Elsevier

Brain Research

Volume 1683, 15 March 2018, Pages 27-35
Brain Research

Research report
Involvement of mGluR I in EphB/ephrinB reverse signaling activation induced retinal ganglion cell apoptosis in a rat chronic hypertension model

https://doi.org/10.1016/j.brainres.2018.01.017Get rights and content

Highlights

  • Spontaneous firing frequency of RGCs is increased in rat COH retinas.

  • EphB/ephrinB reverse signaling activation induces RGC hyperexcitability.

  • mGluR I antagonists block the RGC hyperexcitability.

  • Co-IP experiments show an interaction between ephrinB2 and mGluR I.

  • mGluR I antagonists inhibit RGC apoptosis in COH retinas.

Abstract

EphB/ephrinB reverse signaling is involved in retinal ganglion cell (RGC) apoptosis in experimental glaucoma. Here, we further investigated the mechanisms underlying EphB/ephrinB reverse signaling activation induced RGC apoptosis in a rat chronic ocular hypertension (COH) model, using patch-clamp techniques in retinal slices. In COH retinas, RGCs showed higher spontaneous firing frequency and much more depolarized membrane potential as compared to control, which was mimicked by intravitreally injection of EphB2-Fc, an activator of ephrinB2. The changes in RGC spontaneous firing and membrane potential could be reversed by the tyrosine kinase inhibitor PP2, suggesting that EphB/ephrinB reverse signaling activation induced RGC hyperexcitability. Intravitreal pre-injection of either LY367385 or MPEP, selective mGluR1 and mGluR5 antagonists, also blocked the changes in RGC spontaneous firing and membrane potential. Co-immunoprecipitation experiments showed an interaction between ephrinB2 and group I metabotropic glutamate receptor (mGluR I) (mGluR1/mGluR5). Furthermore, intravitreal pre-injection of the mixture of L-NAME (an NO synthase inhibitor) and XPro1595 (a selective inhibitor of soluble TNF-α) could reduce the EphB2-Fc injection induced increase in RGC firing, suggesting that Müller cells might be involved in EphB/ephrinB reverse signaling activation induced change in RGC hyperexcitability. In addition, LY367385/MPEP reduced the numbers of TUNEL-positive RGCs both in EphB2-Fc injected and COH retinas. All results suggest that activation of EphB/ephrinB reverse signaling induces RGC hyperexcitability and apoptosis by interacting with mGluR I in COH rats. Appropriate reduction of EphB/ephrinB reverse signaling could alleviate the loss of RGCs in glaucoma.

Introduction

Erythropoietin-producing hepatocyte receptors (Ephs) are the largest family of receptor tyrosine kinases (RTKs), which consist of two subtypes, EphA (A1-A10) and EphB (B1-B6). The corresponding ligands are ephrinA (A1-A5) and ephrinB (B1-B3) (Pasquale, 2005, Pasquale, 2008). Ephs and ephrins are reciprocally expressed in two neighboring cells. When ephrin binds to its receptor, bi-directional signals will be generated on receptor-expressing cells (forward signaling) and ephrin-expressing cells (reverse signaling), both of which result in tyrosine kinase activation and play important roles in modulating a variety of physiological and pathological processes in the central nervous system (CNS) (Calò et al., 2005a, Calò et al., 2005b, Chen et al., 2012, Dalva et al., 2000, Dong et al., 2015, Ethell et al., 2001, Grunwald et al., 2004, Henderson et al., 2001, Henkemeyer et al., 2003, Hruska and Dalva, 2012, Kania and Klein, 2016, Kayser et al., 2006, Kayser et al., 2008, Klein, 2009, Nolt et al., 2011, Takasu et al., 2002).

Increasing evidence shows that ephrinB/EphB signalings, both forward and reverse, are closely related to retinal ganglion cell (RGC) axon degeneration and neuronal apoptosis in experimental glaucoma models (Dong et al., 2015, Du et al., 2007, Fu et al., 2010, Fu and Sretavan, 2012, Schmidt et al., 2007). In a previous study, we found that EphB/ephrinB reverse signaling was activated in RGCs in a rat chronic ocular hypertension (COH) model, which resulted in RGC apoptosis through increasing the Ca2+-impermeable GluA2-containing AMPA receptor trafficking (Dong et al., 2015). However, it is interesting that Naspm, a selective antagonist of Ca2+ permeable AMPA receptors, only partially blocked EphB/ephrinB reverse signaling activation induced RGC apoptosis, suggesting that other mechanisms may be involved, in addition to AMPA receptors (Dong et al., 2015). It was reported that ephrinB/EphB signaling may directly interact with NMDA receptors and/or metabotropic glutamate receptors (mGluR1) (Calò et al., 2005a, Calò et al., 2005b, Dalva et al., 2000, Grunwald et al., 2004, Takasu et al., 2002). Activation of EphB/ephrinB reverse signaling in rat cultured cortical neurons boosted the NMDA toxicity, which was mediated by mGlu1 receptors (Calò et al., 2005a), thus contributing to excitotoxic neuronal death (Calò et al., 2006, Grunwald et al., 2004). In addition, we have demonstrated that in the physiological condition glutamate endogenously released from bipolar cells increased RGC excitability by activating postsynaptic mGluR I (Li et al., 2017). These studies suggest that mGluR I may be one of key targets when EphB/ephrinB reverse signaling is activated, and over-activation of mGluR I may contribute to RGC apoptosis in COH retina. In the present study, we show that activation of EphB/ephrinB reverse signaling increases RGC excitability in COH retina through interacting with mGluR I, thus increasing RGC apoptosis.

Section snippets

Involvement of activated EphB/ephrinB reverse signaling in intraocular pressure (IOP) elevation induced change of RGC firing

We have previously demonstrated that elevated IOP in COH rats induced an activation of EphB/ephrinB reverse signaling in RGCs (Dong et al., 2015). We first examined whether the activated EphB/ephrinB reverse signaling may change RGC excitability by recording spontaneous firing of RGCs in retinal slices. Since EphB/ephrinB reverse signaling activation in COH rats was observed as early as 1 day (G1d) after IOP elevation (Dong et al., 2015), the recordings were done in retinal slices obtained from

EphB/ephrinB reverse signaling activation induces RGC hyperexcitability in COH retina

Previous studies have demonstrated that neuronal hyperexcitability, which is characterized by the enhanced number of action potentials, is associated with subsequent apoptosis (Ashpole et al., 2012, Pasantes-Morales and Tuz, 2006, Turovsky et al., 2013, Zucchini et al., 2008). In the present study, we found that the frequency of spontaneous firing in RGCs was increased in COH retinas (Fig. 1), which could be mimicked by activating EphB/ephrinB reverse signaling and be blocked by PP2 (Fig. 2,

Animals

All experimental procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals and were approved by the Institutes of Brain Science at Fudan University. Male Sprague-Dawley rats, weighing 100–120 g, obtained from SLAC Laboratory Animal Co., Ltd (Shanghai, China), were housed on a 12 h light/dark schedule.

Rat COH model

COH rats were produced following the procedure previously described in details (Chen et al., 2017, Samsel et al.,

Acknowledgements

We would like to thank Dr. Xiong-Li Yang for his helpful discussion and critical comments on the manuscript. We also thank Dr. David E. Szymkowski (Xencor) for the donation of XPro1595. This work was supported by grants from the National Natural Science Foundation of China (81790642; 31671078; 81430007), and the International Science & Technology Cooperation Program of China (2015DFA31340).

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