PLA(2) signaling is involved in calpain-mediated degradation of synaptic dihydropyrimidinase-like 3 protein in response to NMDA excitotoxicity.

Dihydropyrimidinase-like 3 (DPYSL3) is believed to play a role in neuronal differentiation, axonal outgrowth and neuronal regeneration, as well as cytoskeleton organization. Recently we have shown that glutamate excitotoxicity and oxidative stress result in calpain-dependent cleavage of DPYSL3, and that NOS plays a role in this process [R. Kowara, Q. Chen, M. Milliken, B. Chakravarthy, Calpain-mediated truncation of dihydropyrimidinase-like 3 protein (DPYSL3) in response to NMDA and H2O2 toxicity, J. Neurochem. 95 (2005) 466-474; R. Kowara, K.L. Moraleja, B. Chakravarthy, Involvement of nitric oxide synthase and ROS-mediated activation of L-type voltage-gated Ca(2+) channels in NMDA-induced DPYSL3 degradation, Brain Res. 1119 (2006) 40-49]. The present study investigates the involvement of PLA(2) signaling in NMDA-induced DPYSL3 degradation. Exposure of rat primary cortical neurons (PCN) to PLA(2) and COX-2 inhibitors significantly prevented NMDA-induced DPYSL3 degradation. Since the metabolic product of PLA(2) signaling, PGE(2), which augments toxic effect of NMDA, is known to stimulate cAMP, the effect of adenyl cyclase activator (forskolin plus IBMX) and inhibitor (MDL12,300) on NMDA-induced DPYSL3 degradation was tested. Our data indicate that the activation of adenyl cyclase contributes to NMDA-induced DPYSL3 degradation. Furthermore, cAMP-dependent protein kinase (PKA) inhibitor PKI (14-22) provided additional evidence of PKA involvement in NMDA-induced DPYSL3 degradation. In summary, the obtained data show the contribution of PLA(2) signaling to NMDA-induced calpain activation and subsequent degradation of synaptic protein DPYSL3.

Involvement of nitric oxide synthase and ROS-mediated activation of L-type voltage-gated Ca2+ channels in NMDA-induced DPYSL3 degradation.

Dihydropyrimidinase-like 3 (DPYSL3), a member of TUC (TOAD-64/Ulip/CRMP), is believed to play a role in neuronal differentiation, axonal outgrowth and possibly in neuronal regeneration. Recently, we have shown that in primary cortical neurons (PCN) NMDA and oxidative stress (H(2)O(2)) caused a calpain-dependent cleavage of DPYSL3 (62 kDa) resulting in the appearance of a lower molecular weight form (60 kDa) of DPYSL3. Our preliminary results had shown that antioxidants significantly reduced NMDA-induced DPYSL3 degradation, indicating involvement of ROS in calpain activation. The aim of this study was to investigate the possible involvement of NOS in NMDA-induced DPYSL3 degradation. We found that NOS inhibitor (L-NAME) significantly prevented NMDA-induced ROS formation, as well as intracellular Ca(2+) increase [Ca(2+)](i), DPYSL3 degradation and cell death. Further, exposure of PCN to NO donor (SNP) resulted in significant [Ca(2+)](i) increase, ROS generation and probable calpain-mediated DPYSL3 truncation. The NMDA- and oxidative stress (ROS)-induced DPYSL3 truncation was totally dependent on extracellular [Ca(2+)](i). While NMDA-induced DPYSL3 truncation was blocked by both NMDA receptor antagonist (MK801) [Kowara, R., Chen, Q., Milliken, M., Chakravarthy, B., 2005. Calpain-mediated degradation of dihydropyrimidinase-like 3 protein (DPYSL3) in response to NMDA and H(2)O(2) toxicity. J. Neurochem. 95 (2), 466-474] and L-VGCC (nimodipine) inhibitors, H(2)O(2)-induced increase in [Ca(2+)](i), ROS generation and DPYSL3 truncation was blocked only by nimodipine. These results indicate that changes in Ca(2+) homeostasis resulting from ROS-dependent activation of L-VGCC are sufficient to induce probable calpain-mediated DPYSL3 truncation and demonstrate for the first time the role of ROS in the mechanism leading to glutamate-induced calpain activation and DPYSL3 protein degradation. The probable calpain-mediated DPYSL3 truncation may have significant impact on its interaction with actin and its assembly, and in turn on growth cone integrity.