Abstract
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.
Similar content being viewed by others
Abbreviations
- CPP:
-
Cell-penetrating peptide
- CARP:
-
Cationic arginine-rich peptide
- HIE:
-
Hypoxic-ischaemic encephalopathy
- MMP:
-
Matrix metalloproteinases
- TAT:
-
‘Trans-activator of transcription’ HIV-1 protein
- TBI:
-
Traumatic brain injury
- R18:
-
Poly-arginine-18 peptide
- O18:
-
Poly-ornithine-18 peptide
References
Meloni BP, Milani D, Edwards AB et al (2015) Neuroprotective peptides fused to arginine-rich cell penetrating peptides: neuroprotective mechanism likely mediated by peptide endocytic properties. Pharmacol Ther 153:36–54. https://doi.org/10.1016/j.pharmthera.2015.06.002
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
Milani D, Cross JL, Anderton RS et al (2017) Neuroprotective efficacy of poly-arginine R18 and NA-1 (TAT-NR2B9c) peptides following transient middle cerebral artery occlusion in the rat. Neurosci Res 114:9–15. https://doi.org/10.1016/j.neures.2016.09.002
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 37:18–19
Chiu LS, Anderton RS, Cross JL et al (2019) Poly-arginine peptide R18D reduces neuroinflammation and functional deficits following traumatic brain injury in the long-evans rat. Int J Pept Res Ther. https://doi.org/10.1007/s10989-018-09799-8
Chiu LS, Anderton RS, Cross JL et al (2017) Assessment of R18, COG1410, and APP96-110 in excitotoxicity and traumatic brain injury. Transl Neurosci 8:147–157. https://doi.org/10.1515/tnsci-2017-0021
Edwards AB, Anderton RS, Knuckey NW, Meloni BP (2018) Assessment of therapeutic window for poly-arginine-18D (R18D) in a P7 rat model of perinatal hypoxic-ischaemic encephalopathy. J Neurosci Res. https://doi.org/10.1002/jnr.24315
Fonar G, Polis B, Meirson T et al (2018) Subcutaneous sustained-release of poly-arginine ameliorates cognitive impairment in a transgenic mouse model of Alzheimer’s disease. Adv Alzheimer’s Dis 7:153–182. https://doi.org/10.4236/aad.2018.74011
Jiang N, Frenzel D, Schartmann E et al (2016) Blood-brain barrier penetration of an Aβ-targeted, arginine-rich, d-enantiomeric peptide. Biochim Biophys Acta Biomembr 1858:2717–2724. https://doi.org/10.1016/j.bbamem.2016.07.002
Davoli E, Sclip A, Cecchi M et al (2014) Determination of tissue levels of a neuroprotectant drug: the cell permeable JNK inhibitor peptide. J Pharmacol Toxicol Methods 70:55–61. https://doi.org/10.1016/j.vascn.2014.04.001
Tang M, Waring AJ, Hong M (2007) Phosphate-mediated arginine insertion into lipid membranes and pore formation by a cationic membrane peptide from solid-state NMR. J Am Chem Soc 129:11438–11446. https://doi.org/10.1021/ja072511s
Meloni BP, Brookes LM, Clark VW et al (2015) Poly-arginine and arginine-rich peptides are neuroprotective in stroke models. J Cereb Blood Flow Metab 35:993–1004. https://doi.org/10.1038/jcbfm.2015.11
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
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
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
Milani D, Clark VW, Cross JL et al (2016) Poly-arginine peptides reduce infarct volume in a permanent middle cerebral artery rat stroke model. BMC Neurosci 17:19. https://doi.org/10.1186/s12868-016-0253-z
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
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
Szeto HH, Liu S, Soong Y et al (2011) Mitochondria-targeted peptide accelerates ATP recovery and reduces ischemic kidney injury. J Am Soc Nephrol 22:1041–1052. https://doi.org/10.1681/ASN.2010080808
Birk AV, Chao WM, Liu S et al (2015) Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia. Biochim Biophys Acta 1847:1075–1084. https://doi.org/10.1016/j.bbabio.2015.06.006
Cameron A, Appel J, Houghten RA, Lindberg I (2000) Polyarginines are potent furin inhibitors. J Biol Chem 275:36741–36749. https://doi.org/10.1074/jbc.M003848200
Cerrato CP, Pirisinu M, Vlachos EN, Langel Ü (2015) Novel cell-penetrating peptide targeting mitochondria. FASEB J 29:4589–4599. https://doi.org/10.1096/fj.14-269225
Hilchie AL, Wuerth K, Hancock REW (2013) Immune modulation by multifaceted cationic host defense (antimicrobial) peptides. Nat Chem Biol 9:761–768. https://doi.org/10.1038/nchembio.1393
Herce HD, Garcia AE, Cardoso MC (2014) Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules. J Am Chem Soc. https://doi.org/10.1021/ja507790z
Åmand HL, Rydberg HA, Fornander LH et al (2012) Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans. Biochim Biophys Acta Biomembr 1818:2669–2678. https://doi.org/10.1016/j.bbamem.2012.06.006
Gonçalves E, Kitas E, Seelig J (2005) Binding of oligoarginine to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide. Biochemistry 44:2692–2702. https://doi.org/10.1021/bi048046i
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
Ziegler A, Li Blatter X, Seelig A, Seelig J (2003) Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis. Biochemistry 42:9185–9190. https://doi.org/10.1021/bi0346805
Schug KA, Lindner W (2005) Noncovalent binding between guanidinium and anionic groups: focus on biological- and synthetic-based arginine/guanidinium interactions with phosph[on]ate and sulf[on]ate residues. Chem Rev 105:67–114. https://doi.org/10.1021/cr040603j
Kawaguchi Y, Takeuchi T, Kuwata K, Chiba J, Hatanaka Y, Nakase I, Futaki S (2016) Syndecan-4 is a receptor for clathrin-mediated endocytosis of arginine-rich cell-penetrating peptides. Bioconjug Chem 27:1119–1130. https://doi.org/10.1021/acs.bioconjchem.6b00082
Bogacheva M, Egorova A, Slita A et al (2017) Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery. Bioorg Med Chem Lett 27:4781–4785. https://doi.org/10.1016/j.bmcl.2017.10.001
Fischer R, Köhler K, Fotin-Mleczek M, Brock R (2004) A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides. J Biol Chem 279:12625–12635. https://doi.org/10.1074/jbc.M311461200
Pantos A, Tsiourvas D, Sideratou Z et al (2004) Interactions of complementary PEGylated liposomes and characterization of the resulting aggregates. Langmuir 20:6165–6172. https://doi.org/10.1021/la040026u
Rothbard JB, Jessop TC, Lewis RS et al (2004) Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells. J Am Chem Soc 126:9506–9507. https://doi.org/10.1021/ja0482536
Vivès E, Brodin P, Lebleu B (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272:16010–16017. https://doi.org/10.1074/jbc.272.25.16010
Mitchell DJ, Steinman L, Kim DT et al (2000) Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 56:318–325. https://doi.org/10.1034/j.1399-3011.2000.00723.x
Brorson JR, Marcuccilli CJ, Miller RJ (1995) Delayed antagonism of calpain reduces excitotoxicity in cultured neurons. Stroke 26:1259–1266. https://doi.org/10.1161/01.str.26.7.1259
Volbracht C, Chua BT, Ng CP et al (2005) The critical role of calpain versus caspase activation in excitotoxic injury induced by nitric oxide. J Neurochem 93:1280–1292. https://doi.org/10.1111/j.1471-4159.2005.03122.x
Jones SW, Christison R, Bundell K et al (2005) Characterisation of cell-penetrating peptide-mediated peptide delivery. Br J Pharmacol 145:1093–1102. https://doi.org/10.1038/sj.bjp.0706279
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
Oh D, Nasrolahi Shirazi A, Northup K et al (2014) Enhanced cellular uptake of short polyarginine peptides through fatty acylation and cyclization. Mol Pharm 11:2845–2854. https://doi.org/10.1021/mp500203e
Mishra A, Lai GH, Schmidt NW et al (2011) Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions. Proc Natl Acad Sci USA 108:16883–16888. https://doi.org/10.1073/pnas.1108795108
Li V, Wang YT (2016) Molecular mechanisms of NMDA receptor-mediated excitotoxicity: implications for neuroprotective therapeutics for stroke. Neural Regen Res 11:1752–1753. https://doi.org/10.4103/1673-5374.194713
Sinai L, Duffy S, Roder JC (2010) Src inhibition reduces NR2B surface expression and synaptic plasticity in the amygdala. Learn Mem 26:364–371. https://doi.org/10.1101/lm.1765710
Cook DR, Gleichman AJ, Cross SA et al (2011) NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury. J Neurochem 118:1113–1123. https://doi.org/10.1111/j.1471-4159.2011.07383.x
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
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
Wang Y, Brittain JM, Wilson SM, Khanna R (2010) Emerging roles of collapsin response mediator proteins (CRMPs) as regulators of voltage-gated calcium channels and synaptic transmission. Commun Integr Biol 3:172–175. https://doi.org/10.4161/cib.3.2.10620
Fotin-Mleczek M (2005) Cationic cell-penetrating peptides interfere with TNF signalling by induction of TNF receptor internalization. J Cell Sci 118:3339–3351. https://doi.org/10.1242/jcs.02460
Weng X-C, Gai X-D, Zheng J-Q, Li J (2003) Agmatine blocked voltage-gated calcium channel in cultured rat hippocampal neurons. Acta Pharmacol Sin 24:746–750
Murphy E, Perlman M, London RE, Steenbergen C (1991) Amiloride delays the ischemia-induced rise in cytosolic free calcium. Circ Res 68:1250–1258. https://doi.org/10.1161/01.RES.68.5.1250
Garcia ML, King VF, Shevell JL et al (1990) Amiloride analogs inhibit l-type calcium channels and display calcium entry blocker activity. J Biol Chem 265:3763–3771
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
Hong L, Kim IH, Tombola F (2014) Molecular determinants of Hv1 proton channel inhibition by guanidine derivatives. Proc Natl Acad Sci USA 11:9971–9976. https://doi.org/10.1073/pnas.1324012111
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
Jang S, Park SH (2018) Antidiabetic drug metformin protects neuronal cells against quinolinic acid-induced excitotoxicity by decreasing intracellular calcium. Chonnam Med J 54:24–30. https://doi.org/10.4068/cmj.2018.54.1.24
Durán-Riveroll LM, Cembella AD (2017) Guanidinium toxins and their interactions with voltage-gated sodium ion channels. Mar Drugs 15:E303. https://doi.org/10.3390/md15100303
Kalia J, Swartz KJ (2011) Elucidating the molecular basis of action of a classic drug: guanidine compounds as inhibitors of voltage-gated potassium channels. Mol Pharmacol 80:1085–1095. https://doi.org/10.1124/mol.111.074989
Bowie D (2018) Polyamine-mediated channel block of ionotropic glutamate receptors and its regulation by auxiliary proteins. J Biol Chem 293:18789–18802. https://doi.org/10.1074/jbc.TM118.003794
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
Williams K (1997) Modulation and block of ion channels: a new biology of polyamines. Cell Signal 9:1–13. https://doi.org/10.1016/s0898-6568(96)00089-7
Zhao K, Zhao GM, Wu D et al (2004) Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem 279:34682–34690. https://doi.org/10.1074/jbc.M402999200
Rigobello MP, Barzon E, Marin O, Bindoli A (1995) Effect of polycation peptides on mitochondrial permeability transition. Biochem Biophys Res Commun 217:144–149. https://doi.org/10.1006/bbrc.1995.2756
Prentice H, Modi JP, Wu J-Y (2015) Mechanisms of neuronal protection against excitotoxicity, endoplasmic reticulum stress, and mitochondrial dysfunction in stroke and neurodegenerative diseases. Oxid Med Cell Longev 2015:1–7. https://doi.org/10.1155/2015/964518
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
O’Donnell LA, Agrawal A, Sabnekar P et al (2007) Apelin, an endogenous neuronal peptide, protects hippocampal neurons against excitotoxic injury. J Neurochem 102:1905–1917. https://doi.org/10.1111/j.1471-4159.2007.04645.x
Yang Y, Zhang X, Cui H et al (2014) Apelin-13 protects the brain against ischemia/reperfusion injury through activating PI3 K/Akt and ERK1/2 signaling pathways. Neurosci Lett 568:44–49. https://doi.org/10.1016/j.neulet.2014.03.037
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
Velier JJ, Ellison JA, Kikly KK et al (1999) Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat. J Neurosci 19:5932–5941
Galluzzi L, Morselli E, Kepp O, Kroemer G (2009) Targeting post-mitochondrial effectors of apoptosis for neuroprotection. Biochim Biophys Acta Bioenerg 1787:402–413. https://doi.org/10.1016/j.bbabio.2008.09.006
Batulu H, Du G, Li D et al (2019) Effect of poly-arginine R18 on neurocyte cell growth via autophagy in traumatic brain injury. Exp Ther Med 17:4109–4115. https://doi.org/10.3892/etm.2019.7423
Chimura T, Launey T, Yoshida N (2015) Calpain-mediated degradation of drebrin by excitotoxicity in vitro and in vivo. PLoS ONE 10:e0125119. https://doi.org/10.1371/journal.pone.0125119
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
Brittain JM, Chen L, Wilson SM et al (2011) Neuroprotection against traumatic brain Injury by a peptide derived from the Collapsin Response Mediator Protein 2 (CRMP2). J Biol Chem 286:37778–37792. https://doi.org/10.1074/jbc.M111.255455
Acknowledgements
The authors would like to acknowledge the Pierce-Armstrong Foundation and the Ian Potter Foundation for funding.
Funding
This work was supported in part by University Postgraduate Award (UPA) from the University of Notre Dame, Australia.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
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.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
MacDougall, G., Anderton, R.S., Ouliel, E. et al. 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. Mol Cell Biochem 464, 27–38 (2020). https://doi.org/10.1007/s11010-019-03646-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11010-019-03646-0