Metabotropic glutamate receptor 5, and its trafficking molecules Norbin and Tamalin, are increased in the CA1 hippocampal region of subjects with schizophrenia

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Abstract

Metabotropic glutamate receptor 5 (mGluR5) is involved in hippocampal-dependent learning and memory, which are processes disrupted in schizophrenia. Recent evidence from human genetic and animal studies suggests that the regulation of mGluR5, including its interaction with trafficking molecules, may be altered in the disorder. However there have been no investigations of hippocampal mGluR5 or mGluR5 trafficking molecules in the postmortem schizophrenia brain to confirm this. In the present study, we investigated whether protein expression of mGluR5, as well as Norbin and Tamalin (modulators of mGluR5 signalling and trafficking), might be altered in the schizophrenia brain, using postmortem samples from the hippocampal CA1 region of schizophrenia subjects and matched controls (n = 20/group). Protein levels of mGluR5 (total: 42%, p < 0.001; monomer: 25%, p = 0.011; dimer: 52%, p < 0.001) and mGluR5 trafficking molecules (Norbin: 47%, p < 0.001; Tamalin: 34%, p = 0.009) were significantly higher in schizophrenia subjects compared to controls. To determine any influence of antipsychotic drug treatment, all proteins were also correlated with lifetime chlorpromazine equivalents in patients, and separately measured in the hippocampus of rats exposed to haloperidol or olanzapine treatment. mGluR5 was negatively correlated with lifetime antipsychotic drug exposure in schizophrenia patients, suggesting antipsychotic drugs could reduce mGluR5 protein in schizophrenia subjects. In contrast, mGluR5 and mGluR5 trafficking molecules were not altered in the hippocampus of antipsychotic drug treated rats. This investigation provides strong support for the hypothesis that mGluR5 is involved in the pathology of schizophrenia, and that alterations to mGluR5 trafficking might contribute to the hippocampal-dependent cognitive dysfunctions associated with this disorder.

Introduction

Metabotropic glutamate receptor subtype 5 (mGluR5) is a postsynaptic G-protein coupled receptor (GPCR), best known for its ability to modulate postsynaptic currents induced by the ionotropic N-methyl-d-aspartate glutamate receptor (NMDAR; Matosin and Newell, 2013). A substantial body of evidence indicates that mGluR5 is critically involved in hippocampal-dependent learning and memory. Specifically, several studies have reported that hippocampal mGluR5 activity is important for long-term potentiation (LTP) and long-term depression (LTD), molecular mechanisms underpinning synaptic plasticity (see Mukherjee and Manahan-Vaughan, 2013). mGluR5 knockout mice display reduced LTP, specifically in the hippocampal cornu ammonis 1 (CA1) region, as well as deficits in spatial learning and memory (Lu et al., 1997). Chronic mGluR5 antagonism in rats similarly induces deficits in LTP in the CA1 region, which correlates with reduced hippocampal mGluR5 protein levels and poorer working and reference memory (Manahan-Vaughan and Braunewell, 2005). Accordingly, mGluR5 positive allosteric modulators (PAMs) enhance the balance of hippocampal LTP and LTD at CA1 synapses, and consequently improve spatial learning in mice (Ayala et al., 2009). Notably, very recent evidence suggests localisation of mGluR5 in this region might modulate the direction or balance of synaptic plasticity (e.g. LTP versus LTD; Purgert et al., 2014).

Despite extensive examination in animal models, the status of hippocampal mGluR5 protein in patients with schizophrenia is largely unknown. Only one study has previously investigated mGluR5 mRNA expression in the hippocampus of schizophrenia subjects (in a small postmortem cohort of 5 schizophrenia subjects and 6 controls), reporting decreased mGluR5 expression in the parahippocampal gyrus, and no alterations in the dentate gyrus, CA1, CA3 or CA4 (Ohnuma et al., 2000). Based on studies indicating the importance of mGluR5 in synaptic plasticity, including reports that suggest mGluR5 deficits lead to schizophrenia-like cognitive dysfunctions, it is important to extend on these works and thoroughly investigate whether hippocampal mGluR5 is altered in schizophrenia, specifically at the protein level, as this might contribute to the manifestation of learning and memory deficits observed in patients with schizophrenia.

Accumulating evidence indicates the importance of mGluR5 protein–protein interactions in the regulation of mGluR5 trafficking, internalisation and signalling (Enz, 2007). The first discovered modulator of mGluR5 was Homer1 (Brakeman et al., 1997), which has since been extensively studied in the context of schizophrenia (Szumlinski et al., 2006). Both Homer1 protein and gene are reported as altered in, or associated with, schizophrenia (Engmann et al., 2011, Spellmann et al., 2011). However, in recent years, other molecules that modulate mGluR5 localisation and traficking have become apparent. The neuron-specific protein Norbin (neurochondrin) plays a critical role in mGluR5 localisation, and positively regulates mGluR5 signalling. Interestingly, Norbin knockout was found to reduce mGluR5-dependent LTP and abolished LTD in CA1 synapses (Wang et al., 2009). In cultured mouse hippocampal CA1/CA3 neurons, the multiscaffold protein Tamalin also proved critical to mGluR5 neuritic localisation processes (Kitano et al., 2002) and deletion of the Tamalin binding site on mGluR5 induced mGluR5 internalisation in cellular assays (Timms et al., 2013). Furthermore, evidence from a schizophrenia pedigree suggests that mGluR5/Tamalin interactions might be disrupted in the disorder (Timms et al., 2013). As mGluR5 localisation appears to impact on mGluR5 functions (Purgert et al., 2014), it is important to consider the possibility of altered mGluR5 trafficking in the context of schizophrenia. However, the status of these mGluR5 trafficking molecules in any neuropsychiatric pathology, including schizophrenia, has not been assessed.

In the present study, we therefore determined whether mGluR5 protein levels are altered in postmortem samples from the CA1 hippocampal region of schizophrenia subjects compared to healthy controls (n = 20/group). We further examined protein levels of the mGluR5 trafficking molecules Norbin and Tamalin, which collectively play an important role in mGluR5 trafficking, internalisation and signalling (Kitano et al., 2003, Wang et al., 2009). The CA1 region was chosen for examination due to evidence that mGluR5-mediated LTP might be specific to CA1 synapses (Lu et al., 1997). Subsequently, we ascertained whether any observed changes in protein levels of mGluR5 or its trafficking molecules were influenced by antipsychotic drug (APD) treatment. For this purpose, we treated rats with first- and second-generation APDs (haloperidol and olanzapine respectively) and measured hippocampal protein levels of mGluR5, Norbin and Tamalin.

Section snippets

Human postmortem brain samples and tissue preparation

Postmortem human CA1 samples from 20 control (no history of psychiatric diagnosis) and 20 schizophrenia subjects, diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV), were obtained from the New South Wales Brain Bank Network (Sydney, Australia). Subjects were matched according to tissue pH, postmortem interval, RNA integrity number (RIN) and age at death (Table 1). APD treatment premortem was standardised to a lifetime chlorpromazine equivalent for each

Protein levels of mGluR5 in schizophrenia subjects compared to controls

We detected a robust increase in mGluR5 protein levels in the CA1 region of schizophrenia subjects compared to controls (total: + 42%, F1,37 = 138.579, p < 0.001; monomer: + 25%, F1,37 = 7.194, p = 0.011; dimer: + 52%, F1,37 = 51.705, p < 0.001; Fig. 1). Demographic and clinical measures that correlated significantly with mGluR5 protein levels (Table 2) were assessed for their influence on the data. After co-varying for freezer storage time and brain pH, which significantly correlated with mGluR5 (total and

Discussion

In the present study, we provide the first evidence that protein expression of mGluR5 is significantly higher (total: 42%; monomer: 25%; dimer: 52%) in the hippocampal CA1 region of schizophrenia subjects relative to healthy controls. Schizophrenia subjects also show a marked increase in Norbin, and Tamalin proteins (47% and 34% respectively), which are endogenous regulators of mGluR5 signalling and trafficking not before analysed in neuropsychiatric pathology. In addition, we found no

Role of funding source

The funding sources had no role in this study, including study design, data collection and publication decisions.

Contributors

All authors contributed to the study design and have contributed to and approved the final manuscript.

Conflict of interest statement

All authors declare that they have no conflicts of interest.

Acknowledgements

This work was supported by the Schizophrenia Research Institute, utilising infrastructure funding from the NSW Ministry of Health. Postmortem brain tissues were received from the NSW Tissue Resource Centre, which is supported by the National Health and Medical Research Council of Australia, Schizophrenia Research Institute and the National Institute of Alcohol Abuse and Alcoholism (NIH (NIAA) R24AA012725). The animal APD study was supported by a National Health and Medical Research Council

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