Altered levels of glutamatergic receptors and Na+/K+ ATPase-α1 in the prefrontal cortex of subjects with schizophrenia
Introduction
Schizophrenia is a complex genetic disorder involving dysfunction in nearly all aspects of higher-order behaviour such as disruption of information processing, altered sensory perception, abnormal mood and affect, profound cognitive impairment and movement abnormalities (Thaker and Carpenter, Jr., 2001). The neuropathological basis of schizophrenia is controversial, but the prefrontal and temporal cortices are believed to be principal sites of putative pathology (Harrison, 1999, Lewis and Lieberman, 2000). Several transmitter systems have been implicated in the pathogenesis of schizophrenia, although so far the primary focus has been on the dopaminergic system (Freedman, 2003). On the other hand, evidence has accumulated over the past years that point to a specific malfunction of the glutamatergic system in the aetiology of schizophrenia (Kim et al., 1980, Javitt, 1987). The glutamatergic hypothesis of schizophrenia is based among others on the assumption that hypofunction of the glutamatergic system in cortico-striatal circuitries provokes a facilitation of thalamo-cortical afferents, producing an augmented sensory input, a decrease in the signal-to-noise ratio and an increase in dopaminergic input because of the disinhibition of the ventral tegmental area in the mesencephalon (Lang et al., 2007).
It is well known that glutamate acts on two major classes of receptors: ionotropic receptors [α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), N-methyl-d-aspartate (NMDA) and Kainate], which are ligand-gated ion channels, and metabotropic receptors (mGluRs) that are coupled to G proteins (Masu et al., 1993). A current model for the pathophysiology of schizophrenia proposes that malfunction of the NMDA receptor system leads to dysregulation of GABAergic fast-spiking interneurons, which eventually disinhibits pyramidal glutamatergic output (Gaspar et al., 2009). Antagonists of NMDA receptors such as phencyclidine and ketamine can induce hallucinations and psychosis in healthy subjects and exacerbate them in patients with schizophrenia (Javitt and Zukin, 1991, Halberstadt, 1995, Steinpreis, 1996). NMDA receptors are located in the postsynaptic density of asymmetric synapses and their modulation involves mGlu and AMPA receptors (Gaspar et al., 2009), thus suggesting a potential multi-level dysregulation of glutamatergic receptors and/or of their intracellular transduction pathways in the pathophysiology of schizophrenia. In fact, it has been shown that positive modulators of the AMPA receptors, called ampakines, improve cognitive function in schizophrenia, and enhancement of AMPA receptor-mediated currents by these compounds may potentiate the activity of antipsychotic medications (Coyle, 1996). In order to corroborate the hypothesis of reduced AMPA receptor activity in schizophrenia, a number of post-mortem studies were carried out to identify abnormalities in AMPA receptor expression in the prefrontal cortex of patients with schizophrenia. These investigations have explored multiple levels of gene expression, ranging from the mRNA for different subunits to receptor binding sites, however data have been often contradictory (Breese et al., 1995, Eastwood et al., 1997, Kerwin et al., 1990, Sokolov, 1998).
NMDA receptors are functionally linked also to mGluR5 via Homer, Shank, and postsynaptic density protein of 95 kDa (PSD-95; Tu et al., 1999). Such interactions propose a model for reciprocal functional potentiation and synergistic activity between these two receptors (Chen et al., 2010). In mice lacking mGluR5, NMDA receptor-dependent long-term potentiation (LTP) was significantly reduced in the CA1 region and dentate gyrus of the hippocampus, whereas NMDA receptor-independent LTP remained intact (Lu et al., 1997). Importantly, mice lacking mGluR5 also showed pre-pulse inhibition (PPI) deficits (Brody et al., 2004, Kinney et al., 2003, Lipina et al., 2007) that were reversed by chronic (Gray et al., 2009), but not acute (Brody et al., 2004), treatment with clozapine. The potential involvement of mGluR5 in psychosis has been confirmed by pharmacological modulation using selective mGluR5 antagonists (MPEP, MTEP), which augmented the behavioural effects induced by NMDA receptor antagonists such as PCP and MK-801 (Campbell et al., 2004, Henry et al., 2002, Homayoun et al., 2004, Kinney et al., 2003, Pietraszek et al., 2005). On the other hand, the use of a mGluR5 agonist, e.g. CHPG (Chan et al., 2008), or positive allosteric modulators (Kinney et al., 2005, Schlumberger et al., 2010) reduced the antipsychotic-like symptoms in animal models of schizophrenia through the enhancement of NMDA receptor currents.
Given the functional implications that glutamate receptors appear to have on psychotic-like activity, it is important to understand how these receptors may be altered in schizophrenia.
In this paper, we have studied by semiquantitative immunoblot analysis the expression of several glutamatergic receptor proteins, namely mGluR5, GluR1 and GluR2, in Brodmann Area 10 (BA10) of patients with schizophrenia compared to control subjects. In addition, we have assessed the protein expression levels of Na+/K+ ATPase-α1, a potential modulator of glutamate uptake that may operate in concert with glutamate transporters to regulate glutamatergic neurotransmission.
Section snippets
Human brain tissue collection
Autopsy brain tissue was obtained from the Cambridge Brain Bank Laboratory. The Cambridge Local Ethical Review Committee approved the collection and storage of tissue, as well as all research procedures carried out in the present study (LREC 99/394). A total of 56 brains were examined in this study, of which 21 were obtained from patients diagnosed with schizophrenia in life and 35 were obtained from people without neurological or psychiatric illness and used as controls. A description of
Antibody specificity
Specificity of respective antibodies for GluR1, GluR2, mGluR5 and Na+/K+ ATPase-α1 were analysed by immunoblot on BA10 extracts of a control subject and representative blots are shown in Fig. 1. A single band of the expected molecular weight was detected with antibodies for Na+/K+ ATPase-α1 (Vasarhelyi et al., 2000), GluR1 and GluR2 (Meng et al., 1997, Wang et al., 2000). The anti-mGluR5 antibody used for this study recognised two bands: the specific monomeric form of mGluR5, at ~ 150 kDa (Fig. 1
Discussion
The present study demonstrates a robust decrease in the protein expression levels of GluR1 and GluR2 subunits (− 24% and − 23%, respectively) in the prefrontal cortical area BA10 of patients with schizophrenia as compared to matched controls. In addition, we show a significant increase of Na+/K+ ATPase-α1 (+ 17%) in BA10 in schizophrenia, whereas mGluR5 protein expression levels were similar between patients with schizophrenia and matched controls.
Conclusions
In conclusion, our data support and expand the hypothesis of glutamatergic dysfunction in prefrontal cortex in the pathophysiology of schizophrenia; a marked reduction in the expression of AMPA receptors (GluR1 and GluR2) confirms the presence of a hypoglutamatergic tone, whereas the increase in the expression levels of Na+/K+ ATPase-α1 corroborates the view of an excessive glutamate release counteracting the reduced number/activity of ionotropic glutamatergic receptors.
Role of Funding Source
Funding for this study was provided in part by GlaxoSmithKline (to J.H.X.) and by the Innsbruck Medical University (to F.F.).
Contributors
Authors C. Corti, J.H. Xuereb and F. Ferraguti designed the study. Author J.H. Xuereb managed the tissue banking and performed neuropathological examinations. Authors C. Corti, L. Crepaldi, M. Corsi and F. Ferraguti contributed to the generation, analysis and interpretation of data. Author F. Michielin undertook the statistical analyses and authors C. Corti and F. Ferraguti wrote the first draught of the manuscript. All authors have contributed to and have approved the final manuscript. The
Conflict of Interest
C. Corti, M. Corsi and F. Michielin are employees of GlaxoSmithKline. All other authors declare that they have no conflicts of interest.
Acknowledgement
We thank Mr. R. Hill for excellent technical support.
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Present address: MRC Laboratory for Molecular and Cell Biology and Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.