Abstract
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.
Highlights
D-aspartate receptors (NMDAR) for glutamate are preferentially coupled to the activation of excitotoxic signaling mechanisms, including the activation of calpains, and were suggested to have a key role in neuronal death.[7,8,9]
We found that the glial cell line-derived neurotrophic factor (GDNF) co-receptor Ret[51] is selectively downregulated in cultured hippocampal neurons subjected to excitotoxic conditions, and in in vitro brain ischemia, by a mechanism dependent on the activation of extrasynaptic NMDAR coupled to stimulation of calpains
In addition to Ret[51], the TrkB receptors for BDNF are downregulated under excitotoxic conditions and after transient middle cerebral artery occlusion (MCAO),[6,44] suggesting that the impairment of the endogenous neuroprotective signaling mechanisms mediated by receptor tyrosine kinases may be a key event in neuronal death in brain ischemia
Summary
D-aspartate receptors (NMDAR) for glutamate are preferentially coupled to the activation of excitotoxic signaling mechanisms, including the activation of calpains, and were 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. We show that the Ret[51]
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