The neuroprotective potential of arginine-rich peptides for the acute treatment of traumatic brain injury

Traumatic brain injury (TBI) encompasses any insult to the brain resulting from external mechanical forces. The subsequent brain damage is complex, spanning a spectrum of symptoms and disabilities. TBI is a significant, but under-appreciated public health and economic concern burdening developed countries. With only preventative and rehabilitative measures currently in place, there is a dire need to develop a therapeutic that preserves brain tissue in the acute stages following TBI. Recent research has highlighted the neuroprotective properties of arginine-rich peptides in vitro following neuronal excitotoxicity and in vivo following stroke. Therefore, based on the in vitro and in vivo neuroprotective actions of arginine-rich peptides these molecules are promising therapeutics to reduce acute TBI.

Brain trauma is a major cause of morbidity and mortality in populations worldwide, usually as a consequence of road traffic accidents, falls, street violence and contact sports. Although this affliction perpetuates developed society, the occurrence of TBI-related hospitalizations is greatest in young adult males [1]. Children, adolescents and adults aged 75 or over are also more susceptible to head trauma because of physical activity, and a risk of falls [1]. More recently, TBI has become a concern for active military personnel, who may be exposed to blast waves and other combat-related traumatic events [2].

Survivors of TBI often suffer from lifelong cognitive, physical, behavioral and communicative deficits that negatively affect families, communities and the economy. Furthermore, TBI sufferers are at a higher risk of developing anxiety and depressive disorders [3], and neurodegenerative diseases such as chronic traumatic encephalopathy, Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis [4].

Current strategies to minimize the impact of TBI focus on preventative measures, acute neuro-critical care and neuro-restorative practices. However, targeting the secondary neuro-damaging processes that follow the primary insult provides an additional and potentially more effective, treatment to complement existing interventions. Therefore, any acute neuroprotective treatment strategy that maximizes preservation of brain tissue provides the best opportunity to improve outcomes following TBI.

Of the multitude of experimental drugs studied for treating TBI, a number have shown preclinical neuroprotective efficacy and advanced to clinical trials. From there, the drug trials have returned neutral, negative or contradictory outcomes. While there may be several reasons why a pharmacological neuroprotective agent has failed clinically, one possibility is that the therapy specifically targets one of the many pathophysiology events activated in the brain following TBI.

Many approaches aimed at mitigating secondary injury after TBI have been directed at attenuating excitotoxicity, a neuro-damaging process caused by the uncontrolled release of the neurotransmitter glutamate into the extracellular space. Clinical studies of glutamate receptor antagonists that target excitotoxicity such as dexanabinol, selfotel and magnesium, reported no statistically significant effects on Glasgow Outcome Score or mortality [5]. Another neuroprotective strategy is to block ion channels, thereby reducing the toxic intracellular influx of calcium and other ions into neurons, and the subsequent production of reactive oxygen species, and activation of calpains and endonucleases [6]. Unfortunately, clinical trials of calcium-channel blockers such as nimodipine and nicardipine have only shown an ability to decrease mortality or severe disability by reducing the onset of vasospasm in a small subset of TBI patients suffering a subarachnoid hemorrhage [5], making this type of medication unsuitable for the general TBI populace. The hormones erythropoietin (Clinicaltrials.gov Identifier: NCT00987454) and progesterone (Clinicaltrials.gov Identifier: NCT00822900), and the immunosuppressive peptide cyclosporine A (CsA) (Clinicaltrials.gov Identifier: NCT02496975) are other therapeutics that have undergone recent clinical trials after demonstrating preclinical efficacy. However, it appears that these agents may be of limited benefit following TBI [5,7].

The jaded nuance felt from the failure of the large number of TBI neuroprotection clinical trials beckons a shift in perspective. Although there have been recent calls for combined therapies of existing drugs, this direction may be ill-advised, as the additive effects could complicate pharmacokinetic interactions to the detriment of the patient [8]. Given the lack of success with previous neuroprotective agents for TBI, and the challenges combined therapy presents, an alternative therapeutic approach involves the use of cationic arginine-rich cell-penetrating peptides (CPP). Cationic CPPs fused to different ‘neuroprotective peptides’ have demonstrated efficacy in numerous acute brain injury models including stroke, perinatal hypoxia-ischemia, epilepsy and TBI. The most commonly used cationic CPP is the HIV-derived TAT peptide (GRKKRRQRRR), which allows delivery of fused cargos (e.g. peptides, proteins, drugs) into the brain and neurons without any apparent toxicity in preclinical studies. TAT-mediated uptake into the brain is attributable to its composition of the basic amino acids lysine (K) and especially arginine (R), which confer a positive charge to the peptide [9]. Interactions between the cationic TAT peptide and anionic cell surface structures induces endocytosis and/or membrane transduction resulting in the transport of TAT and its cargo across the blood–brain barrier and cell membranes.

However, of even greater potential significance was the demonstration that the TAT peptide itself possessed neuroprotective properties and that other cationic arginine-rich CPPs displayed considerably greater neuroprotective efficacy than the TAT peptide, following in vitro excitotoxicity [10]. Furthermore, studies revealed that arginine residues were the critical elements for neuroprotection, with peptide efficacy increasing with arginine content [11]. In addition, arginine-rich peptides were shown to be neuroprotective in a rat stroke model and also capable of reducing neuronal calcium influx following glutamate excitotoxicity [11]. Finally, mounting evidence suggests that the neuroprotective actions of TAT-fused neuroprotective peptides is largely mediated by the arginine residues and to a lesser extent lysine and tryptophan residues within the TAT and/or cargo peptide [12].

Unlike previously explored therapeutics, arginine-rich peptides are not believed to act as direct glutamate receptor and/or calcium channel antagonists. Rather, it has been proposed that at least one mechanism by which these arginine-rich peptides exert their neuroprotective effects is by inducing the internalization of neuronal cell surface structures, as a result of endocytosis [10,12,13], thereby reducing the effects of excitotoxicity and its down-stream neuro-damaging signaling pathways. Additionally, poly-arginine and other cationic arginine-rich peptides are potent inhibitors of proprotein convertase enzymes, including furin [14], a ubiquitously expressed calcium-dependent convertase involved in both physiological and pathological processes such as matrix metalloproteinase activation.

Moreover, once internalized, arginine-rich peptides may target mitochondria [15] and exert beneficial actions on mitochondrial membranes, thus behaving as a multipotent therapeutic with both extracellular and intracellular neuroprotective mechanisms. In targeting the mitochondria, cationic arginine-containing peptides have been demonstrated to reduce reactive oxygen species production, preserve ATP synthesis, inhibit membrane permeability transition, limit calcium influx and prevent cytochrome c release [16,17].

Interestingly, the neuroprotective efficacy of peptides containing arginine and lysine residues such as those derived from apolipoprotein E (APOE) and the amyloid precursor protein (APP) has been investigated in TBI and other brain injury models. These peptides have demonstrated reduced axonal, ischemic and hypoxic injury, in addition to anti-inflammatory effects and functional improvements in animal models of TBI [18–22]. Although these studies hypothesize that these peptides are neuroprotective through other mechanisms, their arginine content and cationic charge makes a compelling argument for a neuroprotective effect similar to that of arginine-rich peptides. Furthermore, it should be noted that in vitro comparisons between these and other peptides containing more arginine residues, found that those derived from APOE and APP were considerably less neuroprotective (unpublished observation).

A TAT-fused peptide, known as TAT-NR2B9c (also known as NA-1) has recently demonstrated preclinical efficacy in rodent and non-human primate stroke models, a disease which shares many pathophysiological similarities with TBI [23]. Although TBI is more complex and heterogeneous than stroke, many neuroprotective therapeutics trialed for TBI were initially investigating for stroke, so it stands to reason that the neuroprotective potential of arginine-rich peptides may also extend to TBI. Moreover, substantial safety and efficacy in the administration of TAT-NR2B9c in humans and non-human primates has been established [24]. To this end, studies in our laboratory have shown that a poly-arginine-18 peptide is more neuroprotective than TAT-NR2B9c in both in vitro [11] and in vivo (unpublished observation) stroke models. Therefore, based on the TAT-NR2B9c, and APOE- and APP-derived peptide studies, plus the superior efficacy of poly-arginine peptides compared with the three aforementioned peptides, it is possible that poly-arginine, or other arginine-rich peptides, may be effective agents following TBI. However, the extent to which arginine-rich peptides have the capacity to influence pathological processes known to exacerbate the negative outcomes of TBI, such as intracranial hypertension, cerebrovascular dysautoregulation, and neuro-biochemical modifications, is yet to be determined.

Conclusions

A collective assessment of previous neuroprotection studies has emphasized the need to reconsider our perspective when developing a therapeutic for TBI. From a pharmacotherapy viewpoint, arginine-rich peptides represent a new and exciting class of neuroprotective agents, which have the capacity to target the secondary damaging events of traumatic injury within the central nervous system. Not only do arginine-rich peptides appear to reduce excitotoxicity, they also have the ability to attenuate mitochondrial dysfunction and inhibit extracellular matrix metalloproteinase activation, thereby assisting the viability of the neurovascular unit within the TBI affected brain. Furthermore, the recent preclinical arginine-rich peptide studies in stroke, a condition that shares many pathophysiological similarities to TBI, provides promise that this class of peptide will be effective following trauma to the brain.

Financial and competing interests disclosure

BP Meloni is a named inventor of several patent applications regarding the use of arginine-rich peptides as neuroprotective agents. NW Knuckey is a named inventor of several patent applications regarding the use of arginine-rich peptides as neuroprotective agents. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.