Signal flow and pathways in response to early Wallerian degeneration after rat sciatic nerve injury

Neuroscience Letters 536 (2013) 56–63

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Signal flow and pathways in response to early Wallerian degeneration after rat sciatic nerve injury夽 Meiyuan Li a,1 , Weimin Guo a,1 , Pingan Zhang a , Huaiqin Li a , Xiaosong Gu a , Dengbing Yao a,b,∗ a b

Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu 226019, PR China School of Life Sciences, Nantong University, Nantong, Jiangsu 226019, PR China

h i g h l i g h t s  Analyzed signal flows and pathways of early Wallerian Degeneration (WD).  Explore the key regulate factors and recurrent neural networks in the process of early WD.  Reported some key factors, which may play a powerful role in regulating gene expressions during early WD.

a r t i c l e

i n f o

Article history: Received 24 November 2012 Accepted 2 January 2013 Keywords: Wallerian degeneration Sciatic nerve Signal pathway Rat

a b s t r a c t Wallerian degeneration (WD) remains a subject of critical research interest in modern neurobiology. WD is a process which a large number of genes are differentially regulated, especially the early response to activate nerve degeneration and regeneration, but the precise mechanisms remain elusive. In this study, we report the signal pathways, key regulate recurrent neural networks and signal flow in the early WD. The data indicated that there are several kinds of up- or down-regulated genes, relating to the regulation of response to stimulus, signal transmission via phosphorylation event, immune response, apoptosis and regulation of cell communication. KEGG pathway analysis revealed activity mainly relating to cytokine–cytokine receptor interaction, MAPK signaling pathway, Jak-STAT signaling pathway, ErbB signaling pathway and TGF-beta signaling pathway involved in the recurrent neural networks that were regulated by the key factors, Cldn-14, Cldn-15, ITG, BID and BIRC3. These results will help to much better understand information relating to the early response to WD and provide us with a firmer basis in future investigations on the molecular mechanisms of WD that regulate nerve degeneration and/or regeneration. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Wallerian degeneration (WD) is one of the most common reactions after peripheral nerve injury and occurs in the peripheral

Abbreviations: ACVR1v, activin A receptor, type 1v; BID, BH3 interacting domain death agonist; BIRC3, baculoviral IAP repeat-containing 3; CCL, chemokine (C-C motif) ligand; CCR, chemokine (C-C motif) receptor; CEACAM, carcinoembryonic antigen-related cell adhesion molecules; EGR, early growth response; JaK, Janus kinase; MAG, myelin associated glycoproteins; MyD88, myeloid differentiation primary response gene 88; Rac, a subfamily of the Rho family of GTPases. 夽 This research was supported by grants from: the National Natural Science Foundation of China (Key Program, Grant No. 81130080); Scientific Research Foundation for Returned Scholars, Ministry of Education of China; Natural Science Foundation of Jiangsu Province (Grant No. BK2010282) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, PAPD. ∗ Corresponding author at: Jiangsu Key Laboratory of Neuroregeneration, School of Life Sciences, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, PR China. Tel.: +86 513 85051850; fax: +86 513 85511585. E-mail address: [email protected] (D. Yao). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.01.008

nervous system (PNS) when nerve fiber is cut or crushed. It occurs rapidly in the PNS, following nerve injury, and involves axonal degeneration and degradation of the myelin sheath [1,3,5,21]. During the early period of WD, the morphological changes of Schwann cells are likely to be mediated by the actin cytoskeleton, which plays a key role in cell shape changes in response to various stimuli. Schwann cells take the major role in myelin cleaning and proliferation and have been observed recruiting macrophages by the release of cytokines and chemokines after nerve injury [2–6]. Understanding the factors that regulate rapid macrophage responses in the PNS during WD, may provide insights into the reasons for slow Wallerian degeneration [7–11]. Although the molecular mechanisms regulating WD have been studied extensively, a large number of genes and signaling molecules are differentially regulated and, especially in the early response of WD, are not completely understood. In this study, we report on gene-expression signal flow and signal pathways in the early response of WD in the distal nerve stump at 0, 1, 6, 12, 24 h after rat sciatic nerve injury using microarray and bioinformatic analysis. These results will help achieve a greater understanding of

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Fig. 1. The pathway analysis of distal sciatic nerve stumps of rats for early response at 0, 1, 6, 12, 24 h carrying WD. Based on the KEGG database, Fisher exact test and chi square test were applied to the differential genes.

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Fig. 2. The signal pathways network analysis of distal sciatic nerve stumps of rats for early response at 0, 1, 6, 12, 24 h carrying WD. The networks are prioritized based on the number of fragments with main parameters of relative enrichment with the uploaded data and relative saturation of networks with canonical pathways.

information relating to the early response of WD and provide us with a firmer basis in future investigations on the molecular mechanisms of WD regulating nerve degeneration and/or regeneration. 2. Materials and methods 2.1. Animals models We used male Sprague-Dawley (SD) rats provided by the Experimental Animal Center of Nantong University. The rats were randomly divided into 5 groups (6 per group) to undergo sciatic neurectomy. We conducted all tests in accordance with “NIH Guidelines for the Care and Use of Laboratory Animals” and anesthetized all rats with complex narcotics. We lifted the sciatic nerve on the lateral aspect of the mid-thigh from the right hind limb and excised a 1 cm segment. We killed one group of rats immediately and the other groups at 0, 1, 6, 12, 24 h following surgery. The 0 h animals received sham-operations. 2.2. RNA isolation and affymetrix microarray scan

Institutes for Biological Sciences, Chinese Academy of Sciences. We analyzed differential genes using the Affymetrix scan system. The expression model profiles were related to the actual or expected number of genes assigned to each model profile and significant profiles demonstrated a higher probability than expected in Fisher’s exact test and multiple comparison tests. We used STC GO to analyze the main functions of the differentially expressed genes according to Gene Ontology. We used pathway analysis to determine the significant pathways of differential genes according to KEGG [4,10,16]. We analyzed gene regulatory networks using a Continuous Time Recurrent Neural Network (CTRNN). Key networks and Signal flow were calculated according to gene-fold expressions and gene interactions in pathways. Networks are built extemporaneously, using networkanalysis algorithms with default settings for biological networks [16,19]. 2.4. Real-time quantitative PCR assay

Total RNA was isolated from the distal nerve stumps according to the manufacturer’s protocols. We conducted Genechip analysis using the Agilent mRNA Microarray System. Labeling and hybridization were performed at Shanghai Biochip Company, Ltd. We analyzed microarray data using GeneSpring GCOS1.2 software. The data were analyzed and differences were considered statistically significant at P < 0.05.

The distal nerve stumps of rats killed at 0, 1, 6, 12, 24 h following injury were dissected. Real-time quantitative PCR was performed with the SYBR Green PCR Master Mix. The primers used in the Realtime quantitative PCR assays are provided in the Supplementary Materials. The relative expression values of each mRNA were calculated using Comparative Ct and were normalized using GAPDH mature mRNA for each data point. All reactions were run in triplicate. All data were expressed as means ± S.D.

2.3. Bioinformatic analysis

2.5. Western blot analysis

Gene screening and bioinformatic analysis were performed by the Bioinformatics Center, Key Lab of Systems Biology, Shanghai

The distal nerve stumps of rats killed at short time points 0, 1, 6, 12, 24 h after injury were directly lysed. We analyzed protein

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Fig. 3. The signal-flow analysis of distal sciatic nerve stumps of rats at 0, 1, 6, 12, 24 h carrying WD. Dots represent genes, straight lines represent relationships of signal transduction between genes included in the KEGG database. The caps of lines represent inhibition, arrows represent activation, phosphorylation or bindings etc.

expressions with monoclonal antibodies against anti-EGR-1, EGR2, CD44, Cldn-14, Cldn-15, c-Jun, c-Fos antibody and conjugated affinity purified goat anti-mouse IgG. We scanned the image with a GS800 Densitometer Scanner, and analyzed the data of optical density using PDQuest 7.2.0 software. Beta-actin was used as an internal control.

2.6. Immunohistochemistry The distal nerve stumps of rats killed at 0 and 24 h after injury. We incubated the samples with mouse monoclonal antiS100 antibodies against Cldn-14 and Cldn-15 then reacted the samples with FITC-labeled goat anti-mouse IgG. We mounted and observed the samples with a confocal laser-scanning microscope. Non-labeled secondary antibodies were used with the control samples. We further reacted the samples with goat anti-mouse IgG and goat anti-rabbit IgG. We viewed the samples under a confocal laser microscope. We treated the control samples similarly (bar = 20 ␮m).

3. Results 3.1. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of differential genes expressed during early WD To screen differentially expressed genes during WD after sciatic nerve injury, we took the union of sets of differential genes at different time points and compared these to the 0 h in each group. The data summarizes all possible expression trends and statistical judgments, in accordance with P < 0.05 as the standard (Supplementary Materials S2). Based on a comparison of our results against the GO database, using BLAST with an E-value cutoff of <10–5, there were significant differential gene matches assigned to KEGG pathways in early WD (Supplementary Materials S3). Included among the identified matches were those relating to cytokine–cytokine receptor interaction, MAPK signaling pathway, toll-like receptor signaling pathway, cell adhesion molecules, Jak-STAT signaling pathway, apoptosis, tight junction, T cell receptor signaling pathway, p53 signaling pathway, Calcium signaling pathway, adherens junction, Gap junction, ECM-receptor interaction, regulation of actin cytoskeleton, and cell cycle (Fig. 1).

2.7. Statistical analysis We used Student’s t-test for comparison between groups. All data were expressed as means ± SD and were analyzed by Scheffe’s Post Hoc Test with the SPSS software package. All values of P < 0.05 were considered significant.

3.2. Key network analysis of differential genes expressed during early WD From the stimuli-induced cellular behavior reflected in protein–protein interactions and expression kinetics, we analyzed

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Fig. 4. Real-time quantitative PCR analysis of distal sciatic nerve stumps of rats for early response at 0, 1, 6, 12, 24 h carrying WD. The relative expression values of each mRNA was normalized using GAPDH mRNA for each point. The average of three independent experiments is shown ±SEM.

network according to gene-fold change expressions and interactions in signal pathways. The relationships of the differential gene expression data were calculated using a Continuous Time Recurrent Neural Network (CTRNN) as a dynamic model for the regulatory network mediating the cell response to a special stimulus. Using a genetic algorithm, we estimated the model parameters. A heat map showed the partition cluster of genes highly expressed in the distal nerve stumps after sciatic nerve injury [12,16,19]. Our study revealed key networks that included IL-6, Cldn, CCR2, IL-1 beta, CCL3, TNF-R1, MyD88 and CCR2. The GO processes included regulation of response to stimulus, signal transmission via phosphorylation event, intracellular protein kinase cascade, regulation of cell communication, regulation of response to stress, cytokine-mediated signaling pathway, initiation of signal transduction, signal initiation by protein/peptide mediator, signal initiation by diffusible mediator and regulation of response to stimulus (Fig. 2). 3.3. Signal-flow analysis of differential genes expressed during early WD Based upon the results of pathway analysis, we built geneexpression values on chips at different time points. Using interactions between genes in the KEGG database relating to signal transmission and control network at various time points, we calculated the weighted value of various network genes. As the

results preliminarily suggest from the relationships between genes in the database (Fig. 3), there are parts of the whole network in which the relationship between genes of a local network on top is comparatively dense, and the weight ratio of each to the others is comparatively high, showing that these parts of the genes may act as a regulated integral network to participate in the organism changes. The KEGG pathway analysis showed a related signaling pathway involved in the recurrent of neural networks that were regulated by the following key factors: Cldn-14, Cldn-15, ITG, BID, CCL and BIRC3.

3.4. Real-time quantitative PCR assay Although we demonstrated the reliability of our array data using a number of genes that have been previously described in response to peripheral nerve injury, we nonetheless performed validation experiments on a number of genes for which expressions or regulations have not been reported. We carried out real-time quantitative PCR for the following genes: Cldn-14, Cldn-15, EGR-1, EGR-2, CD44 and c-Fos in early WD. We used real-time quantitative PCR to statistically analyze our comparison of expression levels from each time point to the 0 h (Fig. 4). These experiments confirmed the transcriptional regulation determined by array hybridization. Our results of Real-time quantitative PCR were mainly consistent with the gene expression tendency of microarray analysis.

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Fig. 5. Western blot analysis of total protein lysates of distal sciatic nerve stumps of rats at 0, 1, 6, 12, 24 h carrying WD (upper) and relative expressions (down). The average of three independent experiments is shown ±SEM. Beta-actin (43 kDa) was used as loading control.

3.5. Western blot analysis To confirm data obtained through microarray and real-time PCR analyses, we employed Western blot with monoclonal antibodies against anti-EGR-1, EGR-2, CD44, Cldn-14, Cldn-15, c-Fos and c-Jun antibody. Beta-actin was used as an internal control. The results were expressed as the mean relative signal intensity, i.e. the ratio of sample signal intensity/positive control signal intensity. The data indicated that the expression tendency was similar to the results of microarray and real-time PCR analyses (Fig. 5). We validated factors that were up- and down-regulated during early WD. EGR proteins function in transcription regulatory activities surrounding cellular growth, differentiation and function. The transcription of c-Fos is upregulated in response to many extracellular signals. Phosphorylation by MAPK, PKA and PKC alters the activity and stability of c-Fos. Members of the Fos family dimerise with c-jun to form the AP-1 transcription factor, which upregulates the transcription of a diverse range of genes involved in proliferation and differentiation to defense against invasion and cell damage [12–15]. 3.6. Immunohistochemistry Cldn-14 and Cldn-15 are proteins encoded by the claudin gene, a member of the claudin family. They play a major role in the tight-junction-specific obliteration of intercellular space through

cell-adhesion activity. We used immunohistochemistry to visualize the location of the Cldn-14 and Cldn-15 antibodies. The distal nerve stumps of rats killed at 0, 24 h after injury were analyzed by immunohistochemistry with anti-S100 antibody, antiCldn-14 and Cldn-15 antibody. Controls were treated similarly, using non-labeled secondary antibodies to confirm the inexistence of nonspecific binding. Double-immunostaining for the Cldn-14, Cldn-15 and S-100 proteins demonstrates that Cldn-14, but not Cldn-15, is co-located in the plasma membrane of Schwann cells (Fig. 6). Immunohistochemistry showed results consistent with real-time PCR and Western blot analysis, confirming the changes of the Cldn-14 and Cldn-15 gene expressions in the distal nerve stumps after nerve injury. 4. Discussion Wallerian degeneration (WD) is a bioprocess following nerve injury that can be described as a clearing process essential for distal-stump repair or reinnervation. Understanding the factors that regulate responses in the PNS during WD and comparing the differences in the expression of these molecules in the lesioned PNS may provide insights into the reasons for slow WD in the PNS. Our data indicated that in early responses, major changes appeared to provide separate signals that were characterized by a high degree of overlapping genes. Within 24 h after nerve injury, we observed a rapid but temporary up- and down-regulation of

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Fig. 6. Immunofluorescence staining for distal sciatic nerve stumps of rats at early response for 24 h carrying WD and normal sciatic nerve. Immunostained for S100, Cldn-14, Cldn-15 and their overlay. Cldn-14 is co-located in the plasma membrane of Schwann cells but not Cldn-15 (bar = 20 ␮m).

the small inducible cytokine genes. These chemokine, is thought to recruit neutrophils [17,18] and may amplify proinflammatory cytokine responses via a phosphatidylinositol 3-kinase/nuclear factor-kappa B pathway. Selective cell death in order to remove supernumerary Schwann cells is not only a characteristic feature of nerve regeneration, but also of successful peripheral nerve repair. Several regulated cell-death-associated genes were re-expressed, such as the Fas receptor/ligand receptor, apoptosis regulator protein Bcl like protein, low affinity NGF receptor and lysozyme were detected [20,21]. Peripheral nerve repair is a result of reactivated regeneration mechanisms in combination with newly activated injurydependent reactions. It regulates initially after nerve injury and repair and the injury induced switch to the genetic response also features a number of degeneration and regeneration-specific genes. During nerve degeneration and regeneration, genes exhibit significant regulation from a baseline. This regulation has not been proven systematically with a direct comparison of the genes of peripheral nerve regeneration. Candidate factors may be required for the adaptation of nerve regeneration, but many of them have not so far been associated with nerve repair after nerve injury [17–21]. Tight junctions represent one mode of cell-to-cell adhesion in epithelial or endothelial cell sheets, forming continuous seals around cells and serving as a physical barrier to prevent solutes and water from passing freely through the paracellular space. These junctions are comprised of sets of continuous networking strands

in the outwardly facing cytoplasmic leaflet, with complementary grooves in the inwardly facing extracytoplasmic leaflet. Cldn-14 and Cldn-15 are two tight-junctional membrane proteins specifically expressed in different type cells that may perform different functions in early WD after sciatic nerve injury. Further studies are necessary to identify these key regulated factors, such as Cldn-14 and Cldn-15, and how they regulate signal pathways in vivo and their functions during early WD after sciatic nerve injury. Conflict of interest The authors declare no financial conflict of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet. 2013.01.008. References [1] T. Araki, R. Nagarajan, J. Milbrandt, Identification of genes induced in peripheral nerve after injury. Expression profiling and novel gene discovery, Journal of Biological Chemistry 276 (36) (2001) 34131–34141. [2] B. Barrette, E. Calvo, N. Vallies, S. Lacroix, Transcriptional profiling of the injured sciatic nerve carrying the Wld(S) mutant gene: identification of genes involved

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