an expanded non-coding hexanucleotide repeat in the C9orf72 gene, a condition commonly referred to as c9FTD/ALS. We and others have demonstrated that the RNA structure of GGGGCC repeats may cause neurodegeneration via their accumulation into discrete structures in the nucleus, termed RNA foci, and by serving as a template for the synthesis of aggregation-prone “c9RAN proteins” by repeat-associated non-ATG (RAN) translation. Expanded repeat carriers also exhibit reduced C9orf72 mRNA levels, suggesting that C9orf72 loss of function may contribute to neurodegeneration. Using a variety of approaches, including the generation of novel antibodies, in vitro and in vivo assays, as well as analyses of human tissue, we examine how bidirectional transcription of the expanded C9orf72 repeat and reduced C9orf72 mRNA expression lead to c9FTD/ALS. We discover antisense transcripts result from bidirectional transcription of the expanded repeat, and we find ectopic expression of CCCCGG repeats, much like GGGGCC repeats, leads to foci formation and RAN translation in cultured and transfected cells. We observe neuronal foci composed of antisense transcripts, as well as cytoplasmic inclusions of expanded (CCCCGG)RAN proteins [poly(PR), poly(GP) and poly(PA)] in various brain tissues in c9FTD/ALS; however, RNA foci and poly(GP) inclusions infrequently co-occur in the same cell suggesting separate disease mechanisms. In other studies, we demonstrate aberrant methylation of histone tail lysine residues, which is detectable in brain tissue, fibroblasts and blood of C9orf72 mutation carriers, mediates C9orf72 mRNA reduced expression and potentially contributes to disease pathogenesis. We identify aberrant histone methylation specific to C9orf72 mutation carriers as a potential disease biomarker. In addition, our findings suggest therapeutic strategies designed to target the RNA structures necessary for RAN translation and foci formation and/or to modulate methylation of C9orf72 -bound histone residues in c9FTD/ALS hold promise.