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Summary A repeat expansion in C9ORF72 causes frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). RNA of the expanded repeat (r(GGGGCC)exp) forms nuclear foci or undergoes repeat-associated non-ATG (RAN) translation producing “c9RAN proteins”. Since neutralizing r(GGGGCC)exp could inhibit these potentially toxic events, we sought to identify small molecule binders of r(GGGGCC)exp. Chemical and enzymatic probing of r(GGGGCC)8 indicate it adopts a hairpin structure in equilibrium with a quadruplex structure. Using this model, bioactive small molecules targeting r(GGGGCC)exp were designed and found to significantly inhibit RAN translation and foci formation in cultured cells expressing r(GGGGCC)66 and neurons trans-differentiated from fibroblasts of repeat expansion carriers. Finally, we show that poly(GP) c9RAN proteins are specifically detected in c9ALS patient cerebrospinal fluid. Our findings highlight r(GGGGCC)exp-binding small molecules as a possible c9FTD/ALS therapeutic, and suggest c9RAN proteins could potentially serve as a pharmacodynamic biomarker to assess efficacy of therapies that target r(GGGGCC)exp.

Individuals carrying (GGGGCC) expanded repeats in the C9orf72 gene represent a significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Elucidating how these expanded repeats cause “c9FTD/ALS” has since become an important goal of the field. Toward this end, we sought to investigate whether epigenetic changes are responsible for the decrease in C9orf72 expression levels observed in c9FTD/ALS patients. We obtained brain tissue from ten c9FTD/ALS individuals, nine FTD/ALS cases without a C9orf72 repeat expansion, and nine disease control participants, and generated fibroblastoid cell lines from seven C9orf72 expanded repeat carriers and seven participants carrying normal alleles. Chromatin immunoprecipitation using antibodies for histone H3 and H4 trimethylated at lysines 9 (H3K9), 27 (H3K27), 79 (H3K79), and 20 (H4K20) revealed that these trimethylated residues bind strongly to C9orf72 expanded repeats in brain tissue, but not to non-pathogenic repeats. Our finding that C9orf72 mRNA levels are reduced in the frontal cortices and cerebella of c9FTD/ALS patients is consistent with trimethylation of these histone residues, an event known to repress gene expression. Moreover, treating repeat carrier-derived fibroblasts with 5-aza-2-deoxycytidine, a DNA and histone demethylating agent, not only decreased C9orf72 binding to trimethylated histone residues, but also increased C9orf72 mRNA expression. Our results provide compelling evidence that trimethylation of lysine residues within histones H3 and H4 is a novel mechanism involved in reducing C9orf72 mRNA expression in expanded repeat carriers. Of importance, we show that mutant C9orf72 binding to trimethylated H3K9 and H3K27 is detectable in blood of c9FTD/ALS patients. Confirming these exciting results using blood from a larger cohort of patients may establish this novel epigenetic event as a biomarker for c9FTD/ALS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1199-1) contains supplementary material, which is available to authorized users.