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Study on the Mechanism of GDNF Biological Effects Mediated by N-cadherin
ABSTRACT
Glia cell line-derived neurotrophic factor (GDNF) is a secreted neurotrophic factor which exerts protective effects in dopaminergic neurons (DAs) as well as motor neurons. Our research aimed to prove that N-cadherin participated in GDNF induced PI3K/AKT activation and was involved in GDNF mediated DAs protection using both Sprague-Dawley (SD) rat and http://www.ukassignment.org/ygkczy/ MN9D cell models. The immunoblotting results showed that Tyr860 and p-Akt were activated by GDNF in a time and dose-dependent manner both in vitro and in vivo. siRNA mediated ablation of N-cadherin significantly increased apoptotic cells in the damaged neurocytes by flow cytometry and Hoechst 33258 staining results. In parrallel, according to the immunobloting results, phosphorylated Akt（p-Akt）level was attenuated while the total Akt quantity remained unchanged. The immunohistochemical staining results demonstrated that the quantities of total N-cadherin, phosphorylated N-cadherin (Tyr860) and p-Akt all decreased, however, the total Akt quantity remained unchanged. Collectively, GDNF can activate the PI3K/AKT pathway via N-cadherin to protect dopaminergic neurons.
N-cadherin-ERα-Src signal models mediate the synergisticpotentiation of activation of PI3K/Akt signal pathway in injureddopaminergic neurons by GDNF and E2
AbstractAccumulating evidence indicates that glial cell line-derived neurotrophic factor (GDNF)synergizes with 17β-estradiol to protect dopaminergic neurons, however, the mechanisms have notyet been elucidated. Based on the fact that either 17β-estradiol or GDNF can activate theintracellular PI3K/Akt signal pathway, we hypothesize that the synergic protection ofdopaminergic neurons exerted by E2 and GDNF is ascribed to enhancing the activation of thecellular PI3K/Akt signal pathway in a certain way. Therefore, we studied the effects of E2 andGDNF on cultured MN9D cells which were injured by 6-OHDA, by using the methods of westernblot and profiles of intracellular Akt phosphorylation. First, we observed that theco-administration of GDNF and E2 significantly increased Akt phosphorylation in injureddopaminergic neurons, compared with the sole administration of GDNF or E2. Then, we explored,under the same condition, the precise pathways underlying the enhanced activation of thePI3K/Akt signal pathway using the methods of co-immunoprecipitation (IP) and immunoblot. Theresults demonstrated that the estrogenic α-receptor (ERα), in injured dopaminergic neurons,accumulated in the inner surface of cells after co-administration of GDNF and E2, and possiblycombined in a complex consisting of intracellular N-cadherin. Further, we observed the adapterprotein also combines with the N-cadherin-ERα complex. The intracellular PI3K/Akt signalpathway could not be activated after inhibiting the activities of the Src family members. Thesefindings indicate the synergistic protection, exerted by GDNF and E2, upon DA neurons may bemediated by enhancing the activation of the PI3K/Akt signal pathway via a signal modelconsisting of intracellular N-cadherin, ERα and Src family adaptor proteins concomitantly.
Synergistic Protection by GDNF and E2 in Injured Dopaminergic Neurons
Accumulating evidence indicates that glial cell line-derived neurotrophic factor (GDNF) and 17β-estradiol (E2) work synergistically to protect dopaminergic (DA) neurons, and N-cadherin appears to be the transmembrane protein that mediates signaling pathway activation. However, it is not clear how N-cadherin participates in this process. We hypothesize that the synergistic protection that GDNF and E2 provide injured DA neurons relates to GDNF’s ability to phosphorylate N-cadherin at Tyr-860 (p-N-cadherin). Therefore, we investigated the effects of E2 and GDNF on MN9D cells treated with 6-hydroxydopamine (6-OHDA). First, the intracellular amount of p-N-cadherin (Tyr-860) was increased with plasmid transfection, and the cells were treated with GDNF and E2. Then, the effect on p-N-cadherin (Tyr-860) in was studied by co-immunoprecipitation, immunoblot, and immunocytochemistry to determine the levels of intracellular N-cadherin and Akt phosphorylation (p-Akt, Ser-473), as well as estrogen receptor-α (ERα) binding with http://www.ukassignment.org/ygkczy/ N-cadherin. The results demonstrated that following treatment with GDNF and E2, the amount of p-Akt (Ser-473) varied in accordance with that of p-N-cadherin (Tyr-860). In the N-cadherin overexpression group, the amount of ERα bound to N-cadherin significantly increased in the membrane but decreased in the cytoplasm, which was significantly different from the Tyr-860 point mutation group. In the absence of GDNF and E2, ERα binding to N-cadherin in the membrane and cytoplasm also varied in accordance with the amount of p-N-cadherin (Tyr-860). Collectively, these studies indicate that N-cadherin mediates the synergistic protection afforded by GDNF and E2 on injured DA neurons via Tyr-860 phosphorylation.
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