Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected, and the functional consequences, have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51), but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD), a model of global brain ischemia, as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity, the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore, GDNF protected hippocampal neurons when present before, during or after OGD, and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF.Neuronal injury induced by brain ischemia is partly owing to an excessive release of excitatory amino acids followed by toxic overactivation of glutamate receptors (excitotoxicity).^{1, 2, 3} The subsequent increase in Ca²⁺]_i activates Ca²⁺-dependent proteolytic enzymes (e.g., calpains), which contribute to ischemic neurodegeneration.^{4, 5, 6} The extrasynaptic N-methyl-d-aspartate receptors (NMDAR) for glutamate are preferentially coupled to the activation of excitotoxic signaling mechanisms, including the activation of calpains, and were therefore suggested to have a key role in neuronal death.^{7, 8, 9}The glial cell line-derived neurotrophic factor (GDNF) protects neurons from excitotoxicity-induced cell death^{10, 11, 12, 13} and from ischemic damage.^{14, 15, 16} GDNF dimers bind to GFRα1 (GDNF family receptor alpha-1) with a high affinity, and this complex signals through the transmembrane Ret receptor tyrosine kinase.^{17, 18} The Ret receptor pre-mRNA is alternatively spliced in three isoforms that differ in the composition and length of the C-terminus tail. Once activated by the complex GDNF–GFRα1, Ret isoforms undergo transphosphorylation on several tyrosine residues, activating the Ras/mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase/AKT, responsible for cell survival or differentiation, and the phospholipase C-γ (PLCγ) pathway.^{19, 20, 21, 22, 23}Cerebral ischemia is known to alter the expression of the GDNF signaling machinery.¹⁶ Thus, the GDNF mRNA and protein were both found to be upregulated in experimental models of transient focal and global ischemia.^{16, 24, 25, 26} An increased expression of the mRNA for GFRα1^{24, 27} and Ret^{27, 28} was observed in damaged areas after transient middle cerebral artery occlusion (MCAO), a model of focal ischemia, and an upregulation of GFRα1 mRNA was also observed in the hippocampus following transient global ischemia in rats.²⁶ However, whether GFRα1 and Ret protein levels are altered in transient brain ischemia, the impact on their signaling activity and the functional consequences, have not been investigated. In this work, we show that the Ret51 protein is selectively downregulated following excitotoxic injury and in brain ischemia, by a calpain-dependent mechanism, with a consequent decrease in GDNF signaling activity. This loss of Ret51 receptors impairs the neuroprotective effects of GDNF after transient in vitro ischemia.