Activation of protease-activated receptor 1 causes Rho kinase pathway-dependent suppression of proliferation of neural progenitor cells generated in injured hippocampal dentate gyrus

It is now clear that there is a continual turnover of the mammalian hippocampal dentate gyrus (DG) neurons throughout life even in adult. Various neurological injuries are widely recognized as promoting endogenous neurogenesis in DG. Thrombin-activated/protease-activated receptor-1 (PAR-1) is known to regulate proliferation of neural cells following brain injury including intracellular hemorrhage. Our previous studies demonstrated that the systemic treatment with trimethyltin chloride (TMT) causes the granule cell loss in the mouse DG, with being regenerated in the dentate granule cell after neuronal loss. To elucidate the roles of PAR-1 in neuroregeneration after neuronal degeneration, we evaluated the expression of PAR-1 in the newly generated cells following neurodegeneration in the DG of adult mouse. In vivo experiments, mice were given TMT to prepare hippocampal slices for immunohistochemical analysis using antibody against PAR-1 and nestin [neural stem/progenitor cells (NPCs) marker]. Cells positive for PAR-1 and nestin markedly increased in the DG on day 3 to 5 after TMT treatment. As vitro experiments, we evaluated the effects of thrombin on proliferative activity of the NPCs isolated from the DG on day 3 post-TMT treatment. NPCs prepared from the dentate gyrus were cultured in the neurobasal medium with B27 supplement, EGF, and bFGF for 14 days in vitro (DIV). After secondary replating nestin-positive cells were cultured for 5 DIV under the same conditions in the absence or presence of thrombin, Y27632, and/or fasudil for assessment of cell proliferation. Immunostaining revealed that PAR-1 was co-localized with most of nestin-positive cells. Exposure of the cells to thrombin significantly attenuated the cell proliferation by bromodeoxyuridine incorporation assay without cell damage. Increased RhoA-GTP (activated RhoA) level was determined after the exposure to thrombin. Thrombin-induced attenuation of proliferative activity was completely abolished by Rho kinase inhibitors such as Y27632 and fasudil. These results suggest that activation of PAR-1 causes Rho kinase pathway-dependent suppression of cell proliferation of the NPCs.