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

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• The C9orf72 gene hexanucleotide repeat expansion (HRE) is a leading cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
• Repeat-associated non-ATG translation (RAN translation) of the HRE generates toxic dipeptide repeat proteins.

Purpose of the Study:

• To investigate the toxic mechanisms of arginine-containing RAN translation products, specifically proline/arginine (PR20) and glycine/arginine (GR20) repeats.
• To determine the impact of PR20 on cellular degradation pathways and motor neuron survival.

Main Methods:

• Primary rat spinal cord neuron cultures were treated with PR20 and GR20.
• Cell viability, ubiquitylated protein levels, and degradation pathway flux were assessed.
• Proteasome association and activity assays were performed.
• The effect of proteasomal activator IU1 on PR20 toxicity was evaluated.

Main Results:

• PR20 demonstrated significant motor neuron toxicity (LD50 = 2 µM) without inducing mitochondrial dysfunction, ER stress, or mTORC downregulation.
• PR20 caused accumulation of ubiquitylated substrates by inhibiting both autophagic and proteasomal degradation pathways.
• PR20 directly binds to and inhibits proteasome activity.
• GR20 did not exhibit these toxic effects.
• Proteasomal activator IU1 rescued motor neurons from PR20-induced toxicity.

Conclusions:

• PR20 toxicity in motor neurons is mediated by direct proteasome inhibition.
• Targeting the proteasome degradation pathway offers a potential therapeutic strategy for C9orf72-related ALS and FTD.