Opposite alterations of 5­HT2A receptor brain density in subjects with schizophrenia: relevance of radiotracers pharmacological profile

This post-mortem study (n=22) quantified the binding density of serotonin 5­HT2A receptors in deceased patients with schizophrenia and found that the active configuration of this receptor, as measured by a two-fold higher agonistic binding of LSD radioligand, had the highest density amongst schizophrenic patients who were not being treated with antipsychotic medication.

Abstract

Introduction: The status of serotonin 5­HT2A receptors (5­HT2ARs) in schizophrenia has been controversial. In vivo positron emission tomography neuroimaging and in vitro post-mortem binding studies have reported conflicting results about 5­HT2AR density. Radiotracers bind different receptor conformations depending on their agonist, antagonist or inverse agonist properties. This study investigates 5­HT2AR density in the post-mortem prefrontal cortex from subjects with schizophrenia and controls using three radiotracers with a different pharmacological profile.

Methods: The specific binding parameters of the inverse agonist [18F]altanserin, the agonist [3H]lysergic acid diethylamide (LSD) and the antagonist [3H]MDL100907 to brain cortex membranes from 20 subjects with schizophrenia and 20 individually matched controls were evaluated under similar methodological conditions. Ten schizophrenia subjects were antipsychotic-free at death. Saturation curve analyses were performed by non-linear regression to obtain a maximal density of binding sites (Bmax) and the affinity of the respective radiotracers (Kd).

Results: In schizophrenia subjects, 5-HT2AR density was decreased when quantified by [18F]altanserin binding, whereas increased when evaluated by [3H]LSD binding. However, [3H]MDL100907 binding was unaltered. A slight loss of affinity (higher Kd) was observed exclusively in [3H]LSD binding. The findings were more evident in antipsychotic-free subjects than in antipsychotic-treated subjects.

Discussion: In conclusion, a higher proportion of the 5-HT2AR-active functional conformation, which is rather identified by agonist radiotracers, was observed in schizophrenia patients. A consequent reduction of the inactive 5-HT2AR conformation, which is preferentially identified by inverse agonist radiotracers, was also obtained. Antagonist radiotracers do not distinguish between molecular conformations of the receptor, and accordingly, the absence of changes was shown. These results are compatible with the proposed increased functional activity of brain cortical 5-HT2ARs in schizophrenia.”

Authors: Rebeca Diez-Alarcia, Carolina Muguruza, Guadalupe Rivero, Aintzane García-Bea, Vanessa Gómez-Vallejo, Luis F. Callado, Jordi Llop, Abraham Martín & J. Javier Meana

Summary

In vivo positron emission tomography neuroimaging and in vitro post-mortem binding studies have reported conflicting results about serotonin 5HT2A receptors in schizophrenia. This study investigated 5-HT2AR density in the post-mortem prefrontal cortex of subjects with schizophrenia and controls using three radiotracers with a different pharmacological profile.

Introduction

Serotonin (5-HT) plays an important role in the pathophysiology and therapeutics of schizophrenia. The 5-HT receptors are the critical target to induce these psychosis-like responses, and second-generation antipsychotics display potent antagonism properties on 5-HT2ARs.

Post-mortem studies have shown that 5-HT2ARs are down-regulated in the frontal cortex of anti-psychotic-naive schizophrenic patients, but some studies have also shown an up-regulation. The differences between in vivo neuroimaging studies and in vitro findings in post-mortem brain have been considered the main explanatory factor. Although unattended, the pharmacological properties of the drugs used as radiotracer tools to identify 5HT2ARs are important determinants for the receptor conformation identified by the radiotracer and, subsequently, for the estimated binding density.

G-protein-coupled receptors, such as 5HT2ARs, display different molecular conformations that are interchangeable and stay in equilibrium17. An imbalance between coupled and uncoupled 5HT2AR conformations might be present in schizophrenia, leading to overactive G-protein-dependent signalling.

Altanserin, a selective antagonist of 5HT2ARs, has been demonstrated to show inverse agonist properties on 5HT2ARs in post-mortem human brain. This finding is in contrast to the findings of [3H]LSD, a partial agonist of 5HT2ARs, which shows enhanced 5HT2AR density in the prefrontal cortex.

The present study used three different radiotracers with different intrinsic activity properties on the 5-HT2AR to investigate the 5HT2AR density in post-mortem prefrontal cortex of subjects with schizophrenia. The results shed light on the unclarified status of brain 5HT2ARs in schizophrenia.

Subjects, materials and methods

Brain samples were obtained at autopsies performed in the Basque Institute of Legal Medicine, Bilbao, Spain, and matched to 20 brains from control subjects in a paired design. Antipsychotic drugs were screened in blood samples from schizophrenia patients and control subjects. Seventeen out of 20 subjects with schizophrenia committed suicide, and matched control subjects mostly died by accidental causes. The mechanism of death indicates that almost all deaths were violent or sudden. Some schizophrenia cases and controls have been used to evaluate the [3H] ketanserin binding density and G-protein activation mediated by 5-HT2ARs.

Materials and drugs

[18F]altanserin was produced by radiofluorination of nitro-altanserin in a TRACERlab FXFN synthesis module. The radiochemical purity was >97% in all cases.

[18F]Altanserin, [3H]LSD and [3H]MDL100907 binding assays

Complete saturation binding assays were performed with [18F]altanserin, [3H]LSD and [3H]MDL100907 in order to determine the density (Bmax) and the affinity (Kd)of5-HT2ARs.

Data and statistical analyses

Data were analysed using non-linear regression, and correlation with independent covariables was tested. A one-way analysis of variance followed by Bonferroni’s post-hoc test was used to compare results between radioligands.

Statistical comparisons between groups were conducted by non-linear curve-fitting coanalysis of all individual binding experiments. Differences in Bmax and/or Kd values between groups were detected by further individual contrasts.

Specific binding sites for [18F]altanserin, [3H]LSD and [3H] MDL100907

The binding of [18F]altanserin and [3H]MDL100907 to 5-HT2ARs was non-linear, and each radioligand bound to a single specific binding site of high affinity. The binding affinities were in the nanomolar range, and there were no significant differences between radioligands.

Effects of demographic parameters and post-mortem conditions

The density of [18F]altanserin, [3H]LSD and [3H]MDL100907 binding sites decreased with age, but not significantly. The binding properties were not affected by post-mortem interval or storage time at 80 °C.

Comparison between schizophrenia and control groups

The co-analysis of saturation curves obtained with [18F] altanserin demonstrated that the density of the binding sites in the schizophrenia group was significantly lower compared to matched controls. No differences in affinity were detected between schizophrenia and control groups.

The co-analysis of saturation curves obtained with [3H] LSD demonstrated that subjects with schizophrenia had increased binding sites and higher Kd values compared to matched controls. The presence of antipsychotic drugs in blood could contribute to the increased Kd values.

The binding sites of [3H] MDL100907 were similar in subjects with schizophrenia and matched controls. Age at death did not influence the results in the analysis of covariance.

Discussion

The present study demonstrated that 5-HT2AR alterations in post-mortem human frontal cortex are dependent on the pharmacological properties of the radiotracer used to identify this receptor. Until recently, a PET study of 5-HT2A/B/CR receptors in post-mortem tissue was not feasible due to the lack of suitable agonist radiotracers. However, recent developments in 5-HT2A/B/CR agonist radiotracers may help overcome these limitations.

The increased density of 5-HT2ARs identified by the agonist radiotracer [3H]LSD confirms the proposed higher functional sensitivity of this receptor in schizophrenia. This finding is in concordance with the increased sensitivity of inhibitory Gi1 proteins in response to the agonist ()DOI (2,5-dimethoxy-4-iodoamphetamine) in post-mortem frontal cortex. Altanserin, a 5-HT2AR antagonist, shows inverse agonist properties in the brain cortex, and this explains the reduced 5-HT2AR density reported in schizophrenia. In brains of subjects with schizophrenia, 5-HT2ARs are imbalanced between G-protein-coupled and G-protein-uncoupled conformations, as indicated by increased density of the agonist [3H]LSD binding and reduced density of the inverse agonist [18F]altanserin binding. Neutral antagonist drugs do not distinguish among molecular conformations of the receptor and are therefore not suitable tools to detect this imbalance.

One apparent inconsistency of the present study is the different receptor binding density obtained between radiotracers. This is because 5-HT2ARs are assembled into homodimeric and heterodimeric structures, and ligands crosstalk to each other. The hallucinogenic 5-HT2AR agonist ()DOB shows a similar affinity for the two binding sites of the dimer43. This suggests that the 5-HT2AR is a homodimeric receptor and that [3H]LSD can label the two binding sites.

Ageing is associated with a decline of 5-HT2AR density, which provides the rationale for individual matching of schizophrenia cases with control.

The presence of antipsychotic drugs in the blood of subjects with schizophrenia represents a confounding factor in radioligand binding studies. Antipsychotic treatment may counterbalance the 5-HT2AR alterations observed in schizophrenia.

Long-term treatment with second-generation antipsychotics modulates 5-HT2AR expression in animals and could modify binding parameters due to residual presence of antipsychotics acting as 5-HT2AR antagonists20. However, the possibility that observed alterations of 5-HT2AR density (Bmax) represent a consequence of the current or past long-term antipsychotic treatment is improbable.

In the present study, the majority of subjects died from violent suicide mechanisms. However, suicide unlikely represents a major confounder in 5-HT2AR binding studies.

The study of 5-HT2AR dysfunctions in schizophrenia requires a deep knowledge of the pharmacological properties of the candidate radiotracers. The development of 5-HT2AR agonist radiotracers in antipsychotic-naive patients should be encouraged to validate the 5-HT2AR overactivity here proposed.

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