M and Kanekura

M and Kanekura. non-penetrating short poly-PR peptide caused cell death, suggesting that modulation of extracellular environment to inhibit the uptake of Arg-rich dipeptides might be a drug target against poly-PR/GR-mediated neurotoxicity. Introduction Dipeptide-repeat proteins (DRPs), translated through repeat-associated non-ATG (RAN) translation from pathogenic G4C2 expansions in C9orf72 gene, are supposed to play a pivotal role in development of C9orf72-related neurological disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)1C4. Among 5 species of DRPs (poly-GA, GP, PR, GR and PA) translated from sense and antisense of G4C2 expansions, especially arginine-rich DRPs (poly-PR and poly-GR) are of interest because they caused neuronal loss and and translation assay and lactate dehydrogenase (LDH) release assay showed that C9orf72-DRPs, but no other CPPs, inhibited protein translation and caused cell death by aberrant conversation with RNA. The cell penetration rate of poly-PR was determined by length of repeats and extracellular pH, and enforced delivery of non-penetrating short poly-PR peptide caused cell death, suggesting that inhibition of intracellular delivery of DRPs might be a novel drug target for C9orf72-mediated ALS. Results Cell penetrating peptides (CPP) which can cross the plasma membrane bilayer are subcategorized by their biochemical characteristics such as hydrophilic CPPs, hydrophobic CPPs and amphipathic CPPs14,15. C9orf72-related Arg-rich dipeptides are classified as the hydrophilic cationic CPP because 50% of their content is usually polar arginine. The most established cationic CPP is usually TAT peptide (GRKKRRQRRRPPQ: 13 a. a.) identified from HIV-1. Since the discovery of TAT, many cationic CPPs including R12 (RRRRRRRRRRRR: 12 a. a.) and FHV peptide (RRRRNRTRRNRRRVR:15 a. a.) were identified and investigated as possible tools for the drug delivery system16. The advantage of these CPPs as a drug delivery carrier is usually their high ability to deliver molecules including proteins and nucleotides across biological membranes without cytotoxicity. Although C9orf72-encoding Arg-rich dipeptides should have comparable biochemical characteristics with these CPPs, C9orf72-related DRPs have been shown to cause cell death once they penetrate the plasma membrane4. To clarify the difference between the C9orf72-related DRPs and non-harmful CPPs, we synthesized well-known cationic CPPs including TAT peptide, R12 peptide and FHV peptide and C9orf72-derived DRPs, (PR)20 peptide (40 a. a.) and (GR)20 peptide (40 a. a.) (Fig.?1A), and examined their penetration to human embryonic kidney (HEK) 293 cells. All of the CPPs examined were uptaken by HEK293 cells as expected (Fig.?1B). The confocal imaging revealed the subcellular localization of FITC-labeled CPPs. TAT made cytosolic large dots, R12 and FHV diffusely BX471 localized to both nuclear and cytosol with some punctate structures in cytosol, (PR)20 mainly localized to the nucleus and especially accumulated to round-shaped nucleoli and (GR)20 localized to both nucleoli and cytosol (Fig.?1B). The nucleolar localization of (PR)20 was also confirmed with Z-stack imaging and colocalization with a nucleolar marker B23/nucleophosmin (Fig.?1C,D) as reported previously4,12. BX471 To deny the possibility that nucleolar distribution of poly-PR and poly-GR was an artifact because these peptides were added extracellularly, we overexpressed green fluorescent protein (GFP)-tagged (PR)50 repeat protein and GFP-tagged (GR)50 repeat protein in HEK293 cells and confirmed that both GFP-(PR)50 and GFP-(GR)50 G-CSF distributed to nucleoli (Fig.?1E,F). Consistent with the subcellular localization of FITC-peptides, GFP-(PR)50 showed almost exclusive distribution to nucleoli and GFP-(GR)50 distributed to both nucleoli and cytosol (Fig.?1E,F). The nucleolar distribution of the FITC-labeled DRPs and GFP-fused DRPs implies existence of molecules with high affinity to C9orf72-DRPs in the nucleolus. The nucleolus is usually a subnuclear membrane-less compartment which is the BX471 site for transcription of ribosomal RNA and assembly of ribosome17. Because the nucleolus contains.