Differential roles of endothelin type A and type B receptor in cofilin-actin rod formation and morphological change or hippocampal neurons

Abstract

Previously, post-mortem studies revealed a higher expression level of endothelin-1 (ET-1) in neurons and reactive astrocyte-like cells of Alzheimer’s disease (AD) patients compared with those of healthy cohorts. To understand the significance of astrocytic ET-1 in neuronal dysfunction, our team had created transgenic mice with overexpression of ET-1 in astrocytes and cross-bred them with the APP (K670/M671) mutant mice. The ET-1/APP (K670/M671) mice demonstrated that overexpression of astrocytic ET-1 caused an earlier onset of memory deficit and exaggerated the impairment in learning and memory task in Morris water maze. To further understand the roles of ET-1 in dysfunction of hippocampal neurons in relevance to AD, primary mouse hippocampal neurons were treated with exogenous ET-1 (1, 10, or 100 nM). Firstly, we showed that both ET receptor (rec) subtypes, ETA rec and ETB rec, were expressed in 14 days in vitro neurons. ET-1 at concentration up to 100 nM did not induce neuronal death or change in phosphorylation of GSK3β or tau. However, ET-1 induced phosphorylation of cofilin, formation of cofilin rods, and increase of filopodial protrusions in dendrites. To determine the differential roles of ETA rec and ETB rec in regulating these cellular changes, hippocampal neurons were fixed or harvested after 24 hrs treatment of 100 nM ET-1 with or without pretreatment of 1 μM antagonist of ETA rec (BQ123) or ETB rec (BQ788), or after 24 hrs treatment of 100 nM ETB rec agonist (IRL1620). Interestingly, the Western blot results suggested that ETA rec mediated inactivation of cofilin by activating LIMK1 and inhibiting SSH-1L. In contrast, ETB rec mediated activation of cofilin by activating SSH-1L. Moreover, ET-1-induced cofilin rod formation was abolished by the pretreatment of BQ788, but not by BQ123. The IRL1620-induced cofilin rod formation was accompanied by the loss of distal dendrites and significant decrease in dendritic length. The IRL1620-induced cofilin rod formation was abolished by the co-treatment of antioxidant N-acetylcysteine (100 μM), or the pretreatment of inhibitor of NADPH oxidase (VAS2870, 5 μM) or calcineurin (FK506, 100 nM). These findings suggested that ETB rec mediated cofilin rod formation and loss of distal dendrites via NADPH oxidase-mediated oxidative stress and calcineurin/SSH-1L-mediated cofilin activation. In addition, ET-1 induced reduction of spine head diameter, loss of mushroom-shaped spines and formation of filopodial protrusions, which were abolished by the pretreatment of BQ788, but not by BQ123, suggesting that ETB rec might mediate the instability of dendritic spines. Taken together, the current study provides a novel mechanism of ET rec involvement in the loss of distal dendrites and mushroom-shaped spines through cofilin dysregulation and rod formation.