Several recent studies have demonstrated that TAT and other arginine-rich cell penetrating peptides (CPPs) have intrinsic neuroprotective properties in their own right. Examples, we have demonstrated that in addition to TAT, poly-arginine peptides (R8 to R18; containing 8-18 arginine residues) as well as some other arginine-rich peptides are neuroprotective in vitro (in neurons exposed to glutamic acid excitotoxicity and oxygen glucose deprivation) and in the case of R9 in vivo (after permanent middle cerebral artery occlusion in the rat). Based on several lines of evidence, we propose that this neuroprotection is related to the peptide's endocytosis-inducing properties, with peptide charge and arginine residues being critical factors. Specifically, we propose that during peptide endocytosis neuronal cell surface structures such as ion channels and transporters are internalised, thereby reducing calcium influx associated with excitotoxicity and other receptor-mediated neurodamaging signalling pathways. We also hypothesise that a peptide cargo can act synergistically with TAT and other arginine-rich CPPs due to potentiation of the CPPs endocytic traits rather than by the cargo-peptide acting directly on its supposedly intended intracellular target. In this review, we systematically consider a number of studies that have used CPPs to deliver neuroprotective peptides to the central nervous system (CNS) following stroke and other neurological disorders. Consequently, we critically review evidence that supports our hypothesis that neuroprotection is mediated by carrier peptide endocytosis. In conclusion, we believe that there are strong grounds to regard arginine-rich peptides as a new class of neuroprotective molecules for the treatment of a range of neurological disorders.

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.