Identifying New Potential Therapeutic Targets for ALS FTD
- RAN translation of C90RF72 repeat expansions generates toxic repeat proteins via a frameshifting mechanism
- RAN translation requires elongation factor eIF4F, suggesting a therapeutic role for eIF4F inhibitors
- Antisense oligonucleotides (ASOs) targeting sequence upstream of the repeats inhibit RAN translation
Nucleotide-repeat expansions in the C90RF72 gene are observed in almost half of familial amyotropic lateral sclerosis (ALS) cases and about 25% of frontotemporal dementia (FTD) cases. These repeat expansions are expressed through an alternative translation pathway (repeat-associated non-AUG or “RAN” translation). In contrast to canonical translation, which uses an AUG start codon, RAN uses a CUG upstream of the repeat as the start codon and then translates in all three frames generating several toxic repeat proteins. The accumulation of some of these proteins has been implicated in neurodegeneration in ALS and FTD.
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Massachusetts General Hospital researchers Clotilde Lagier-Tourenne, MD, PhD, assistant in Neuroscience, and Ricardos Tabet, PhD, research fellow in Neurology, reported in Nature Communications, their findings from a study aimed at identifying potential therapeutic targets in the RAN translation pathway to block the accumulation of these toxic molecules. They found that factors in the RAN translation pathway may be new therapeutic targets to treat ALS caused by repeat expansions and an additional mechanism of toxicity associated with repeat expansions in the C90RF72 gene.
Since RAN translation of C90RF72 repeats involves a frameshifting mechanism that generates several toxic proteins, the Mass General team employed a C90RF72 construct designed to add frame-specific labels to the proteins generated by RAN translation. This allowed them to monitor the efficiency of RAN translation under various conditions in a cell-free assay. This construct helped determine what factors are necessary for RAN translation and how these factors may be modulated to block toxic protein production. They tested various antisense oligonucleotides (ASO) targeted to sequences upstream of the repeats to block initiation of translation. The team also looked for differences in RAN translation efficiency across each frame in the context of different repeat lengths.
The researchers found indications that although RAN translation shares many aspects and requirements of canonical translation, there are factors in the RAN pathway that can be targeted to modulate it. They showed that antisense oligonucleotides (ASO) targeted to the RAN translation CUG start codon 24 nucleotides upstream of the repeat expansion downregulated RAN translation. They also found that the presence of an upstream open reading frame (uORF) sequence previously reported in abnormally spliced C90RF72 also inhibited translation.
Next, they found that repeat expansion size strongly influences expression of one of the toxic repeat proteins compared to the others.
Last, they showed that RNA transcripts with repeat expansions form complexes that sequester ribosomal subunits independent of translation, a gain-of-function that may contribute to the toxicity of C90RF72 repeat expansions.
This study revealed several key components of RAN translation that could be targeted in therapeutic intervention strategies in much of the ALS or FTD patient population. Therapeutic strategies may involve using existing inhibitors of elongation factor eIF4F or employ ASOs targeted to sequence upstream of the repeats to block RAN translation of repeat expansions in the C90RF72 gene.
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