An intronic hexanucleotide repeat expansion (HRE) mutation in the C9ORF72 gene is the most common cause of familial amyotrophic lateral sclerosis (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 non-canonical 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 buildup 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 C9ORF72 HRE.
Statement of Significance: Peptides made up of two alternating amino acids, proline/arginine (PR) or glycine/arginine (GR), are thought to contribute to the pathophysiology of familial amyotrophic lateral sclerosis and frontotemporal dementia caused by expansion of the intronic microsatellite repeat sequence GGGGCC in the C9ORF72 gene. Here we show that proline/arginine repeated twenty times (PR20) is toxic to motor neurons and inhibits substrate flux through the proteasome and the lysosomal-autophagy pathway. Stimulation of the proteasome alleviates this toxicity suggesting that targeting the PR20-proteasome interaction may have therapeutic potential.
Authors report no conflict of interest.
This work is supported by NIH Grants: NS087077 and NS052325 to R.G.K., NS072233 and R01-NS097542 to S.B., the National Research Foundation of Korea (2016R1A2B2006507) and the Brain Research Program (2016M3C7A1913895) to M.J.L., and the Roy & Diana Vagelos Scholars Program in the Molecular Life Sciences (M.L.).
↵* R.G. and M.L. co-first authors