The Proline/Arginine Dipeptide from Hexanucleotide Repeat Expanded<i>C9ORF72</i>Inhibits the Proteasome

Abstract

An intronic hexanucleotide repeat expansion (HRE) mutation in the <i>C9ORF72</i> gene is the most common cause of familial ALS and frontotemporal dementia (FTD) and is found in ∼7% of individuals with apparently sporadic disease. Several different diamino acid peptides can be generated from the HRE by noncanonical translation (repeat-associated non-ATG translation, or RAN translation), and some of these peptides can be toxic. Here, we studied the effects of two arginine containing RAN translation products [proline/arginine repeated 20 times (PR₂₀) and glycine/arginine repeated 20 times (GR₂₀)] in primary rat spinal cord neuron cultures grown on an astrocyte feeder layer. We find that PR₂₀ kills motor neurons with an LD₅₀ of 2 µM, but in contrast to the effects of other ALS-causing mutant proteins (i.e., SOD or TDP43), PR₂₀ does not evoke the biochemical signature of mitochondrial dysfunction, ER stress, or mTORC down-regulation. PR₂₀ does result in a time-dependent build-up of ubiquitylated substrates, and this is associated with a reduction of flux through both autophagic and proteasomal degradation pathways. GR₂₀, however, does not have these effects. The effects of PR₂₀ on the proteasome are likely to be direct because (1) PR₂₀ physically associates with proteasomes in biochemical assays, and (2) PR₂₀ inhibits the degradation of a ubiquitylated test substrate when presented to purified proteasomes. Application of a proteasomal activator (IU1) blocks the toxic effects of PR₂₀ on motor neuron survival. This work suggests that proteasomal activators have therapeutic potential in individuals with <i>C9ORF72</i> HRE.