Abstract
Poly-arginine peptide-18 (R18) has recently emerged as a highly effective neuroprotective agent in experimental stroke models, and is particularly efficacious in protecting cortical neurons against glutamic acid excitotoxicity. While we have previously demonstrated that R18 can reduce excitotoxicity-induced neuronal calcium influx, other molecular events associated with R18 neuroprotection are yet to investigated. Therefore, in this study we were particularly interested in protein expression changes in R18 treated neurons subjected to excitotoxicity.Proteomic analysis was used to compare protein expression patterns in primary cortical neuronal cultures subjected to: (i) R18-treatment alone (R18); (ii) glutamic acid excitotoxic injury (Glut); (iii) R18-treatment and glutamic acid injury (R18 + Glut); (iv) no treatment (Cont). Whole cell lysates were harvested 24 h post-injury and subjected to quantitative proteomic analysis (iTRAQ), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and subsequent bioinformatic analysis of differentially expressed proteins (DEPs).Relative to control cultures, R18, Glut, and R18 + Glut treatment resulted in the detection of 5, 95 and 14 DEPs respectively. Compared to Glut alone, R18 + Glut revealed 98 DEPs, including 73 proteins whose expression was also altered by treatment with Glut and/or R18 alone, as well as 25 other uniquely regulated proteins. R18 treatment reversed the up- or down-regulation of all 73 Glut-associated DEPs, which included proteins involved in mitochondrial integrity, ATP generation, mRNA processing and protein translation. Analysis of protein-protein interactions of the 73 DEPs showed they were primarily associated with mitochondrial respiration, proteasome activity and protein synthesis, transmembrane trafficking, axonal growth and neuronal differentiation, and carbohydrate metabolism. Identified protein pathways associated with proteostasis and energy metabolism, and with pathways involved in neurodegeneration.Collectively, the findings indicate that R18 neuroprotection following excitotoxicity is associated with preservation of neuronal protein profiles, and differential protein expression that assists in maintaining mitochondrial function and energy production, protein homeostasis, and membrane trafficking.Graphical abstract
Highlights
A major pathophysiological mechanism responsible for ischaemic stroke injury is excitotoxicity, which is trigged by the excessive release of the excitatory neurotransmitter glutamic acid in response to reduced cerebral blood flow and compromised ATP synthesis
While we have previously demonstrated that R18-treatment alone (R18) has the capacity to reduce glutamic acid-induced excitotoxic neuronal death and intracellular calcium influx, and reduce neuronal NMDA receptor levels [22], cationic arginine-rich peptides (CARPs) have cell-penetrating properties and can target mitochondria [23]
The False Discovery Rate (FDR) was automatically calculated by the Proteomics System Performance Evaluation Pipeline (PSPEP) feature in the ProteinPilotTM software (AB Sciex, Foster, CA, USA; Version 5.0.1) using the reversed version of the protein sequences contained in the search database
Summary
A major pathophysiological mechanism responsible for ischaemic stroke injury is excitotoxicity, which is trigged by the excessive release of the excitatory neurotransmitter glutamic acid in response to reduced cerebral blood flow and compromised ATP synthesis. We have demonstrated that poly-arginine-18 (R18, 18-mer of arginine) is neuroprotective in in vitro neuronal excitotoxicity models and in vivo in rodent models of stroke [11,12,13,14,15,16,17,18], hypoxic-ischaemic encephalopathy (HIE) [19], and traumatic brain injury (TBI) [20, 21]
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