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First published online 2 January 2007
doi: 10.1242/jcs.03322

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Cdc2-mediated Schwann cell migration during peripheral nerve regeneration

In Sun Han^1,*, Tae Beom Seo^1,*, Kwan-Hoi Kim², Jin-Hwan Yoon³, Sung-Jin Yoon⁴ and Uk Namgung^1,

¹ Department of Oriental Medicine, Daejeon University, Daejeon 300-716, Korea,
² Department of Pharmacology, School of Medicine, Pusan National University, Busan, Korea
³ Department of Sports and Leisure Studies, Hannam University, Daejeon 300-791, Korea
⁴ Department of Physical Education, Korea University, Seoul, Korea

View larger version (41K): [in this window] [in a new window]   Fig. 1. Induction of Cdc2 protein in regenerating sciatic nerves. (A) Western analysis showed a strong but transient increase of Cdc2 protein in 2 cm nerve segments proximal and distal to the injury site. (B) Distribution of Cdc2 protein around the injury site. Vertical arrow indicates the injury site. (C) RT-PCR for Cdc2 mRNA expression in 1 cm nerve segment proximal (P) or distal (D) to the injury site. (D) FISH analysis of Cdc2 mRNA in the sciatic nerve. Sciatic nerve sections were used for FISH with antisense or sense riboprobe and immunofluorescence staining with anti-S100 antibody. (E) Immunofluorescence views show that signals for S100 and Cdc2 proteins are colocalized in the nerve sections. In D and E, transverse nerve sections (20 µm) 0.5 cm distal to the injury site were prepared at 7 d.p.c. Actin was detected in A-C as internal loading controls. Bars, 50 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Cdc2 expression in cultured Schwann cells. (A) Western blot analysis of Cdc2 in cultured Schwann cells. Schwann cells were prepared from sciatic nerves given crush injury for 0-14 days or from the sciatic nerve of the rat of postnatal day 3 (pnd3). Cells were incubated for 2 days in 10% serum and 2 µM forskolin (FSK) (+) or 0.5% serum and 0 µM forskolin (–). Cdc2 protein in Schwann cells was increased by injury preconditioning of the sciatic nerve and further upregulated by serum and forskolin treatment. Actin was detected as an internal loading control. (B) Merged views for the localization of S100- or Cdc2-positive cells in the total cell population (Hoechst 33258 stained) or Cdc2-positive cells in S100-positive cell population. Schwann cells were prepared from sciatic nerves with or without injury preconditioning for 7 days. Cells positive to S100 and/or Cdc2 were increased by injury preconditioning, and forskolin and serum treatment. Bar, 20 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Cdc2 activity is required for Schwann cell migration. (A) Immunofluorescence staining of explant culture for the detection of S100-positive Schwann cells (red) and Cdc2-positive cells (green). Enlarged image of the rectangle is shown in the inset. The merged image indicates that most migrating Schwann cells in injury-preconditioned explants express Cdc2. Quantification data of Schwann cell migration in the explant culture with or without injury preconditioning (Injury-pre. vs. Non-pre) are shown on the right. The arrow indicates the direction of migrating Schwann cells from the sciatic nerve center. (B) Comparison of Schwann cell migration in culture between injury-preconditioned and non-preconditioned groups. The data represent Schwann cell migration as percentage of total cells on the filter culture recovered from injury-preconditioned or non-preconditioned sciatic nerves. (C) Dose-dependent decrease of Schwann cell migration by roscovitine treatment. Injury-preconditioned Schwann cells were cultured under the different concentrations of roscovitine for 2 days. Statistical comparisons were made compared with the cell group treated with 0 µM roscovitine (*P<0.05; ***P<0.001). In B,C, migrated Schwann cells on the coverslip were immunostained with anti-S100 antibody, and the cells on the top of the filter were stained with Cresyl Violet. The cells from ten random fields were counted. (n=3 for A and n=4 for B,C). Bars, 50 µm (A); 20 µm (inset in A).

View larger version (34K): [in this window] [in a new window]   Fig. 4. Facilitated migration of implanted Schwann cells infected with Ad-Cdc2 viral vectors in the injured sciatic nerve. (A) Representative images of Schwann cells infected with Ad-wt-Cdc2 and Ad-GFP in the sciatic nerve sections. Seven days after injury, infected Schwann cells (marked in circles) were identified by co-infected GFP reporter above the background staining with NF-200 or with Hoechst 33258 nuclear staining. (B) Distribution of implanted Schwann cells along the nerve. In 3 d.p.c. and 7 d.p.c. groups, Schwann cells infected with wt-Cdc2 virus were observed in the more distal portion of the nerve compared with those infected with dn-Cdc2 virus. Arrow indicates site of injury. (C) Quantitative comparison of infected cells in the distal portion of the sciatic nerves. In the sciatic nerves prepared at 3 d.p.c. or 7 d.p.c., statistical comparisons were made between the groups infected with wt-Cdc2 and dn-Cdc2 viruses (asterisks marked to the left of the symbols indicating wt-Cdc2 viral infection; *P<0.05, **P<0.01, *P<0.001; n=4, mean ± s.e.m.). Bars, 100 µm (A); 300 µm (B).

View larger version (40K): [in this window] [in a new window]   Fig. 5. Cdc2 phosphorylation of caldesmon in injury-preconditioned Schwann cells. (A) Western blot analysis of phospho-caldesmon in cultured Schwann cells. Phospho-caldesmon protein was greatly increased in cultured Schwann cells with injury preconditioning for 7 days. As a control, Schwann cell lysate was prepared from the sciatic nerve in rats at postnatal day 3 (pnd3). (B) Identification of phospho-caldesmon protein in Schwann cells by western blot analysis. Caldesmon pulled down by immunoprecipitation of actin was used as substrate for the kinase reaction by exogenous Cdc2 enzyme (lane 1). Kinase reaction in the presence of roscovitine is shown in lane 2 and the reaction without Cdc2 in lane 3. (C) In vitro kinase assay for caldesmon phosphorylation by exogenous Cdc2. Caldesmon pulled down by immunoprecipitation of actin was used as a substrate for the kinase reaction. Inclusion or exclusion of roscovitine (rosco) or histone H1 in the incubation mixture are indicated respectively by + or –, respectively. Caldesmon phosphorylation by Cdc2 was decreased by the cdk inhibitor, roscovitine or by competition with the alternative substrate, histone H1. (D) Inhibition of caldesmon phosphorylation by dn-Cdc2 expression. Schwann cells were cotransfected with pGFP and pCMV5 vector or pGFP and pCMVdn-Cdc2. A cell group transfected with dn-Cdc2 was less positive for phospho-caldesmon immunostaining than the vector control. The graph shows a significant reduction of phospho-caldesmon-positive (+) cells among the GFP-positive (+) transfected cell population by dn-Cdc2-transfection. (n=3, mean ± s.e.m.). Western blot analysis shows significant suppression of phospho-caldesmon immunopositivity in Schwann cells that had been injury preconditioned and transfected with dn-Cdc2. (E) Subcellular localization of caldesmon in injury-preconditioned Schwann cells. The perinuclear distribution of caldesmon in vehicle-treated cultures contrasts with the peripheral distribution in cultures treated with 10 µM roscovitine (arrow). Bars, 30 µm (D); 20 µm (E). Actin was detected as an internal loading control.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Schwann cell migration is facilitated by activation of the Cdc2-caldesmon pathway. Cultures of Schwann cells from nerves with (A) or without (B) injury preconditioning were transfected with plasmids expressing dn-Cdc2, wt-Cdc2, caldesmon 7th mutant (Cald 7th), and pGFP. All GFP-positive cells that migrated to coverslips were counted. Horizontal lines in A and B indicate the mean number of cells (**P<0.01, ***P<0.001; n=5).

View larger version (34K): [in this window] [in a new window]   Fig. 7. Effect of Cdc2 activity on neurite outgrowth of DRG sensory neurons. (A) Representative images of neurite outgrowth of DRG sensory neurons under different culture conditions. Dissociated cells were prepared from DRGs (lumbar 4-5) and sciatic nerves of animals that had or had not been subjected to preconditioning injury in the sciatic nerves (7 d.p.c.). Pre, injury preconditioned; Non-pre, non-preconditioned; SN, sciatic nerve. The primary sensory neurons and Schwann cells so derived were co-cultured. Cells were visualized by NF-200 immunostaining. (B) Quantification of neurite outgrowth of DRG sensory neurons. The number of neurite branch points in DRG sensory neurons co-cultured with Schwann cells of the sciatic nerve was significantly decreased by 10 µM roscovitine treatment. (**P<0.01; n=4). Bar, 50 µm.

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