Up-regulation of the Trace Amine Receptor, TAAR-1, in the Prefrontal Cortex of Individuals Affected by Schizophrenia

Abstract

Background and Hypothesis

Type-1 trace amine-associated receptors (TAAR1) modulate dopaminergic and glutamatergic neurotransmission and are targeted by novel antipsychotic drugs. We hypothesized that schizophrenia (SCZ) causes adaptive changes in TAAR1 expression in the prefrontal cortex.

Study Design

We measured TAAR1 mRNA and protein levels by quantitative PCR and immunoblotting in post-mortem prefrontal cortical samples obtained from 23 individuals affected by SCZ and 23 non-schizophrenic controls (CTRL). Data were correlated with a number of variables in both groups.

Study Results

TAAR1 mRNA levels were largely increased in the SCZ prefrontal cortex, and did not correlate with age, age at onset and duration of SCZ, or duration of antipsychotic treatment. For the analysis of TAAR1 protein levels, CTRL and SCZ were divided into 2 subgroups, distinguished by the extent of neuropathological burden. CTRL with low neuropathological burden (LNB) had lower TAAR1 protein levels than CTRL with high neuropathological burden (HNB), whereas no changes were found between LNB and HNB in the SCZ group. TAAR1 protein levels were lower in CTRL with LNB with respect to all SCZ samples or to SCZ samples with LNB. In the SCZ group, levels showed an inverse correlation with the duration of antipsychotic treatment and were higher in individuals treated with second-generation antipsychotics as compared with those treated with first-generation antipsychotics.

Conclusions

The up-regulation of TAAR1 observed in the SCZ prefrontal cortex supports the development of TAAR1 agonists as new promising drugs in the treatment of SCZ.

Introduction

Trace amines, such as β-phenylethylamine, p-tyramine, p-octopamine, tryptamine, p-synephrine, and tyronamines, are present in low amounts in the CNS and modulate synaptic transmission by interacting with G protein-coupled receptors called trace amine-associated receptors (TAARs).^1–4 TAARs form a large family of receptors, of which TAAR1-4 and TAAR5-9 are activated by primary and tertiary amines, respectively.⁵ TAAR1 expression in experimental animals is high in brain regions involved in the pathophysiology of schizophrenia (SCZ), such as the prefrontal cortex, basal ganglia, ventral tegmental area (VTA), and dorsal raphe nuclei.^6–8 In humans, TAAR1 is encoded by an intronless gene located on the long arm of chromosome 6 (6q23.1).⁹ Polymorphic variants of the TAAR1 gene have been associated with SCZ and bipolar disorder.^10–12

Studies performed in rodent lines lacking or overexpressing TAAR1 have disclosed a key role for TAAR1 in the regulation of monoaminergic neurotransmission and in the response to natural or drug rewards. For example, mice with genetic deletion of TAAR1 showed a greater motor response to amphetamine, whereas the opposite was observed in mice overexpressing TAAR1.^8,13,14

In the CNS, TAAR1 is expressed in both axon terminals and postsynaptic elements and modulates dopaminergic transmission by acting at different levels. It has been suggested that TAAR1 is primarily localized in the endoplasmic reticulum and mitochondrial membranes. In response to agonist activation, TAAR1 translocates to the plasma membrane, where it associates with D2 dopamine receptors.⁴ In presynaptic terminals, TAAR1 negatively regulates the activity of the dopamine (DA)-synthesizing enzyme, tyrosine hydroxylase (TH), by reducing TH phosphorylation. Accordingly, enhanced TH phosphorylation at serine (Ser) residues 19, 31, and 40 was found in the striatum of TAAR1 knockout mice,¹⁵ whereas treatment with the TAAR agonists, β-phenylethylamine and p-tyramine, reduced Ser40 TH phosphorylation.¹⁶ In addition, presynaptic TAAR1 may inhibit the activity of the high-affinity dopamine transporter (DAT) although this is a matter of controversy.⁴ In postsynaptic elements, TAAR1 activation negatively modulates one of the 2 major signaling pathways triggered by D2 receptor activation. D2 receptors are coupled to G_i/o proteins and inhibit adenylyl cyclase activity. In response to agonist activation, phosphorylation of D2 receptors by G protein-coupled receptor kinases recruits β–arrestin-2 to the receptor-G protein complex. This, in turn, causes D2 receptor uncoupling, internalization, and intracellular signaling resulting in the activation of glycogen synthase kinase-3β (GSK3β).¹⁷ The latter pathway is inhibited in response to TAAR1 activation.¹⁸ The ensuing deactivation of GSK3β may be relevant to the treatment of SCZ.⁴

In the mouse prefrontal cortex, genetic deletion of TAAR1 reduces the expression of the GluN1 and GluN2B subunits of N-methyl-d-aspartate (NMDA) receptors and NMDA receptor-mediated synaptic responses in layer V pyramidal neurons.¹⁹ In contrast, pharmacological activation of TAAR1 enhances GluN1 expression in cultured cortical neurons.¹⁹ These findings have a high translational value considering the hypoglutamatergic hypothesis of SCZ.²⁰

The ability of TAAR1 activation to restrain meso-striatal dopaminergic transmission and enhance NMDA receptor function in the prefrontal cortex makes TAAR1 a potential drug target for the treatment of SCZ. A number of TAAR1 full and partial agonists have shown activity in preclinical models that are predictive of drug efficacy in improving positive and negative symptoms of SCZ.^14,21–27 One of these molecules, the dual TAAR1-5TH1a receptor agonist, SEP363856 or ulotaront, holds promise as one of the next drugs entering the market for the treatment of SCZ.²⁷ In a randomized, placebo-controlled, 4-week phase 2 clinical study, ulotaront reduced both positive and negative symptoms measured by the Positive and Negative Syndrome Scale (PANSS). In the open-label extension study, ulotaront maintained good therapeutic efficacy up to 26 weeks.²⁸ A phase 3 clinical trial with ulotaront is currently ongoing.²⁹ Remarkably, ulotaront did not cause extrapyramidal adverse effects or increases in body weight,^28,30 as opposed to first- and second-generation antipsychotics, respectively.

To our knowledge, there are no studies on the expression or function of TAAR1 in human brain regions in SCZ or other psychiatric disorders. We now report that TAAR1 mRNA and protein levels are up-regulated in the prefrontal cortex of individuals affected by SCZ, and TAAR1 protein levels are inversely related to the duration of antipsychotic medication.

Material and Methods

Human Brain Tissue

We examined TAAR1 mRNA and protein levels in the dorsolateral prefrontal cortex (Brodmann area 10) of 23 individuals affected by SCZ and 23 non-schizophrenic control subjects obtained from the Harvard Brain Tissue Resource Center, funded through NIH-NeuroBiobank HHSN-271-2013-00030C (see also ref. ³¹). We obtained information on age, sex, the time elapsed from death and tissue removal (“post-mortem interval”) for both individuals affected by SCZ and non-schizophrenic controls. For individuals affected by SCZ, we also obtained information on the duration of the agonal period, presence of hypoxia and/or infection during the agonal state, duration of the disease, and type and duration of antipsychotic medication. We also obtained information on other drugs used by individuals affected by SCZ and controls. The neuropathological characteristics of all samples are also reported (see supplementary table 1). We included samples with no sign of neuropathology, or with a mild/moderate vascular pathology in the subgroup of “low neuropathological burden” (LNB), and samples with tau or amyloid pathology and/or severe vascular pathology in the subgroup of “high neuropathological burden” (HNB).

Real-time PCR

RNA was extracted from 50 to 100 mg of brain tissues with Trizol reagent (Life Technologies) according to the manufacturer’s protocol in conjunction with the Pure link RNA mini kit (Cat. No. 12183018A) Thermo Fisher Scientific. Total RNA was treated with DNase and a reverse transcriptase reaction was carried out from 500 ng of total RNA with VILO kit (Invitrogen). The real-time PCR was performed on 50 ng of cDNA by TaqMan gene expression assay from Applied Biosystems using GAPDH (Hs02786624_g1) and TAAR1 (Hs00373229_s1). The reactions were performed on a Quantstudio System Q5 Applied Biosystem instrument with the following thermal cycler conditions: 2 min at 50°C, 10 min at 95°C, 45 cycles of denaturation (15 s at 95°C), and combined annealing/extension (1 min at 60°C). mRNA copy number of TAAR1 was normalized with GAPDH using the 2^−ΔΔCT method.^32,33

Western Blot Analysis

Tissue was dissected out and homogenized at 4°C in ice-cold lysis buffer, and 1 µl of homogenates were used for protein determinations. Proteins (10 µg) were resuspended in sodium dodecyl sulfate (SDS)-bromophenol blue reducing buffer containing 5% 2-mercaptoethanol and separated by electrophoresis on 10% SDS-polyacrylamide gels.³⁴ Samples were incubated at 65°C for 5 min before loading. All samples were loaded at least 2 times in different blots.

The following primary antibodies were used: rabbit polyclonal anti-TAAR1 (Novus Biologicals, NBP2-24714; 1:1000); mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology, sc-32233; 1:1000). Immunostaining was revealed by the Chemidoc computerized densitometer (Bio-Rad), and quantified by ImageLab 3.0 software (Bio-Rad). Values were always expressed as percent of CTRL LNB, except in figure 2B where they were expressed as percent of SCZ LNB.

Fig. 2.

TAAR1 protein levels in the prefrontal cortex of CTRL or SCZ with low or high neuropathological burden (LNB or HNB). Comparisons between LNB and HNB in CTRL and SCZ are shown in (A) and (B), respectively. Densitometric values are means ± SEM (in A and B, n = 9 and 14 in the LNB and HNB, respectively). *P < .05 vs the respective CTRL LNB subgroup (Student’s t-test). All immunoblots are shown in the figure.

Statistical Analysis

Statistical analysis was performed with GraphPad Prism software (Version 8; San Diego, CA, USA). Data were analyzed by Student’s t-test with Welch’s correction. The non-parametric Mann–Whitney test was applied for analysis data of figures 1C, 3D, 3F. The correlation between TAAR1 protein levels, and age, age at onset of SCZ, duration of SCZ, duration of antipsychotic medication, and post-mortem intervals was examined by linear regression analysis. A P value <.05 was considered significant.

Fig. 1.

TAAR1 mRNA levels in the prefrontal cortex of individuals affected by schizophrenia (SCZ) and no-schizophrenic controls (CTRL). Comparison between all samples of the CTRL and SCZ groups are shown in (A); comparison between samples from male and female subjects are shown in (B) and (C), respectively. Values are means ± SEM (A: n = 23 in both groups; B: n = 18 CTRL and 15 SCZ; C: n = 4 CTRL and 7 SCZ). *P < .05 vs the respective CTRL group (Student’s t-test). Correlation analysis between TAAR1 mRNA levels and age in both groups, age at onset, duration of disease, and duration of treatment in SCZ group and post-mortem intervals (PMI) in both groups is shown in (D–J), respectively.

Fig. 3.

TAAR1 protein levels are increased in the prefrontal cortex of individuals affected by schizophrenia (SCZ) as compared with CTRL subjects with low neuropathological burden (LNB). Comparison between the entire SCZ group or the SCZ LNB subgroup and the CTRL LNB subgroup is shown in (A) and (B), respectively. Comparison between the entire SCZ group and the entire CTRL group is shown in (C). Comparison between men and women in the CTRL group and in SCZ group are shown in (D) and (E), respectively. Comparison between treatment with first- and second-generation antipsychotics (FGA, SGA) in the SCZ group is shown in (F). Values are means ± SEM (A: n = 9 in CTRL LNB and 23 in SCZ; B: n = 9 in CTRL LNB and 9 in SCZ LNB, C: n = 23 in both CTRL and SCZ; D: n = 19 and 4 in CTRL men and women respectively; E: n = 16 and 7 in SCZ men and women, respectively; F: n = 4 and 11 in SCZ treated with first- and second-generation antipsychotics, respectively). *P < .05 (Student’s t-test) vs the respective CTRL LNB subgroup (A and B), SCZ men (D), or SCZ treated with first-generation antipsychotics (F). Values in (C–F) were extrapolated by immunoblots shown in figures 2A, 3A, and 3B, and expressed as percent of CTRL LNB.

Results

The analysis was carried out in dorsolateral prefrontal cortical samples obtained from 23 individuals affected by SCZ (16 males and 7 females; age, 25–62 years; mean ± SEM, 48.87 ± 2.2) and 23 non-schizophrenic controls (CTRL, 19 males, and 4 females; age, 36–64 years; mean ± SEM, 55.39 ± 1.53) (SCZ vs CTRL, Student’s t-test t_39.66 = 2.47; P = .018). Information on the duration of the disease and type of antipsychotic medication was available for 16 individuals in the SCZ group. Age at onset was reported for 15 individuals of the SCZ group (supplementary table 1). Duration of disease ranged from 1 to 46 years; mean ± SEM, 25.6 ± 3.57, and duration of treatment from 1 to 40 years; mean ± SEM, 10.72 ± 3.95. The post-mortem intervals ranged from 6.1 to 19.9 h in the SCZ group; mean ± SEM, 15.25 ± 0.9, and from 4.75 to 18.15 h in the CTRL group (mean ± SEM, 14.85 ± 0.76) (SCZ vs CTRL, Student’s t-test t_42.85 = 0.34; P = .73). Supplementary table 1 also shows records of treatments for disorders other than SCZ (mainly drugs for cardiovascular disorder and diabetes) in both groups. In addition, neuropathological characteristics of all samples, including signs of cerebrovascular disorders, amyloid burden, and tau pathology are reported in supplementary table 1. All controls and individuals affected by SCZ were subdivided into the LNB subgroup with no or LNB (9 CTRL and 9 SCZ), and the HNB subgroup with severe neuropathology (14 CTRL and 14 SCZ) (see Methods section for further details). This subdivision was made as an attempt to minimize the bias linked to a potential effect of concurrent CNS pathologies on TAAR1 expression.

Comparing all samples regardless of the neuropathological burden, we found a >2-fold increase in TAAR1 mRNA levels in the prefrontal cortex of the SCZ group (t_26.74 = 2.627; P = .0141) (figure 1A). The increase remained significant when samples from male CTRL ad SCZ were compared (t_16.66 = 2.454; P = .0255) (figure 1B). The difference between female CTRL and SCZ was not statistically significant (t₉ = 2.159; P = .0592), probably because of the low sample size in the CTRL group (n = 4) (figure 1C). There was no significant correlation between TAAR1 mRNA levels and age in both groups (CTRL, r² = 0.00563; r = 0.07503; P = .7399; SCZ, r² = 0.006135; r = 0.16025; P = .729) (figure 1D and 1E).

In the SCZ group, TAAR1 mRNA levels did not correlate with age at onset (r² = 0.02568; r = 0.16025; P = .5842), duration of the disease (r² = 0.006597; r = 0.081222; P = .8123), or duration of treatment (r² = 0.001907; r = 0.04367; P = .8772) (figure 1F–H). There was no significant correlation between TAAR1 mRNA levels and post-mortem intervals in both CTRL (r² = 0.03636; r = 0.19068; P = .3953) and SCZ (r² = 0.03810; r = 0.1952; P = .3840) (figure 1I and 1J).

We extended the analysis to TAAR1 protein levels. Immunoblots of dorsolateral prefrontal cortices showed a band at 34 kDa, corresponding to the molecular size of TAAR1 monomers. Because tau or amyloid aggregates might have affected protein synthesis in both CTRL and SCZ, we examined TAAR1 protein levels in the subgroups with LNB and HNB. In CTRL, TAAR1 protein levels were higher in the HNB subgroup (t_17.37 = 2.621; P = .0177) (figure 2A), whereas there was no difference between LNB and HNB in the SCZ group (t_20.88 = 0.3542; P = .7268) (figure 2B). TAAR1 protein levels were higher in the entire SCZ group (t_25.9 = 2.263; P = .0322) or in the SCZ LNB subgroup (t_15.58 = 2.212; P = .0423), when compared with the CTRL LNB subgroup (figure 3A and 3B). However, there was no significant difference between CTRL and SCZ when all samples were included in the analysis regardless of the neuropathological burden (t_26.74 = 1.471; P = .153) (figure 3C).

TAAR1 protein levels did not differ between men and women (t_6.83 = 1.784; P = 0.1187) in the CTRL group but were significantly higher in women than in men in the SCZ group (t_19.21 = 2.169; P = .0428) (figure 3D and 3E). TAAR1 protein levels were significantly higher in individuals affected by SCZ who had been treated exclusively with second-generation antipsychotics (clozapine, olanzapine, quetiapine, aripiprazole, and risperidone) than in those receiving first-generation antipsychotics (haloperidol, thioridazine, or fluphenazine) alone or in combination with second-generation antipsychotics (t_7.082 = 2.517; P = .0396) (figure 3F).

TAAR1 levels in CTRL and SCZ showed no significant correlation with post-mortem intervals (CTRL, r² = 0.03456; r = 0.18590; P = .3957; SCZ, r² = 0.01344; r = 0.11593; P = .5983) (figure 4A and 4B), indicating the stability of the TAAR1 protein. In addition, in the SCZ group, TAAR1 protein levels were not affected by the presence of an agonal state (score 0 = fast death/terminal phases ≤1 h; score 1 = agonal time ranging from 1 to 24 h),³⁵ or by the presence of hypoxia and/or infection during the agonal state (supplementary figure 1A–C). There was no significant difference between TAAR1 protein levels and age in both CTRL (r² = 0.1318; r = 0.3630; P = .0886) and SCZ (r² = 0.00447; r = 0.06685; P = .7618) (figure 4C and 4D) regardless of the neuropathological burden. In the SCZ group, TAAR1 protein levels did not correlate with the age at the onset of SCZ (r² = 0.01491; r = 0.1221; P = .6524) (figure 4E), and with the duration of disease (r² = 0.05962; r = 0.24417; P = .3805) (figure 4F) but showed an inverse correlation with the duration of treatment (r² = 0.412; r = 0.64187; P = .0332) (figure 4G).

Fig. 4.

Correlation between TAAR1 protein levels and number of variables in the prefrontal cortex of CTRL and SCZ. Correlation analysis of TAAR1 protein levels with post-mortem intervals in CTRL and SCZ, age in CTRL and SCZ, and age at onset, duration of disease, and duration of treatment are shown in (A–G), respectively.

Discussion

Our findings suggest that SCZ is associated with an up-regulation of TAAR1 in the prefrontal cortex, presumably as a result of an increased expression of the TAAR1-encoding gene or an increased stability of TAAR1 mRNA. We wish to highlight that our results are exploratory because there were no specified hypotheses and no corrections for multiple comparisons. The increased TAAR1 expression in the prefrontal cortex supports the development of TAAR1 ligands in the treatment of SCZ and suggests to extend the analysis to other brain regions that are relevant to the pathophysiology of SCZ.

Because TAAR1 agonists consistently showed therapeutic efficacy in clinical trials (see Introduction and References therein), the up-regulation of TAAR1 might represent a compensatory mechanism aimed at counterbalancing the primary defect in glutamatergic neurotransmission associated with SCZ. Moving from data obtained in TAAR1 knockout mice and cultured cortical neurons,¹⁹ it can be predicted that TAAR1 supports NMDA receptor expression and function in the prefrontal cortex, and, therefore, the up-regulation of TAAR1 might be considered as an attempt to correct alterations in cortical network oscillations associated with SCZ.³⁶

Measurements of the transcript or protein levels in human brain samples are biased by the different times elapsed between death and tissue removal, the neuropathological burden, the history of the disease, and the time and duration of drug treatments, including treatments for associated disorders, such as diabetes or cardiovascular disorders (see supplementary table 1). TAAR1 protein levels appeared to be relatively stable in the post-mortem period and did not change as a function of age or sex, with the exception of a slight increase found in samples obtained from women affected by SCZ. However, in the CTRL groups, TAAR1 levels were affected by the neuropathological burden and were lower in the prefrontal cortex of subjects with no or mild neuropathology. This did not occur in the SCZ group, suggesting that the increase in TAAR1 protein levels is intrinsically associated with SCZ, and there is no cumulative effect with other pathological conditions.

There was an apparent dichotomy between TAAR1 transcript and protein levels because mRNA levels were >2-fold higher in the SCZ group with respect to the entire CTRL group, whereas an increase in TAAR1 protein levels in the SCZ group could only be demonstrated when compared with the CTRL subgroup with no or mild neuropathological burden. Most CTRL with HNB showed tau or amyloid pathology, and the presence of protein aggregates might have triggered the unfolded protein response, with ensuing alterations in mRNA translation and protein synthesis.³⁷ This might contribute to explain the difference between mRNA and protein levels in our study.

Another variable is the type and duration of drug treatment, which lasted for many years in the majority of individuals in the SCZ group. Interestingly, TAAR1 levels were significantly higher in samples from patients affected by SCZ who had been treated with second-generation antipsychotics, although this finding should be interpreted with caution because of the low number of patients who received first-generation antipsychotics.

There was an inverse correlation between the duration of antipsychotic medication and TAAR1 protein levels. This suggests (but does not prove) that the increase in TAAR1 expression found in the SCZ group does not represent a long-term adaptation mechanism caused by antipsychotic medication (in this case, we would have expected a positive correlation between the duration of treatment and protein levels). The use of antipsychotics in preclinical models of SCZ, or the detection of TAAR1 in drug-naïve patients affected by SCZ (by PET imaging or analysis of CNS-derived exosomes) may help to establish the role of antipsychotics on TAAR1 expression.

In conclusion, we have shown for the first time that TAAR1, which is targeted by novel antipsychotic medications, is up-regulated in the dorsolateral prefrontal cortex of individuals affected by SCZ. Comparisons between CTRL and SCZ subgroups with low or HNB suggest that TAAR1 is highly responsive to pathology and that alterations found in SCZ are not additive to those found in response to tau or amyloid pathology. The increased expression of TAAR1 supports the use of TAAR1 agonists in SCZ, which might act to reinforce a compensatory mechanism that is otherwise insufficient to correct the glutamatergic deficit in the prefrontal cortex. We cannot exclude that the presence of TAAR1 gene variants associated with SCZ^10–12 might have contributed to the up-regulation of TAAR1 in the SCZ group. A much larger sample size is required to examine this possibility.

Funding

The study has been funded by the Italian Ministry of Health within the context of “Ricerca Corrente” of the IRCCS Neuromed.