The Proline/Arginine Dipeptide from Hexanucleotide Repeat ExpandedC9ORF72Inhibits the Proteasome

Abstract

An intronic hexanucleotide repeat expansion (HRE) mutation in the C9ORF72 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 C9ORF72 HRE.

Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells

“…This outcome is partially consistent with multiple previous reports that arginine-rich DPRs are cytotoxic when delivered either by way of intracellular expression or through exogenous incubation [15,[21][22][23][24][25][26][29][30][31][32]38]. In particular, these results echo findings that have shown that exogenous delivery of PR dipeptide repeat proteins to cells results in cytotoxicity [29][30][31][32]. Furthermore, these results are similar to previous findings suggesting that exogenous polyPR was toxic to primary rat neurons, but not to astrocytes [31].…”

“…In particular, these results echo findings that have shown that exogenous delivery of PR dipeptide repeat proteins to cells results in cytotoxicity [29][30][31][32]. Furthermore, these results are similar to previous findings suggesting that exogenous polyPR was toxic to primary rat neurons, but not to astrocytes [31].…”

“…First, different cell lines and incubation times used might account for different degrees of sensitivity; indeed, the different sensitivity between CHO cells and NSC-34 cells is one of our key observations. Secondly, various groups have studied DRPs of different lengths [15,[21][22][23][24][25][26][29][30][31][32][33]38] with one group providing clear evidence for changes in toxicity as a function of DRP size [30]. It is possible that results observed in our experiments with 15-mer DRP treatment on cells may not be representative of DRP modes of function at other dipeptide repeat lengths.…”

“…Uncovering the mechanisms of action of C9-DRPs and developing assay systems to test possible anti-DRP therapeutics remains imperative to understanding, and perhaps treating, C9ALS/FTD. Recently, multiple groups have conducted studies exploring the effects of cell-line incubation in the presence of synthesized C9-DRPs, typically between 10 and 20 repeats, and revealed signs of cytotoxicity and implicated various impaired cellular processes driving cell death [29][30][31][32][33].In this study, we developed multiple cell-based assay systems to assess changes in cellular function and health caused by exogenous treatment with C9-DRPs. Using these assay systems, we reveal increased DRP toxicity in a neuron-like cell line, as well as in primary neurons, when compared to non-neuronal cells.…”

Regulation of mRNA Translation in Neurons—A Matter of Life and Death

“…However, disruption of proteostasis may be a common mechanism of toxicity for many of these aberrant translation products. Consistent with this hypothesis, several RAN products from C9ORF72 (polyGly-Ala, -Gly-Pro, -Gly-Arg, and -Pro-Arg) and FMR1polyGly have been linked to proteasomal dysfunction (Gupta et al, 2017; Oh et al, 2015; Yamakawa et al, 2015; Zhang et al, 2014b). C9ORF72polyPro-Arg is also associated with impaired autophagy (Gupta et al, 2017).…”