In vitro cellular uptake and neuroprotective efficacy of poly-arginine-18 (R18) and poly-ornithine-18 (O18) peptides: critical role of arginine guanidinium head groups for neuroprotection
- 60 Downloads
We have previously demonstrated that Cationic Arginine-Rich Peptides (CARPs) and in particular poly-arginine-18 (R18; 18-mer of arginine) exhibit potent neuroprotective properties in both in vitro and in vivo neuronal injury models. Based on the current literature, there is a consensus that arginine residues by virtue of their positive charge and guanidinium head group is the critical element for imparting CARP neuroprotective properties and their ability to traverse cell membranes. This study examined the importance of guanidinium head groups in R18 for peptide cellular uptake, localization, and neuroprotection. This was achieved by using poly-ornithine-18 (O18; 18-mer of ornithine) as a control, which is structurally identical to R18, but possesses amino head groups rather than guanidino head groups. Epifluorescence and confocal fluorescence microscopy was used to examine the cellular uptake and localization of the FITC-conjugated R18 and O18 in primary rat cortical neurons and SH-SY5Y human neuroblastoma cell cultures. An in vitro cortical neuronal glutamic acid excitotoxicity model was used to compare the effectiveness of R18 and O18 to inhibit cell death and intracellular calcium influx, as well as caspase and calpain activation. Fluorescence imaging studies revealed cellular uptake of both FITC-R18 and FITC-O18 in neuronal and SH-SY5Y cells; however, intracellular localization of the peptides differed in neurons. Following glutamic acid excitotoxicity, only R18 was neuroprotective, prevented caspases and calpain activation, and was more effective at reducing neuronal intracellular calcium influx. Overall, this study demonstrated that for long chain cationic poly-arginine peptides, the guanidinium head groups provided by arginine residues are an essential requirement for neuroprotection but are not required for entry into neurons.
KeywordsNeuroprotection Poly-arginine Poly-ornithine Excitotoxicity R18 Guanidinium
Cationic arginine-rich peptide
‘Trans-activator of transcription’ HIV-1 protein
Traumatic brain injury
The authors would like to acknowledge the Pierce-Armstrong Foundation and the Ian Potter Foundation for funding.
This work was supported in part by University Postgraduate Award (UPA) from the University of Notre Dame, Australia.
Compliance with ethical standards
Conflict of interest
B.P. Meloni and N.W. Knuckey are named inventors of several patent applications (Provisional Patents: 2013904197; 30/10/2013 and 2014902319; 17/6/2014 and PCT/AU2014/050326; 30/10/2104) regarding the use of arginine-rich peptides as neuroprotective agents. The other authors declare they have no conflict of interest.
- 2.Meloni BP, Milani D, Cross JL et al (2017) 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. NeuroMolecular Med 19:271–285. https://doi.org/10.1007/s12017-017-8441-2 CrossRefPubMedGoogle Scholar
- 4.Edwards A, Feindel K, Cross J et al (2017) Neuroprotective efficacy of poly-arginine-18 (R18) peptides using an in vivo model of perinatal hypoxic ischaemic encephalopathy (HIE). J Cereb Blood Flow Metab 37:18–19Google Scholar
- 13.Meloni BP, Craig AJ, Milech N et al (2014) The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures. Cell Mol Neurobiol 34:173–181. https://doi.org/10.1007/s10571-013-9999-3 CrossRefPubMedGoogle Scholar
- 14.MacDougall G, Anderton RS, Mastaglia FL et al (2018) Mitochondria and neuroprotection in stroke: cationic arginine-rich peptides (CARPs) as a novel class of mitochondria-targeted neuroprotective therapeutics. Neurobiol Dis 121:17–33. https://doi.org/10.1016/j.nbd.2018.09.010 CrossRefPubMedGoogle Scholar
- 15.Edwards AB, Cross JL, Anderton RS et al (2018) Poly-arginine R18 and R18D (d-enantiomer) peptides reduce infarct volume and improves behavioural outcomes following perinatal hypoxic-ischaemic encephalopathy in the P7 rat. Mol Brain 11:1–12. https://doi.org/10.1186/s13041-018-0352-0 CrossRefGoogle Scholar
- 17.Milani D, Cross JL, Anderton RS et al (2017) Delayed 2-h post-stroke administration of R18 and NA-1 (TAT-NR2B9c) peptides after permanent and/or transient middle cerebral artery occlusion in the rat. Brain Res Bull 135:62–68. https://doi.org/10.1016/j.brainresbull.2017.09.012 CrossRefPubMedGoogle Scholar
- 18.MacDougall G, Anderton RS, Edwards AB et al (2017) 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. J Mol Neurosci 61:235–246. https://doi.org/10.1007/s12031-016-0861-1 CrossRefPubMedGoogle Scholar
- 27.Console S, Marty C, García-Echeverría C et al (2003) Antennapedia and HIV transactivator of transcription (TAT) “protein transduction domains” promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. J Biol Chem 278:35109–35114. https://doi.org/10.1074/jbc.M301726200 CrossRefPubMedGoogle Scholar
- 40.Marshall J, Wong KY, Rupasinghe CN et al (2015) Inhibition of N-Methyl-d-aspartate-induced retinal neuronal death by polyarginine peptides is linked to the attenuation of stress-induced hyperpolarization of the inner mitochondrial membrane potential. J Biol Chem 290:22030–22048. https://doi.org/10.1074/jbc.M115.662791 CrossRefPubMedPubMedCentralGoogle Scholar
- 46.Brittain JM, Piekarz AD, Wang Y et al (2009) An atypical role for collapsin response mediator protein 2 (CRMP-2) in neurotransmitter release via interaction with presynaptic voltage-gated calcium channels. J Biol Chem 284:31375–31390. https://doi.org/10.1074/jbc.M109.009951 CrossRefPubMedPubMedCentralGoogle Scholar
- 47.Chi XX, Schmutzler BS, Brittain JM et al (2009) Regulation of N-type voltage-gated calcium channels (Cav2.2) and transmitter release by collapsin response mediator protein-2 (CRMP-2) in sensory neurons. J Cell Sci 122:4351–4362. https://doi.org/10.1242/jcs.053280 CrossRefPubMedPubMedCentralGoogle Scholar
- 53.Keana JF, McBurney RN, Scherz MW et al (1989) Synthesis and characterization of a series of diarylguanidines that are noncompetitive N-methyl-d-aspartate receptor antagonists with neuroprotective properties. Proc Natl Acad Sci USA 86:5631–5635. https://doi.org/10.1073/pnas.86.14.5631 CrossRefPubMedGoogle Scholar
- 55.Reddy NL, Hu LY, Cotter RE et al (1994) Synthesis and structure-activity studies of N, N′-Diarylguanidine Derivatives. N-(1-Naphthyl)-N′-(3-ethylphenyl)-N′-methylguanidine: a new, selective noncompetitive NMDA receptor antagonist. J Med Chem 37:260–267. https://doi.org/10.1021/jm00028a009 CrossRefPubMedGoogle Scholar
- 60.Bowie D, Lange GD, Mayer ML (2018) Activity-dependent modulation of glutamate receptors by polyamines. J Neurosci 18:8175–8185. https://doi.org/10.1523/jneurosci.18-20-08175.1998 CrossRefGoogle Scholar
- 65.Zhang YM, Bhavnani BR (2006) Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor (AIF) and this process is inhibited by equine estrogens. BMC Neurosci 15:49. https://doi.org/10.1186/1471-2202-7-49 CrossRefGoogle Scholar
- 68.Courderot-Masuyer C, Dalloz F, Maupoil V, Rochette L (1999) Antioxidant properties of aminoguanidine. Fundam Clin Pharmacol 13:535–540. https://doi.org/10.1111/j.1472-8206.1999.tb00358.x CrossRefPubMedGoogle Scholar
- 73.Nath R, Raser KJ, Stafford D et al (2015) Non-erythroid α-spectrin breakdown by calpain and interleukin 1 β-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem J 1:683–690. https://doi.org/10.1042/bj3190683 CrossRefGoogle Scholar