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. 2014 Jun 25;34(7):1023–1036. doi: 10.1007/s10571-014-0078-1

Involvement of Upregulated SYF2 in Schwann Cell Differentiation and Migration After Sciatic Nerve Crush

Zhengming Zhou 1,5, Yang Liu 2,4, Xiaoke Nie 2,5, Jianhua Cao 3, Xiaojian Zhu 3, Li Yao 2,5, Weidong Zhang 4, Jiang Yu 4, Gang Wu 5, Yonghua Liu 2,5,✉, Huiguang Yang 1,5,✉
PMCID: PMC11488921  PMID: 24962097

Abstract

SYF2 is a putative homolog of human p29 in Saccharomyces cerevisiae. It seems to be involved in pre-mRNA splicing and cell cycle progression. Disruption of SYF2 leads to reduced α-tubulin expression and delayed nerve system development in zebrafish. Due to the potential of SYF2 in modulating microtubule dynamics in nervous system, we investigated the spatiotemporal expression of SYF2 in a rat sciatic nerve crush (SNC) model. We found that SNC resulted in a significant upregulation of SYF2 from 3 days to 1 week and subsequently returned to the normal level at 4 weeks. At its peak expression, SYF2 distributed predominantly in Schwann cells. In addition, upregulation of SYF2 was approximately in parallel with Oct-6, and numerous Schwann cells expressing SYF2 were Oct-6 positive. In vitro, we observed enhanced expression of SYF2 during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation. SYF2-specific siRNA-transfected Schwann cells did not show significant morphological change in the process of Schwann cell differentiation. Also, we found shorter and disorganized microtubule structure and a decreased migration in SYF2-specific siRNA-transfected Schwann cells. Together, these findings indicated that the upregulation of SYF2 was associated with Schwann cell differentiation and migration following sciatic nerve crush.

Keywords: SYF2, α-Tubulin, Schwann cells, Differentiation, Migration, Sciatic nerve crush

Introduction

Injury to the peripheral nerve, which may cause significant morbidity, such as movement disorders, sensory disturbances, and neuropathic pain, often results from traffic accidents, diseases, or surgical procedures (Fricker and Bennett 2011; Hokfelt et al. 1994; Robinson 2000a). In the adult mammalian, while severed axons fail to regenerate and their cell bodies die or undergo atrophy in the central nervous system, severed peripheral nerve system neurons survive and regenerate their axons (Lee and Wolfe 2000; Lundborg 2003). Following peripheral nerve injury, axons readily regenerate and the denervated Schwann cells begin to dedifferentiate, proliferate, migrate from the band of Büngner, wrap, and remyelinate axons, whose migration and terminal differentiation seem to recapitulate physiological development (Feneley et al. 1991; Mirsky and Jessen 1996; Salzer and Bunge 1980; Zorick and Lemke 1996). All these changes suggest that Schwann cells play a predominant supportive role in the repair of peripheral nervous system after injury. Thus, to learn the molecular and cellular mechanisms that underlie Schwann cell differentiation and identification of proteins involved will definitely contribute to our understanding of neuroregeneration. Also, investigating the mechanisms of axonal regeneration and improving recovery following nervous system injury have always been a momentous goal of the neuroscience and medical community.

SYF2, also known as Ntc31, is a putative homolog of human p29 in Saccharomyces cerevisiae belonging to the SYF2 family. SYF2 is likely to be part of the cell cycle and splicing complex, which is involved in both pre-mRNA splicing and cell cycle progression (Ben-Yehuda et al. 2000). p29 was found to interact with GCIP in yeast two-hybrid screening. GCIP associates with Cyclin D and p29 was functionally related with Cyclin D1 in the process of LPS-induced neuroinflammation (Chang et al. 2000; Xu et al. 2014b). Depletion of p29 using small interfering RNA duplexes has induced disordered cell cycle progression and DNA damage checkpoint responses (Chu et al. 2006). Mutation of both SYF2 and ISY1 resulted in low levels of α-tubulin, which induced cell cycle arrest via activation of the spindle checkpoint in S. cerevisiae (Dahan and Kupiec 2002a). Disruption of murine SYF2 leads to embryonic lethality along with reduction of α-tubulin and Chk1 expression. Interruption of zebrafish SYF2 brings about significantly attenuated acetylated α-tubulin and less neurons/neuritis (Chen et al. 2012b). To date, however, researches specifically addressing the significance of SYF2 following sciatic nerve injury remain barren.

Due to the potential of regulating cell cycle and α-tubulin level in Schwann cells, we conducted this study to evaluate the spatiotemporal expression of SYF2 on adult rat sciatic nerve crush (SNC) model. We observed significantly upregulated protein level of SYF2 following SNC, and the expression of SYF2 was in parallel with Oct-6. Through double immunofluorescent staining, we also found the enhanced colocalization of SYF2 and Oct-6 in Schwann cells. In the cAMP-induced Schwann cell differentiation model system, expressions of both SYF2 and P0 were increased and they were colocalized in Schwann cells. Interruption of SFY2 by small interfering RNA confirmed the involvement of SYF2 in the process of Schwann cell differentiation. Further we showed that SFY2 rolling in differentiation and migration of Schwann cells may be the consequence of its modulation on α-tubulin expression.

Materials and Methods

Animals and Surgery

Adult male Sprague–Dawley rats (weighing 250–300 g) were used in our study. We randomly divided the rats into two groups: a normal group (n = 4) and the crushed sciatic nerve group (n = 32). The rats were anesthetized with pentobarbital (50 mg/kg, i.p.). Using aseptic technique, the normal sciatic nerve was exposed 1.0 cm distal to the sciatic notch, then crushed by a small hemostat at the mid-point for 10 s, and then unclamped for 10 s, repeated three times. These processes were performed as described before (Long et al. 2013). The rats were allowed to recover from the surgery. Animals were housed under a 12 h light–dark cycle and the room temperature (RT) was kept at 22 ± 0.5 °C. The animals were anesthetized to harvest the sciatic nerves at several time points after surgery (6, 12 h, 1, 3, 5 days, 1, 2, and 4 weeks; n = 4 for each time point). One-centimeter-long sciatic nerve segments centered on the lesion site from above time points and corresponding segments from the normal group were excised and snap frozen at −80 °C until use. Additional experimental animals (n = 3 per time point) for sections were anesthetized and perfused through the ascending aorta with saline, followed by 4 % paraformaldehyde at each time point. All surgical interventions and postoperative animal care were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council 1996, USA) and were approved by the Chinese National Committee to the Use of Experimental Animals for Medical Purposes, Jiangsu Branch. All efforts were made to minimize the number of animals used and their suffering.

Western Blot Analysis

Western blot analysis was performed in accordance with the previous reports (Liu et al. 2012; Wang et al. 2009). Normal sciatic nerve or injured sciatic nerve at different time points post-operation were homogenized in tissue homogenization buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 % sodium deoxycholate, 0.2 % Triton X-100, 1 % NP-40, and 1× complete protease inhibitor cocktail [Roche Diagnostics, Basel, Switzerland]). The Schwann cell samples were lysed with RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, and 1× complete protease inhibitor cocktail) on ice for 30 min. The lysates were then centrifuged for 15 min at 13,000 g at 4 °C. Next, the supernatant was applied to determine protein concentration with the Bradford assay (BioRad, Hercules, CA). The protein samples were supplemented with SDS sample buffer, boiled for 10 min, and separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). The separated proteins were transferred to Polyvinylidene difluoride filter (PVDF) membranes (Millipore). The membranes were blocked with 5 % nonfat milk. After 2 h at RT, the membranes were washed and incubated with primary antibodies as follows: rabbit polyclonal anti-SYF2 (1:500; Santa Cruz), rabbit polyclonal anti-OCT6 (1:500; Sigma), goat polyclonal anti-myelin protein zero (P0, 1:1,000; Sigma), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1,000; Santa Cruz), and mouse monoclonal anti-α-tubulin (1:500; Santa Cruz) at 4 °C overnight. The protein bands were visualized with an Odyssey infrared Western Blot Imager (Licor, Lincoln, NE).

Immunohistochemistry

At the scheduled time points, the normal and injured rats were terminally anesthetized and perfused through the ascending aorta with physiological saline followed by 4 % paraformaldehyde. After perfusion, the normal or crushed proximal and distal stumps of the sciatic nerve were removed and fixed in the same fixative for 24 h and then replaced with 20 % sucrose for 1 day, followed by 30 % sucrose for 2–3 days. Transverse sciatic nerve sections (7 μm) were cut using a cryostat microtome and processed for immunochemical analysis. All sections were blocked using 10 % donkey serum with 0.3 % Triton X-100 and 1 % bovine serum albumin (BSA) for 2 h at RT. After blocking, the membranes were incubated overnight at 4 °C with rabbit polyclonal antibody against SYF2 (1:200; Santa Cruz) followed by incubating with a biotinylated secondary antibody. Staining was visualized using DAB. A negative control was performed with control rabbit IgG (1:100; Bioworld Technology, St. Louis Park, MN) on the injured nerve sections. Slides were examined at 10 or 40× magnifications on a Leica light microscope (Germany). Cells were counted from each group separately at higher magnified images. Cells with strong or moderate brown staining were counted as positive; cells with no staining were counted as negative; cells with weak staining were scored separately. The numbers of SYF2-positive cells were counted in a 500 × 500 μm measuring frame from 24 slices of four animals.

Double Immunofluorescent Staining

After being air-dried for 1 h at RT, the sciatic nerve sections were washed three times in 0.1 M PBS for 10 min, then the sections were first blocked with 10 % normal serum-blocking solution species the same as the secondary antibody, containing 3 % (w/v) BSA, 0.1 % TritonX-100, and 0.05 % Tween-20 for 2 h at RT in order to avoid unspecific staining. Then, the sections were incubated overnight with primary antibody specific for SYF2 (rabbit polyclonal, 1:100; Santa Cruz) and different markers such as mouse monoclonal antibodies anti-S100 (Schwann cells marker, 1:100; Sigma), mouse monoclonal antibodies against Neurofilament NF200 (neuronal marker, 1:100; Sigma), mouse monoclonal antibodies anti-CD11b (macrophage marker, 1:100; BD Pharmingen) and rabbit polyclonal antibodies anti-OCT6 (1:100; Sigma), and also goat polyclonal anti-myelin protein zero (P0, 1:100; Sigma) at 4 °C. On the next day, a mixture of FITC- and TRITC-conjugated secondary antibodies was added in a dark room and incubated for 2–3 h at 4 °C. The stained sections were examined with Leica confocal microscope or Leica fluorescence microscope. A negative control for SYF2 was performed with control rabbit IgG (1:100; Bioworld Technology, St. Louis Park, MN) on the normal nerve sections.

After treatment with cAMP for proper time, Schwann cells were fixed with cold PBS containing 4 % paraformaldehyde at 4 °C for 20 min, permeabilized with 0.1 % Triton X-100 for 10 min, and then blocked by 1 % BSA for 2 h. After washing in PBS, the cells were incubated with rabbit polyclonal anti-SYF2 antibody (1:100; Santa Cruz) and followed by incubation with TRITC-conjugated anti-rabbit IgG and DAPI. Finally, the cells were washed with PBS and reversed on glass slides with glycerol and PBS (1:1), and then examined using a Zeiss Confocal Laser Scanning Microscope (Li et al. 2011).

Isolation, Purification and Culture of Schwann Cells

The procedures were in accordance with that described before (Zhu et al. 2014). Both sciatic nerves were removed from 3-day-old Sprague–Dawley rat pups, shredded using fine forceps, and dissociated by incubating at 37 °C for 30 min, with occasional mixing, in 10 mL of phosphate-buffered saline (Sigma) containing 0.1 % collagenase A and 0.25 % trypsin (Sigma). The dissociated cells were washed twice by resuspending in 10 mL of DMEM-HG containing 10 % FCS followed by centrifugation (5 min at 190 × g). Finally, the cells were resuspended in 5 mL of fresh Schwann growth medium without the addition of heregulin. The cells were plated onto a poly-l-lysine-coated, 6-cm culture dish and allowed to adhere for 1 day at 37 °C. Non-Schwann cells were then eliminated as follows: For purification, the cells were treated with cytosine arabinoside (10 μM; Sigma) twice for 24 h and subjected to immunopanning with an antibody against THY1.1 (Sigma). We obtained a Schwann cell culture of >95 % purity by these procedures. For differentiation experiments, the purified cells were cultured in DMEM supplemented with 10 % FBS for 3 days then in serum-free, defined media, which consisted of a 1:1 mixture of DMEM supplemented with 10 % FBS and Ham’s F-12 with N2 supplement (Invitrogen), for another 2 days before treatment with cyclic adenosine monophosphate (cAMP; 10 μM, Sigma).

SYF2 siRNA Vector Construction and Transfection

Double-stranded oligonucleotides corresponding to the target sequence for the human SYF2 (Genbank Accession No. NM_015484.4) gene were cloned into the pSilencer 4.1-CMV siRNA plasmid (Invitrogen). According to the manufacturer’s instructions, Schwann cells were transfected with the SYF2 siRNA plasmids using Lipofectamine 2,000 (Invitrogen). 4 μg pSilencer 4.1-CMVSYF2 siRNA was used together with 1 μg control and non-specific siRNA (Invitrogen) to transfect Schwann cells with 10 μl Lipofectamine 2,000. 24 h after transfection, 10 μM cAMP was used for stimulating the cells for 3 days, then proteins were extracted for interference efficiency analysis. Then the cells were further stimulated for differentiation for 1–5 days with 10 μM cAMP.

In Vitro Migration Assays

The process of in vitro migration assays was performed as described before (Yao et al. 2014). Transwell filters (Millipore) and a 24-well Transwell plate (8 μm pore size, Corning) were used to measure the migratory ability of each transfectants. 5 × 104 cells were plated in serum-free medium in the upper chamber, which was lined with a noncoated membrane, while medium containing 10 % FBS in the lower chamber. After incubation for 24 h, cells in the chambers were fixed with 4 % paraformaldehyde following stained with toluidine blue. Cells on the membrane in the upper chamber were wiped away with a swab and cells on the other side were counted under an inverted microscope. The mean values of triplicate assays for each experimental condition were used. A series of transwell migration assays were performed to find the most flourishing migration of Schwann cells which we can detect before ascertaining the incubation time of 24 h.

Quantitative Analysis

Cell quantification was performed in an unbiased manner in accordance with the principles described before (Konigsmark and Murphy 1970). Cells were counted every fifth section (50 μm apart) for fear of counting the same cell in more than one section. The number of SYF2-positive cells in the sciatic nerve at the injury site was counted at 400× magnification. Three separate sciatic nerve regions were examined for each section. The total number of SYF2-positive cells per square millimeter was determined by the cell counts in the three or four sections. The number of cells double-labeled for SYF2 and the other phenotypic markers such as S100 and NF200 was quantified. To identify the proportion of NF200-positive cells expressing SYF2, a minimum of 200 NF200 positive cells were counted in each section. Then, double-labeled cells for SYF2 and S100 were recorded. Two or three adjacent sections per animal were sampled.

Statistical Analysis

All data were analyzed with Stata 7.0 statistical software. All values were expressed as mean ± SEM. The statistical significance of differences between groups was determined by the one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc multiple comparison tests in Figs. 1, 5, 6, 8. Data were compared using the Student’s t test in Figs. 2, 3, 7. P values less than 0.05 were considered statistically significant. Each experiment consisted of at least three replicates per condition.

Fig. 1.

Fig. 1

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Analysis of SYF2 expression after SNC by Western blot. a Total protein level of SYF2 in uninjured adult rat sciatic nerve (N) and 6 and 12 h, 1, 3, and 5 days, and 1, 2, and 4 weeks after a crush lesion. b Bar chart represents the SYF2: GAPDH intensity ratio. Results are the mean ± SEM of three independent sets of analyses (the asterisk indicates significant difference at P < 0.01, compared with the normal group)

Fig. 5.

Fig. 5

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Association of SFY2 with promyelinating of Schwann cells after SNC. a Western blot analysis of Oct-6 in sciatic nerve following SNC showed a significant upregulation from 3 days to 1 week, which was in parallel with SYF2. b Quantification graphs for Oct-6. Oct-6 had an upregulation, peaked at 1 week and then decreased to the normal level (P < 0.01). Error bars represent SEM of three independent experiments. Asterisks indicate significant differences compared with the normal group. c–h Double immunofluorescence staining for SYF2 (green), and Oct-6 (red) in normal (c–e) and crushed sciatic nerves (f–h) showed that many SYF2-positive Schwann cells especially after SNC expressed Oct-6. Scale bar 50 μm

Fig. 6.

Fig. 6

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SYF2 expression is upregulated in cAMP-induced Schwann cell differentiation. a Phase contrast microscopy images showed that Schwann cells became polygonal, longer, fine, and tapering after treatment with 10 μM cAMP for 3 days. Scale bars 50 μm. b Western blot analysis showed the upregulation of SYF2 and P0 expression in Schwann cells after cAMP treatment. c The quantitative graphs demonstrated the ratio of SYF2 and P0 protein relative to GAPDH for each time point as measured using densitometry. The data are the mean ± SEM (N = 3; the *, # indicate significant differences compared with the normal group, P < 0.01). d–g Immunofluorescent staining of SYF2 (green), P0 (red), DAPI (blue), and the merged image indicated the colocation of SYF2 and P0 in Schwann cells. Scale bars 50 μm

Fig. 8.

Fig. 8

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SYF2 affects α-tubulin and promotes Schwann cell migration. a Interruption of SYF2 expression suppressed cAMP-induced Schwann cell morphological changes. Arrows showed that the nonspecific siRNA-transfected Schwann cells had a significant microtubule structure which forms cellule surface tension and prusion shape was noticeable. Arrowheads showed that SYF2-specific siRNA-transfected Schwann cells had a shorter and disorganized microtubule structure. S100 stained green while α-tubulin stained red. b Transwell assay of Schwann cells transfected with SYF2-specific siRNA and non-specific and also the normal control. Schwann cells stained pink. SYF2-specific siRNA transfected Schwann cells showed significantly attenuated migration efficiency compared to the non-specific siRNA transfectants. Scale bars 50 μm. The data are the mean ± SEM (N = 3; the asterisk indicates a significant difference compared with the normal group, P < 0.01)

Fig. 2.

Fig. 2

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Immunohistochemical staining of SYF2 expression in the adult rat sciatic nerve after SNC. Transverse cryosections of normal and crushed sciatic nerve, 0.5 mm distal to the epicenter of injury, were immunostained with anti-SYF2 antibody. a, b Normal sciatic nerve showed weak immunoreactivity for SYF2 antibody and the brown staining was visible in Schwann cell crescents (b). c, d At day 5 following SNC, significantly stronger brown staining was observed in Schwann tubules (d). e Negative control of injured nerves in which the primary antibody to SYF2 was displaced by control rabbit IgG. f Quantitative analysis of SYF2-positive cells per square millimeter between the normal and crushed groups. The number of SYF2 expressing cells was markedly increased at 5 days after injury. Asterisk indicated significant difference at P < 0.01 compared with the normal group. Error bars represent SEM. Scale bars 100 (a, c, e) and 40 μm (b, d)

Fig. 3.

Fig. 3

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Double immunofluorescence analysis of localization of SYF2 5 days after SNC. Double-label IF was performed on normal (a–c, g–i) and crushed (d–f, j–l) sections using antibodies specific for SYF2 (green, a, d, g, j), and different cell markers, such as S100 (a–f) and NF200 (g–l). SYF2 (a, d, g, h) colocalized with S100-IR Schwann cells (b, e), but barely overlapped with NF-200-labeled axons (h, k), which was obviously showed in the merge of double exposures for each (c, f, i, l). Negative controls (m, n). o Quantitative analysis of S100-positive cells expressing SYF2 (in percent) in normal and injured nerves. *P < 0.05, significant difference compared with the normal group. Error bars represent the SEM. Scale bar 50 μm

Fig. 7.

Fig. 7

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Effects of SYF2-specific siRNA on cAMP-induced Schwann cell differentiation. a SYF2-specific siRNA was transfected into Schwann cells with the mentioned plasmids for 48 h; then, the knock-down efficiency was estimated using immunoblotting for SYF2. b Suppression of SYF2 inhibited cAMP-induced Schwann cell morphological changes. Compared with the control, siRNA transfected Schwann cells had little morphology changes following 3 days of the treatment of cAMP. c Average protrusion lengths were quantified, and the data are the mean values ± SEM. The asterisk indicates a significant difference compared with the control group at P < 0.05. Scale bar 20 μm. d Compared with the control, the protein level of P0 remained low after treatment with cAMP in SYF2-specific siRNA Schwann cells

Results

The Protein Expression Changes of SYF2 After SNC

We used Western blot to investigate the temporal pattern of SYF2 expression in sciatic nerves after crush. SYF2 protein level which was relatively lower in the normal sciatic nerves, markedly increased from 3 d after crush, peaked at day 5 and then gradually decreased to the normal level (Fig. 1). These data suggested substantial alteration of SYF2 after rat sciatic nerve injury.

The Changes of SYF2 Immunohistochemistry Staining in Rat Sciatic Nerves After Crush

To assess the changes in SYF2 staining and distribution after SNC, we carried out immunohistochemistry on transverse cryosections of the sciatic nerves. The SYF2 staining was relatively low in the normal sciatic nerve (Fig. 2a, b). Five days after crush, SYF2 staining increased obviously (Fig. 2c, d). Under higher magnification, we can see the typical brown crescent immunoreactivity concentration of Schwann cells (Fig. 2b, d). And we also noticed both the nuclear and the cytoplasm distribution of SYF2 in Schwann cells. Quantitative analysis of SYF2-positive cells per square millimeter between the normal and crushed groups was in parallel with Western blot results for SYF2 expression following SNC (Fig. 2f). No staining was observed in the negative control of sciatic nerve (Fig. 2e).

Cellular Colocalization of SYF2 with Different Phenotype Specific Markers by Double Immunofluorescence Staining

Now that the results of immunohistochemistry staining indicated SYF2’s location in Schwann cells, we performed double immunofluorescent staining to further trace SYF2 inside Schwann cells. Immunostain signals of SYF2 (green), S100 (Schwann cells marker, red), and NF-200 (neuronal marker, red) were examined in transverse cryosections from normal and the injured nerve. We observed widely expressed SYF2 in Schwann cells (Fig. 3d–f) and with a relatively low level in normal nerve (Fig. 3a–c). While the pattern of SYF2 and NF-200 immunofluorescent staining showed that there was no colocalization in the axons of normal nerve (Fig. 3g–i) and showed just a small amount of colocalization in the axons of crushed nerve (Fig. 3j–l). To identify the proportion of S100-positive cells expressing SYF2, a minimum of 200 phenotype-specific marker positive cells were counted between normal and 5 days following crush. SYF2 expression increased obviously in Schwann cells at day 5 after SNC (Fig. 3o). The results here were in accordance with the results of Western blot and immunohistochemistry and all these data indicated that SYF2 might be associated with differentiation of Schwann cells during the period of Wallerian degeneration after sciatic nerves crush.

Of great intrigue, we could distinguish round SYF2-positive cell-like composition among numerous Schwann cells (Fig. 3f, l, arrows). Based on the fact that the round SYF2-positive signal has a typical morphological character of activated phagocytic cells and macrophages play a great important role during peripheral nerve regeneration after injury (Brück 1997; Kiefer et al. 2001), we attributed these signals to immunofluorescent staining of macrophages. To confirm this hypothesis, we performed immunofluorescent staining to detect the CD11b-positive macrophages (Lou et al. 2012). In addition to the Schwann cellular localizations, SYF2 had also colocalizated with round DAPI-labeled nucleoli which do not belong to fusiform nucleoli of Schwann cells (Fig. 4e–h, arrows). At day 5 following injury, the round SYF2 labeling induced by SNC had a great colocation with CD11b on longitudinal sciatic nerve sections (Fig. 4i–l, arrows). Enlarged images of cells were shown within a white rectangle frame (Fig. 4d, h, l). These observations suggested that SYF2 was expressed in macrophages as well as in the Schwann cells. We also noted that many SYF2-positive macrophages had vacuole-like immunofluorescent-staining defect (Fig. 4i–l, arrows). All these suggested that SYF2-positive macrophages may be activated in response to sciatic nerve crush.

Fig. 4.

Fig. 4

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Colocalization of SYF2 with CD11b in the injured sciatic nerve. Longitudinal cryosections of normal sciatic nerve (a–d) and crushed nerve (e–l), were immunostained with SYF2 (green), S100 (red), CD11b (red), and DAPI (blue). Besides normal cellular localizations in Schwann cells, SYF2 had colocalization with round DAPI-labeled nucleoli in crushed nerve (e–h, arrows). These round Hoechst-labeled nucleoli were found in CD11b-positive cells, which colocalized with SYF2 (i–l, arrows). Enlarged images of cells were showed by white rectangle frame (d, h, l). Scale bar 60 μm

Expression of Oct-6 After Sciatic Nerves Crush

Experiments explained above suggested the possibility of SYF2’s association with Schwann cell differentiation. To analyze the relationship between SYF2 and Schwann cells, using Western blots, we assessed the expression of Oct-6, a promyelinating Schwann cell marker (Arroyo et al. 1998). In adult sciatic nerve, Oct-6 expression significantly increased post-injury and reached a peak at 1 week. And the expression change of Oct-6 nearly simulated SYF2 protein expression (Fig. 5a, b). Further we performed the double labeling immunofluorescent staining of SYF2 together with Oct-6 (Fig. 5c–h). We found that SYF2 coexisted with the Oct-6-expressed Schwann cells in the cyctoplasm and the colocalization was enhanced at the top expression of SYF2 at about 1 week following SNC. All these suggested that changes in SYF2 expression following SNC were associated with Schwann cell differentiation.

Upregulated SYF2 Expression in cAMP-Induced Schwann Cell Differentiation Model

To further evaluate SYF2 regarding Schwann cell differentiation, we conducted the cAMP-induced Schwann cell differentiation model system. Schwann cells became polygonal, longer, fine, and tapering following treatment with 10 μM cAMP for 3 days (Fig. 6a). We confirmed the differentiated Schwann cells by detecting the enhanced protein expression of P0 (Myelin protein zero, the myelin gene) by western blotting (Fig. 6b). Meanwhile the protein level of SYF2 was markedly elevated during the process of differentiation. Standardized densitometry against GAPDH could be clearly observed (P < 0.01) (Fig. 6c). Immunofluorescent staining showed the colocation between SYF2 and P0 in Schwann cells (Fig. 6d–g), which suggested the involvement of SYF2 in the process of Schwann cell remyelination. Results up to now strongly indicate that SYF2 might play a positive role in Schwann cell differentiation.

Effects of SYF2-Specific siRNA on cAMP-Induced Schwann Cell Differentiation

To further evaluate the function of SYF2 in the process of Schwann cell differentiation, we constructed SYF2-specific siRNA to interrupt SYF2 expression in Schwann cells. Western blot analysis showed >50 % reduction of the SYF2 protein level in SYF2-specific siRNA-transfected Schwann cells than in the non-specific siRNA transfected Schwann cells (Fig. 7a). We found that transfection with SYF2-specific siRNA disturbed cAMP-induced Schwann cell morphological changes 3 days after cAMP treatment, but transfection with non-specific siRNA did not (Fig. 7b). Using Western blot analysis, we assessed the expressions of P0 in non-specific siRNA and SYF2-specific siRNA-transfected Schwann cells with or without cAMP treatment. We found that P0 was upregulated after cAMP stimulation in non-specific siRNA-transfected Schwann cells. Whereas in SYF2-specific siRNA-transfected Schwann cells, the protein level of P0 remained low (Fig. 7d). Together, these results regarded SYF2 as an important regulator of Schwann cell differentiation.

SYF2 Affects α-Tubulin and Promotes Schwann Cell Migration

Previous study showed the reduced mRNA and protein level of α-tubulin on account of the disruption of SYF2 in rodents (Chen et al. 2012b). Combining with our observation above, we hypothesized that the involvement of SYF2 in the process of Schwann cell differentiation may be the consequence of its affection on α-tubulin. So we performed double immunofluorescent staining to assess the effect of SYF2-specific siRNA on α-tubulin together with the differentiation of Schwann cells. Similar to the above, SYF2-specific siRNA-transfected Schwann cells showed hysteretic morphological change compared to that of non-specific siRNA (Fig. 8a). At day 3 in the process of Schwann cell differentiation we detected filamentous microtubule structure by the red staining of α-tubulin in both groups (Fig. 8a, arrows and arrowheads). Yet SYF2-specific siRNA-transfected Schwann cells exerted arresting shorter and disorganized microtubule structure (Fig. 8a, arrowheads) compared to the non-specific control (Fig. 8a, arrows). Next we also observed decreased migration of SYF2-specific siRNA-transfected Schwann cells through transwell assay (Fig. 8b). All these manifest that SYF2 may affect α-tubulin and participate in the process of Schwann cell differentiation and migration.

Discussion

In the present study, for the first time, we provide some evidence that the protein expression of SYF2 changes after the crush of sciatic nerve. Western blot analysis showed that SYF2 had a significant upregulation from 3 days, peaked at day 5 and then decreased gradually to the normal level at 4 weeks, similarly to the promyelinating Schwann cells marker Oct-6. Immunohistochemistry showed apparently stronger staining of SYF2 in crushed nerves than in normal nerves. Immunofluorescent staining revealed that SYF2 was widely expressed in Schwann cells and SYF2 co-labeled with Oct-6 and P0. As indicated by the results of cell culture experiments, the protein level of SYF2 rose during the process of cAMP-induced Schwann cell differentiation. Depletion of SYF2 inhibited cAMP-induced Schwann cell morphological changes and the expression of P0. Also, Schwann cells transfected with SYF2-specific siRNA showed shorter and disorganized microtubule structure and a decreased migration as compared with the non-specific control. All these results suggested that SYF2 might be associated with differentiation and migration of Schwann cells after SNC.

The success of peripheral nerve repair relies heavily on the ability of Schwann cells to proliferate and to provide trophic support for regenerating axons. After injury to the rat sciatic nerve, axons distal to the injury site conduct Wallerian degeneration which is characterized by proteolytic degradation of axonal proteins like neurofilaments (Karlsson et al. 1993). During this axonal degeneration, Schwann cell myelin sheath in the distal nerve stump breaks down and the debris is cleared by Schwann cells and macrophages. Up to 48 h following injury, Schwann cells cease producing myelin proteins (Trapp et al. 1988; White et al. 1989) and upregulate regeneration-associated genes (GAP-43, neuregulin and its receptors, and neurotrophic factors and their receptors) (Liu et al. 1995; Murinson et al. 2005; Pellegrino et al. 1986). Then, Schwann cells proliferate and redifferentiate about 5 days following injury, providing a permissive environment for nerve regeneration (Cheng et al. 2007; Fawcett and Keynes 1990; Ji et al. 2010; Yang et al. 2008). As to cell differentiation, Schwann cells stem from neural crest cells, and experience extensive migration, proliferation, and maturation before they terminally differentiate (Jessen and Mirsky 2005). Evidence points to the fact that the recapitulation of remyelination of Schwann cells and development in terms of the timing and sequence of gene-expression pattern (Scherer et al. 1994; Zorick et al. 1996). Thus, we speculate the occurrence and significance of Schwann cell migration and eventually differentiation following injury that is in accordance with the Schwann cells natural development.

During the process of peripheric nerve repair following injury, cross talk between axons and Schwann cells is indispensable. While Schwann cells act as a supporter for axons regeneration, axons affect Schwann cells phenotypes by interaction with them (Aguayo et al. 1976; Spencer et al. 1981; Weinberg and Spencer 1976). As epithelial cells, Schwann cells display various phenotypes caused by different modes of axons' contact and also other factors (Kidd et al. 1996). The myelinating phenotype of Schwann cells is launched by physical contact with appropriate axons (Aguayo et al. 1976; Spencer et al. 1981; Weinberg and Spencer 1976). Myelination is a complex process in which glial cells grow processes that enclose axons and generate compact myelin (Morell et al. 1999). Functional nerve regeneration requires not only axonal elongation and sprouting, but also new myelin synthesis. Remyelination is indispensable for the replacement of normal nerve conduction and for the protection of axons against new neurodegenerative immunologic attacks (Horner and Gage 2000). Schwann cells morphological changes and migration are obviously two essential aspects for successful remyelination, during the process of which the organization of the cytoskeleton is indispensable. The cell cytoskeleton is mainly composed of actin microfilaments, microtubules, and intermediate filaments (Tang et al. 2013). It is believed that these three cytoskeletons are definitely precisely coordinated and tightly regulated in order to maintain cellular homeostasis at all levels, including cell movement, cell proliferation, endocytic vesicle-mediated protein trafficking, and others (Tang et al. 2013). Of these three, microtubules are particularly essential for cellular processes such as mitosis, cell motility, neuronal differentiation (Etienne-Manneville 2010; Meunier and Vernos 2012; Su et al. 2012; Drewes et al. 1995) as key components of the cytoskeleton of eukaryotic cells (Wade and Hyman 1997). Microtubules constitute alpha and beta tubulin heterodimers that bind head-to-tail to make up protofilaments, 13 of which form a hollow tubule. This architecture endows microtubules an intrinsic polarity with assembly and disassembly occurring exclusively at their ends. The quantity of either alpha or beta tubulin protein contribute inevitably to the dynamics of microtubules. Disruption of SYF2 leads to decreased α-tubulin expression in mice, and interruption of zebrafish SYF2 results in significantly attenuated acetylated α-tubulin and damaged nervous development (Chen et al. 2012a). Here in our study, we found the upregulated expression of SYF2 (Fig. 1) in Schwann cells after sciatic nerve crush. The expression pattern of SYF2 was similar to the promyelinating Schwann cell marker Oct-6 (Fig. 5). Also, in the cAMP-induced Schwann cell differentiation model system, we found the elevated level of the colocalized SYF2 and P0 during Schwann cell differentiation (Fig. 6). Disruption of SYF2 using the SYF2-specific siRNA obstructed cAMP-induced Schwann cell morphological change and also the expression of P0 (Fig. 7). All these suggested that SYF2 might positively regulate Schwann cell differentiation. Well-organized microtubule dynamics play a critical role in cell motility and differentiation (Etienne-Manneville 2010). As one of the two microtubule components, α-tubulin was reported to be positively correlated with SYF2 (Chen et al. 2012a; Dahan and Kupiec 2002b). We observed shorter and disorganized microtubule structure and also the impaired cell morphology changes in SYF2-specific siRNA transfected Schwann cells by immunofluorescent staining during the process of Schwann cell differentiation (Fig. 8a). Also, Schwann cell migration was interfered when SYF2 was knock-down (Fig. 8b). Thus, we speculated here that SYF2 may contribute to Schwann cell differentiation and migration following SNC via its modulation on α-tubulin. Of course, the exact mechanism underlying the SYF2 influence on Schwann cell differentiation and migration needs further investigation.

Upregulated SYF2 was shown to be involved in the process of LPS-induced central nervous system inflammation (Xu et al. 2014a). Macrophages are vital immune cells that respond to peripheral nervous injury (Gaudet et al. 2011). It has been shown that macrophages play an important role in the process of Wallerian degeneration after peripheral nerve injury (PNI) (Brück 1997; Kiefer et al. 2001). After nerve crush injury or axotomy, macrophages are recruited to the injury sites, and along with Schwann cells, contribute to axonal- and myelin-derived fragments clearance (Chen et al. 2007). During the process of regeneration following injury, the interaction between macrophages and Schwann cells may be indispensable (Martini et al. 2008). It was reported that oxidized galectin-1 binds to an unidentified receptor on macrophages, leading to the secretion of a factor that promotes Schwann cell migration and axon regrowth (Horie et al. 2004). Hematogenous macrophages are essential for effective myelin phagocytosis (Brück et al. 1996; Beuche and Friede 1984) and generating cytokines that activate Schwann cells (La Fleur et al. 1996). Further, macrophage emigration was needed to complete the phagocytosis of degenerating myelin, during the process of which macrophages must penetrate Schwann cell basal lamina tubes (Martini et al. 2008). In the present study, we found that SYF2 is expressed in also the CD11b-positive macrophages 5 days after SNC. Further, round SYF2-positive macrophages have a typical morphological character of activated phagocytic cells, and we speculated that SYF2-positive macrophages might be activated. Similar to that in the Schwann cells, SYF2 may contribute to the migration of macrophages during the process of Wallerian degeneration following PNI described above. However, mountains of work remain to do before verifying the precise mechanism of SYF2’s contribution to the inflammation induced by PNI.

Traumatic lesion to the peripheral nerve is a worldwide problem (Robinson 2000b). The injuries of peripheral nerve give rise to major social and economic burdens for their general occurrence in the most productive age group (Eser et al. 2009). So it is quite important for us to investigate the mechanism of nerve regeneration and repair following injury. Here our study demonstrated for the first time that upregulation of SYF2 was involved in the differentiation and migration of Schwann cells in the process of peripheral nervous repair following crush injury, which might be mediated via its modulation on α-tubulin. However, the precise mechanism and other roles of SYF2 playing in peripheral nerve regeneration remain to be further researched. A better understanding of its contribution in future investigations may extend our knowledge in improving peripheral nerve regeneration.

Acknowledgments

This work was supported by the National Natural Scientific Foundation of China Grant (No. 31300902), the Colleges and Universities in Natural Science Research Project of Jiangsu Province (13KJB310009), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Conflict of interest

All the authors of this study declare no conflict of interest.

Abbreviations

SNC

Sciatic nerve crush

PNI

Peripheral nerve injury

cAMP

Cyclic adenosine monophosphate

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

P0

Myelin protein zero

Footnotes

Zhengming Zhou, Yang Liu, Yonghua Liu and Huiguang Yang contributed equally to this work.

Contributor Information

Yonghua Liu, Email: liuyonghua_ntu@163.com.

Huiguang Yang, Email: yanghuiguang_ntu@163.com.

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