RESUMO
Schwann cells (SCs) are unique glial cells in the peripheral nerve and may secrete multiple neurotrophic factors, adhesion molecules, extracellular matrix molecules to form the microenvironment of peripheral nerve regeneration, guiding and supporting nerve proliferation and migration. Cdc42 plays an important regulatory role in dynamic changes of the cytoskeleton. However, there is a little study referred to regulation and mechanism of Cdc42 on glial cells after peripheral nerve injury. The present study investigated the role of Cdc42 in the proliferation and migration of SCs after sciatic nerve injury. Cdc42 expression was tested, showing that the mRNA and protein expression levels of Cdc42 were significantly up-regulated after sciatic nerve injury. Then, we isolated and purified SCs from injuried sciatic nerve at day 7. The purified SCs were transfected with Cdc42 siRNA and pcDNA3.1-Cdc42, and the cell proliferation, cell cycle and migration were assessed. The results implied that Cdc42 siRNA remarkably inhibited Schwann cell proliferation and migration, and resulted in S phase arrest. While pcDNA3.1-Cdc42 showed a contrary effect. Besides, we also observed that Cdc42 siRNA down-regulated the protein expression of ß-catenin, Cyclin D1, c-myc and p-p38, which were up-regulated by pcDNA3.1-Cdc42. Meanwhile, the inhibitor of Wnt/ß-catenin and p38 MAPK signaling pathway IWP-2 and SB203580 significantly inhibited the effect of pcDNA3.1-Cdc42 on cell proliferation and migration. Overall, our data indicate that Cdc42 regulates Schwann cell proliferation and migration through Wnt/ß-catenin and p38 MAPK signaling pathway after sciatic nerve injury, which provides further insights into the therapy of the sciatic nerve injury.
Assuntos
Células de Schwann/fisiologia , Neuropatia Ciática/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Proteína cdc42 de Ligação ao GTP/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Células de Schwann/efeitos dos fármacos , Neuropatia Ciática/tratamento farmacológico , Neuropatia Ciática/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética , Proteína cdc42 de Ligação ao GTP/administração & dosagem , Proteína cdc42 de Ligação ao GTP/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
A challenge in the field of neural stem cell biology is the mechanistic dissection of single stem cell behavior in tissue. Although such behavior can be tracked by sophisticated imaging techniques, current methods of genetic manipulation do not allow researchers to change the level of a defined gene product on a truly acute time scale and are limited to very few genes at a time. To overcome these limitations, we established microinjection of neuroepithelial/radial glial cells (apical progenitors) in organotypic slice culture of embryonic mouse brain. Microinjected apical progenitors showed cell cycle parameters that were indistinguishable to apical progenitors in utero, underwent self-renewing divisions and generated neurons. Microinjection of single genes, recombinant proteins or complex mixtures of RNA was found to elicit acute and defined changes in apical progenitor behavior and progeny fate. Thus, apical progenitor microinjection provides a new approach to acutely manipulating single neural stem and progenitor cells in tissue.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Neurais/fisiologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Diferenciação Celular/genética , Embrião de Mamíferos , Proteínas Imediatamente Precoces/genética , Técnicas In Vitro , Proteínas Luminescentes/administração & dosagem , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microinjeções/métodos , Mutação/genética , Células-Tronco Neurais/efeitos dos fármacos , RNA Mensageiro/farmacologia , Rombencéfalo/citologia , Rombencéfalo/embriologia , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Proteína cdc42 de Ligação ao GTP/administração & dosagem , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Spinal cord injury (SCI) often results in permanent functional loss. This physical trauma leads to secondary events, such as the deposition of inhibitory chondroitin sulfate proteoglycan (CSPG) within astroglial scar tissue at the lesion. METHODOLOGY/PRINCIPAL FINDINGS: We examined whether local delivery of constitutively active (CA) Rho GTPases, Cdc42 and Rac1 to the lesion site alleviated CSPG-mediated inhibition of regenerating axons. A dorsal over-hemisection lesion was created in the rat spinal cord and the resulting cavity was conformally filled with an in situ gelling hydrogel combined with lipid microtubes that slowly released constitutively active (CA) Cdc42, Rac1, or Brain-derived neurotrophic factor (BDNF). Treatment with BDNF, CA-Cdc42, or CA-Rac1 reduced the number of GFAP-positive astrocytes, as well as CSPG deposition, at the interface of the implanted hydrogel and host tissue. Neurofilament 160kDa positively stained axons traversed the glial scar extensively, entering the hydrogel-filled cavity in the treatments with BDNF and CA-Rho GTPases. The treated animals had a higher percentage of axons from the corticospinal tract that traversed the CSPG-rich regions located proximal to the lesion site. CONCLUSION: Local delivery of CA-Cdc42, CA-Rac1, and BDNF may have a significant therapeutic role in overcoming CSPG-mediated regenerative failure after SCI.
