The proline/arginine dipeptide from hexanucleotide repeat expanded C9ORF72 inhibits the proteasome

Rahul Gupta, Matthews Lan, Jelena Mojsilovic-Petrovic, Won Hoon Choi, Nathaniel Safren, Sami Barmada, Min Jae Lee, Robert Kalb

Research output: Contribution to journalArticle

17 Citations (Scopus)

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.

Original languageEnglish
Article numbere0249-16.2017
JournaleNeuro
Volume4
Issue number1
DOIs
StatePublished - 1 Jan 2017

Fingerprint

Dipeptides
Proteasome Endopeptidase Complex
Proline
Arginine
Poisons
Motor Neurons
Glycine
Feeder Cells
Frontotemporal Dementia
Peptides
Lethal Dose 50
Mutant Proteins
Astrocytes
Dementia
Spinal Cord
Down-Regulation
Neurons

Keywords

  • ALS
  • Frontotemporal dementia
  • Lysosome-autophagy
  • Motor neuron
  • Proteasome

Cite this

Gupta, R., Lan, M., Mojsilovic-Petrovic, J., Choi, W. H., Safren, N., Barmada, S., ... Kalb, R. (2017). The proline/arginine dipeptide from hexanucleotide repeat expanded C9ORF72 inhibits the proteasome. eNeuro, 4(1), [e0249-16.2017]. https://doi.org/10.1523/ENEURO.0249-16.2017
Gupta, Rahul ; Lan, Matthews ; Mojsilovic-Petrovic, Jelena ; Choi, Won Hoon ; Safren, Nathaniel ; Barmada, Sami ; Lee, Min Jae ; Kalb, Robert. / The proline/arginine dipeptide from hexanucleotide repeat expanded C9ORF72 inhibits the proteasome. In: eNeuro. 2017 ; Vol. 4, No. 1.
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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.",
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Gupta, R, Lan, M, Mojsilovic-Petrovic, J, Choi, WH, Safren, N, Barmada, S, Lee, MJ & Kalb, R 2017, 'The proline/arginine dipeptide from hexanucleotide repeat expanded C9ORF72 inhibits the proteasome', eNeuro, vol. 4, no. 1, e0249-16.2017. https://doi.org/10.1523/ENEURO.0249-16.2017

The proline/arginine dipeptide from hexanucleotide repeat expanded C9ORF72 inhibits the proteasome. / Gupta, Rahul; Lan, Matthews; Mojsilovic-Petrovic, Jelena; Choi, Won Hoon; Safren, Nathaniel; Barmada, Sami; Lee, Min Jae; Kalb, Robert.

In: eNeuro, Vol. 4, No. 1, e0249-16.2017, 01.01.2017.

Research output: Contribution to journalArticle

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

PY - 2017/1/1

Y1 - 2017/1/1

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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Gupta R, Lan M, Mojsilovic-Petrovic J, Choi WH, Safren N, Barmada S et al. The proline/arginine dipeptide from hexanucleotide repeat expanded C9ORF72 inhibits the proteasome. eNeuro. 2017 Jan 1;4(1). e0249-16.2017. https://doi.org/10.1523/ENEURO.0249-16.2017