Poly-arginine-18 (R18) Confers Neuroprotection through Glutamate Receptor Modulation, Intracellular Calcium Reduction, and Preservation of Mitochondrial Function

Abstract

Recent studies have highlighted that a novel class of neuroprotective peptide, known as cationic arginine-rich peptides (CARPs), have intrinsic neuroprotective properties and are particularly effective anti-excitotoxic agents. As such, the present study investigated the mechanisms underlying the anti-excitotoxic properties of CARPs, using poly-arginine-18 (R18; 18-mer of arginine) as a representative peptide. Cortical neuronal cultures subjected to glutamic acid excitotoxicity were used to assess the effects of R18 on ionotropic glutamate receptor (iGluR)-mediated intracellular calcium influx, and its ability to reduce neuronal injury from raised intracellular calcium levels after inhibition of endoplasmic reticulum calcium uptake by thapsigargin. The results indicate that R18 significantly reduces calcium influx by suppressing iGluR overactivation, and results in preservation of mitochondrial membrane potential (ΔΨm) and ATP production, and reduced ROS generation. R18 also protected cortical neurons against thapsigargin-induced neurotoxicity, which indicates that the peptide helps maintain neuronal survival when intracellular calcium levels are elevated. Taken together, these findings provide important insight into the mechanisms of action of R18, supporting its potential application as a neuroprotective therapeutic for acute and chronic neurological disorders.

Keywords: ROS; cationic arginine-rich peptides (CARPs); ionotropic glutamate receptors; mitochondrial membrane potential (ΔΨm); neuroprotection; poly-arginine-18 (R18).

Figure 1

Ionotropic glutamate receptor agonists induce dose-dependent neuronal death at 24 h post-exposure. Dose–response studies of DIV10 primary cortical neurons exposed to ionotropic glutamate receptor agonists (10–200 µM) for 5 min. Neuronal viability and cell death were measured using MTS and LDH assays, respectively, at 24 h post-exposure to (a,b) NMDA, (c,d) KA, and (e,f) AMPA. Absorbance values were adjusted to represent cell viability (untreated control as 100%) and fold change in LDH release. Values are means ± SEM; n = 3, with a minimum of eight biological replicates per sample in each experiment; * p < 0.05. Experiments were conducted at least twice with independent neuronal cultures.

Figure 2

R18 protects primary cortical neurons against excitotoxicity induced by various ionotropic glutamate receptor agonists. Neuronal cultures were pre-treated with R18 (1, 2, and 5 µM) for 10 min and exposed to a 100 µM final concentration of specific iGluR agonists for 5 min. The medium was then replaced, and neuronal viability and death were measured at 24 h post-insult via MTS and LDH assays, respectively. Receptor agonists included (a,b) glutamate, (c,d) NMDA, (e,f) kainic acid, and (g,h) AMPA. The receptor antagonists, MK801 (10 µM) and CNQX (10 µM), were used as positive controls. Absorbance values were adjusted to represent cell viability (untreated control as 100%). Values are means ± SEM; n = 3, with a minimum of eight biological replicates per sample in each experiment; * p < 0.05. Experiments were conducted at least three times with independent neuronal cultures.

Figure 3

R18 protects primary cortical neurons against iGluR-mediated calcium influx. Cortical neurons were loaded with Fura-2 AM (5 µM in MEM/B27) for 30 min at 37 °C, and the medium was replaced with 50 µL phenol-free Hank’s Balanced Salt Solution (HBSS). Baseline calcium levels were measured every 30 s for 1.5 min prior to the addition of (a) iGluR agonists, glutamate, NMDA, AMPA, and KA (100 µM final concentration). Subsequent calcium measurements were made every 30 s for 5 min. To assess the neuroprotective ability of R18 against receptor-mediated calcium influx, neurons were treated with R18 (2 and 5 µM) or receptor blockers (MK801 and/or CNQX; 10 µM) for 10 min prior to stimulation with individual iGluR agonists, (b) glutamate, (c) NMDA, (d) kainic acid, (e) or AMPA, and calcium levels were collected every 30 s for a further 5 min. Values are means ± SD; n = 3, with a minimum of four biological replicates per sample in each experiment; * p < 0.05. Experiments were conducted at least three times with independent neuronal cultures. Fluorescent readings were adjusted to remove background signal and displayed as ΔF ratio (340/380 nm) relative to the untreated control at each timepoint.

Figure 4

R18 does not disrupt mitochondrial bioenergetics and preserves mitochondrial bioenergetics post-glutamate excitotoxic insult in primary cortical neurons. Cultures received a 5 min glutamate exposure (100 µM final concentration) following a 10 min pre-treatment with R18 (2 or 5 µM). Parameters of mitochondrial bioenergetics were measured fluorometrically post-insult and are represented as fold change in fluorescent intensity. (a,b) Membrane potential was measured with tetramethylrhodamine ethyl ester (TMRE) immediately post-insult. (c,d) ATP production and (e,f) were measured at 24 h post-insult. Background fluorescent values were removed. Background fluorescent values were removed, and experiments were conducted at least three times with independent neuronal cultures. Values are means ± SEM; n = 3, with a minimum of eight biological replicates per sample in each experiment; * p < 0.05. FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; 20 µM) was used as a positive control for mitochondrial membrane depolarization.

Figure 5

R18 protects primary cortical neurons against thapsigargin-induced neuronal death and ER-mediated calcium release. A 24 h exposure of primary cortical neuronal cultures to thapsigargin (TPG) causes (a) a significant reduction in MTS metabolism and (b) increased LDH release in a dose-dependent manner. Neuronal cultures were pre-treated with R18 (1, 2, and 5 µM) and exposed to 10 µM final concentration of TPG for 24 h exhibited (c) significantly improved MTS metabolism and (d) reduced LDH release compared to TPG control at 24 h post-insult. Absorbance values were adjusted to represent cell viability (untreated control as 100%). Values are means ± SEM; n = 3, with a minimum of eight biological replicates per sample in each experiment; * p < 0.05. Experiments were conducted at least twice with independent neuronal cultures.