Elsevier

Neuropharmacology

Volume 112, Part A, January 2017, Pages 113-123
Neuropharmacology

Conditional deletion of Eps8 reduces hippocampal synaptic plasticity and impairs cognitive function

https://doi.org/10.1016/j.neuropharm.2016.07.021Get rights and content

Highlights

  • Eps8 deletion results in NMDA receptor hyperfunction.

  • Eps8 deletion promotes decay of long-term potentiation.

  • Eps8 deletion leads to cognitive deficits.

Abstract

Epidermal growth factor receptor substrate 8 (Eps8) is a multifunctional protein involved in actin cytoskeleton regulation and is abundantly expressed in many brain regions. However, the functional significance of Eps8 in the brain has only just begun to be elucidated. Here, we demonstrate that genetic deletion of Eps8 (Eps8−/−) from excitatory neurons leads to impaired performance in a novel object recognition test. Consistently, Eps8−/− mice displayed a deficit in the maintenance of long-term potentiation in the CA1 region of hippocampal slices, which was rescued by bath application of N-methyl-d-aspartate receptor (NMDAR) antagonist 2-amino-5-phosphonopentanoate. While Eps8−/− mice showed normal basal synaptic transmission, a significant increase in the amplitude and a significantly slower decay kinetic of NMDAR-mediated excitatory postsynaptic currents (EPSCs) were observed in hippocampal CA1 neurons. Furthermore, a significant increase in the expression of ifenprodil-sensitive NMDAR-mediated EPSCs was observed in neurons from Eps8−/− mice compared with those from wild-type mice. Eps8 deletion led to decreased mature mushroom-shaped dendritic spine density but increased complexity of basal dendritic trees of hippocampal CA1 pyramidal neurons. These results implicate NMDAR hyperfunction in the cognitive deficits observed in Eps8−/− mice and demonstrate a novel role for Eps8 in regulating hippocampal long-term synaptic plasticity and cognitive function.

This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’.

Introduction

Epidermal growth factor (EGF) receptor substrate 8 (Eps8) is the prototype of an Eps8-family proteins that was originally isolated as a substrate for the EGF receptor tyrosine kinase (Fazioli et al., 1993). Eps8 protein typically contains an N-terminal phosphotyrosine-binding (PTB) domain, a central Src homology 3 (SH3) domain and a C-terminal effector domain, each being a potential site for protein-protein interactions (Di Fiore and Scita, 2002). Previous studies have revealed that Eps8 participates, via its SH3 domain, in the formation of distinct protein complexes that either transduce signals from Ras to Rac leading to actin remodeling or regulate EGF receptor endocytosis through its interaction with RN-tre, a GTPase-activating protein for Rab5 (Lanzetti et al., 2000, Di Fiore and Scita, 2002, Auciello et al., 2013). In addition, Eps8 can regulate actin dynamics through its ability to activate Rac by forming an active molecular complex with the guanine nucleotide exchange factor Sos-1, the adaptor Abi-1, and the p85 regulatory subunit of phosphoinositide 3-kinase (Disanza et al., 2004), the stability of which is enhanced by association with insulin receptor substrate p53 (IRSp53) (Funato et al., 2004). Moreover, Eps8 also directly controls actin cytoskeleton dynamics and architecture through its actin barbed-end capping and bundling activities, which reside in its C-terminal effector domain (Disanza et al., 2004). While the molecular characteristics of Eps8 have been elucidated in the past several years, most functional studies of this protein were performed using cell line systems. The functional relevance of Eps8 in vivo; however, has only just begun to be elucidated.

By using in situ hybridization and immunohistochemical analysis, Eps8 was found to be abundantly expressed in many brain regions, including the prefrontal cortex, hippocampus, amygdala and cerebellum. It is ubiquitously expressed in the somatodendritic and axonal compartments of granule cells and unipolar brush cells in the rat cerebellum (Sekerková et al., 2007). Furthermore, Eps8 is enriched in both synaptosomal and postsynaptic density components, where it is tightly associated with the postsynaptic density (PSD) proteins, PSD-95, chapsyn 110/PSD-93 and N-methyl-d-aspartate receptor (NMDAR) subunits, in cerebellar granule cells (Offenhäuser et al., 2006, Sekerková et al., 2007). Importantly, a recent study has revealed the role of Eps8 in dendritic spine formation and activity-mediated synaptic plasticity in cultured hippocampal neurons (Stamatakou et al., 2013). Consistently, deficits in dendritic spine formation and learning-dependent spinogenesis have been reported in hippocampal neurons of Eps8 knockout mice (Menna et al., 2013). Furthermore, Eps8 has also been shown to regulate the formation of axonal filopodia in cultured hippocampal neurons in response to brain-derived neurotrophic factor (Menna et al., 2009). These results strongly imply the potential role of Eps8 in the regulation of hippocampal neuronal structure and function. To address this idea, we have taken advantage of Cre/loxP recombinase-based strategy to delete Eps8 exclusively from excitatory neurons of the mouse forebrain in a conditional manner to examine its in vivo relevance. We found that conditional deletion of Eps8 from excitatory neurons leads to impaired performance in novel object recognition test and a deficit in the maintenance of hippocampal CA1 long-term potentiation (LTP).

Section snippets

Animals

Floxed Eps8 (Eps8loxP/loxP) mice were generated by standard techniques using a targeting containing a neomycin resistance (Neo) selection cassette flanked by FRT sites. The conditional knockout vector that targets exon 5 of mouse Eps8 gene was constructed by cloning a 5′ (4664 bp) and 3′ (4193 bp) homology fragment into the pD223_DTA_spec vector using the recombineering methods. Exon 5 of the Eps8 gene was inserted into two flanking loxP sites. For selection purpose, the LacZ reporter and Neo

Conditional deletion of Eps8 from hippocampal excitatory neurons

We generated an Eps8 mutant allele by flanking the fifth coding exon with loxP sites. To conditionally delete Eps8 from hippocampal excitatory neurons, Eps8flox/flox mice were crossed with calcium/calmodulin-dependent protein kinase II α (CaMKIIα)-cre transgenic mice to generate cell type-specific Eps8 knockout mice using the cre-loxP recombination approach (Fig. 1A). Polymerase chain reaction (PCR) screening of mouse genomic tail DNA confirmed heterozygous (Eps8+/−) and homozygous Eps8 (Eps8−/−

Discussion

The results of recent in vitro neuronal culture studies have underscored the importance of Eps8 in regulating spine morphogenesis and activity-mediated synaptic plasticity (Menna et al., 2013, Stamatakou et al., 2013). In cultured rat hippocampal neurons, knockdown of Eps8 decreases spine formation but promotes filopodium formation through its actin-capping activity (Stamatakou et al., 2013). Similarly, excessive synaptic growth and abnormal spine characteristics were observed in primary

Author contributions

Y.T.W., C.C.H. and K.S.H. planned the experiments. Y.T.W., C.C.H., Y.S.L., W.F.H., C.Y.Y., C.C.L. and C.M.Y. performed experiments and gathered data. Y.T.W., C.C.H. C.M.Y. and C.Y.Y. analyzed data and prepared figures. Y.T.W., C.C.H. and K.S.H. wrote the manuscript. All authors reviewed the manuscript.

Disclosure

The authors declare no conflict of interest.

Acknowledgements

This work was supported by research grants from the Ministry of Science and Technology (NSC100-2321-B-006-001-MY4 and MOST 104-2320-B-006-040-MY3) and the Ministry of Education (Aim for the Top University Project to the NCKU), Taiwan.

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