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.

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.

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.

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.

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.

Evidence that a working memory cognitive phenotype within schizophrenia has a unique underlying biology.

It is suggested studying phenotypes within the syndrome of schizophrenia will accelerate understanding the complex molecular pathology of the disorder. Supporting this hypothesis, we have identified a sub-group within schizophrenia with impaired working memory (WM) and have used Affymetrix™ Human Exon 1.0 ST Arrays to compare their blood RNA levels (n=16) to a group of with intact WM (n=18). Levels of 72 RNAs were higher in blood from patients with impaired WM, 11 of which have proven links to the maintenance of different aspects of working memory (cognition). Overall, changed gene expression in those with impaired WM could be linked to cognition through glutamatergic activity, olfaction, immunity, inflammation as well as energy and metabolism. Our data gives preliminary support to the hypotheses that there is a working memory deficit phenotype within the syndrome of schizophrenia with has a biological underpinning. In addition, our data raises the possibility that a larger study could show that the specific changes in gene expression we have identified could prove to be the biomarkers needed to develop a blood test to identify those with impaired WM; a significant step toward allowing the use of personalised medicine directed toward improving their impaired working memory.

Changes in cortical gene expression in the muscarinic M1 receptor knockout mouse: potential relevance to schizophrenia, Alzheimer's disease and cognition.

Postmortem and neuroimaging studies show low levels of cortical muscarinic M1 receptors (CHRM1) in patients with schizophrenia which is significant because CHRM signalling has been shown to change levels of gene expression and cortical gene expression is altered in schizophrenia. We decided to identify CHRM1-mediated changes in cortical gene expression by measuring levels of RNA in the cortex of the Chrm1-/- mouse (n = 10), where there would be no signalling by that receptor, and in wild type mouse (n = 10) using the Affymetrix Mouse Exon 1.0 ST Array. We detected RNA for 15,501 annotated genes and noncoding RNA of which 1,467 RNAs were higher and 229 RNAs lower in the cortex of the Chrm1-/- mouse. Pathways and proteins affected by the changes in cortical gene expression in the Chrm1-/- are linked to the molecular pathology of schizophrenia. Our human cortical gene expression data showed 47 genes had altered expression in Chrm1-/- mouse and the frontal pole from patients with schizophrenia with the change in expression of 44 genes being in opposite directions. In addition, genes with altered levels of expression in the Chrm1-/- mouse have been shown to affect amyloid precursor protein processing which is associated with the pathophysiology of Alzheimer's disease, and 69 genes with altered expression in the Chrm1-/- mouse are risk genes associated with human cognitive ability. Our findings argue CHRM1-mediated changes in gene expression are relevant to the pathophysiologies of schizophrenia and Alzheimer's disease and the maintenance of cognitive ability in humans.

Cortical expression of the RAPGEF1 gene in schizophrenia: investigating regional differences and suicide.

Rap guanine nucleotide exchange factor 1 (RAPGEF1) is involved in cell adhesion and neuronal migration. Previously we found lower RAPGEF1 mRNA levels in Brodmann's area (BA) 9 in subjects with schizophrenia compared to controls. This study aimed to determine whether RAPGEF1 expression was altered in other brain regions implicated in schizophrenia and whether this was associated with suicide. Using qPCR, we measured the levels of RAPGEF1 in post-mortem BA 8 and 44 from 27 subjects with schizophrenia and 26 non-psychiatric control subjects. To address the effect of antipsychotic treatments, Rapgef1 mRNA levels were measured in the cortex from rats treated with typical antipsychotic drugs. There was no difference in RAPGEF1 normalised relative expression levels in BA 8 or 44. However, in BA 8, schizophrenia subjects had higher raw Ct RAPGEF1 levels compared to controls. There were higher RAPGEF1 levels in suicide completers compared to non-suicide schizophrenia subjects in BA 8. Rapgef1 expression levels in the rat cortex did not vary with antipsychotic treatment. Our findings suggest changes in RAPGEF1 expression may be limited to the dorsolateral prefrontal cortex from subjects with schizophrenia. Further investigation of the function of RAPGEF1 may lead to a greater understanding of the pathophysiology of schizophrenia.

Higher levels of α7 nicotinic receptors, but not choline acetyltransferase, in the dorsolateral prefrontal cortex from a sub-group of patients with schizophrenia.

It has been suggested the study of sub-groups within the syndrome of schizophrenia will assist in elucidating the complex pathophysiology of the syndrome. Hence, we have studied a number of cholinergic markers in the cortex from a sub-group of subjects with schizophrenia that have a marked decrease in levels of muscarinic M1 receptors (MRDS). The displacement of [3H]NMS by cortical extracts was used to measure tissue anticholinergic load, [125I]α bungarotoxin binding was used to measure levels of the α7 nicotinic receptor (CHRNA7) and western blotting was used to measure levels of choline acetyltransferase (ChAT) 68 and 82 as well as synaptosome nerve-associated protein 25 (SNAP25). In comparing schizophrenia, MRDS and non-MRDS to controls, there were no differences in levels of ChAT 68 or 82, SNAP 25 or cholinergic load in BA 9. However, levels of CHRNA7 were higher in BA 9, but not BA 6 or 44, from subjects with MRDS. These data argue that there is no change in cholinergic innovation (measured using ChAT), presynaptic neurons (measured using SNAP25) or cholinergic load in schizophrenia, MRDS or non-MRDS. However, increased levels of CHRNA7 may be contributing to a breakdown in cholinergic homeostasis in BA 9, but not BA 6 or 44, in subjects with MRDS.

Muscarinic M1 and M4 receptors: Hypothesis driven drug development for schizophrenia.

The finding that the drug KarXT, a formulation of xanomeline and tropsium which targets muscarinic receptors, has given a positive result in reducing the positive and negative symptoms of schizophrenia in a phase II trial suggests targeting muscarinic receptors is a new approach to treating the disorder. This review will detail the synergistic interplay between studies to understand the role of muscarinic receptors in the aetiology of schizophrenia and drug development and how this has supported the hypothesis that activating the muscarinic M1 and M4 receptors is critical to the efficacy of KarXT, in schizophrenia. The discovery of an intermediate phenotype within schizophrenia which is characterised by the presence of a marked loss of cortical muscarinic M1 receptors will be reviewed. Highlighted will be progress in understanding the biochemistry of that intermediate phenotype and evidence to suggest that those with the intermediate phenotype may resist treatment with agonist to the orthosteric site on the muscarinic M1 and M4 receptor. Finally, the possibility of using drugs targeting the allosteric binding sites on muscarinic receptors to treat schizophrenia will be discussed. This timely review will therefore highlight how research can influence hypothesis driven drug discovery that should produce new treatments for schizophrenia.

The use of a gene expression signature and connectivity map to repurpose drugs for bipolar disorder.

OBJECTIVES: To create a gene expression signature (GES) to represent the biological effects of a combination of known drugs for bipolar disorder (BD) on cultured human neuronal cells (NT2-N) and rat brains, which also has evidence of differential expression in individuals with BD. To use the GES to identify new drugs for BD using Connectivity Map (CMap).Methods: NT2-N (n = 20) cells and rats (n = 8) were treated with a BD drug combination (lithium, valproate, quetiapine and lamotrigine) or vehicle for 24 and 6 h, respectively. Following next-generation sequencing, the differential expression of genes was assessed using edgeR in R. The derived GES was compared to differentially expressed genes in post-mortem brains of individuals with BD. The GES was then used in CMap analysis to identify similarly acting drugs.Results: A total of 88 genes showed evidence of differential expression in response to the drug combination in both models, and therefore comprised the GES. Six of these genes showed evidence of differential expression in post-mortem brains of individuals with BD. CMap analysis identified 10 compounds (camptothecin, chlorambucil, flupenthixol, valdecoxib, rescinnamine, GW-8510, cinnarizine, lomustine, mifepristone and nimesulide) acting similarly to the BD drug combination.Conclusions: This study shows that GES and CMap can be used as tools to repurpose drugs for BD.

Changes in cortical protein markers of iron transport with gender, major depressive disorder and suicide.

Objectives: The objective of this study was to determine whether a breakdown in proteins regulating cortical iron homeostasis could be involved in the pathophysiology of mood disorders.Methods: Levels of select proteins responsible for cortical iron transport were quantitated by Western blotting of Brodmann's (BA) areas 6 and 10 from patients with major depressive disorder (n = 13), bipolar disorder (n = 12) and age/sex matched controls (n = 13).Results: We found the inactive form of ceruloplasmin was lower in BA 6 from males compared to females. Levels of copper containing ceruloplasmin was lower in BA 6 from suicide completers whilst levels of amyloid precursor protein, TAU and transferrin were higher in BA 10 from those individuals. The level of prion protein was lower in BA 6 from subjects with major depressive disorder.Conclusions: Our data suggests that perturbation in cortical iron transport proteins is not prevalent in mood disorders. By contrast, our data suggests changes in iron transport proteins in BA 6 and BA 10 are present after suicide completion. If these changes were present before death, they could have had a role in the genesis of the contemplation and completion of suicide.

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.

Cortical biometals: Changed levels in suicide and with mood disorders.

BACKGROUND: Changes in levels of metals have been suggested to contribute to the pathophysiologies of several neurodegenerative disorders but to our knowledge this is the first metallomic study in CNS from patients with mood disorders. The focus of this study was on cortical regions affected by the pathophysiologies of bipolar disorders and major depressive disorders. METHODS: Levels of metals were measured using inductively coupled plasma mass spectrometry in Brodmann's areas (BA) 6, 10 and 17 from patients with major depressive disorders (n = 13), bipolar disorders (n = 12) and age / sex matched controls (n = 13). RESULTS: There were lower levels of cortical strontium (BA 6 & 10), ruthenium (BA 6 & 17) and cadmium (BA 10) from patients with major depressive disorder as well as lower levels of strontium in BA 10 from patients with bipolar disorders. Unexpectedly, there were changes in levels of 16 metals in the cortex, mainly BA 6, from suicide completers compared to those who died of other causes. LIMITATIONS: Cohort sizes were relatively small but comparable with many studies using human postmortem CNS. Like all studies on non-treatment naïve patients, drug treatment was a potential confound in our experiments. CONCLUSIONS: Our exploratory study suggests changes in levels of metals in bipolar disorders and major depressive disorders could be affecting cortical oxidative balance in patients with mood disorders. Our data raises the possibility that measuring levels of specific biometals in the blood could be used as a biomarker for increased risk of suicide.

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.

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.

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.

Changed frontal pole gene expression suggest altered interplay between neurotransmitter, developmental, and inflammatory pathways in schizophrenia.

Schizophrenia (Sz) probably occurs after genetically susceptible individuals encounter a deleterious environmental factor that triggers epigenetic mechanisms to change CNS gene expression. To determine if omnibus changes in CNS gene expression are present in Sz, we compared mRNA levels in the frontal pole (Brodmann's area (BA) 10), the dorsolateral prefrontal cortex (BA 9) and cingulate cortex (BA 33) from 15 subjects with Sz and 15 controls using the Affymetrix™ Human Exon 1.0 ST Array. Differences in mRNA levels (±≥20%; p < 0.01) were identified (JMP Genomics 5.1) and used to predict pathways and gene x gene interactions that would be affected by the changes in gene expression using Ingenuity Pathway Analysis. There was significant variation in mRNA levels with diagnoses for 566 genes in BA 10, 65 genes in BA 9 and 40 genes in BA 33. In Sz, there was an over-representation of genes with changed expression involved in inflammation and development in BA 10, cell morphology in BA 9 and amino acid metabolism and small molecule biochemistry in BA 33. Using 94 genes with altered levels of expression in BA 10 from subjects with Sz, it was possible to construct an interactome of proven direct gene x gene interactions that was enriched for genes in inflammatory, developmental, oestrogen, serotonergic, cholinergic and NRG1 regulated pathways. Our data shows complex, regionally specific changes in cortical gene expression in Sz that are predicted to affect homeostasis between biochemical pathways already proposed to be important in the pathophysiology of the disorder.

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.