Prevention of thrombin-induced motoneuron degeneration with different neurotrophic factors in highly enriched cultures

Abstract

Previous reports have shown that neuronal and glial cells express functionally active thrombin receptors. The thrombin receptor (PAR-1), a member of a growing family of protease activated receptors (PARs), requires cleavage of the extracellular amino-terminus domain by thrombin to induce signal transduction. Studies from our laboratory have shown that PAR-1 activation following the addition of thrombin or a synthetic thrombin receptor activating peptide (TRAP) induces motoneuron cell death both in vitro and in vivo. In addition to increasing motoneuron cell death, PAR- 1 activation leads to decreases in the mean neurite length and side branching in highly enriched motoneuron cultures. It has been suggested that motoneuron survival depends on access to sufficient target-derived neurotrophic factors through axonal branching and synaptic contacts. However, whether the thrombininduced effects on motoneurons can be prevented by neurotrophic factors is still unknown. Using highly enriched avian motoneuron cultures, we show here that alone, soluble chick skeletal muscle extracts (CMX), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF) significantly increased motoneuron survival compared to controls, whereas nerve growth factor (NGF) did not have a significant effect on motoneuron survival. Furthermore, cotreatment with muscle-derived agents (i.e., CMX, BDNF, GDNF) significantly prevented the death of motoneurons induced by alpha-thrombin. Yet, non-muscle-derived agents (CNTF and NGF) had little or no significant effect in reversing thrombin-induced motoneuron death. CMX and CNTF significantly increased the mean length of neurites, whereas NGF, BDNF, and GDNF failed to enhance neurite outgrowth compared to controls. Furthermore, CMX and CNTF significantly prevented thrombin-induced inhibition of neurite outgrowth, whereas BDNF and GDNF only partially reversed thrombin-induced inhibition of neurite outgrowth. These findings show differential effects of neurotrophic factors on thrombin-induced motoneuron degeneration and suggest specific overlaps between the trophic and stress pathways activated by some neurotrophic agents and thrombin, respectively.