title: The Neuroprotective Peptide Poly-Arginine-12 (R12) Reduces Cell Surface Levels of NMDA NR2B Receptor Subunit in Cortical Neurons; Investigation into the Involvement of Endocytic Mechanisms.
abstract: We have previously reported that cationic poly-arginine and arginine-rich cell-penetrating peptides display high-level neuroprotection and reduce calcium influx following in vitro excitotoxicity, as well as reduce brain injury in animal stroke models. Using the neuroprotective peptides poly-arginine R12 (R12) and the NR2B9c peptide fused to the arginine-rich carrier peptide TAT (TAT-NR2B9c; also known as NA-1), we investigated the mechanisms whereby poly-arginine and arginine-rich peptides reduce glutamate-induced excitotoxic calcium influx. Using cell surface biotin protein labeling and western blot analysis, we demonstrated that R12 and TAT-NR2B9c significantly reduced cortical neuronal cell surface expression of the NMDA receptor subunit NR2B. Chemical endocytic inhibitors used individually or in combination prior to glutamate excitotoxicity did not significantly affect R12 peptide neuroprotective efficacy. Similarly, pretreatment of neurons with enzymes to degrade anionic cell surface proteoglycans, heparan sulfate proteoglycan (HSPG), and chondroitin sulfate proteoglycan (CSPG), as well as sialic acid residues, did not significantly affect peptide neuroprotective efficacy. While the exact mechanisms responsible for R12 peptide-mediated NMDA receptor NR2B subunit cell surface downregulation were not identified, an endocytic process could not be ruled out. The study supports our hypothesis that arginine-rich peptides reduce excitotoxic calcium influx by reducing the levels of cell surface ion channels.
authors.affiliation: Department of Neurosurgery, Sir Charles Gairdner Hospital, QEII Medical Centre, Nedlands, WA, Australia. gabriella.macdougall1@my.nd.edu.au.|Western Australian Neuroscience Research Institute, A Block, 4th Floor, QEII Medical Centre, Verdun St, Nedlands, WA, 6009, Australia. gabriella.macdougall1@my.nd.edu.au.|School of Heath Sciences, The University Notre Dame, Fremantle, WA, Australia. gabriella.macdougall1@my.nd.edu.au.
authors.forename: Gabriella
authors.identifier: http://orcid.org/0000-0002-6432-4675
authors.initials: G
authors.lastname: MacDougall
authors.affiliation: Western Australian Neuroscience Research Institute, A Block, 4th Floor, QEII Medical Centre, Verdun St, Nedlands, WA, 6009, Australia.|School of Heath Sciences, The University Notre Dame, Fremantle, WA, Australia.
authors.forename: Ryan S
authors.initials: RS
authors.lastname: Anderton
authors.affiliation: Department of Neurosurgery, Sir Charles Gairdner Hospital, QEII Medical Centre, Nedlands, WA, Australia.|Western Australian Neuroscience Research Institute, A Block, 4th Floor, QEII Medical Centre, Verdun St, Nedlands, WA, 6009, Australia.|School of Heath Sciences, The University Notre Dame, Fremantle, WA, Australia.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Edwards
authors.affiliation: Department of Neurosurgery, Sir Charles Gairdner Hospital, QEII Medical Centre, Nedlands, WA, Australia.|Western Australian Neuroscience Research Institute, A Block, 4th Floor, QEII Medical Centre, Verdun St, Nedlands, WA, 6009, Australia.|Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Nedlands, Australia.
authors.forename: Neville W
authors.initials: NW
authors.lastname: Knuckey
authors.affiliation: Department of Neurosurgery, Sir Charles Gairdner Hospital, QEII Medical Centre, Nedlands, WA, Australia.|Western Australian Neuroscience Research Institute, A Block, 4th Floor, QEII Medical Centre, Verdun St, Nedlands, WA, 6009, Australia.|Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Nedlands, Australia.
authors.forename: Bruno P
authors.initials: BP
authors.lastname: Meloni
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004705:Endocytosis; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*
chemical_list: C121001:NR2B NMDA receptor; D018696:Neuroprotective Agents; D010455:Peptides; D016194:Receptors, N-Methyl-D-Aspartate; C015462:polyarginine
keywords: Calcium influx; Endocytosis; Glutamate excitotoxicity; NR2B receptor subunit; Neuroprotection; Poly-arginine peptides