Ryan S. Anderton

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties

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 peptides 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.

Poly-Arginine and Arginine-Rich Peptides are Neuroprotective in Stroke Models

Using cortical neuronal cultures and glutamic acid excitotoxicity and oxygen-glucose deprivation (OGD) stroke models, we demonstrated that poly-arginine and arginine-rich cell-penetrating peptides (CPPs), are highly neuroprotective, with efficacy increasing with increasing arginine content, have the capacity to reduce glutamic acid-induced neuronal calcium influx and require heparan sulfate preotoglycan-mediated endocytosis to induce a neuroprotective effect. Furthermore, neuroprotection could be induced with immediate peptide treatment or treatment up to 2 to 4 hours before glutamic acid excitotoxicity or OGD, and with poly-arginine-9 (R9) when administered intravenously after stroke onset in a rat model. In contrast, the JNKI-1 peptide when fused to the (non-arginine) kFGF CPP, which does not rely on endocytosis for uptake, was not neuroprotective in the glutamic acid model; the kFGF peptide was also ineffective. Similarly, positively charged poly-lysine-10 (K10) and R9 fused to the negatively charged poly-glutamic acid-9 (E9) peptide (R9/E9) displayed minimal neuroprotection after excitotoxicity. These results indicate that peptide positive charge and arginine residues are critical for neuroprotection, and have led us to hypothesize that peptide-induced endocytic internalization of ion channels is a potential mechanism of action. The findings also question the mode of action of different neuroprotective peptides fused to arginine-rich CPPs.

Neuroprotective efficacy of poly-arginine R18 and NA-1 (TAT-NR2B9c) peptides following transient middle cerebral artery occlusion in the rat

We examined the efficacy of R18 in a transient MCAO model and compared its effectiveness to the well-characterized neuroprotective NA-1 peptide. R18 and NA-1 peptides were administered intravenously (30, 100, 300, 1000nmol/kg), 60min after the onset of 90min of MCAO. Infarct volume, cerebral swelling and functional outcomes (neurological score, adhesive tape and rota-rod) were measured 24h after MCAO. R18 reduced total infarct volume by 35.1% (p=0.008), 24.8% (p=0.059), 12.2% and 9.6% for the respective 1000 to 30nmol/kg doses, while the corresponding doses of NA-1 reduced lesion volume by 26.1% (p=0.047), 16.6%, 16.5% and 7%, respectively. R18 also reduced hemisphere swelling by between 46.1% (1000 and 300nmol/kg; p=0.009) and 24.4% (100nmol/kg; p=0.066), while NA-1 reduced swelling by 25.7% (1000nmol/kg; p=0.054). In addition, several R18 and NA-1 treatment groups displayed a significant improvement in at least one parameter of the adhesive tape test. These results confirm the neuroprotective properties of R18, and suggest that the peptide is a more effective neuroprotective agent than NA-1. This provides strong justification for the continuing development of R18 as a neuroprotective treatment for stroke.

The R18 Polyarginine Peptide Is More Effective Than the TAT-NR2B9c (NA-1) Peptide When Administered 60 Minutes after Permanent Middle Cerebral Artery Occlusion in the Rat

We examined the dose responsiveness of polyarginine R18 (100, 300, and 1000 nmol/kg) when administered 60 minutes after permanent middle cerebral artery occlusion (MCAO). The TAT-NR2B9c peptide, which is known to be neuroprotective in rodent and nonhuman primate stroke models, served as a positive control. At 24 hours after MCAO, there was reduced total infarct volume in R18 treated animals at all doses, but this reduction only reached statistical significance at doses of 100 and 1000 nmol/kg. The TAT-NR2B9c peptide reduced infarct volume at doses of 300 and 1000 nmol/kg, but not to a statistically significant extent, while the 100 nmol/kg dose was ineffective. The reduction in infarct volume with R18 and TAT-NR2B9c peptide treatments was mirrored by improvements in one or more functional outcomes (namely, neurological score, adhesive tape removal, and rota-rod), but not to a statistically significant extent. These findings further confirm the neuroprotective properties of polyarginine peptides and for R18 extend its therapeutic time window and dose range, as well as demonstrating its greater efficacy compared to TAT-NR2B9c in a severe stroke model. The superior neuroprotective efficacy of R18 over TAT-NR2B9c highlights the potential of this polyarginine peptide as a lead candidate for studies in human stroke.

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

We have previously reported that cationic poly-arginine and arginine-rich cell-penetrating peptides display high-level neuroprotection and reduce calcium influx following in vitro excitotoxicity, as well as reduce brain injury in animal stroke models. Using the neuroprotective peptides poly-arginine R12 (R12) and the NR2B9c peptide fused to the arginine-rich carrier peptide TAT (TAT-NR2B9c; also known as NA-1), we investigated the mechanisms whereby poly-arginine and arginine-rich peptides reduce glutamate-induced excitotoxic calcium influx. Using cell surface biotin protein labeling and western blot analysis, we demonstrated that R12 and TAT-NR2B9c significantly reduced cortical neuronal cell surface expression of the NMDA receptor subunit NR2B. Chemical endocytic inhibitors used individually or in combination prior to glutamate excitotoxicity did not significantly affect R12 peptide neuroprotective efficacy. Similarly, pretreatment of neurons with enzymes to degrade anionic cell surface proteoglycans, heparan sulfate proteoglycan (HSPG), and chondroitin sulfate proteoglycan (CSPG), as well as sialic acid residues, did not significantly affect peptide neuroprotective efficacy. While the exact mechanisms responsible for R12 peptide-mediated NMDA receptor NR2B subunit cell surface downregulation were not identified, an endocytic process could not be ruled out. The study supports our hypothesis that arginine-rich peptides reduce excitotoxic calcium influx by reducing the levels of cell surface ion channels.

Spinal Muscular Atrophy and the Antiapoptotic Role of Survival of Motor Neuron (SMN) Protein

Spinal muscular atrophy (SMA) is a devastating and often fatal neurodegenerative disease that affects spinal motor neurons and leads to progressive muscle wasting and paralysis. The survival of motor neuron (SMN) gene is mutated or deleted in most forms of SMA, which results in a critical reduction in SMN protein. Motor neurons appear particularly vulnerable to reduced SMN protein levels. Therefore, understanding the functional role of SMN in protecting motor neurons from degeneration is an essential prerequisite for the design of effective therapies for SMA. To this end, there is increasing evidence indicating a key regulatory antiapoptotic role for the SMN protein that is important in motor neuron survival. The aim of this review is to highlight key findings that support an antiapoptotic role for SMN in modulating cell survival and raise possibilities for new therapeutic approaches.

Co-regulation of survival of motor neuron and Bcl-xL expression: Implications for neuroprotection in spinal muscular atrophy

Spinal muscular atrophy (SMA), a fatal genetic motor disorder of infants, is caused by diminished full-length survival of motor neuron (SMN) protein levels. Normally involved in small nuclear ribonucleoprotein (snRNP) assembly and pre-mRNA splicing, recent studies suggest that SMN plays a critical role in regulating apoptosis. Interestingly, the anti-apoptotic Bcl-x isoform, Bcl-xL, is reduced in SMA. In a related finding, Sam68, an RNA-binding protein, was found to modulate splicing of SMN and Bcl-xL transcripts, promoting SMNΔ7 and pro-apoptotic Bcl-xS transcripts. Here we demonstrate that Bcl-xL expression increases SMN protein by ∼2-fold in SH-SY5Y cells. Conversely, SMN expression increases Bcl-xL protein levels by ∼6-fold in SH-SY5Y cells, and ∼2.5-fold in the brains of transgenic mice over-expressing SMN (PrP-SMN). Moreover, Sam68 protein levels were markedly reduced following SMN and Bcl-xL expression in SH-SY5Y cells, suggesting a feedback mechanism co-regulating levels of both proteins. We also found that exogenous SMN expression increased full-length SMN transcripts, possibly by promoting exon 7 inclusion. Finally, co-expression of SMN and Bcl-xL produced an additive anti-apoptotic effect following PI3-kinase inhibition in SH-SY5Y cells. Our findings implicate Bcl-xL as another potential target in SMA therapeutics, and indicate that therapeutic increases in SMN may arise from modest increases in total SMN.

Survival of motor neuron protein over-expression prevents calpain-mediated cleavage and activation of procaspase-3 in differentiated human SH-SY5Y cells

Spinal muscular atrophy (SMA), a neurodegenerative disorder primarily affecting motor neurons, is the most common genetic cause of infant death. This incurable disease is caused by the absence of a functional SMN1 gene and a reduction in full length survival of motor neuron (SMN) protein. In this study, a neuroprotective function of SMN was investigated in differentiated human SH-SY5Y cells using an adenoviral vector to over-express SMN protein. The pro-survival capacity of SMN was assessed in an Akt/PI3-kinase inhibition (LY294002) model, as well as an oxidative stress (hydrogen peroxide) and excitotoxic (glutamate) model. SMN over-expression in SH-SY5Y cells protected against Akt/phosphatidylinositol 3-kinase (PI3-kinase) inhibition, but not oxidative stress, nor against excitotoxicity in rat cortical neurons. Western analysis of cell homogenates from SH-SY5Y cultures over-expressing SMN harvested pre- and post-Akt/PI3-kinase inhibition indicated that SMN protein inhibited caspase-3 activation via blockade of calpain-mediated procaspase-3 cleavage. This study has revealed a novel anti-apoptotic function for the SMN protein in differentiated SH-SY5Y cells. Finally, the cell death model described herein will allow the assessment of future therapeutic agents or strategies aimed at increasing SMN protein levels.

Is cholesterol and amyloid-β stress induced CD147 expression a protective response? evidence that extracellular cyclophilin a mediated neuroprotection is reliant on CD147

The CD147 protein is a ubiquitous multifunctional membrane receptor. Expression of CD147, which is regulated by sterol carrier protein, reportedly modulates amyloid-β (Aβ), the neurotoxic peptide implicated in neuronal degeneration in Alzheimers disease (AD). Given that high fat/cholesterol is linked to amyloid deposition in AD, we investigated if cholesterol and/or Aβ can alter CD147 expression in rat cortical neuronal cultures. Water-soluble cholesterol and Aβ42 dose-dependently increased CD147 protein expression, but reduced FL-AβPP protein expression. Cholesterol and Aβ42 treatment also increased lactate dehydrogenase release but to varying degrees. Upregulation of CD147 expression was probably mediated by oxidative stress, as H2O2 (3 μM) also induced CD147 protein expression in neuronal cultures. In light of these findings, we investigated if CD147 induction was cytoprotective, a compensatory response to injury, or alternatively, a cell death signal. To this end, we used recombinant adenovirus to overexpress human CD147 (in SH-SY5Y cells and primary cortical neurons), and pre-treated cultures with or without recombinant cyclophilin A (rCYPA) protein, prior to Aβ42 exposure. We showed that increased CD147 expression protected against Aβ42, only when rCYPA protein was added to neuronal cultures. Together, our findings reveal potentially important relationships between cholesterol loading, CD147 expression, Aβ toxicity, and the putative involvement of CYPA protein in neuroprotection in AD.

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 underappreciated 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 TBIrelated 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 neurorestorative 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 hypoxiaischemia, 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. EXPERT REVIEW OF NEUROTHERAPEUTICS, 2016 VOL. 16, NO. 4, 361–363 http://dx.doi.org/10.1586/14737175.2016.1150180