Resveratrol inhibits respiratory syncytial virus replication by targeting heparan sulfate proteoglycans.

Resveratrol, renowned as an antioxidant, also exhibits significant potential in combatting severe respiratory infections, particularly the respiratory syncytial virus (RSV). Nevertheless, the specific mechanism underlying its inhibition of RSV replication remains unexplored. Heparan sulfate proteoglycans (HSPGs) play a pivotal role as attachment factors for numerous viruses, offering a promising avenue for countering viral infections. Our research has unveiled that resveratrol effectively curbs RSV infection in a dose-dependent manner. Remarkably, resveratrol disrupts the early stages of RSV infection by engaging with HSPGs, rather than interacting with RSV surface proteins like fusion (F) protein and glycoprotein (G). Resveratrol's affinity appears to be predominantly directed towards the negatively charged sites on HSPGs, thus impeding the binding of viral receptors. In an in vivo study involving RSV-infected mice, resveratrol demonstrates its potential by ameliorating pulmonary pathology. This improvement is attributed to the inhibition of pro-inflammatory cytokine expression and a reduction in viral load within the lungs. Notably, resveratrol specifically alleviates inflammation characterized by an abundance of neutrophils in RSV-infected mice. In summation, our data first shows how resveratrol combats RSV infection through interactions with HSPGs, positioning it as a promising candidate for innovative drug development targeting RSV infections. Our study provides insight into the mechanism of resveratrol antiviral infection.

Heat stress combined with lipopolysaccharide induces pulmonary microvascular endothelial cell glycocalyx inflammatory damage in vitro.

Heat stroke is a life-threatening disease with high mortality and complications. Endothelial glycocalyx (EGCX) is essential for maintaining endothelial cell structure and function as well as preventing the adhesion of inflammatory cells. Potential relationship that underlies the imbalance in inflammation and coagulation remains elusive. Moreover, the role of EGCX in heat stroke-induced organ injury remained unclear. Therefore, the current study aimed to illustrate if EGCX aggravates apoptosis, inflammation, and oxidative damage in human pulmonary microvascular endothelial cells (HPMEC). Heat stress and lipopolysaccharide (LPS) were employed to construct in vitro models to study the changes of glycocalyx structure and function, as well as levels of heparansulfate proteoglycan (HSPG), syndecan-1 (SDC-1), heparansulfate (HS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, Von Willebrand factor (vWF), endothelin-1 (ET-1), occludin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and reactive oxygen species (ROS). Here, we showed that heat stress and LPS devastated EGCX structure, activated EGCX degradation, and triggered oxidative damage and apoptosis in HPMEC. Stimulation of heat stress and LPS decreased expression of HSPG, increased levels of SDC-1 and HS in culture supernatant, promoted the production and release of proinflammation cytokines (TNF-α and IL-6,) and coagulative factors (vWF and ET-1) in HPMEC. Furthermore, Expressions of E-selection, VCAM-1, and ROS were upregulated, while that of occludin was downregulated. These changes could be deteriorated by heparanase, whereas they meliorated by unfractionated heparin. This study indicated that EGCX may contribute to apoptosis and heat stroke-induced coagulopathy, and these effects may have been due to the decrease in the shedding of EGCX.

Effect of Amino Acid Substitution on Cell Adhesion Properties of Octa-arginine.

Octa-arginine (R8) is a cell-permeable peptide with excellent cell adhesion properties. Surface-immobilized R8 mediates cell attachment via cell surface receptors, such as heparan sulfate proteoglycans and integrin ß1, and promotes cell spreading and proliferation. However, it is not clear how these properties are affected by specific peptide composition and if they could be improved. Here, we synthesized XR8 peptides, in which half of the original R8 arginine residues were replaced with another amino acid (X). We then aimed to investigate the effect of the substitution on cell adhesion and proliferation on XR8-conjugated agarose matrices. The XR8-matrix showed slightly better cell attachment when X was a hydrophobic or aromatic amino acid. However, hydrophobic XR8-matrices tended to promote cell proliferation to a less extent. Eventually, YR8-matrix most efficiently promoted cell adhesion, spreading, and proliferation among the XR8-matrices tested. Collectively, these observations indicate that the properties of residue X play a major role in the biological activity of XR8-matrices and shed light on the interaction between small peptides and the cell membrane. Further, YR8 is a promising cell-adhesive peptide for the development of cell culture substrates and biomaterials.

The role of the glypican and syndecan families of heparan sulfate proteoglycans in cardiovascular function and disease.

Heparan sulfate proteoglycans (HSPGs) are proteoglycans formed by a core protein to which one or multiple heparan sulfate chains are covalently bound. They are ubiquitously expressed in cellular surfaces and can be found in the extracellular matrix and secretory vesicles. The cellular effects of HSPGs comprehend multiple functionalities that include 1) the interaction with other membrane surface proteins to act as a substrate for cellular migration, 2) acting as a binding site for circulating molecules, 3) to have a receptor role for proteases, 4) to act as a coreceptor that can provide finetuning of growth factor receptor activity threshold, and 5) to activate intracellular signaling pathways (Sarrazin S, Lamanna WC, Esko JD. Cold Spring Harb Perspect Biol 3: a004952, 2011). Among the different families of HSPGs, the syndecan and glypican families of HSPGs have gained increased attention in relation to their effects on cardiovascular cells and potential role in disease progression. In this review, we will summarize the effects of syndecan and glypican homologs on the different cardiovascular cell types and discuss their contribution to common processes found in cardiovascular diseases (inflammation, hypertrophy, and vascular remodeling) as well as their potential role in the development and progression of specific diseases including hypertension, heart failure, and atherosclerosis.

Heparin-binding VEGFR1 variants as long-acting VEGF inhibitors for treatment of intraocular neovascular disorders.

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

Heparan Sulfate Proteoglycans Can Promote Opposite Effects on Adhesion and Directional Migration of Different Cancer Cells.

Heparan sulfate proteoglycans take part in crucial events of cancer progression, such as epithelial-mesenchymal transition, cell migration, and cell invasion. Through sulfated groups on their glycosaminoglycan chains, heparan sulfate proteoglycans interact with growth factors, morphogens, chemokines, and extracellular matrix (ECM) proteins. The amount and position of sulfated groups are highly variable, thus allowing differentiated ligand binding and activity of heparan sulfate proteoglycans. This variability and the lack of specific ligands have delayed comprehension of the molecular basis of heparan sulfate proteoglycan functions. Exploiting a tumor-targeting peptide tool that specifically recognizes sulfated glycosaminoglycans, we analyzed the role of membrane heparan sulfate proteoglycans in the adhesion and migration of cancer cell lines. Starting from the observation that the sulfated glycosaminoglycan-specific peptide exerts a different effect on adhesion, migration, and invasiveness of different cancer cell lines, we identified and characterized three cell migration phenotypes, where different syndecans are associated with alternative signaling for directional cell migration.

Perlecan Facilitates Neuronal Nitric Oxide Synthase Delocalization in Denervation-Induced Muscle Atrophy.

Perlecan is an extracellular matrix molecule anchored to the sarcolemma by a dystrophin-glycoprotein complex. Perlecan-deficient mice are tolerant to muscle atrophy, suggesting that perlecan negatively regulates mechanical stress-dependent skeletal muscle mass. Delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the cytosol triggers protein degradation, thereby initiating skeletal muscle atrophy. We hypothesized that perlecan regulates nNOS delocalization and activates protein degradation during this process. To determine the role of perlecan in nNOS-mediated mechanotransduction, we used sciatic nerve transection as a denervation model of gastrocnemius muscles. Gastrocnemius muscle atrophy was significantly lower in perinatal lethality-rescued perlecan-knockout (Hspg2-/--Tg) mice than controls (WT-Tg) on days 4 and 14 following surgery. Immunofluorescence microscopy showed that cell membrane nNOS expression was reduced by denervation in WT-Tg mice, with marginal effects in Hspg2-/--Tg mice. Moreover, levels of atrophy-related proteins-i.e., FoxO1a, FoxO3a, atrogin-1, and Lys48-polyubiquitinated proteins-increased in the denervated muscles of WT-Tg mice but not in Hspg2-/--Tg mice. These findings suggest that during denervation, perlecan promotes nNOS delocalization from the membrane and stimulates protein degradation and muscle atrophy by activating FoxO signaling and the ubiquitin-proteasome system.

Heparan sulfate proteoglycans as targets for cancer therapy: a review.

Heparan sulfate proteoglycans (HSPGs) play important roles in cancer initiation and progression, by interacting with the signaling pathways that affect proliferation, adhesion, invasion and angiogenesis. These roles suggest the possibility of various strategies of regulation of these molecules. In this review, we demonstrated that the anticancer drugs can regulate the heparan sulfate proteoglycans activity in different ways: some act directly in core protein, and can bind to a specific type of HSPG. Others drugs interact with glycosaminoglycans chains, and others can act directly in enzymes that regulate HSPGs levels. We also demonstrated that the HSPGs drug targets can be divided into four groups: monoclonal antibodies, antitumor antibiotic, natural products, and mimetics peptide. Interestingly, many drugs demonstrated in this review are approved by FDA and is used in cancer therapy (Food and Drug Administration) like trastuzumab, panitumumab, bleomycin and bisphosphonate zoledronic acid (ASCO) or are in clinical trials like codrituzumab and genistein. This review should help researchers to understand the mechanism of action of anticancer drugs existing and also may inspire the discovery of new drugs that regulate the heparan sulfate proteoglycans activity.

Autophagic degradation of HAS2 in endothelial cells: A novel mechanism to regulate angiogenesis.

Hyaluronan plays a key role in regulating inflammation and tumor angiogenesis. Of the three transmembrane hyaluronan synthases, HAS2 is the main pro-angiogenic enzyme responsible for excessive hyaluronan production. We discovered that HAS2 was degraded in vascular endothelial cells via autophagy evoked by nutrient deprivation, mTOR inhibition, or pro-autophagic proteoglycan fragments endorepellin and endostatin. Using live-cell and super-resolution confocal microscopy, we found that protracted autophagy evoked a dynamic interaction between HAS2 and ATG9A, a key transmembrane autophagic protein. This regulatory axis of HAS2 degradation occurred in various cell types and species and in vivo upon nutrient deprivation. Inhibiting in vivo autophagic flux via chloroquine showed increased levels of HAS2 in the heart and aorta. Functionally, autophagic induction via endorepellin or mTOR inhibition markedly suppressed extracellular hyaluronan production in vascular endothelial cells and inhibited ex vivo angiogenic sprouting. Thus, we propose autophagy as a novel catabolic mechanism regulating hyaluronan production in endothelial cells and demonstrate a new link between autophagy and angiogenesis that could lead to potential therapeutic modalities for angiogenesis.

Heparan sulfate proteoglycan promotes fibroblast growth factor-2 function for ischemic heart repair.

It is well known that the basic fibroblast growth factor (bFGF) promotes angiogenesis after myocardial infarction (MI), but its biological functions decrease in the event of diffusion, enzymolysis, and weak binding with co-receptors in vivo. Heparan sulfate proteoglycans (HSPG) are a major component of extracellular matrices and have been shown to regulate a wide range of cellular functions and bioprocesses by acting as a co-receptor for bFGF and affecting its bioactivities. However, the influence of HSPG on the function of bFGF after myocardial infarction is unknown. Here, exogenous HSPG along with bFGF was injected into the hearts of rats to deliver the angiogenic growth factor for ischemic heart repair following induced MI. The specific binding of HSPG with bFGF protein was demonstrated, which was about 6-fold stronger than the binding of bFGF with heparin. The biological mechanisms of HSPG binding with bFGF were further studied by cell adhesion assay, and assays of bFGF and matrix metalloproteinase 2 (MMP2) activities demonstrated that HSPG enhances cell adhesion, promotes the bioactivity of bFGF in angiogenesis, and protects bFGF from enzymolysis. Our results indicate that HSPG has potential clinical utility as a delivery agent for heparin-binding growth factors. Additionally, HSPG shows high binding affinities with different ECM proteins which also help to anchor bFGF to heart tissue. Therefore, extracellular proteins that mimic the bio-scaffold of the extracellular matrix could promote the activities of bFGF to facilitate ischemic heart repair.

Multivalent Flexible Nanogels Exhibit Broad-Spectrum Antiviral Activity by Blocking Virus Entry.

The entry process of viruses into host cells is complex and involves stable but transient multivalent interactions with different cell surface receptors. The initial contact of several viruses begins with attachment to heparan sulfate (HS) proteoglycans on the cell surface, which results in a cascade of events that end up with virus entry. The development of antiviral agents based on multivalent interactions to shield virus particles and block initial interactions with cellular receptors has attracted attention in antiviral research. Here, we designed nanogels with different degrees of flexibility based on dendritic polyglycerol sulfate to mimic cellular HS. The designed nanogels are nontoxic and broad-spectrum, can multivalently interact with viral glycoproteins, shield virus surfaces, and efficiently block infection. We also visualized virus-nanogel interactions as well as the uptake of nanogels by the cells through clathrin-mediated endocytosis using confocal microscopy. As many human viruses attach to the cells through HS moieties, we introduce our flexible nanogels as robust inhibitors for these viruses.

Substitution-Inert Polynuclear Platinum Complexes as Metalloshielding Agents for Heparan Sulfate.

Cleavage of heparan sulfate proteoglycans (HSPGs) by the enzyme heparanase modulates tumour-related events including angiogenesis, cell invasion, and metastasis. Metalloshielding of heparan sulfate (HS) by positively charged polynuclear platinum complexes (PPCs) effectively inhibits physiologically critical HS functions. Studies using bacterial P. heparinus heparinase II showed that a library of Pt complexes varying in charge and nuclearity and the presence or absence of a dangling amine inhibits the cleavage activity of the enzyme on the synthetic pentasaccharide, Fondaparinux (FPX). Charge-dependent affinity of PPC for FPX was seen in competition assays with methylene blue and ethidium bromide. The dissociation constant (Kd ) of TriplatinNC for FPX was directly measured by isothermal titration calorimetry (ITC). The trend in DFT calculated interaction energies with heparin fragments is consistent with the spectroscopic studies. Competitive inhibition of TAMRA-R9 internalization in human carcinoma (HCT116) cells along with studies in HCT116, wildtype CHO and mutant CHO-pgsA745 (lacking HS/CS) cells confirm that HSPG-mediated interactions play an important role in the cellular accumulation of PPCs.

Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism.

Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (∼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism.  These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.

Recombinant Domain V of Human Perlecan Is a Bioactive Vascular Proteoglycan.

The C-terminal domain V of the extracellular matrix proteoglycan perlecan plays unique and often divergent roles in a number of biological processes, including angiogenesis, vascular cell interactions, wound healing, and autophagy. Recombinant forms of domain V have been proposed as therapeutic agents for the treatment of cancer, stroke, and the development of cardiovascular devices and bioartificial tissues. However, the effect of domain V appears to be related to the differences in domain V structure and function observed in different expression systems and environments and exactly how this occurs is not well understood. In this study, the sequence from amino acid 3626 to 4391 of the perlecan protein core, which includes domain V, is expressed in HEK-293 cells and purified as a secreted product from conditioned media. This recombinant domain V (rDV) is expressed as a proteoglycan decorated with heparan sulfate and chondroitin sulfate chains and supports endothelial cell interactions to the same extent as full-length perlecan. This expression system serves as an important model of recombinant proteoglycan expression, as well as a source of biologically active rDV for therapeutic applications.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

Integrins and heparan sulfate proteoglycans on hepatic stellate cells (HSC) are novel receptors for HSC-derived exosomes.

Exosomes mediate intercellular microRNA delivery between hepatic stellate cells (HSC), the principal fibrosis-producing cells in the liver. The purpose of this study was to identify receptors on HSC for HSC-derived exosomes, which bind to HSC rather than to hepatocytes. Our findings indicate that exosome binding to HSC is blocked by treating HSC with RGD, EDTA, integrin αv or ß1 siRNAs, integrin αvß3 or α5ß1 neutralizing antibodies, heparin, or sodium chlorate. Furthermore, exosome cargo delivery and exosome-regulated functions in HSC, including expression of fibrosis- or activation-associated genes and/or miR-214 target gene regulation, are dependent on cellular integrin αvß3, integrin α5ß1, or heparan sulfate proteolgycans (HSPG). Thus, integrins and HSPG mediate the binding of HSC-derived exosomes to HSC as well as the delivery and intracellular action of the exosomal payload.

Impaired wound healing in bleomycin-induced murine scleroderma: a new model of wound retardation.

Bleomycin-induced scleroderma in mice is an established model for human scleroderma. Making use of this, we have established a new model for wound retardation. After inducing dermal sclerosis by local bleomycin treatment in nude mice, a full-thickness wound was made by punch excision on the bleomycin application site. Mice pretreated with bleomycin showed a significant delay in wound closure, as compared with mice pretreated with phosphate-buffered saline. Proliferation of keratinocytes was significantly inhibited and the number of Ki-67-positive keratinocytes was significantly lower in the bleomycin-pretreated skin. Also, the number of CD31-positive blood vessels was markedly reduced in the bleomycin-treated skin. The topical daily application of basic fibroblast growth factor (bFGF) significantly promoted wound closure, while increasing blood vessel formation and reducing transforming growth factor-ß and alpha-smooth muscle actin mRNA levels. Furthermore, only two applications of PG-FGF1, a fusion protein of FGF1 with heparan sulfate proteoglycan, overcame the delay in wound closure. Wound delay in this model mainly occurred as a result of decreased vessel formation and keratinocyte migration following bleomycin treatment. It is expected that this model will provide novel insights into the pathogenesis of wound healing and the exploration of possible candidate drugs for refractory or chronic wounds in the clinical setting.

Novel heparan sulfate-binding peptides for blocking herpesvirus entry.

Human cytomegalovirus (HCMV) infection can lead to congenital hearing loss and mental retardation. Upon immune suppression, reactivation of latent HCMV or primary infection increases morbidity in cancer, transplantation, and late stage AIDS patients. Current treatments include nucleoside analogues, which have significant toxicities limiting their usefulness. In this study we screened a panel of synthetic heparin-binding peptides for their ability to prevent CMV infection in vitro. A peptide designated, p5+14 exhibited ~ 90% reduction in murine CMV (MCMV) infection. Because negatively charged, cell-surface heparan sulfate proteoglycans (HSPGs), serve as the attachment receptor during the adsorption phase of the CMV infection cycle, we hypothesized that p5+14 effectively competes for CMV adsorption to the cell surface resulting in the reduction in infection. Positively charged Lys residues were required for peptide binding to cell-surface HSPGs and reducing viral infection. We show that this inhibition was not due to a direct neutralizing effect on the virus itself and that the peptide blocked adsorption of the virus. The peptide also inhibited infection of other herpesviruses: HCMV and herpes simplex virus 1 and 2 in vitro, demonstrating it has broad-spectrum antiviral activity. Therefore, this peptide may offer an adjunct therapy for the treatment of herpes viral infections and other viruses that use HSPGs for entry.

Diverse functions of perlecan in central nervous system cells in vitro.

Therapeutic treatment targeting one cell type is considered ineffective in remedying any injury to the central nervous system (CNS). Perlecan, a multi-functional, heparan sulfate proteoglycan, shows diverse effects on distinct cell types, suggesting that it is one of the candidates that can augment the regenerative mechanisms in the injured CNS. Therefore, we examined the functions of perlecan in CNS cells in vitro by using perlecan purified from bovine kidney. Perlecan-coated cell culture plates, unlike their type I/III collagen-coated counterparts, did not inhibit the adhesion of neural stem/progenitor cells (NS/PCs) and neurons. The coated perlecan and the perlecan added to the culture medium suppressed astrocyte proliferation; however, perlecan added to the medium promoted NS/PC proliferation. Neurons were promoted to extend their neurites on the perlecan-coated substrate, and perlecan added to the medium also showed a similar effect. NS/PC proliferation and neurite extension is a major regenerative reaction in CNS injury, whereas excess proliferation of astrocytes cause hypertrophy of glial scars, which repels neurons. Our in vitro study suggests that perlecan is an attractive candidate to promote regenerative mechanisms and to suppress reactions that hamper regenerative processes in cases of CNS injury.

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.