Cdc2-mediated Schwann cell migration during peripheral nerve regeneration

Abstract

Schwann cell migration facilitates peripheral nerve regeneration after injury. We have recently found increased activation of Cdc2 kinase in regenerating sciatic nerves. Here we show that Cdc2 phosphorylation of caldesmon regulates Schwann cell migration and nerve regeneration. A robust but transient increase in Cdc2 expression was found in cultured Schwann cells prepared from the sciatic nerve in rats that had undergone crush injury for 7 days. These `injury-preconditioned' Schwann cells exhibited enhanced migration compared with non-preconditioned control cells and treatment with the cdk inhibitor roscovitine prevented cell migration. After transduction with recombinant Cdc2 DNA adenoviral vectors, Schwann cells were implanted into sciatic nerves; those expressing wild-type Cdc2 migrated further in the distal direction than those expressing dominant-negative Cdc2. We identified caldesmon as a downstream substrate of Cdc2 in Schwann cells and its phosphorylation by Cdc2 changed its subcellular localization. Overexpression of dominant-negative caldesmon significantly counteracted the migration effect caused by Cdc2. Finally, neurite outgrowth of cultured DRG sensory neurons, facilitated by co-culture with injury-preconditioned Schwann cells, was suppressed by roscovitine treatment. The results indicate that activation of the Cdc2-caldesmon pathway is necessary for Schwann cell migration and suggest a role for this pathway in peripheral axonal growth.

Shengmai-san–Mediated Enhancement of Regenerative Responses of Spinal Cord Axons After Injury in Rats

“…There is evidence demonstrating that Cdc2 is expressed in astrocytes after SCI (unpublished data). As has been implicated by Cdc2-mediated migration of Schwann cells and cancer cells (13,45), locally upregulated Cdc2 activity by SMS in non-neuronal cells (e.g., astrocytes) could promote their migration into the injury cavity as observed by the increased number of Hoechststained nuclei by SMS treatment. Cells that migrated into the injury cavity could be involved in trabecula formation and contribute to axonal regeneration.…”

“…We have recently found that Erk1/2 and Cdc2 pathways are activated during axonal regeneration in peripheral sciatic nerves (9,13). Here, regulation of phospho-Erk1/ 2 and Cdc2 proteins by SMS treatment was investigated in the dorsal spinal cord and in the motor cortex where the cell bodies of CST axons are located.…”

“…For DRG sensory neuron culture, DRG at lumbar 4 -5 were prepared from rats that had undergone sciatic nerve injury for 3 days. The procedures of primary DRG neurons and spinal cord non-neuronal cells are essentially the same as described previously (13) Digital images of neuronal process were captured and transferred to the Adobe Photoshop program (version 5.5; Adobe). The number and length of neurite processes exhibiting clear outgrowth (longer than cell body size) from the cell body were analyzed by the iSolution software program (Image and Microscope Technology, Goleta, CA, USA).…”

“…The sections (20 μm) were cut using a cryostat and mounted on positively charged slides (Fisher, Pittsburgh, PA, USA). Pretreatment, primary and secondary antibody reactions, and microscopic analysis of sections were performed as described previously (13). The primary antibodies used in the present study were anti-GFAP antibody (rabbit polyclonal, Chemicon), anti-CSPG antibody (mouse monoclonal, CS56; Sigma), anti-NF-200 antibody (mouse monoclonal, 1:200; Sigma), and anti-CD11β antibody (mouse monoclonal; BD Bioscience, San Jose, CA, USA); and the secondary antibody was fluorescein-goat anti-mouse (Molecular Probes, Eugene, OR, USA) or rhodamine-goat antirabbit secondary antibodies (Molecular Probes).…”

“…Phosphorylated Erk1/2 protein is increased in regenerating peripheral neurons and also associated with regenerative responses after SCI (9 -12). Finally, we have recently demonstrated that cell division cycle 2 (Cdc2), a prototypical cyclin-dependent kinase (Cdk) family protein, is involved in peripheral nerve regeneration via promoting Schwann cell migration (13); yet, its function in the injured spinal cord is not known. Thus, molecules favoring axonal regeneration in the PNS can be considered for therapeutic strategies to induce axonal growth and functional recovery after SCI.…”