Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties
Introduction
In recent years there has been an increased interest in the use of specifically designed peptides targeting cyto-damaging or cyto-protective pathways as neuroprotective agents. There are several reasons why this interest arose, including: i) peptide sequences critical for neurodamaging or neuroprotective intracellular protein–protein interactions can be easily identified and used as competitive inhibitors of target proteins (e.g. JNKI-1 peptide); ii) small peptides (2–40 amino acids) can be synthesised relatively cheaply using commercial sources; and iii) the development of cell penetrating peptides (CPPs), also referred to as protein or peptide transduction domains (PTDs), has provided a way to deliver peptides and other cargos (incl. proteins, nucleic acids and drugs) into cells and across the blood–brain barrier.
The discovery of CPPs has led to studies on the ability of a number of peptides and proteins to act as neuroprotection agents, as well as providing a means to explore the role of protein/protein interactions in brain function in health and disease (viz. neurological and non-neurological disorders). The main focus of this review is the use of arginine-rich CPPs (mainly TAT) for the delivery of neuroprotective peptides (<40 amino acids) particularly in cerebral ischaemia and stroke. The recent observation that CPPs have intrinsic neuroprotective properties in their own right has led us to question the conclusions of other studies. Here, we critically reappraise previous studies that have used putative neuroprotective peptides fused to CPPs as agents in cerebral ischaemia and other models of CNS injury, and examine the mechanism whereby arginine rich-peptides exert their neuroprotective effects. Importantly, we highlight that many past studies on neuroprotective peptides that have used cationic CPPs for CNS delivery may need to be reinterpreted in the light of the intrinsic neuroprotective effects of the carrier-peptide.
Cell penetrating peptides (CPPs) are small peptides (typically 5–25 amino acids) that are commonly used to facilitate the delivery of normally non-permeable cargo molecules such as other peptides, proteins, nucleic acids or drugs into cells, and across the blood–brain barrier. The development of CPPs as drug vehicles was sparked by the discovery of the PTD within the human immunodeficiency virus-type 1 trans-activator of transcription (HIV-TAT) protein (Frankel and Pabo, 1988, Green and Loewenstein, 1988). The active transporting peptide sequence within the HIV-TAT protein was isolated (TAT48–57: GRKKRRQRRR) and is now referred to as the TAT peptide or TAT (Becker-Hapak et al., 2001). Subsequently, over 100 CPPs have been identified (Milletti, 2012).
By far the most commonly used CPP peptide is TAT, especially to deliver various cargo molecules to the brain, including neuroprotective peptides and proteins. Other CPPs include penetratin (also known as antennapedia), poly-arginine peptides (R8 to R12; where R refers to arginine residues), Pep-1 and transportan. The amino acid sequences for these peptides, as well as of some less commonly used CPPs, are shown in Table 1. TAT, poly-arginine and penetratin are cationic arginine-rich CPPs.
Potential neuroprotective peptides fused to CPPs have been assessed in cultured neurons and animal models that mimic neural injury mechanisms seen in a variety of disorders, including cerebral ischaemia, spinal cord injury, traumatic brain injury, epilepsy, Parkinson's disease and Alzheimer's disease (Arthur et al., 2007, Colombo et al., 2007, Lai et al., 2005, Liu et al., 2006, Meade et al., 2009, Nagel et al., 2008). However, several years ago, we and others demonstrated that TAT possesses intrinsic neuroprotective properties both in vitro in neurons exposed to excitotoxicity and oxygen–glucose deprivation (OGD) and in vivo following cerebral ischaemia in P12 rats after intraventricular injection (Craig et al., 2011, Meade et al., 2010a, Vaslin et al., 2009b, Xu et al., 2008). We subsequently showed that poly-arginine-9 (R9), penetratin and Pep-1 also display neuroprotective actions in in vitro excitotoxic and/or OGD models (Meloni et al., 2014). Furthermore, our data showed that R9 and penetratin were 17- and 4.6-fold respectively more neuroprotective than TAT (Meloni et al., 2014).
The higher potency of R9 relative to TAT and penetratin led us to explore the in vitro neuroprotective potency of other poly-arginine peptides (R1, R3, R6–R15 and R18), as well as, other arginine-rich peptides (Meloni et al., 2015). These studies confirmed that poly-arginine and arginine-rich peptides as a group are highly neuroprotective, with efficacy increasing with increasing arginine content, peaking at R15 (Meloni et al., 2015). We also showed that arginine-rich peptides have the capacity to reduce glutamic acid-induced neuronal calcium influx and are neuroprotective with a single treatment several hours before glutamic acid or OGD exposure. Furthermore, neuroprotective efficacy was shown to be directly related to peptide positive net charge conferred by the positively charged arginine (R) and lysine (K) amino acids residues, which could be blocked by fusion with a negatively charged glutamic acid (E9) poly-peptide (e.g. R9/E9 peptide) or by incubation with the highly negatively charged molecule heparin. The latter finding strongly suggests that peptides bind to negatively charged cell surface molecules such as heparin sulphate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs) or sialic acid residues present in glycosphingolipids to initiate and stimulate peptide endocytosis (Favretto et al., 2014, Kim et al., 2012, Ravindran et al., 2013, Wallbrecher et al., 2014) a process crucial for neuroprotection (Meloni et al., 2015). In this context, others have demonstrated that the nature of the peptide interaction with HSPGs determines CPPs endocytic properties (Wallbrecher et al., 2014).
With respect to endocytosis, studies have demonstrated that peptide charge conferred by arginine and lysine residues (note: arginine and lysine are the only two strongly positively charged amino acids, with histidine being only weakly positively charged, whereas glutamic acid and aspartic acid are the only two negatively charged amino acids) facilitate HSPG binding, and that mainly arginine residues trigger the endocytic process (Amand et al., 2012, Wallbrecher et al., 2014, Yang et al., 2014). Consistent with our proposed endocytic neuroprotective mechanism, we have demonstrated that poly-lysine (K10) is only weakly neuroprotective in a cortical neuronal glutamic acid excitotoxicity model (Meloni et al., 2015). It is also likely that other amino acids can influence the endocytic properties of cationic peptides in both a positive and negative manner as has been demonstrated for tryptophan (W; Rydberg et al., 2012, Bechara et al., 2013) and alanine (A; Yang et al., 2014), respectively. Indeed, we have now confirmed that tryptophan and alanine amino acids within arginine-rich peptides respectively increase and decrease neuroprotective efficacy in a glutamic acid excitotoxicity model (Fig. 1).
Based on our recent findings we hypothesised that arginine-rich peptides exert their neuroprotection effects by inducing the endocytic internalisation of cell surface ion channels, thereby reducing the damaging effects of excitotoxicity (see Fig. 2). This is a novel hypothesis that essentially identifies arginine-rich peptides as a new class of neuroprotective molecule. There are several lines of evidence based on our findings and those of others that support our endocytosis hypothesis. Arginine-rich peptides, including so called “neuroprotective peptides” fused to TAT have been shown to: i) reduce neuronal calcium influx (Meloni et al., 2015) and interfere with ion channel function (NMDA receptor: Ferrer-Montiel et al., 1988, Tu et al., 2010, Sinai et al., 2010, Brittain et al., 2011b, Brustovetsky et al., 2014, VR1: Planells-Cases et al., 2000, CaV2.2: Brittain et al., 2011a, Brittain et al., 2011b, Feldman and Khanna, 2013, Brustovetsky et al., 2014; sodium calcium exchanger [NCX], CaV3.3: García-Caballero et al., 2014); ii) cause internalisation of neuronal ion channels (Brustovetsky et al., 2014, Sinai et al., 2010); and iii) require endocytosis as a prerequisite for neuroprotection (Meloni et al., 2015, Vaslin et al., 2011). Interestingly, other TAT-fused peptides have also been shown to interfere with the function of neuronal receptors (D1R–D2R; Pei et al., 2010; PTPσ: Lang et al., 2015). In this context, it is important to note that endocytosis is a known mechanism used by cells to internalise cell surface receptors (Höller and Dikic, 2004, Marchese, 2014, Maxfield and McGraw, 2004).
Neuroprotective efficacy, at least for poly-arginine peptides (Meloni et al., 2015), appears to correlate with peptide transduction efficacy (Mitchell et al., 2000), a process known to occur by endocytosis (Appelbaum et al., 2012, Bechara et al., 2013, El-Sayed et al., 2009). Furthermore, it is important to note that the rapid and transient (lasting up to 4 h with peptide pre-treatment) nature of the neuroprotection induced by poly-arginine peptides (Meloni et al., 2015) corresponds closely to the timeframes of endocytosis and endosomal receptor re-cycling (Gundelfinger et al., 2003, Maxfield and McGraw, 2004, Yashunsky et al., 2009). Importantly, it is known that TAT, penetratin and R9 can induce the internalisation of EGFR and TNFR in HeLa cells (Fotin-Mleczek et al., 2005). Our hypothesis also links endocytosis as a common neuroprotective mechanism of action for a diverse range of arginine-rich peptides (including TAT-fused peptides), all of which are likely to have endocytic inducing properties.
This neuroprotective mechanism that we propose is also consistent with the link between neuronal cell surface-HSPGs (Litwack et al., 1994) and endocytic activity (Vaslin et al., 2009a), which are known to promote endosomal uptake of cationic CPPs (Nakase et al., 2007, Vaslin et al., 2009a, Vaslin et al., 2011). It is also possible that other negatively charged cell surface receptors such as CSPGs and glycosphingolipids can promote cationic CPP endocytosis and neuroprotection. As mentioned above, positively charged poly-arginine and arginine-rich peptides are known to bind negatively charged HSPGs to initiate endocytosis. It is important to note that any neuroprotective peptide fused to a CPP and internalised by endocytosis must escape the endosome to interact with its intended cytoplasmic target. However, endosomal escape appears to be a highly inefficient process (Appelbaum et al., 2012, Qian et al., 2014) (and rarely confirmed) and as a result, due to the cargo's inability to engage with its intracellular target it is unlikely to have a significant impact within the cytoplasm.
In light of our recent findings, the aim of this review is to critically re-examine studies in the literature that have used neuroprotective peptides fused to cationic CPPs (i.e. TAT, R9) and present evidence supporting our hypothesis that the neuroprotective actions of these peptides are primarily, if not exclusively, due to the endocytic properties of the peptide per se.
Section snippets
Examination of studies using CPP-fused to neuroprotective peptides in neuronal injury models
To date, over a dozen of neuroprotective peptides fused to CPPs have been described (Table 2, Table 3, Table 4). Three of the most intensely studied peptides developed as potential neuroprotective agents for stroke/cerebral ischaemia are NR2B9c, JNKI-1 and CBD3 (Table 2, Table 3, Table 4). This review in particular critically examines the use of these three peptides in a neurological setting, and provides evidence suggesting that the critical neuroprotective and functional structural elements
Examination neuroprotective arginine- (and lysine-) rich peptides used in neuronal injury models
It is beyond the scope of this review to include every study that has used a neuroprotective TAT-fused peptide. For completeness, other in vivo studies using TAT-fused peptides are listed in Table 5. Evidently, several studies have identified arginine- and lysine-rich peptides not fused to TAT or a CPP as being neuroprotective. We argue that it is possible that these peptides are intrinsically neuroprotective via mechanisms unrelated to their proposed action on a specific cell surface receptor
Discussion and concluding remarks
The main purpose of this review is to describe the neuroprotective properties of peptides fused to the arginine-rich CPP, TAT and in doing so provide evidence supportive of our hypothesis that neuroprotection is mediated not by the cargo molecule but largely by TAT itself. This hypothesis crystallised for us following the analysis of the neuroprotective and calcium influx inhibiting properties of a diverse set of peptides including: 1) arginine-rich CPPs (TAT, penetratin); 2) poly-arginine
Conflict of interest
B.P. Meloni and N.W. Knuckey are named inventors of several patent applications regarding the use of arginine-rich peptides as neuroprotective agents. The other authors declare no conflict of interest.
Acknowledgments
This study in part was supported by the Department of Neurosurgery, Sir Charles Gairdner Hospital and by a Neurotrauma Research Program of Western Australia research grant.
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