Proteomic analysis of cortical neuronal cultures treated with poly-arginine peptide-18 (R18) and exposed to glutamic acid excitotoxicity

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 Electronic supplementary material The online version of this article (10.1186/s13041-019-0486-8) contains supplementary material, which is available to authorized users.


Introduction
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. Excitotoxicity initiates a range of forward-feeding biochemical events known as the 'ischaemic cascade' , which if not inhibited eventually lead to neuronal death and cerebral infarction [1]. Furthermore, as glutamic acid is the most prominent excitatory neurotransmitter in the CNS [2], the detrimental effects of glutamic acid excitotoxicity also play a role in other acute brain disorders such as traumatic brain injury and epilepsy, as well as chronic neurodegenerative disorders, such as Alzheimer's disease (AD) [3,4], Huntington's disease (HD) [5,6], Parkinson's disease (PD) [7,8], and amyotrophic lateral sclerosis (ALS) [9,10].
Given the neuroprotective properties of R18, it is imperative that the molecular pathways that underlie its neuroprotective action are fully elucidated in order to gauge its therapeutic potential. While we have previously demonstrated that R18 has the capacity to reduce glutamic acid-induced excitotoxic neuronal death and intracellular calcium influx, and reduce neuronal NMDA receptor levels [22], CARPs also have cell-penetrating properties and can target mitochondria [23]. Therefore, it is likely that R18 and other CARPs have additional intracellular neuroprotective mechanisms of action. In addition, it is also important to examine the ability of R18 to preserve intracellular protein expression and biochemical pathways following a neurodamaging insult. As such, in this study we performed iTRAQ proteomics and bioinformatic analysis ( Fig. 1) of protein cell lysates collected from primary cortical neuronal cultures subjected to glutamic acid excitotoxicity with and without treatment with R18.

Peptides
Poly-arginine-18 (R18; H-RRRRRRRRRRRRRRRRRR-OH) was synthesized by Mimotopes (Australia) and purified to 98% by HPLC. Peptides were prepared as 500 μM stocks in Baxter water (Australia) and stored at − 20°C prior to use.

Glutamic acid excitotoxicity model and assessment of cell viability
Cortical neuronal cultures were subjected to glutamic acid excitotoxicity and R18 treatment as previously described [22]. R18 treatment consisted of adding the peptide to culture wells 10 min prior to glutamic acid (L-glutamic acid; Sigma-Aldrich) exposure by removing media and adding 50 μL of Minimal Essential Media (MEM)/2% B27 containing peptide (2 μM). To induce excitotoxicity, 50 μL of MEM/2% B27 containing glutamic acid (200 μM; final concentration 100 μM) was added to the culture wells and incubated at 37°C in the CO 2 incubator for 5 min (note: peptide concentration reduced to 1 μM during this step). Following the 5-min exposure, media was replaced with 100 μL of MEM/2% B27 and cultures incubated for a further 24 h at 37°C in the CO 2 incubator. Untreated controls with or without glutamic acid treatment underwent the same incubation steps and media additions.
At 24 h post-injury, cell viability was assessed qualitatively by light microscopy, and quantitatively using the CellTiter 96 Aqueous Cell Proliferation MTS assay (Promega, Australia), which determines metabolic capacity of cells through the reduction of the tetrazolium salt (MTS), forming a brown formazan salt that is measured spectrophotometrically at 490 nm.
Lysates were clarified by centrifugation at 14,000 g for 5 min at 4°C, and protein concentration determined via Bradford's assay (Bio-Rad). Aliquots (3.5 mg/mL) of each treatment group were prepared for subsequent iTRAQ analysis and stored at − 20°C prior to use.

Protein sample preparation and iTRAQ labelling
Quantitative 4-plex iTRAQ proteomics analysis was performed on four independent protein samples for each treatment. Protein sample preparation and iTRAQ labeling was as previously described [25]. Briefly, the protein samples were de-salted, reduced, alkylated, and trypsin-digested according to the iTRAQ protocol [Sciex]. The resulting peptide samples were labeled with iTRAQ reagents as follows: 114: Untreated control (Cont); 115: glutamic acid treated (Glut); 116: R18 treated (R18); 117: R18 and glutamic acid treated (R18 + Glut). All labeled samples were combined to make a pooled sample. Peptides were desalted on a Strata-X 33 μM polymeric reversed phase column (Phenomenex) and dissolved in a buffer containing 2% acetonitrile 0.1% formic acid before separation by High pH on an Agilent 1100 HPLC system using a Zorbax C18 column (2.1 × 150 mm). Peptides were eluted with a linear gradient of 20 mM ammonium formate, 2% ACN to 20 mM ammonium formate, 90% ACN at 0.2 mL/ min. The 95 fractions were concatenated into 12 fractions and dried down. Each fraction was analyzed by electrospray ionization mass spectrometry using the Shimadzu Prominence nano HPLC system [Shimadzu] coupled to a 5600 TripleTOF mass spectrometer [Sciex]. Samples were loaded onto an Agilent Zorbax 300SB-C18, 3.5 μm [Agilent Technologies] and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). Fourteen percent of the labeled sample was loaded on the mass spectrometer.

Proteomic data analysis: qualification and quantitation
Spectral data was qualified using ProteinPilot™ 5.0 software [Sciex] against the SwissProt database, utilizing the Rattus norvegicus (Rat) taxonomy (Version: April 2017, 7,985 sequences; https://www.uniprot.org/proteomes/ UP000002494). The False Discovery Rate (FDR) was automatically calculated by the Proteomics System Performance Evaluation Pipeline (PSPEP) feature in the ProteinPilot™ software (AB Sciex, Foster, CA, USA; Version 5.0.1) using the reversed version of the protein sequences contained in the search database. For quantitative protein analysis, a fold change in protein expression > ±1.3-fold with a p < 0.05 was considered to be a 'differentially expressed protein' (DEP). Protein expression changes with R18, Glut, and R18 + Glut treatment were compared to the control (Cont). In addition, protein changes with Glut treatment were compared with R18 + Glut treatment using Cont protein expression as baseline.

Proteomic data analysis: bioinformatics
Gene ontology analysis with the 'Protein ANalysis THrough Evolutionary Relationships' (PANTHER; Version 14.0, released 2018-12-03; http://pantherdb.org/) classification system was utilized to categorize the collective DEPs in the R18, Glut, or R18 + Glut samples, relative to Cont sample, as well as Glut sample, relative to the Glut + R18 sample. These proteins were functionally categorized according to the domains of 'biological processes' , 'molecular functions' , and 'cellular components' [26].
Protein-protein interaction networks were identified using STRING (Version 11.0, released 2017-05-14; http://www.string-db.org/). STRING is a database of known and predicted physical and functional proteinprotein interaction, generated through computational prediction from five key databases: 'Genomic Context Predictions' , 'High-throughput Lab Experiments' , '(Conserved) Co-Expression' , ' Automated Textmining' , and 'Previous Knowledge in Databases'. Cytoscape (Version 3.7.1) was subsequently utilized to construct and analyze the protein-protein interaction networks, and 'Cluster with overlapping Neighbourhood Expansion' (Cluster ONE; Version 14) was used for network clustering of protein-protein interactions, to identify densely connected and overlapping protein networks.
Identified DEPs were also imported into the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database (http://www.genome.jp/kegg/pathway.html) for analysis of common biological pathways and diseases associated with the DEPs.

Statistical analysis
Statistical analysis was conducted with the Prism 8.0 GraphPad statistical software package. Cell viability data were expressed as mean ± S.E.M. of biological replicates, and multiple comparisons were conducted with one-way ANOVA and Bonferroni's post hoc test to assess significance, with significance taken as p < 0.05. For Cytoscape network analysis, network cut-offs of > 3 proteins were utilized, and a one-sided Mann-Whitney U test was used to identify significant common networks (p < 0.05).

R18 improves cell viability in uninjured and glutamic acid-treated neuronal cells
In line with previous studies [12], R18 exhibited potent neuroprotection against glutamic excitotoxic injury in cortical neuronal cultures. In addition, as has been previously reported, cell viability was also significantly increased in neuronal cultures treated with R18 compared to control cultures (Fig. 2).

Proteins regulated by glutamic acid injury alone (glut vs Cont)
Of the 95 DEPs identified in the Glut sample, 21 were uniquely regulated ( Table 1). The greatest magnitude fold-change in a down-regulated protein was observed for brain-specific angiogenesis inhibitor 1-associated protein 2 (Baiap2; − 90.09), while the greatest foldchange in an up-regulated protein was for fatty acidbinding protein, brain (Fabp7; 4.41).

Proteins regulated by R18 plus glutamic acid injury (R18 + Glut vs Cont)
Of the 14 DEPs identified in the R18 + Glut sample none were uniquely regulated (Additional file 2: Table S2). The greatest magnitude fold-change in an up-regulated protein was observed for UV excision repair protein RAD23 homolog B (Rad23b; 3.251), while the greatest fold-change in a down-regulated protein was for Keratin, type I cytoskeletal 10 (Krt10; − 33.333).

Proteins regulated by R18 plus glutamic acid injury vs glutamic acid injury alone (R18 + Glut vs glut)
Of the 98 DEPs identified after R18 + Glut treatment (R18 + Glut vs Glut), 73 of the proteins were also regulated by R18 and/or Glut treatments alone, and 25 were uniquely regulated (Table 1; uniquely regulated proteins indicated by *). In addition, R18 treatment reversed the up-or down-regulation of all 73 DEPs (Table 1, Fig. 3). Of the 25 uniquely regulated proteins, the greatest magnitude fold-change in protein expression observed was with Brain-specific angiogenesis inhibitor 1-associated protein 2 (Baiap2; − 99.08).

Functional categorization of differentially regulated proteins (DEPs)
For further functional characterization of DEPs we focused on protein changes in the R18 and R18 + Glut treatments groups as we were most interested in the effects of R18 on proteins regulated in uninjured and injured neurons. PANTHER Gene Ontology analysis was utilized to categorize the DEPs regulated by R18 alone (5 proteins; R18 vs Cont), and the DEPs regulated by R18 + Glut treatment (73 proteins; R18 + Glut vs Glut) according to 'cellular component' (Fig. 4a and d) 'biological process' (Fig. 4b and e), and 'molecular function' (Fig. 4c and f ) (Full data provided in Additional file 3: Table S3). The 5 DEPs regulated by R18 treatment included proteins located in the nucleus and ribosomes which catalyze purine nucleotide catabolic activity (e.g. Nudt3), and modulate rRNA binding (e.g. Rplp0). Other proteins regulated by R18 are involved in intracellular ion trafficking (e.g. Atp1a3) and cytoskeletal structure (e.g. Krt1) ( Table 1).
The 73 DEPs regulated by R18 + Glut treatment included proteins involved in mitochondrial respiration and function (e.g. Aco2 and Atp5a1), proteasomal regulation and protein synthesis (e.g. Psmc1 and Cct3), proteostasis/protein modification in the endoplasmic reticulum (e.g. Hspa9 and Uba1), and RNA trafficking/ processing (e.g. Eif5a and Hnrnpa1), as well as cytoskeletal rearrangement and axonal growth (e.g. Map2 and Dcx) and vesicular/membrane trafficking (e.g. Atp1a3 and Camk2a). Significant changes of key neuronal proteins are summarized in the schematic detailed in Fig. 5 (full data available in Additional file 2: Table S2 and Additional file 3: Table S3).
Protein-protein interaction network analysis of DEPs regulated by R18 in uninjured and glutamic acid injured neurons STRING analysis was used to identify potential proteinprotein interactions across the 5 DEPs regulated by R18  (Fig. 6a). Two hundred and twenty-two nodes representing direct and indirect protein-protein interactions were identified for the 73 DEPs regulated by R18 + Glut treatment (R18 + Glut vs Glut) (Fig. 6b).

Discussion
In recent years, CARPs have emerged as a novel class of potential neuroprotective therapeutics for a broad range of acute brain injuries and chronic neurodegenerative disorders. These CARPs include short-chained polyarginine peptides [11,13,27], SS-peptides [28,29], APOE-derived peptides [27,30], and TAT-fused peptides, including TAT-NR2B9c (NA-1) and JNK1-TAT [11,31]. Such CARPs have been shown to exert their neuroprotective action through a variety of targets, which include structural and functional preservation of mitochondria [32], reduced ROS generation [33], inhibition of protein aggregation [34], modulation of glutamate or calcium ion receptors (excitotoxicity/calcium influx) [35], and activation of pro-survival signaling [36,37]. Given the diverse biochemical and cellular effects CARPs can exert on cells, it is likely that other cytoprotective processes are also involved, which have yet to be fully elucidated. To this end, the present study is the first to employ an iTRAQ proteomics approach to gain insight into protein expression changes after polyarginine-18 (R18) treatment of uninjured neuronal cultures and neuronal cultures subjected to glutamic acid excitotoxic injury. The effects of R18 treatment on uninjured neurons Proteomics analysis of neuronal cultures 24-h after a 10-min exposure to R18 identified 5 DEPs. The small number of detected protein expression changes was surprising given that CARPs can induce a variety of biological effects on cells [38]. However, it is likely that the small number of proteins detected was in part due to the 24-h post-R18 treatment time point used to analyze protein expression changes, as majority of the protein changes elicited by R18 treatment may potentially occur within the first few hours, and as such, may no longer have been detectable or did The proteins that were identified as being affected by R18 were largely associated with protein synthesis and transmembrane protein and cationic ion transport, and did not significantly map onto KEGG pathways, suggesting that the R18 peptide does not exert long-term biological effects in uninjured neurons. This is in line with the proposed notion that neuroprotective agents should preferentially interact with and/or modulate cellular targets activated following pathological events to minimize the chance of offtarget side effects. Such protective agents are deemed 'pathologically-activated' therapeutics, which are thought to have a particularly useful application in neurological disorders, as brain tissue is especially susceptible to drug-induced disruptions and unwanted drug side-effects [38,39]. However, to provide a more comprehensive assessment of the biological effects of R18 on uninjured neurons, future studies should examine protein expression at earlier time points.  Table 1 R18 treatment reduces glutamic acid-induced changes in protein expression An important finding of this study was the ability of R18 treatment to reverse the majority (74.5%; 73 of 98) of the protein changes induced by glutamic acid excitotoxicity, and thereby preserve the protein expression profiles of cortical neurons post-insult (Table  1; Additional file 4: Table S4). Further analysis revealed that these protein changes underpin key cellular functions, such as mitochondrial respiration and energy production, proteostasis, neuronal transmembrane trafficking, and RNA processing, which are dysregulated by excitotoxicity. Moreover, KEGG analysis of protein-protein interactions indicated predominant involvement of pathways pertaining to the proteasome and oxidative phosphorylation, which also represent two central biological processes underpinning aspects of neurodegenerative pathophysiology. This likely contributed to the identification of enriched protein-protein interactions pertaining to Parkinson's disease, Alzheimer's disease and Huntington's disease.
Severe and/or prolonged disruptions in the ubiquitinproteasome system have been implicated in both acute A B Fig. 6 Enriched STRING PPI network analysis of DEPs regulated by a. R18 treatment in healthy neurons (vs Cont), and b. DEPs commonly regulated by Glut and R18 + Glut (vs Glut), demonstrating molecular actions of direct and indirect protein-protein interactions between significantly regulated proteins. STRING parameters were set to high confidence (0.700), with only query proteins shown (ischaemic stroke, TBI) and chronic (AD, PD, motor neuron disease) neurological disorders [39,40]. Previous studies have shown that CARPs exhibit proteasomal modulatory activity and could potentially conserve protein expression profiles through inhibition of injury-induced proteasomal protein degradation. For example, the arginine-rich PR-11 (H-RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPR FP-OH; net charge + 11) and PR-39 (H-RRRPRPPY LPR-OH; net charge + 5) peptides attenuate inflammation induced by ischaemia-reperfusion injury through inhibition of proteasomal degradation of IκBα; a NFκB inhibitory protein [41,42]. Taken together, it appears CARPs can influence the function of the proteasome, and thereby exert neuroprotective benefits during times of cellular stress.
Proteomic analysis also revealed that R18 preserved protein expression profiles pertaining to mitochondrial bioenergetics and structural integrity. Mitochondria are central mediators of intracellular calcium signaling events during excitotoxicity, and as such, are considered the "judge, jury, and executioner" of the cell [31,43].
During excitotoxic injury, mitochondria act as a buffer for toxic intracellular calcium accumulation, however excessive mitochondrial calcium uptake can disrupt their structural and functional integrity, resulting in the release of pro-death signaling proteins from the mitochondrial inter-membrane space [44,45]. Therefore, the ability of R18 treatment to attenuate excitotoxicityinduced protein changes underlying loss of mitochondrial integrity, provides evidence that the peptide helps preserve the function of the organelle in times of cellular stress. In line with the ability of CARPs to maintain mitochondrial function and energy generation, in this and previous studies R18 was demonstrated to increase MTS metabolism in uninjured neurons and in neurons after exposure to glutamic acid [14].
Bioinformatic analysis of the DEPs identified in the present study largely focused on the 73 proteins up-or down-regulated by glutamic acid excitotoxic injury. However, it is important to note that 25 other DEPs were also identified to be uniquely regulated by combined R18 + Glut treatment, which may represent additional proteins influenced by R18 and associated with Fig. 7 Cytoscape ClusterONE analysis of enriched STRING protein-protein interaction network of shared DEPs regulated by both Glut and R18 + Glut. Cytoscape ClusterONE analysis was used to group protein clusters based on their involvement in 'Mitochondrial respiration', 'Axonal growth and neuronal differentiation', 'Transmembrane trafficking', 'Endoplasmic reticulum proteostasis', 'Glycolysis and carbohydrate metabolism', 'Mitochondrial fatty acid synthesis', or 'RNA trafficking and processing'. Clusters represent statistical significance cut-offs of p < 0.05, and empty nodes represent proteins that do not share statistically significant functions with other proteins neuroprotection. Alternatively, these proteins could reflect non-specific changes in protein expression unrelated to neuroprotection.

Limitations and future directions
The proteomics methodology used in this study does not provide insight into other forms of protein modification, such as post-translational changes (e.g. phosphorylation, acetylation, and glycosylation), which may influence protein functions important for neuroprotection. In addition, only a 24-h timepoint was examined and therefore it would also be of interest to examine protein expression changes, as well as post-translational modifications at earlier timepoints after R18 treatment. Further studies are also required to confirm if the DEPs and the biochemical and disease pathways influenced by R18 treatment after glutamic acid excitotoxicity in vitro are also affected by the peptide in animal models of acute brain injury (e.g. stroke, TBI) and chronic neurodegenerative disorders (AD, PD).

Conclusion
This exploratory study has demonstrated for the first time that the poly-arginine peptide R18 exerts significant effects in attenuating the protein expression changes associated with neuronal excitotoxicity in vitro, while inducing minimal changes in uninjured neurons. Collectively, our findings indicate that the neuroprotective effects of R18 following excitotoxicity are associated predominantly with preservation of neuronal proteostasis, together with positive effects on mitochondrial and proteasomal function. The findings of this study provide further evidence supporting the role of poly-arginine peptides as a potential neuroprotective therapeutic for both acute and chronic neurodegenerative disorders.

Additional files
Additional file 1: Table S1.  Table S3. PANTHER gene-ontology functional categorization. PANTHER gene-ontology functional categorization of DEPs significantly regulated by R18 treatment alone (R18 vs Cont), glutamic acid exposure (Glut vs Cont), and R18 pre-treatment with glutamic acid Fig. 8 Enriched KEGG pathway analysis of shared DEPs common to both Glut and R18 + Glut (vs Glut), demonstrating the top 20 pathways. Shown above is the percentage of proteins mapped to the respective significantly regulated pathways, with the fraction of regulated proteins mapping onto the total number of proteins in each pathway provided in white. The top 5 pathways are outlined in red exposure (R18 + Glut vs Glut). Note: proteins may have multiple functions, and as such, the total number of proteins in each category may be greater than the sum of DEPs across each treatment group. (DOCX 49 kb) Additional file 4: Table S4. Cytoscape quantitative analysis. Cytoscape quantitative analysis of STRING data cluster strength for 73 shared DEPs across Glut and R18 + Glut treatment groups. (DOCX 56 kb) Additional file 5: Table S5. Quantitative data and full gene list of KEGG pathway analysis. Quantitative data and full gene list of KEGG pathway analysis of 73 shared DEPs across Glut and R18 + Glut treatment groups, with details provided on term ID, overserved gene count vs. background gene count, and FDR. (DOCX 17 kb) Abbreviations AD: Alzheimer's disease; ALS: Amyotrophic lateral sclerosis; Cont: 'No treatment' group; DEP: Differentially expressed proteins; Glut: 'Glutamic acid excitotoxic injury' treatment group; HD: Huntington's disease; LC-MS/ MS: Liquid chromatography-tandem mass spectrometry; PD: Parkinson's disease; R18 + Glut: 'R18-treatment and glutamic acid injury' treatment group; R18: Poly-arginine-18 peptide