Identification of the role of C/EBP in neurite regeneration following microarray analysis of a L. stagnalis CNS injury model

Abstract

Background: Neuronal regeneration in the adult mammalian central nervous system (CNS) is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. In contrast, the CNS of adult Lymnaea stagnalis (L. stagnalis), a freshwater pond snail, is capable of spontaneous regeneration following neuronal injury. Thus, L. stagnalis has served as an animal model to study the cellular mechanisms underlying neuronal regeneration. However, the usage of this model has been limited due to insufficient molecular tools. We have recently conducted a partial neuronal transcriptome sequencing project and reported over 10,000 EST sequences which allowed us to develop and perform a large-scale high throughput microarray analysis.

Results: To identify genes that are involved in the robust regenerative capacity observed in L. stagnalis, we designed the first gene chip covering ~15, 000 L. stagnalis CNS EST sequences. We conducted microarray analysis to compare the gene expression profiles of sham-operated (control) and crush-operated (regenerative model) central ganglia of adult L. stagnalis. The expression levels of 348 genes were found to be significantly altered (p < 0.05) following nerve injury. From this pool, 67 sequences showed a greater than 2-fold change: 42 of which were up-regulated and 25 down-regulated. Our qPCR analysis confirmed that CCAAT enhancer binding protein (C/EBP) was up-regulated following nerve injury in a time-dependent manner. In order to test the role of C/EBP in regeneration, C/EBP siRNA was applied following axotomy of cultured Lymnaea PeA neurons. Knockdown of C/EBP following axotomy prevented extension of the distal, proximal and intact neurites. In vivo knockdown of C/EBP postponed recovery of locomotory activity following nerve crush. Taken together, our data suggest both somatic and local effects of C/EBP are involved in neuronal regeneration.

Conclusions: This is the first high-throughput microarray study in L. stagnalis, a model of axonal regeneration following CNS injury. We reported that 348 genes were regulated following central nerve injury in adult L. stagnalis and provided the first evidence for the involvement of local C/EBP in neuronal regeneration. Our study demonstrates the usefulness of the large-scale gene profiling approach in this invertebrate model to study the molecular mechanisms underlying the intrinsic regenerative capacity of adult CNS neurons.

Figure 1

Experimental model and changes in gene expression following injury. (A) The dorsal surface of L. stagnalis CNS and CNS injury protocol: both right parietal (RPa) nerves and right cerebral nerves (RCe) were crushed with fine forceps. Inset: a schematic diagram of the ganglia and location of injury (black lines). (B) Heat map representing the hierarchical clustering of 67 genes showing significant differential expression (P < 0.05). The expression pattern of each gene in the sham and crashed groups is displayed as a horizontal strip. For each gene, the expression ratio of the crush-operated to the sham-operated experiments is represented by the green and red scale at the bottom of the figure. The genes are numbered on the right, and the experimental groups are labelled on the bottom. (C) Ratio of the regulated EST sequences 3 hrs following CNS injury in L. stagnalis. (C1) The signal intensity of 348 genes or ESTs was differentially regulated. Open pie (80.7%) represents genes/EST's changes < 2.0-fold in signal intensity but significant (P < 0.05); grey pie (19.3%): changes ≥ 2.0-fold (P < 0.05). Dark grey: up-regulated genes (12%), and light grey: down regulated genes (7%). (C2) Ratio of the 67 differentially regulated genes, 16 (23.8%) genes have orthologous with known functions related to development, survival or signal transduction, whereas 51 (76.2%) genes have no orthologue.

Figure 2

Protein sequence alignment of C/EBP. Amino acid alignment between CCAAT enhancer binding protein from L. stagnalis [GenBank accession#: BAD16556], A. kurodia [GenBank#: AAG61258], R. norvegicus [GenBank#: AAI29072] and H. sapian [GenBank#: EAW75629] C/EBP. The high degree of sequence similarity at the DNA-binding domain indicates that C/EBP has a conserved functional domain across species.

Figure 3

Time-dependent changes in C/EBP mRNA expression following nerve injury. Real-time PCR was performed with specific primers for C/EBP between sham-operated and crush-operated L. stagnalis CNS at 1 hr, 3 hrs, and 5 hrs post injury. (A) Relative mRNA levels of C/EBP vs GADPH increased significantly following CNS injury as compared to the sham-operated controls at 3 hrs (2.35 ± 0.26, n = 5) (t = -3.5, df = 8, pP < 0.05). (B) Standard curves of C/EBP and GAPDH from 6 independent samples including sham-operated and crush-operated L. stagnalis CNS at 1 hr, 3 hrs, and 5 hrs post injury. The PCR efficiency of the primers was estimated by the slope (m) = -1/log(efficiency). The expression GAPDH was unchanged following CNS injury, and therefore CAPDH was used as an internal control. (C) Representative Ct-Ct correlation plots between C/EBP and the control gene, GADPH, at three different time points following injury. C1: 1 hr; C2: 3 hrs; C3: 5 hrs. Relative expression ratio between C/EBP and GADPH was estimated as Y_intercept = -log(ratio)/log(efficiency C/EBP). (D) Relative gene expression of C/EBP vs. GADPH normalized to corresponding control samples. C/EBP increased in a time-dependent manner. 1 hr: 7.36 ± 0.73 (n = 5); 3 hrs: 2.35 ± 0.26 (n = 5), and 5 hrs: 1.98 ± 0.75 (n = 5). * indicates significant differences: ANOVA: F_(2,12) = 17.4, p < 0.05. All data are presented as mean ± S.E.M.

Figure 4

Knockdown of C/EBP reduced net growth of the distal, proximal, and intact neurites following axotomy. Representatives of transected axons in culture. Immediately following axotomy (t = 0), either CM, control siRNA, C/EBP siRNA #1, or C/EBP siRNA #2 was added into culture medium at a final concentration of 7 nM, immediately after neurite transection. The neurons were observed over additional 36 hours and images were taken at 10, 24 and 36 hours as indicated. Arrows denote the transection site.

Figure 5

Differential effects of C/EBP knockdown on the distal, proximal, and intact neurites following axotomy. The length of the distal, proximal, or intact neurite of injured cells was measured at three time points (10 hrs, 24 hrs, 36 hrs) after control or C/EBP siRNA treatment, which was given immediately after axotomy (t = 0). (A) The distal neurite. The net changes in the C/EBP siRNA #1 or #2 treated cells were significantly reduced as compared to that in the control CM or control siRNA treated cells (n = 14; ANOVA: at 10 hrs, F_(3,52) = 14.4, p < 0.05; at 24 hrs, F_(3,52) = 20.1, P < 0.05; at 36 hrs, F_(3,52) = 29.7, p < 0.05). (B) The proximal neurite. A significant reduction in outgrowth was observed only at 36 hrs after axotomy in the C/EBP siRNA #1 or #2 treated cells as compared to control CM and control siRNA treated cells (n = 14; ANOVA: F_(3,52) = 5.1, p < 0.05). (C) The intact neurite. A significant reduction in outgrowth was only observed at 36 hrs in the C/EBP siRNA #1 or #2 treated cells as compared to control CM and control siRNA treated cells (n = 11; ANOVA: F_(3,40) = 6.5, p < 0.05). (D) Relative gene expression level of C/EBP vs GAPDH was significantly reduced in the C/EBP siRNA #1 (63.34 ± 8.8%) and #2 (56.25 ± 9.4%) (n = 5, ANOVA: F_(2,12) = 5.7, p < 0.05) groups as compared to control siRNA group. All data are presented as mean ± S.E.M.. * indicates p < 0.05.

Figure 6

C/EBP siRNA treatment hinders recovery of locomotion activity of the snails following CNS injury. Sham-operated or crush-operated snails were injected with either saline, control siRNA or C/EBP siRNA. The distance that the injured snails crawled in 10 min was measured at various time points (1 day, 5 days, and 10 days) following the nerve crush procedure. On the 10^th day, the crush-operated C/EBP siRNA group crawled an average of 4.99 ± 0.71 cm (n = 5) per 10 min which was reduced as compared to the sham-operated saline (10.28 ± 1.28 cm, n = 6), control siRNA (12.23 ± 1.36 cm, n = 6), or C/EBP siRNA (11.56 ± 2.75 cm, n = 5), and crush-operated saline (10.65 ± 2.84 cm, n = 4) or control siRNA treated snails (10.17 ± 1.0 cm, n = 5). Data are presented as mean ± S.E.M. * indicates p < 0.05.

Figure 7

Schematic illustration of the potential mechanisms of C/EBP action in maintaining distal axon integrity following axotomy. C/EBP mRNA is expressed in both the soma and axon [34,56]. In the soma, C/EBP functions as a transcription factor and results in the transcription of regenerative-associated genes, such as α-tubulin and GAP-43, via a cAMP/PKA/CREB dependent signalling pathway [56]. Axon injury also activates an ERK-dependent pathway that results in the phosphorylation of C/EBP and further activation of pro-regenerative genes [59]. In both proximal and distal axons, C/EBP mRNA is stabilized by DLK-1 following axotomy [34]. Local protein synthesis of C/EBP in the distal axon may be required for regeneration or to prevent degeneration of distal axons following injury.