Altered neuroligin expression is involved in social deficits in a mouse model of the fragile X syndrome

doi:10.1016/j.bbr.2009.11.019

Behavioural Brain Research

Volume 208, Issue 1, 17 March 2010, Pages 96-105

https://doi.org/10.1016/j.bbr.2009.11.019 Get rights and content

Abstract

The fragile X syndrome (FXS) is the most common form of inherited mental retardation. Caused by a transcriptional silencing of the fragile X mental retardation protein (FMRP), a mRNA binding protein itself, misregulated translation is thought to be the leading cause of the fragile X syndrome. Interestingly, recent results indicated several neuroligin interacting proteins to be affected by this misregulation, including neurexin1 and PSD95, which have also been implicated in autism spectrum disorders.

Using co-immunoprecipitation assays and RT-PCR, FMRP is shown to interact with neuroligin1- and 2-mRNA, while no interaction with neuroligin3–mRNA is observed. In line with FMRP's role in translation regulation, Western blot as well as immunohistochemistry analysis reveal changes in protein expression levels suggesting impaired synaptic function. As increasing evidence indicates neuroligin expression to be critical for synapse maturation and function, consequences of impaired neuroligin1 expression in FXS are assessed by overexpressing HA-neuroligin1 in FMR1−/− mice, a model for FXS.

Behavioural assessments demonstrate that enhanced neuroligin1 expression improves social behaviour in FMR1−/− mice, whereas no positive effect on learning and memory is seen. These results provide for the first time evidence for an involvement of a neuroligin–neurexin protein network in core symptoms of FXS.

Introduction

In all human populations and ethnic groups, FXS is the most common form of inherited mental retardation, affecting approximately 1:4000 males and 1:8000 females [1]. Impaired individuals display a variety of intellectual deficits from mild learning impairments to abnormal social behaviours and autism (ASD). The disease is caused by a transcriptional silencing of FMRP, a mRNA binding protein that is most abundant in the brain, and which functions in the regulation of synaptic activity related local protein synthesis (reviewed in [2]). Under normal conditions, activation of metabotropic glutamate receptors (mGluRs) induces a rapid dephosphorylation of FMRP, which results in a derepression of local protein synthesis and allows for immediate expression of FMRP associated mRNAs. The ability of FMRP to also modulate the stability of ligand mRNAs in an activity dependent manner further contributes to the regulation of local translation and FMRP dependent plasticity. In FXS, the loss of FMRP leads to a lack of the activity dependent increase in local translation of FMRP target mRNAs.

Interestingly, FMRP has been found to interact with a substantial sub-population of mRNAs, approximately 4% of all mRNAs in the mammalian brain. A number of FMRP ligand mRNAs encode proteins that affect α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, e.g. MAP1b, App or Arc and elevated levels of Arc may be responsible for the persistent internalisation of AMPARs and exaggerated LTD observed in FMR1−/− mice [2]. Furthermore, several mRNAs of synaptic key proteins including those of Munc13 [3], [4], synaptotagmin1/5 [5], neurexin1 [5], PSD95 [6], [7], [8] and SAPAP3/4 [3], [9], [10], [11], have recently been indicated to be regulated by FMRP.

Neuroligins (NLs) are a small family of postsynaptic trans-membrane proteins, which interact with neurexins (NXs), presynaptic cell-adhesion proteins, in a Ca²⁺ dependent manner [12], [13]. By simultaneously binding to PSD95, NLs link the postsynaptic density and control of ionotropic neurotransmitter receptors to the exocytotic machinery of the presynaptic terminal [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. Remarkably, mutations in NL3/4 involved in autism spectrum disorders (ASD) have been shown to destabilize the structure of NL by mapping the mutations on crystallography models of the NL–NX complex and comprised viability of NL is thought to be brought about [25], [26], [27], [28]. In addition, a retention of NL in the endoplasmatic reticulum as well as a decrease in it's affinity for NX have been associated with some of those mutations [29], [30], thereby rising the idea that impaired trafficking and protein binding properties of NL might be related to the disease.

Given that all the previously listed synaptic proteins are direct or indirect binding partners of the NL–NX complex and that three central members, PSD95, NL and NX, have recently been related to ASD [31], [32], [33], [34], [35], [36], we hypothesize that a NL–NX protein network is involved in deficits observed in FXS. To test this hypothesis, we have expressed HA–NL1 under the thy1-promotor in FMR1−/− mice, a model for FXS.

Section snippets

Breeding of transgenic mice

All mice were kept at the University of British Columbia Animal Resource Unit and the University of British Columbia Zoology Facility for Small Mammals according to protocols approved by the Animal Care Committee of the University of British Columbia. Breeding of the different transgenic strains (FMR1−/− (C57BL/6), TgNL1 (C57BL/6 [37]) and TgN1F (FMR1−/− × TgNL1) was implemented according to supplemental Fig. 1a. Mice were housed under a 12 h light–dark cycle with constant temperature in groups of

Expression of HA–NL1 in the brain of FMR1−/− mice does not impair their general health

FMR1−/− mice expressing an HA tagged construct of full length NL1 (TgN1F mice) under the thy1-promotor were generated by cross-breeding of FMR1−/− and TgNL1 animals [37]. Immunohistochemistry and WB analysis demonstrate that HA–NL1 is present throughout the brain, and is particularly high expressed in the cortex and the hippocampus (Fig. 1a), while expression levels in the olfactory bulb or the cerebellum are lower. More specifically, HA–NL1 is found in neurons positive for CamKII as well as in

Discussion

The presented study focused on the relation of a postsynaptic cell adhesion protein involved in synapse maturation and implicated in ASD, NL1, and social behaviour of FMR1−/− mice, a model for FXS. By applying a broad spectrum of biochemical as well as behavioural methods, FMRP was found to interact with NL1-mRNA, resulting in misregulated NL1 expression and abnormal synapse specificities, while enhanced NL1 expression specifically improved social behaviours in FMR1−/− mice. In addition, an

Acknowledgments

This project is funded by grants from CIHR to AEH. AEH was a Michael Smith Foundation for Health Research Senior Scholar. Since his tragic death at Christmas 2007, Prof. Steven Vincent and Prof. Yu Tian Wang have been kindly facilitating continuation of this project. The authors also thank Dr. Hauke Juranek for designing analysis software. RD received support from the FXRFC in 2007 and a postdoctoral fellowship award from the FXRFC and CIHR partnership program in 2008. This work is dedicated to

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Research reportAltered neuroligin expression is involved in social deficits in a mouse model of the fragile X syndrome

Abstract

Introduction

Section snippets

Breeding of transgenic mice

Expression of HA–NL1 in the brain of FMR1−/− mice does not impair their general health

Discussion

Acknowledgments

Cell

Cell

Mol Brain Res

Dev Cell

Genomics

Genomics

Neuron

Neuron

Chemico-Biol Interact

Curr Biol

Behav. Brain Res

Neurosci Lett

Mol Cell Neurosci

Brain Res Dev Brain Res

Cell

Behav Brain Res

Neuron

Neuron

Neuron

Cell

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

Trends Neurosci

Neurosci Lett

Neuron

A decade of molecular studies on the fragile X syndrome

Annu Rev Neurosci

Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function

Neuron

Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice

PNAS

The fragile X mental retardation protein is required for type-I metabotropic glutamate receptor-dependent translation of PSD-95

PNAS

A new function for the fragile X mental retardation protein in regulation of PSD-95mRNA stability

Nat Neurosci

Dysregulated metabotropic glutamate receptor-dependent translation of AMPA receptor and postsynaptic density-95 mRNAs at synapses in a mouse model of fragile X syndrome

J Neurosci

FMRP phosphorylation reveals an immediate-early signalling pathway triggered by group I mGluR and mediated by PP2A

J Neurosci

PSD95 involvement in maturation of excitatory synapses

Science

Neuroligation: building synapses around the neurexin–neuroligin link

Nat Neurosci

Direct interactions between PSD95 and stargazin control synaptic AMPA receptor number

PNAS

PSD-95 regulates synaptic transmission and plasticity in rat cerebral cortex

J Physiol

Neurexin mediates the assembly of presynaptic terminals

Nat Neurosci

Postsynaptic density-95 mimics and occludes hippocampal long-term potentiation and enhances long-term depression

J Neurosci

Postsynaptic density 95 controls AMPA receptor incorporation during long-term potentiation and experience-driven synaptic plasticity

J Neurosci

Research report
Altered neuroligin expression is involved in social deficits in a mouse model of the fragile X syndrome