Dysregulated 5-HT2A receptor binding in postmortem frontal cortex of schizophrenic subjects
Introduction
5-Hydroxytryptamine (serotonin, 5-HT) receptors constitute a large family of evolutionary conserved seven transmembrane G protein-coupled receptors (GPCRs) (Millan et al., 2008). One of the serotonin receptor subtypes, the 5-HT2AR, has been involved in the symptoms of schizophrenia, and proposed to contribute to the molecular mechanisms by which atypical antipsychotic drugs, such as clozapine, olanzapine and risperidone, induce their clinical effects (Gonzalez-Maeso and Sealfon, 2009, Fribourg et al., 2011, Kurita et al., 2012). Second generation, or atypical, antipsychotic drugs all have in common a high-affinity for the 5-HT2AR, and a lower affinity for the dopamine D2 receptor (Miyamoto et al., 2005). Hallucinogenic 5-HT2AR agonists, such as mescaline, psilocybin and lysergic acid diethylamide (LSD), produce in normal subjects symptom profiles comparable to those seen in acutely ill unmedicated or first-episode schizophrenics (Young, 1974, Hermle et al., 1992, Vollenweider et al., 1998, Gouzoulis-Mayfrank et al., 2005, Quednow et al., 2011). Hallucinogenic drugs aggravate psychosis in schizophrenia patients (Hoch et al., 1952). Of particular interest is also the finding of hallucinogenic (LSD)-induced psychotic symptoms in relatives of schizophrenic patients (Anastasopoulos and Photiades, 1962), suggesting that individuals with greater predisposition to schizophrenia are more susceptible to the psychotic responses that require activation of the 5-HT2AR. In murine models, we and others have demonstrated that 5-HT2AR-regulated pathways on cortical pyramidal neurons are necessary to mediate the signaling pattern and behavioral responses to hallucinogenic drugs (see Gonzalez-Maeso and Sealfon, 2009 for review).
Theory and experimental evidence suggest that GPCRs adopt multiple structural conformations when bound to different ligands. Drugs that increase or decrease the function of GPCRs are thought to modulate the proportion of receptors that are in the structurally active conformations relative to those in inactive, non-signaling conformations. Agonists bind with higher affinity to the active conformations of the receptor, whereas inverse agonists are ligands that preferentially bind and stabilize inactive conformational states. Interestingly, recent observations suggest that most clinically effective antipsychotic drugs are, in fact, 5-HT2AR inverse agonists rather than simply neutral antagonists—ligands that compete for the same orthosteric binding site and prevent the cellular responses induced by agonists and inverse agonists (Egan et al., 1998, Fribourg et al., 2011). Since activation and decrease in the basal activity of the receptor represent a common feature of all hallucinogenic and atypical antipsychotic drugs, respectively, these findings suggest that the 5-HT2AR may be involved in the mechanisms responsible for psychotic symptoms in schizophrenic patients.
Frontal cortex plays an important role in cognition and perception, and has been implicated more recently in schizophrenia and other psychotic disorders (Gonzalez-Maeso et al., 2008, Gonzalez-Maeso and Sealfon, 2009, Kurita et al., 2012). Many laboratories, including ours, have investigated the level of expression of 5-HT2AR protein in frontal cortex of schizophrenic subjects. Most of this work has been performed in postmortem tissue samples with the use of radioligands, including [3H]ketanserin (Reynolds et al., 1983, Mita et al., 1986, Laruelle et al., 1993, Burnet et al., 1996, Dean and Hayes, 1996, Dean et al., 1996, Dean et al., 1998, Dean et al., 1999, Dean et al., 2008, Marazziti et al., 2003, Matsumoto et al., 2005, Gonzalez-Maeso et al., 2008, Kang et al., 2009) and [3H]LSD (Bennett et al., 1979, Whitaker et al., 1981, Joyce et al., 1993, Gurevich and Joyce, 1997), as well as in untreated first-episode schizophrenic patients by positron emission tomography (PET) with [18F]altanserin (Rasmussen et al., 2010). Remarkably, there are striking differences in the results obtained: some studies suggested up-regulation of 5-HT2AR binding sites, whereas others pointed toward absence of alterations or down-regulation in the number of binding sites. Recent reviews discuss the possible role of demographic and clinical measures, such as age, treatment with antipsychotic or other psychotropic drugs, and suicide as cause of death, in these discrepant results between studies (Dean, 2003, Dean et al., 2008, Gonzalez-Maeso and Sealfon, 2009). Here, we extend our previous observations with evidence that [3H]ketanserin binding sites are up-regulated in postmortem frontal cortex of antipsychotic-free schizophrenic subjects, and demonstrate that the density of [3H]ketanserin binding sites is affected by antipsychotic drug treatment and aging. Our findings also suggest that this up-regulation may not be related to suicidal behavior, since the number of [3H]ketanserin binding sites was unchanged in suicide victims with other neuropsychiatric disorders. With the use of ligands that preferentially bind different structural and/or functional receptor conformations, our findings could also provide a pharmacological explanation that may unify previous efforts on the quantification of 5-HT2AR density in frontal cortex of schizophrenic subjects and controls.
Section snippets
Postmortem human brain samples
Human brains were obtained at autopsies performed in the Basque Institute of Legal Medicine, Bilbao, Spain, in compliance with policies of research and ethical boards for postmortem brain studies between 1992 and 2008. Deaths were subjected to retrospective searching for previous medical diagnosis and treatment using examiner's information and records of hospitals and mental health centers. After searching of antemortem information was fulfilled, 45 subjects who had met inclusion criteria of
[3H]Ketanserin binding is increased in postmortem frontal cortex of antipsychotic-free schizophrenic subjects
[3H]Ketanserin binds with high-affinity to both 5-HT2A and 5-HT2C receptors. Using the 5-HT2 receptor ligand methysergide (10 μM) to define non-specific binding, we found that [3H]ketanserin binding sites are abolished in frontal cortex of 5-HT2A knockout (KO) mice as compared to wild type littermate controls (Supplementary Figure 1). These findings validate the use of specific [3H]ketanserin biding as a tool to measure 5-HT2AR density.
We first investigated the number of [3H]ketanserin binding
Discussion
In the present study we characterized the number of [3H]ketanserin binding sites in postmortem frontal cortex of antipsychotic-free schizophrenic subjects, schizophrenic subjects treated with antipsychotic drugs, and control subjects individually matched by gender, age, and postmortem delay. We found that [3H]ketanserin binding was increased in frontal cortex of antipsychotic-free schizophrenic subjects, with absence of changes in antipsychotic-treated schizophrenics. Since specific binding was
Concluding remarks
In summary, we find that [3H]ketanserin binding is increased in postmortem frontal cortex of antipsychotic-free schizophrenic subjects. We demonstrate up-regulation of high-affinity binding sites for the hallucinogenic 5-HT2AR agonist DOI in schizophrenia frontal cortex. Our findings also suggest that altanserin behaves as a 5-HT2AR inverse agonist in schizophrenic subjects, but not in controls. These data may help explain the differences previously reported with use of ligands that bind with
Role of funding source
This study was supported by Spanish MINECO and FEDER Founds (SAF2009-08460) (JJM), Basque Government (IT199/07) (JJM), UPV/EHU (UFI11/35) (JJM), National Institutes of Health (R01 MH084894) (JGM), Dainippon Sumitomo Pharma (JGM), NARSAD (JGM), and The Mortimer D. Sackler Foundation (JGM). CM was supported by a predoctoral fellowship from the University of the Basque Country UPV/EHU. The sponsors had no further role in the study design; in the sample obtaining; in the collection, analysis and
Contributors
CM, JLM and AU carried out the neurochemical assays, statistical analyses and designed the figures. LFC and JJM collected the human brain samples and associated clinical and toxicological information. JJM and JGM conceived the study, planned the experiments, supervised the study and designed the first draft of the manuscript. All authors contributed to data interpretation and writing of the manuscript. All authors approved the final version.
Conflict of interest
The authors declare that they have no conflicts of interest to report.
Acknowledgments
The authors wish to thank staff members, and especially Dr. B. Morentin, of the Basque Institute of Legal Medicine, Bilbao, Spain, for their cooperation in the study.
References (50)
- et al.
Brain 5-HT1A, 5-HT1D, and 5-HT2 receptors in suicide victims
Biol. Psychiatry
(1994) - et al.
5-HT1A and 5-HT2A receptor mRNAs and binding site densities are differentially altered in schizophrenia
Neuropsychopharmacology
(1996) - et al.
Brain 5-HT2 receptor binding sites in depressed suicide victims
Brain Res.
(1988) - et al.
Neurotransmitter receptors and monoamine metabolites in the brains of patients with Alzheimer-type dementia and depression, and suicides
Neuropharmacology
(1984) - et al.
Evidence for altered post-receptor modulation of the serotonin 2a receptor in schizophrenia
Schizophr. Res.
(2008) - et al.
Decreased frontal cortical serotonin2A receptors in schizophrenia
Schizophr. Res.
(1996) - et al.
Reliability of psychiatric diagnosis in postmortem research
Biol. Psychiatry
(2005) - et al.
Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs
Cell
(2011) - et al.
Psychedelics and schizophrenia
Trends Neurosci.
(2009) - et al.
Autoradiographic analysis of [3H]ketanserin binding in the human brain postmortem: effect of suicide
Brain Res.
(1990)