Associations of psychiatric disease and ageing with FKBP5 expression converge on superficial layer neurons of the neocortex.

Identification and characterisation of novel targets for treatment is a priority in the field of psychiatry. FKBP5 is a gene with decades of evidence suggesting its pathogenic role in a subset of psychiatric patients, with potential to be leveraged as a therapeutic target for these individuals. While it is widely reported that FKBP5/FKBP51 mRNA/protein (FKBP5/1) expression is impacted by psychiatric disease state, risk genotype and age, it is not known in which cell types and sub-anatomical areas of the human brain this occurs. This knowledge is critical to propel FKBP5/1-targeted treatment development. Here, we performed an extensive, large-scale postmortem study (n = 1024) of FKBP5/1, examining neocortical areas (BA9, BA11 and ventral BA24/BA24a) derived from subjects that lived with schizophrenia, major depression or bipolar disorder. With an extensive battery of RNA (bulk RNA sequencing, single-nucleus RNA sequencing, microarray, qPCR, RNAscope) and protein (immunoblot, immunohistochemistry) analysis approaches, we thoroughly investigated the effects of disease state, ageing and genotype on cortical FKBP5/1 expression including in a cell type-specific manner. We identified consistently heightened FKBP5/1 levels in psychopathology and with age, but not genotype, with these effects strongest in schizophrenia. Using single-nucleus RNA sequencing (snRNAseq; BA9 and BA11) and targeted histology (BA9, BA24a), we established that these disease and ageing effects on FKBP5/1 expression were most pronounced in excitatory superficial layer neurons of the neocortex, and this effect appeared to be consistent in both the granular and agranular areas examined. We then found that this increase in FKBP5 levels may impact on synaptic plasticity, as FKBP5 gex levels strongly and inversely correlated with dendritic mushroom spine density and brain-derived neurotrophic factor (BDNF) levels in superficial layer neurons in BA11. These findings pinpoint a novel cellular and molecular mechanism that has potential to open a new avenue of FKBP51 drug development to treat cognitive symptoms in psychiatric disorders.

Changed cortical risk gene expression in major depression and shared changes in cortical gene expression between major depression and bipolar disorders.

BACKGROUND: Mood disorders likely occur in someone with a genetic predisposition who encounters a deleterious environmental factor leading to dysregulated physiological processes due to genetic mutations and epigenetic mechanisms altering gene expression. To gain data to support this hypothesis, we measured levels of gene expression in three cortical regions known to be affected by the pathophysiologies of major depression and bipolar disorders. METHODS: Levels of RNA were measured using the Affymetrix™ Human Exon 1.0 ST Array in Brodmann's areas 9, 10 and 33 (left hemisphere) from individuals with major depression, bipolar disorder and age- and sex-matched controls with changed expression taken as a fold change in RNA ⩾1.2 at p < 0.01. Data were analysed using JMP® genomics 6.0 and the probable biological consequences of changes in gene expression determined using Core and Pathway Designer Analyses in Ingenuity Pathway Analysis. RESULTS: There were altered levels of RNA in Brodmann's area 9 (major depression = 424; bipolar disorder = 331), Brodmann's area 10 (major depression = 52; bipolar disorder = 24) and Brodmann's area 33 (major depression = 59 genes; bipolar disorder = 38 genes) in mood disorders. No gene was differentially expressed in all three regions in either disorder. There was a high correlation between fold changes in levels of RNA from 112 genes in Brodmann's area 9 from major depression and bipolar disorder (r2 = 0.91, p < 0.001). Levels of RNA for four risk genes for major depression were lower in Brodmann's area 9 in that disorder. CONCLUSION: Our data argue that there are complex regional-specific changes in cortical gene expression in major depression and bipolar disorder that includes the expression of some risk genes for major depression in those with that disorder. It could be hypothesised that the common changes in gene expression in major depression and bipolar disorder are involved in the genesis of symptoms common to both disorders.

Catechol-O-methyltransferase (COMT) genotypes are associated with varying soluble, but not membrane-bound COMT protein in the human prefrontal cortex.

Catechol-O-methyltransferase (COMT) is an enzyme that catalyses the O-methylation, and thereby the inactivation, of catechol-containing molecules. In humans, it has been suggested that COMT modulates cognitive ability, possibly by regulating degradation of dopamine in the prefrontal cortex. Hence, it is significant that two COMT SNPs, rs4680 (c.472 G > A, p.Val158Met) and rs4818 (c.408 C > G), have been associated with cognitive ability in humans. We have shown these SNPs to be associated with levels of muscarinic M1 receptor mRNA in human cortex, which is significant as that receptor also regulates cognitive ability. We decided to determine if COMT genotype was associated with varying levels of COMT protein, as this could be a mechanism by which COMT genotype could be associated with changes in muscarinic M1 receptor mRNA levels. Hence, we measured COMT levels in prefrontal cortex obtained postmortem from 199 subjects, some of whom had a history of schizophrenia, major depressive disorders or bipolar disorders. Our data show, independent of diagnostic status, that genotype at rs4680 and rs4818, but not at rs737865 and rs165599, is associated with differing levels of soluble COMT (S-COMT), but not membrane-bound COMT (MB-COMT). These findings suggest that the association between COMT polymorphisms and cognitive functioning could be, at least in part, due to their association with varying levels of S-COMT. This is important as, unlike MB-COMT, the substrates targeted by S-COMT are likely to be intra-cellular rather than, like dopamine, located mainly in the synaptic vesicles or the extra-cellular space.

Studies on Prostaglandin-Endoperoxide Synthase 1: Lower Levels in Schizophrenia and After Treatment with Antipsychotic Drugs in Conjunction with Aspirin.

Background: Antipsychotic drugs plus aspirin (acetylsalicylic acid), which targets prostaglandin-endoperoxide synthase 1 (PTGS1: COX1), improved therapeutic outcomes when treating schizophrenia. Our microarray data showed higher levels of PTGS1 mRNA in the dorsolateral prefrontal cortex from subjects with schizophrenia of long duration of illness, suggesting aspirin plus antipsychotic drugs could have therapeutic effects by lowering PTGS1 expression in the cortex of subjects with the disorder. Methods: We used Western blotting to measure levels of PTSG1 protein in human postmortem CNS, rat and mouse cortex, and cells in culture. Results: Compared with controls, PTGS1 levels were 41% lower in the dorsolateral prefrontal cortex (P<.01), but not the anterior cingulate or frontal pole, from subjects with schizophrenia. Levels of PTGS1 were not changed in the dorsolateral prefrontal cortex in mood disorders or in the cortex of rats treated with antipsychotic drugs. There was a strong trend (P=.05) to lower cortical PTGS1 10 months after mice were treated postnatally with polyinosinic-polycytidylic acid sodium salt (Poly I:C), consistent with cortical PTGS1 being lower in adult mice after exposure to an immune activator postnatally. In CCF-STTG1 cells, a human-derived astrocytic cell line, aspirin caused a dose-dependent decrease in PTGS1 that was decreased further with the addition of risperidone. Conclusions: Our data suggest low levels of dorsolateral prefrontal cortex PTGS1 could be associated with the pathophysiology of schizophrenia, and improved therapeutic outcome from treating schizophrenia with antipsychotic drugs augmented with aspirin may be because such treatment lowers cortical PTGS1.

Low levels of muscarinic M1 receptor-positive neurons in cortical layers III and V in Brodmann areas 9 and 17 from individuals with schizophrenia.

BACKGROUND: Results of neuroimaging and postmortem studies suggest that people with schizophrenia may have lower levels of muscarinic M1 receptors (CHRM1) in the cortex, but not in the hippocampus or thalamus. Here, we use a novel immunohistochemical approach to better understand the likely cause of these low receptor levels. METHODS: We determined the distribution and number of CHRM1-positive (CHRM1+) neurons in the cortex, medial dorsal nucleus of the thalamus and regions of the hippocampus from controls (n = 12, 12 and 5, respectively) and people with schizophrenia (n = 24, 24 and 13, respectively). RESULTS: Compared with controls, levels of CHRM1+ neurons in people with schizophrenia were lower on pyramidal cells in layer III of Brodmann areas 9 (-44%) and 17 (-45%), and in layer V in Brodmann areas 9 (-45%) and 17 (-62%). We found no significant differences in the number of CHRM1+ neurons in the medial dorsal nucleus of the thalamus or in the hippocampus. LIMITATIONS: Although diagnostic cohort sizes were typical for this type of study, they were relatively small. As well, people with schizophrenia were treated with antipsychotic drugs before death. CONCLUSION: The loss of CHRM1+ pyramidal cells in the cortex of people with schizophrenia may underpin derangements in the cholinergic regulation of GABAergic activity in cortical layer III and in cortical/subcortical communication via pyramidal cells in layer V.

Low levels of muscarinic M1 receptor-positive neurons in cortical layers III and V in Brodmann areas 9 and 17 from individuals with schizophrenia.

BACKGROUND: Results of neuroimaging and postmortem studies suggest that people with schizophrenia may have lower levels of muscarinic M1 receptors (CHRM1) in the cortex, but not in the hippocampus or thalamus. Here, we use a novel immunohistochemical approach to better understand the likely cause of these low receptor levels. METHODS: We determined the distribution and number of CHRM1-positive (CHRM1+) neurons in the cortex, medial dorsal nucleus of the thalamus and regions of the hippocampus from controls (n = 12, 12 and 5, respectively) and people with schizophrenia (n = 24, 24 and 13, respectively). RESULTS: Compared with controls, levels of CHRM1+ neurons in people with schizophrenia were lower on pyramidal cells in layer III of Brodmann areas 9 (-44%) and 17 (-45%), and in layer V in Brodmann areas 9 (-45%) and 17 (-62%). We found no significant differences in the number of CHRM1+ neurons in the medial dorsal nucleus of the thalamus or in the hippocampus. LIMITATIONS: Although diagnostic cohort sizes were typical for this type of study, they were relatively small. As well, people with schizophrenia were treated with antipsychotic drugs before death. CONCLUSION: The loss of CHRM1+ pyramidal cells in the cortex of people with schizophrenia may underpin derangements in the cholinergic regulation of GABAergic activity in cortical layer III and in cortical/subcortical communication via pyramidal cells in layer V.

Higher levels of different muscarinic receptors in the cortex and hippocampus from subjects with Alzheimer's disease.

It has been suggest that drugs specifically targeting muscarinic receptors will be useful in treating Alzheimer's disease. We decided to determine if the response to such drugs may be altered, because of changes in the levels of muscarinic receptors in the CNS from subjects with the disorder. We used in situ radioligand binding with autoradiography to measure the levels of [3H]pirenzepine binding to muscarinic M1 receptors, [3H]AF-DX 386 binding to muscarinic M1, M2, and M4 receptors, and [3H]4-DAMP binding to muscarinic M1 and M3 receptors in the dorsolateral prefrontal cortex and hippocampus from subjects with Alzheimer's and age/sex-matched controls. Compared with controls, [3H]pirenzepine binding was higher in the dentate gyrus from subjects with Alzheimer's disease. [3H]AF-DX 386 binding was higher in the subiculum and parahippocampal gyrus from subjects with the disorder. In Alzheimer's disease, [3H]-DAMP binding was higher in the dorsolateral prefrontal cortex but not different in the hippocampus. Our data show complex changes in the levels of muscarinic receptors in the CNS from subjects with Alzheimer's disease which may affect clinical response to treatment with drugs-targeting these receptors.

Muscarinic receptor binding changes in postmortem Parkinson's disease.

Parkinson's disease (PD) is a devastating disorder, affecting approximately 2% of people aged 60 and above. It is marked by progressive neurodegeneration that has long been known to impact dopaminergic cells and circuits, but more recently the acetylcholine system has also been implicated in the complex aetiology and symptomatology of the disease. While broad changes in cholinergic markers have been described, insight into the contribution of specific acetylcholine receptors is less clear. To address this important unknown, in this study we performed [3H] pirenzepine, [3H] 4DAMP, and [3H] AF-DX 384 in situ radioligand binding on postmortem tissues from Brodmann's area 6, 9, 46, and the caudate putamen, from PD and matched controls to detect muscarinic M1, M3, and M1/2/4 receptors, respectively. We found no difference in [3H] pirenzepine binding between PD and controls across all regions assessed. [3H] 4DAMP binding was found to be higher in PD CPu and BA9 than in controls. [3H] AF-DX 384 was higher in BA9 of PD compared with controls. In sum, we show selective increase in M3 receptors in cortical and subcortical regions, as well as increased M2/M4 in cortical area BA9, which together support a role for cholinergic dysfunction in PD.

Changes in Muscarinic M2 Receptor Levels in the Cortex of Subjects with Bipolar Disorder and Major Depressive Disorder and in Rats after Treatment with Mood Stabilisers and Antidepressants.

BACKGROUND: Increasingly, data are implicating muscarinic receptors in the aetiology and treatment of mood disorders. This led us to measure levels of different muscarinic receptor-related parameters in the cortex from people with mood disorders and the CNS of rats treated with mood stabilisers and antidepressant drugs. METHODS: We measured [(3)H]AF-DX 384 binding in BA 46 and BA 24 from subjects with bipolar disorders (n = 14), major depressive disorders (n = 19), as well as age- and sex-matched controls (n = 19) and the CNS of rats treated with fluoxetine or imipramine. In addition, we used Western blots to measure levels of CHRM2 protein and oxotremorine-M stimulated [(35)S]GTPγS binding as a measure of CHRM 2 / 4 signaling. RESULTS: Compared with controls, [(3)H]AF-DX 384 binding was lower in BA 24 and BA 46 in bipolar disorders and major depressive disorders, while CHRM2 protein and oxotremorine-M stimulated [(35)S]GTPγS binding was only lower in BA 24. Compared with vehicle, treatment with mood stabilisers, antidepressant drugs for 10 days, or imipramine for 28 days resulted in higher levels of in [(3)H]AF-DX 384 binding select regions of rat CNS. CONCLUSIONS: Our data suggest that levels of CHRM2 are lower in BA 24 from subjects with mood disorders, and it is possible that signalling by that receptor is also less in this cortical region. Our data also suggest increasing levels of CHRM2 may be involved in the mechanisms of action of mood stabilisers and tricyclic antidepressants.

Validating reference genes using minimally transformed qpcr data: findings in human cortex and outcomes in schizophrenia.

BACKGROUND: It is common practice, when using quantitative real time polymerase chain reaction (qPCR), to normalise levels of mRNA to reference gene mRNA which, by definition, should not vary between tissue, with any disease aetiology or after drug treatments. The complexity of human CNS means it unlikely that any gene could fulfil these criteria. METHODS: To address this issue we measured levels of mRNA for six potential reference genes (GAPDH, PPIA, SNCA, NOL9, TFB1M and SKP1) in three cortical regions (Brodmann's areas (BA) 8, 9 and 44) from 30 subjects with schizophrenia and 30 age and sex matched controls. We used a structured statistical approach to examine the characteristics of these data to determine their suitability as reference genes. We also analysed our data using reference genes selected by rank as defined using the average of the standard deviation of pair-gene ΔCt and the BestKeeper, NormFinder and geNorm algorithms to determine if they suggested the same reference genes. RESULTS: Our minimally derived data showed that levels of mRNA for all of the six genes varied between cortical regions and therefore no gene fulfilled the absolute requirements for use as reference genes. As levels of some mRNA for some genes did not vary with diagnoses within a cortical region from subjects with schizophrenia compared to controls, we normalised levels of mRNA for all the other genes to mRNA for one, two or three reference genes in each cortical region. This showed that using the geometric mean of at least two reference genes gave more reproducible results. Finally, using the reference gene ranking protocols the average of the standard deviation of pair-gene ΔCt, BestKeeper, NormFinder and geNorm we showed that these approaches ranked potential reference genes differently. We then showed that outcomes of comparing data from subjects with schizophrenia and controls varied depending on the reference genes chosen. CONCLUSIONS: Our data shows that the selection of reference genes is a significant component of qPCR study design and therefore the process by which reference genes are selected must be clearly listed as a potential confound in studying gene expression in human CNS. This should include showing that, using minimally derived qPCR data, levels of mRNA for proposed reference genes does not vary with variables such as diagnoses and CNS region.

Changes in cortical N-methyl-D-aspartate receptors and post-synaptic density protein 95 in schizophrenia, mood disorders and suicide.

OBJECTIVES: In humans, depending on dose, blocking the N-methyl-D-aspartate receptor (NMDAR) with ketamine can cause psychomimetic or antidepressant effects. The overall outcome for drugs such as ketamine depends on dose and the number of its available binding sites in the central nervous system, and to understand something of the latter variable we measure NMDAR in the frontal pole, dorsolateral prefrontal, anterior cingulate and parietal cortices from people with schizophrenia, bipolar disorder, major depressive disorders and age/sex matched controls. METHOD: We measured levels of NMDARs (using [(3)H]MK-801 binding) and NMDAR sub-unit mRNAs (GRINs: using in situ hybridisation) as well as post-synaptic density protein 95 (anterior cingulate cortex only; not major depressive disorders: an NMDAR post-synaptic associated protein) in bipolar disorder, schizophrenia and controls. RESULTS: Compared to controls, levels of NMDAR were lower in the outer laminae of the dorsolateral prefrontal cortex (-17%, p = 0.01) in people with schizophrenia. In bipolar disorder, levels of NMDAR binding (laminae IV-VI; -19%, p < 0.01) and GRIN2C mRNA (laminae I-VI; -27%, p < 0.05) were lower in the anterior cingulate cortex and NMDAR binding was lower in the outer lamina IV of the dorsolateral prefrontal cortex (-19%, p < 0.01). In major depressive disorders, levels of GRIN2D mRNA were higher in frontal pole (+22%, p < 0.05). In suicide completers, levels of GRIN2B mRNA were higher in parietal cortex (+20%, p < 0.01) but lower (-35%, p = 0.02) in dorsolateral prefrontal cortex while post-synaptic density protein 95 was higher (+26%, p < 0.05) in anterior cingulate cortex. CONCLUSION: These data suggest that differences in cortical NMDAR expression and post-synaptic density protein 95 are present in psychiatric disorders and suicide completion and may contribute to different responses to ketamine.

COMT genotype is associated with differential expression of muscarinic M1 receptors in human cortex.

Catechol-O-methyltransferase (COMT) genotype has been associated with varying levels of cognitive functioning and an altered risk of schizophrenia. COMT regulates the breakdown of catecholamines, particularly dopamine, which is thought critical in maintaining cognitive function and the aetiology of schizophrenia. This hypothesis gained support from reports that the VAL allele at rs4680 was associated with poorer performance on cognitive tests and a slightly increased risk of schizophrenia. More recently, genotype at rs4818, part of a hapblock with rs4680, has been shown to impact on cognitive ability more than genotype at rs4680 but, as yet, not the risk for schizophrenia. Here, we determined if COMT genotype at rs4680 or rs4818, as well as rs165519 and rs737865, two synonymous single nucleotide polymorphisms (SNPs) with no known functional consequences, were associated with an altered risk of schizophrenia and if genotype at the four COMT SNPs was related to expression of the cortical muscarinic M1 receptor (CHRM1) because the expression of the cortical CHRM1 has been reported to be lower in schizophrenia and is important in maintaining cognitive functioning in humans. We report that the variation in gene sequence at the four COMT SNPs studied was not associated with an altered the risk of schizophrenia but genotype at rs4680 and rs4818, but not rs165519 and rs737865, were associated with varying levels of cortical CHRM1 expression in the human dorsolateral prefrontal cortex (DLPFC). These data are the first to suggest that levels of CHRM1 in the human DLPFC are, in part, determined by COMT gene sequence. © 2016 Wiley Periodicals, Inc.

Biomarkers for Psychiatry: The Journey from Fantasy to Fact, a Report of the 2013 CINP Think Tank.

BACKGROUND: A think tank sponsored by the Collegium Internationale Neuropsychopharmacologium (CINP) debated the status and prospects of biological markers for psychiatric disorders, focusing on schizophrenia and major depressive disorder. METHODS: Discussions covered markers defining and predicting specific disorders or domains of dysfunction, as well as predicting and monitoring medication efficacy. Deliberations included clinically useful and viable biomarkers, why suitable markers are not available, and the need for tightly-controlled sample collection. RESULTS: Different types of biomarkers, appropriate sensitivity, specificity, and broad-based exploitability were discussed. Whilst a number of candidates are in the discovery phases, all will require replication in larger, real-life cohorts. Clinical cost-effectiveness also needs to be established. CONCLUSIONS: Since a single measure is unlikely to suffice, multi-modal strategies look more promising, although they bring greater technical and implementation complexities. Identifying reproducible, robust biomarkers will probably require pre-competitive consortia to provide the resources needed to identify, validate, and develop the relevant clinical tests.

Lower cortical serotonin 2A receptors in major depressive disorder, suicide and in rats after administration of imipramine.

We have attempted to replicate studies showing higher levels of serotonin 2A receptors (HTR2A) in the cortex of people with mood disorders and to determine the effects of treating rats with antidepressant drugs on levels of that receptor. In situ [3H]ketanserin binding and autoradiography was used to measure levels of HTR2A in Brodmann's area (BA) 46 and 24 from people with major depressive disorders (MDD, n = 16), bipolar disorders (BD, n = 14) and healthy controls (n = 14) as well as the central nervous system (CNS) of rats (20 per treatment arm) treated for 10 or 28 d with fluoxetine (10 mg/kg/d) or imipramine (20 mg/kg/d). Compared with controls, HTR2A were lower in BA 24, but not BA 46, from people with MDD (p = 0.005); HTR2A were not changed in BD. Levels of HTR2A were lower in BA 24 (p = 0.007), but not BA 46, from people who had died by suicide. Finally, levels of HTR2A were lower in the CNS of rats treated with imipramine, but not fluoxetine, for 28 d, but not 10 d. From our current and previous data we conclude cortical HTR2A are lower in schizophrenia, MDD, people with mood disorders who died by suicide, rats treated with some antipsychotic or some antidepressant drugs. As levels of cortical HTR2A can be affected by the aetiologies of different disorders and mechanisms of action of different drugs, a better understanding of how such changes can occur needs to be elucidated.

Problems and solutions to filling the drying drug pipeline for psychiatric disorders: a report from the inaugural 2012 CINP Think Tank.

The inaugural Collegium Internationale Neuro-Psychopharmacologicum (CINP) Think Tank, a small open meeting sponsored by the CINP, discussed impediments to developing new drugs for psychiatric disorders and approaches to overcome these impediments. Whilst neuropsycharmacology has a rich pharmacopeia (current treatments benefiting many individuals), issues of treatment resistance, sub-optimal response and unwanted side effects remain problematic. Many scientific, economic and social issues are impeding the development of drugs (e.g. higher risk of failure, placebo effects, problematic regulatory environments, pressures imposed by patent protection, downward pressure on reimbursements and financial, legal and social risk aversion). A consensus of the meeting was that efforts to understanding the core pathophysiology of psychiatric disorders are fundamental to increasing the chance of developing new drugs. However, findings from disorders such as Huntington's chorea, have shown that knowing the cause of a disorder may not reveal new drug targets. By contrast, clinically useful biomarkers that define target populations for new drugs and models that allow findings to be accurately translated from animals to humans will increase the likelihood of developing new drugs. In addition, a greater accent on experimental medicine, creative clinical investigations and improved communication between preclinical neuropsychopharmacologists, clinicians committed to neuropsychopharmacological research, industry and the regulators would also be a driver to the development of new treatments. Finally, it was agreed that the CINP must continue its role as a conduit facilitating vibrant interactions between industry and academia as such communications are a central component in identifying new drug targets, developing new drugs and transitioning new drugs into the clinic.

Common changes in rat cortical gene expression after valproate or lithium treatment particularly affect pre- and post-synaptic pathways that regulate four neurotransmitters systems.

OBJECTIVES: We have postulated that common changes in gene expression after treatment with different therapeutic classes of psychotropic drugs contribute to their common therapeutic mechanisms of action. METHODS: To test this hypothesis, we measured levels of cortical coding and non-coding RNA using GeneChip® Rat Exon 1.0 ST Array after treatment with vehicle (chow only), chow containing 1.8 g lithium carbonate/kg (n = 10) or chow containing 12 g sodium valproate/kg (n = 10) for 28 days. Differences in levels of RNA were identified using JMP Genomics 13 and the Panther Gene Ontology Classification System was used to identify potential consequences of RNA. RESULTS: Compared to vehicle treatment, levels of cortical RNA for 543 and 583 coding and non-coding RNAs were different after treatment with valproate and lithium, respectively. Moreover, levels of 323 coding and non-coding RNAs were altered in a highly correlated way by treatment with valproate and lithium, changes that would impact on cholinergic, glutamatergic, serotonergic and dopaminergic neurotransmission as well as on voltage gated ion channels. CONCLUSIONS: Our study suggests that treating with mood stabilisers cause many common changes in levels of RNA which will impact on CNS function, particularly affecting post-synaptic muscarinic receptor functioning and the release of multiple neurotransmitters.

Common changes in rat cortical gene expression after antidepressant drug treatment: Impacts on metabolism of polyamines, mRNA splicing, regulation of RAS by GAPs, neddylation and GPCR ligand binding.

OBJECTIVES: This study sought to identify pathways affected by rat cortical RNA that were changed after treatment with fluoxetine or imipramine. METHODS: We measured levels of cortical RNA in male rats using GeneChip® Rat Exon 1.0 ST Array after treatment with vehicle (0.9% NaCl), fluoxetine (10 mg/kg/day) or imipramine (20 mg/kg/day) for 28 days. Levels of coding and non-coding RNA in vehicle treated rats were compared to those in treated rats using ANOVA in JMP Genomics 13 and the Panther Gene Ontology Classification System was used to identify pathways involving the changed RNAs. RESULTS: 18,876 transcripts were detected; there were highly correlated changes in 1010 levels of RNA after both drug treatments that would principally affect the metabolism of polyamines, mRNA splicing, regulation of RAS by GAPs, neddylation and GPCR ligand binding. Using our previously published data, we compared changes in transcripts after treatment with antipsychotic and mood stabilising drugs. CONCLUSIONS: Our study shows there are common, correlated, changes in coding and non-coding RNA in the rat cortex after treatment with fluoxetine or imipramine; we propose the pathways affected by these changes are involved in the therapeutic mechanisms of action of antidepressant drugs.

Lower levels of soluble ß-amyloid precursor protein, but not ß-amyloid, in the frontal cortex in schizophrenia.

We identified a sub-group (25%) of people with schizophrenia (muscarinic receptor deficit schizophrenia (MRDS)) that are characterised because of markedly lower levels of cortical muscarinic M1 receptors (CHRM1) compared to most people with the disorder (non-MRDS). Notably, bioinformatic analyses of our cortical gene expression data shows a disturbance in the homeostasis of a biochemical pathway that regulates levels of CHRM1. A step in this pathway is the processing of ß-amyloid precursor protein (APP) and therefore we postulated there would be altered levels of APP in the frontal cortex from people with MRDS. Here we measure levels of CHRM1 using [3H]pirenzepine binding, soluble APP (sAPP) using Western blotting and amyloid beta peptides (Aß1-40 and Aß1-42) using ELISA in the frontal cortex (Brodmann's area 6: BA 6; MRDS = 14, non-MRDS = 14, controls = 14). We confirmed the MRDS cohort in this study had the expected low levels of [3H]pirenzepine binding. In addition, we showed that people with schizophrenia, independent of their sub-group status, had lower levels of sAPP compared to controls but did not have altered levels of Aß1-40 or Aß1-42. In conclusion, whilst changes in sAPP are not restricted to MRDS our data could indicate a role of APP, which is important in axonal and synaptic pruning, in the molecular pathology of the syndrome of schizophrenia.

Widespread decreases in cortical muscarinic receptors in a subset of people with schizophrenia.

These studies were undertaken to investigate the selectivity of cortical muscarinic receptor radioligand binding in muscarinic M(1) and M(4) receptor knockout mice and to determine whether a marked decrease in [(3)H]pirenzepine binding in Brodmann's area (BA) 9 from a subset of people with schizophrenia was predictive of decreased muscarinic receptors in other central nervous system (CNS) regions. Our data show that, under the conditions used, [(3)H]pirenzepine binding was highly selective for the muscarinic M(1) receptor whereas both [(3)H]AF-DX 386 and [(3)H]4DAMP had less discriminatory power. In addition, the data suggest that a marked decrease in [(3)H]pirenzepine binding in BA 9 from a subset of people with schizophrenia is predictive of decreases in muscarinic receptors in other CNS regions. However, there were some region-specific decreases in muscarinic receptors in tissue from people with schizophrenia who were outside this subset. These data add to a growing body of evidence suggesting there are widespread decreases in muscarinic receptors in the CNS of some subjects with schizophrenia, as demonstrated by neuroimaging. Our data have implications for understanding the potential clinical utility of drugs directed at the orthosteric and allosteric sites of muscarinic receptors to treat schizophrenia.

Lower levels of cortical [3H]pirenzepine binding to postmortem tissue defines a sub-group of older people with schizophrenia with less severe cognitive deficits.

Multiple lines of evidence argue for lower levels of cortical muscarinic M1 receptors (CHRM1) in people with schizophrenia which is possibly due to a sub-group within the disorder who have a marked loss of CHRM1 (muscarinic receptor deficit sub-group (MRDS)). In this study we sought to determine if the lower levels of CHRM1 was apparent in older people with schizophrenia and whether the loss of CHRM1 was associated with symptom severity by measuring levels of cortical [3H]pirenzepine binding to CHRM1 from 56 people with schizophrenia and 43 controls. Compared to controls (173 ± 6.3 fmol / mg protein), there were lower levels of cortical [3H]pirenzepine binding in the people with schizophrenia (mean ± SEM: 153 ± 6.0 fmol / mg protein; p = 0.02; Cohen's d = - 0.46). [3H]pirenzepine binding in the people with schizophrenia, but not controls, was not normally distributed and best fitted a two-population solution. The nadir of binding separating the two groups of people with schizophrenia was 121 fmol / mg protein and levels of [3H]pirenzepine binding below this value had a 90.7 % specificity for the disorder. Compared to controls, the score from the Clinical Dementia Rating Scale (CDR) did not differ significantly in MRDS but were significantly higher in the sub-group with normal radioligand binding. Positive and Negative Syndrome Scale scores did not differ between the two sub-groups with schizophrenia. Our current study replicates and earlier finding showing a MRDS within schizophrenia and, for the first time, suggest this sub-group have less severe cognitive deficits others with schizophrenia.