Assessment of the Neuroprotective Effects of Arginine-Rich Protamine Peptides, Poly-Arginine Peptides (R12-Cyclic, R22) and Arginine–Tryptophan-Containing Peptides Following In Vitro Excitotoxicity and/or Permanent Middle Cerebral Artery Occlusion in Rats

Abstract

We have demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties with arginine content and peptide positive charge being particularly critical for neuroprotective efficacy. In addition, the presence of other amino acids within arginine-rich peptides, as well as chemical modifications, peptide length and cell-penetrating properties also influence the level of neuroprotection. Against this background, we have examined the neuroprotective efficacy of arginine-rich protamine peptides, a cyclic (R12-c) poly-arginine peptide and a R22 poly-arginine peptide, as well as arginine peptides containing tryptophan or other amino acids (phenylalanine, tyrosine, glycine or leucine) in in vitro glutamic acid excitotoxicity and in vivo rat permanent middle cerebral artery occlusion models of stroke. In vitro studies demonstrated that protamine and poly-arginine peptides (R12-c, R22) were neuroprotective. Arginine-tryptophan-containing peptides were highly neuroprotective, with R12W8a being the most potent arginine-rich peptide identified in our laboratory. Peptides containing phenylalanine or tyrosine substituted in place of tryptophan in R12W8a were also highly neuroprotective, whereas leucine, and in particular glycine substitutions, decreased peptide efficacy. In vivo studies with protamine administered intravenously at 1000 nmol/kg 30 min after MCAO significantly reduced infarct volume and cerebral oedema by 22.5 and 38.6%, respectively. The R12W8a peptide was highly toxic when administered intravenously at 300 or 100 nmol/kg and ineffective at reducing infarct volume when administered at 30 nmol/kg 30 min after MCAO, unlike R18 (30 nmol/kg), which significantly reduced infarct volume by 20.4%. However, both R12W8a and R18 significantly reduced cerebral oedema by 19.8 and 42.2%, respectively. Protamine, R12W8a and R18 also reduced neuronal glutamic acid-induced calcium influx. These findings further highlight the neuroprotective properties of arginine-rich peptides and support the view that they represent a new class of neuroprotective agent.

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

“…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]. Importantly, an arginine and cationic charge mediated neuroprotective mechanism of action of CARPs, including CPP-fused to putative neuroprotective peptides (e.g., TAT-NR2B9c and TAT-NR2Bct), is supported in studies by Marshall et al [65] and more recently McQueen et al [66].…”

“…We subsequently demonstrated that in an in vitro cortical neuronal excitotoxic model, the CPPs poly-arginine-9 (RRRRRRRRR-NH 2 ; net charge +10) and penetratin (RQIKIWFQNRRMKWKK-NH 2 ; net charge +8) were even more potent than TAT [12]. The high potency of R9 led us to explore other poly-arginine peptides (e.g., R1, R3, R6–R15, R18, R22), as well as several other CARPs, including protamine in the in vitro excitotoxic and OGD neuronal injury models [27,28,29]. These studies confirmed that CARPs are highly neuroprotective, with efficacy increasing with arginine content and peptide positive charge; peaking at R15 to R18 for poly-arginine peptides [27,29] [Note arginine and lysine (K) are the only positively charged amino acids, while histidine has a low positive charge].…”

“…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].…”

“…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]. …”

“…Based on the recent application of cationic CPPs for the delivery of putative neuroprotective peptides in animal models of HIE and the recent discovery of the intrinsic neuroprotective properties of CARPs, the objectives of this review are: (i) catalogue studies that have used peptides as neuroprotective agents in HIE, especially those employing a CPP delivery system; and (ii) to evaluate the potential neuroprotective effects of both CPP and cargo molecules in terms of their arginine content and cationic charge, as well as the content of other potentially neuroprotective amino acids such as tryptophan [29]. We will propose that the neuroprotective properties of most, if not all putative neuroprotective peptides fused to cationic CPPs are likely to be mediated in a large part by the actions of the carrier CPP (e.g., TAT) rather than the cargo molecule itself, with potency being further enhanced according to the amino acid content of the cargo (e.g., number of arginine residues) [19].…”