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.

Mitochondria and neuroprotection in stroke: Cationic arginine-rich peptides (CARPs) as a novel class of mitochondria-targeted neuroprotective therapeutics

“…peptide (TAT-NR2B9c), have the capacity to not only reduce glutamic acid induced excitotoxic neuronal death and intracellular calcium influx [160,161,[218][219][220], but also reduce neuronal surface expression of the NMDA receptor subunit protein NR2B [221].…”

Perinatal Hypoxic-Ischemic Encephalopathy and Neuroprotective Peptide Therapies: A Case for Cationic Arginine-Rich Peptides (CARPs)

“…Our laboratory has also demonstrated that CARPs and in particular the poly-arginine peptide R18 are neuroprotective in in vivo experimental brain ischemic and/or hypoxic injury models [18,20,21,22,24,26,27,29], and that CARPs can reduce neuronal cell surface glutamate receptor levels and excitotoxic calcium influx [19,22,23,25,27,28]. As highlighted previously [19], based on our findings, we have proposed that the neuroprotective properties of most, if not all, putative neuroprotective peptides fused to cationic CPPs (e.g., TAT, R9 and penetratin), as well as other CARPs, are determined by the arginine content and positive charge of the peptide, with potency also influenced by other amino acid residues (e.g., lysine, tryptophan and phenylalanine) [19,29].…”

“…One mechanism whereby CARPs have the capacity to reduce intracellular calcium influx is the reduction of neuronal cell surface ion channels and receptors; thereby reducing excitotoxic calcium influx. To this end, CARPs have been demonstrated to reduce the cell surface expression or function of neuronal NMDA receptors [23,63,70,71,72,73], voltage gated calcium channels (CaV2.2 and CaV3.3) [68,70,74,75], a voltage gated sodium channel (NaV1.7) [76] and the sodium calcium exchanger 3 (NCX3) [70]. Given their effects on different receptors, and the positive charge of CARPs, they may also be antagonizing ion channel receptor function directly and/or by affecting calcium or sodium ion influx via an electrostatic mechanism.…”

“…It has been demonstrated that CARPs inhibit neuronal excitotoxic calcium [19,22,23,25,27,28] and potassium [64,75] influx and in doing so inhibit many of the pathophysiological processes downstream from the ischemic excitotoxic cascade (e.g., calpain activation, mitochondrial dysfunction and ROS production, calcium induced cell signaling and lipase and DNase activation). Therefore, given that the vast majority of the proposed mechanisms of action of developed TAT-fused and other neuroprotective CARPs are molecular targets downstream from excitotoxicity (e.g., NF-κB, JNK and Cdk5), it is not surprising that they are effective and appear to be inhibiting their intended targets.…”

“…However, several years ago, our and other laboratories demonstrated that the TAT peptide has modest intrinsic neuroprotective properties in its own right [12,13,14,15,16,17]. We subsequently demonstrated that other cationic arginine-rich peptides [CARPs; e.g., penetratin, protamine, sodium calcium exchanger inhibitory peptide (XIP), poly-arginine peptides (e.g., R9, R12 and R18; R indicates amino acid arginine and number indicates mer)], possess potent neuroprotective properties in both in vitro and animal models of cerebral ischemia and/or hypoxia [12,18,19,20,21,22,23,24,25,26,27,28,29], and demonstrated that the arginine content and peptide positive charge are critical determinants of neuroprotection [19,27,28]. …”