THROMBIN PERTURBS NEURITE OUTGROWTH AND INDUCES APOPTOTIC CELL-DEATH IN ENRICHED CHICK SPINAL MOTONEURON CULTURES THROUGH CASPASE ACTIVATION

Increasing evidence indicates several roles for thrombin-like serine p roteases and their cognate inhibitors (serpins) in normal development and/or pathology of the nervous system. In addition to its prominent r ole in thrombosis and/or hemostasis, thrombin inhibits neurite outgrow th in neuroblastoma and primary neuronal cells in vitro, prevents stel lation of glial cells, and induces cell death in glial and neuronal ce ll cultures. Thrombin is known to act via a cell surface protease-acti vated receptor (PAR-I), and recent evidence suggests that rodent neuro ns express PAR-I. Previously, we have shown that the thrombin inhibito r, protease nexin-1, significantly prevents neuronal cell death both i n vitro and in vivo. Here we have examined the effects of human alpha- thrombin and the presence and/or activation of PAR-1 on the survival a nd differentiation of highly enriched cultures of embryonic chick spin al motoneurons. We show that thrombin significantly decreased the mean neurite length, prevented neurite branching, and induced motoneuron d eath by an apoptosis-like mechanism in a dose-dependent manner. These effects were prevented by cotreatment with hirudin, a specific thrombi n inhibitor. Treatment of the cultures with a synthetic thrombin recep tor-activating peptide (SFLLRNP) mimicked the deleterious effects of t hrombin on motoneurons. Furthermore, cotreatment of the cultures with inhibitors of caspase activities completely prevented the death of mot oneurons induced by either thrombin or SFLLRNP These findings indicate that (1) embryonic avian spinal motoneurons express functional PAR-1 and (2) activation of this receptor induces neuronal cell degeneration and death via stimulation of caspases. Together with previous reports , our results suggest that thrombin, its receptor(s), and endogenous t hrombin inhibitors may be important regulators of neuronal cell fate d uring development, after injury, and in pathology of the nervous syste m.