TY - JOUR
T1 - The proline/arginine dipeptide from hexanucleotide repeat expanded C9ORF72 inhibits the proteasome
AU - Gupta, Rahul
AU - Lan, Matthews
AU - Mojsilovic-Petrovic, Jelena
AU - Choi, Won Hoon
AU - Safren, Nathaniel
AU - Barmada, Sami
AU - Lee, Min Jae
AU - Kalb, Robert
N1 - Funding Information:
Received August 19, 2016; accepted January 12, 2017; First published January 24, 2017. The authors declare no competing financial interests. Author contributions: R.G., R.K., and M.L. designed research; R.K., W.H.C., N.S., S.B., and M.J.L., performed research; R.G., M.L., J. M.-P., and N.S., contributed unpublished reagents/analytic tools; R.K. analyzed data; R.G., M.L., W.H.C., N.S., S.B., M.J.L, and R.K. wrote the paper. This work is supported by National Institutes of Health Grants NS087077 and NS052325 (to R.G.K.) and NS072233 and R01-NS097542 (to S.B.), the National Research Foundation of Korea Grant 2016R1A2B2006507 and the Brain Research Program Grant 2016M3C7A1913895 (to M.J.L.), and the Roy & Diana Vagelos Scholars Program in the Molecular Life Sciences (M.L.). *R.G. and M.L. are co-first authors. Acknowledgements: We thank the generosity of Steve McKnight (University of Texas, Southwestern) for sharing synthetic peptides for initial studies and Daniel Finley (Harvard Medical School) for thoughtful discussions. Correspondence should be addressed to either of the following: Robert Kalb at the above address, E-mail: kalb@email.chop.edu; or Min Jae Lee at the above address, E-mail: minjlee@snu.ac.kr. DOI:http://dx.doi.org/10.1523/ENEURO.0249-16.2017 Copyright © 2017 Gupta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
Funding Information:
This work is supported by National Institutes of Health Grants NS087077 and NS052325 (to R.G.K.) and NS072233 and R01-NS097542 (to S.B.), the National Research Foundation of Korea Grant 2016R1A2B2006507 and the Brain Research Program Grant 2016M3C7A1913895 (to M.J.L.), and the Roy & Diana Vagelos Scholars Program in the Molecular Life Sciences (M.L.). *R.G. and M.L. are co-first authors. Acknowledgements: We thank the generosity of Steve McKnight (University of Texas, Southwestern) for sharing synthetic peptides for initial studies and Daniel Finley (Harvard Medical School) for thoughtful discussions.
Publisher Copyright:
© 2017 Gupta et al.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - ALS
KW - Frontotemporal dementia
KW - Lysosome-autophagy
KW - Motor neuron
KW - Proteasome
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U2 - 10.1523/ENEURO.0249-16.2017
DO - 10.1523/ENEURO.0249-16.2017
M3 - Article
C2 - 28197542
AN - SCOPUS:85021337721
SN - 2373-2822
VL - 4
JO - eNeuro
JF - eNeuro
IS - 1
M1 - e0249-16.2017
ER -