DNA methylation patterns of FKBP5 regulatory regions in brain and blood of humanized mice and humans.

Humanized mouse models can be used to explore human gene regulatory elements (REs), which frequently lie in non-coding and less conserved genomic regions. Epigenetic modifications of gene REs, also in the context of gene x environment interactions, have not yet been explored in humanized mouse models. We applied high-accuracy measurement of DNA methylation (DNAm) via targeted bisulfite sequencing (HAM-TBS) to investigate DNAm in three tissues/brain regions (blood, prefrontal cortex and hippocampus) of mice carrying the human FK506-binding protein 5 (FKBP5) gene, an important candidate gene associated with stress-related psychiatric disorders. We explored DNAm in three functional intronic glucocorticoid-responsive elements (at introns 2, 5, and 7) of FKBP5 at baseline, in cases of differing genotype (rs1360780 single nucleotide polymorphism), and following application of the synthetic glucocorticoid dexamethasone. We compared DNAm patterns in the humanized mouse (N = 58) to those in human peripheral blood (N = 447 and N = 89) and human postmortem brain prefrontal cortex (N = 86). Overall, DNAm patterns in the humanized mouse model seem to recapitulate DNAm patterns observed in human tissue. At baseline, this was to a higher extent in brain tissue. The animal model also recapitulated effects of dexamethasone on DNAm, especially in peripheral blood and to a lesser extent effects of genotype on DNAm. The humanized mouse model could thus assist in reverse translation of human findings in psychiatry that involve genetic and epigenetic regulation in non-coding elements.

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.

Neuronal hyperexcitability in Alzheimer's disease: what are the drivers behind this aberrant phenotype?

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to loss of cognitive abilities and ultimately, death. With no cure available, limited treatments mostly focus on symptom management. Identifying early changes in the disease course may provide new therapeutic targets to halt or reverse disease progression. Clinical studies have shown that cortical and hippocampal hyperactivity are a feature shared by patients in the early stages of disease, progressing to hypoactivity during later stages of neurodegeneration. The exact mechanisms causing neuronal excitability changes are not fully characterized; however, animal and cell models have provided insights into some of the factors involved in this phenotype. In this review, we summarize the evidence for neuronal excitability changes over the course of AD onset and progression and the molecular mechanisms underpinning these differences. Specifically, we discuss contributors to aberrant neuronal excitability, including abnormal levels of intracellular Ca2+ and glutamate, pathological amyloid ß (Aß) and tau, genetic risk factors, including APOE, and impaired inhibitory interneuron and glial function. In light of recent research indicating hyperexcitability could be a predictive marker of cognitive dysfunction, we further argue that the hyperexcitability phenotype could be leveraged to improve the diagnosis and treatment of AD, and present potential targets for future AD treatment development.

Alterations in Astrocytic Regulation of Excitation and Inhibition by Stress Exposure and in Severe Psychopathology.

Dysregulation of excitatory and inhibitory signaling is commonly observed in major psychiatric disorders, including schizophrenia, depression, and bipolar disorder, and is often targeted by psychological and pharmacological treatment methods. The balance of excitation and inhibition is highly sensitive to severe psychological stress, one of the strongest risk factors for psychiatric disorders. The role of astrocytes in regulating excitatory and inhibitory signaling is now widely recognized; however, the specific involvement of astrocytes in the context of psychiatric disorders with a history of significant stress exposure remains unclear. In this review, we summarize how astrocytes regulate the balance of excitation and inhibition in the context of stress exposure and severe psychopathology, with a focus on the PFC, a brain area highly implicated in psychopathology. We first focus on preclinical models to demonstrate that the duration of stress (particularly acute vs chronic stress) is key to shaping astrocyte function and downstream behavior. We then provide a hypothesis for how astrocytes are involved in stress-associated cortical signaling imbalance, discuss how this directly contributes to phenotypes of psychopathologies, and provide suggestions for future research. We highlight that astrocytes are a key target to understand and treat the dysregulation of cortical signaling associated with stress-related psychiatric disorders.

How stress physically re-shapes the brain: Impact on brain cell shapes, numbers and connections in psychiatric disorders.

Severe stress is among the most robust risk factors for the development of psychiatric disorders. Imaging studies indicate that life stress is integral to shaping the human brain, especially regions involved in processing the stress response. Although this is likely underpinned by changes to the cytoarchitecture of cellular networks in the brain, we are yet to clearly understand how these define a role for stress in human psychopathology. In this review, we consolidate evidence of macro-structural morphometric changes and the cellular mechanisms that likely underlie them. Focusing on stress-sensitive regions of the brain, we illustrate how stress throughout life may lead to persistent remodelling of the both neurons and glia in cellular networks and how these may lead to psychopathology. We support that greater translation of cellular alterations to human cohorts will support parsing the psychological sequalae of severe stress and improve our understanding of how stress shapes the human brain. This will remain a critical step for improving treatment interventions and prevention outcomes.

Understanding the pathology of psychiatric disorders in refugees.

Displacement of people from their homes, families and countries is a current global crisis, with over 70 million people forcibly on the move. A substantial proportion of these people will end up in regions with a different language and culture, where they are registered as refugees or asylum seekers. Due to the underlying reasons for displacement (including conflicts, persecution or violation of human rights), displaced people are severely stress-exposed, which continues into their post-migration life and increases risk for developing psychiatric disorders such as post-traumatic stress disorder and other anxiety disorders and mood disorders. While landmark studies have illustrated the increased prevalence of psychopathology in asylum seeker and refugee populations following pre-/post-displacement stress, few studies add to our understanding of the basic biological mechanisms underpinning risk to psychiatric disorders in these populations. Additionally, the mechanisms underlying resilience despite significant adversity remain unclear. Understanding the molecular mechanisms underpinning the development of psychiatric disorders in refugees can propel treatments (both drug and non-drug) that are capable of influencing biology at the molecular level, and the design of interventions. In the following review, we summarise the status quo of research investigating the pathophysiology of psychiatric disorders in refugees, and propose new ways to address gaps in knowledge with multidisciplinary research.

Severe childhood and adulthood stress associates with neocortical layer-specific reductions of mature spines in psychiatric disorders.

Severe stress exposure causes the loss of dendritic spines on cortical pyramidal neurons and induces psychiatric-like symptoms in rodent models. These effects are strongest following early-life stress and are most persistent on apical dendrites. However, the long-term impacts and temporal effects of stress exposure on the human brain remain poorly understood. Using a novel postmortem cohort of psychiatric cases with severe stress experienced in childhood, adulthood, or no severe stress, and matched controls, we aimed to determine the impact of stress timing on pyramidal neuron structure in the human orbitofrontal cortex (OFC). We performed Golgi Cox staining and manually measured the morphology and density of over 22,000 dendritic spines on layer-specific pyramidal neuron apical dendrites. We also quantified glucocorticoid receptor mRNA and protein as a marker of stress dysregulation. Both childhood and adulthood stress were associated with large reductions in mature mushroom spine density (up to 56% loss) in both the superficial (II/III) and deeper layers (V) of the OFC. However, childhood stress caused more substantial reductions to both total and mature mushroom spines. No difference in glucocorticoid receptor mRNA and protein were seen between groups, although both negatively correlated with total spine density within the whole cohort. These findings indicate that severe stress, especially when experienced during childhood, persistently affects the fine morphological properties of neurons in the human OFC. This may impact on cell connectivity in this brain area, and at least partly explain the social and emotional symptoms that originate in the OFC in psychiatric disorders.

The Role of Cathepsins in Memory Functions and the Pathophysiology of Psychiatric Disorders.

Cathepsins are proteases with functions in cellular homeostasis, lysosomal degradation and autophagy. Their role in the development of neurodegenerative diseases has been extensively studied. It is well established that impairment of proper cathepsin function plays a crucial role in the pathophysiology of neurodegenerative diseases, and in recent years a role for cathepsins in mental disorders has emerged given the involvement of cathepsins in memory function, hyperactivity, and in depression- and anxiety-like behavior. Here we review putative cathepsin functions with a special focus on their role in the pathophysiology of psychiatric diseases. Specifically, cathepsins are crucial for maintaining cellular homeostasis, particularly as part of the autophagy machinery of neural strategies underlying acute stress response. Disruption of cathepsin functions can lead to psychiatric diseases such as major depressive disease (MDD), bipolar disorder, and schizophrenia. Specifically, cathepsins can be excreted via a process called secretory autophagy. Thereby, they are able to regulate extracellular factors such as brain-derived neurotrophic factor and perlecan c-terminal fragment LG3 providing maintenance of neuronal homeostasis and mediating neuronal plasticity in response to acute stress or trauma. In addition, impairment of proper cathepsin function can result in impaired synaptic transmission by compromised recycling and biogenesis of synaptic vesicles. Taken together, further investigations on cathepsin functions and stress response, neuroplasticity, and synaptic transmission will be of great interest in understanding the pathophysiology of psychiatric disorders.

Chronic Adolescent CDPPB Treatment Alters Short-Term, but not Long-Term, Glutamatergic Receptor Expression.

Dysfunction of the glutamatergic system is believed to underlie many neurodevelopmental disorders including autism, Rett syndrome and schizophrenia. Metabotropic glutamate receptor (mGluR5) positive allosteric modulators (PAM) potentiate glutamatergic signaling, particularly indirectly via the NMDA receptor. Preclinical studies report mGluR5 PAMs can improve schizophrenia-relevant behaviours. Furthermore, adolescent administration has shown to prevent cognitive induced deficits in adult rodents. However, there is limited understanding of the short- and long-term neurochemical effects of mGluR5 PAMs, which may underlie their therapeutic effects. We examined the effect of 7-day adolescent (PN28-34) treatment with the mGluR5 PAM, CDDPB (30 mg/kg), on glutamatergic receptor expression at adolescence (PN35) and adulthood (PN96). Immunoblot analysis revealed that 7-day adolescent CDPPB treatment increased protein expression of glutamatergic receptors including the NMDA receptor subunits, NR1 and NR2A and the AMPA subunits (GluA1 and GluA2) in the adolescent hippocampus, changes that did not extend to adulthood. In contrast, there were no changes in the adolescent frontal cortex, however elevated mGluR5 protein expression was observed at adulthood following adolescent CDPPB treatment. The present study indicates adolescent CDPPB treatment may cause brain region dependent effects on the glutamatergic system, which do not persist into adulthood. These findings may have implications for the preclinical development of mGluR5 PAMs for the treatment of neurodevelopmental disorders.

Understanding the Molecular Mechanisms Underpinning Gene by Environment Interactions in Psychiatric Disorders: The FKBP5 Model.

Epidemiologic and genetic studies suggest common environmental and genetic risk factors for a number of psychiatric disorders, including depression, bipolar disorder, and schizophrenia. Genetic and environmental factors, especially adverse life events, not only have main effects on disease development but also may interact to shape risk and resilience. Such gene by adversity interactions have been described for FKBP5, an endogenous regulator of the stress-neuroendocrine system, conferring risk for a number of psychiatric disorders. In this review, we present a molecular and cellular model of the consequences of FKBP5 by early adversity interactions. We illustrate how altered genetic and epigenetic regulation of FKBP5 may contribute to disease risk by covering evidence from clinical and preclinical studies of FKBP5 dysregulation, known cell-type and tissue-type expression patterns of FKBP5 in humans and animals, and the role of FKBP5 as a stress-responsive molecular hub modulating many cellular pathways. FKBP5 presents the possibility to better understand the molecular and cellular factors contributing to a disease-relevant gene by environment interaction, with implications for the development of biomarkers and interventions for psychiatric disorders.

Perinatal administration of phencyclidine alters expression of Lingo-1 signaling pathway proteins in the prefrontal cortex of juvenile and adult rats.

Postnatal administration of phencyclidine (PCP) in rodents causes major brain dysfunction leading to severe disturbances in behavior lasting into adulthood. This model is routinely employed to model psychiatric disorders such as schizophrenia, as it reflects schizophrenia-related brain disturbances including increased apoptosis, and disruptions to myelin and plasticity processes. Leucine-rich repeat and Immunoglobin-like domain-containing protein 1 (Lingo-1) is a potent negative regulator of both axonal myelination and neurite extension. The Nogo receptor (NgR)/tumor necrosis factor (TNF) receptor orphan Y (TROY) and/or p75 neurotrophin receptor (p75) complex, with no lysine (K) (WNK1) and myelin transcription factor 1 (Myt1) are co-receptors or cofactors in Lingo-1 signaling pathways in the brain. We have examined the developmental trajectory of these proteins in a neurodevelopmental model of schizophrenia using PCP to determine if Lingo-1 pathways are altered in the prefrontal cortex throughout different stages of life. Sprague-Dawley rats were injected with PCP (10 mg/kg) or saline on postnatal days (PN)7, 9, and 11 and killed at PN12, 5 or 14 weeks for measurement of Lingo-1 signaling proteins in the prefrontal cortex. Myt1 was decreased by PCP at PN12 (P=0.045), and at 14 weeks PCP increased Lingo-1 (P=0.037), TROY (P=0.017), and WNK1 (P=0.003) expression. This is the first study reporting an alteration in Lingo-1 signaling proteins in the rat prefrontal cortex both directly after PCP treatment in early development and in adulthood. We propose that Lingo-1 pathways may be negatively regulating myelination and neurite outgrowth following the administration of PCP, and that this may have implications for the cortical dysfunction observed in schizophrenia.

Effects of common GRM5 genetic variants on cognition, hippocampal volume and mGluR5 protein levels in schizophrenia.

GRM5 (coding for metabotropic glutamate receptor 5, mGluR5) is a promising target for the treatment of cognitive deficits in schizophrenia, but there has been little investigation of its association with cognitive and brain phenotypes within this disorder. We examined the effects of common genetic variation in GRM5 with cognitive function, hippocampal volume, and hippocampal mGluR5 protein levels in schizophrenia patients relative to healthy controls. Two independent GRM5 variants rs60954128 [C>T] and rs3824927 [G>T] were genotyped in a schizophrenia case/control cohort (n=249/261). High-resolution anatomical brain scans were available for a subset of the cohort (n=103 schizophrenia /78 control). All participants completed a standard set of neuropsychological tests. In a separate postmortem cohort (n=19 schizophrenia/20 controls), hippocampal mGluR5 protein levels were examined among individuals of different GRM5 genotypes. Schizophrenia minor allele carriers of rs60954128 had reduced right hippocampal volume relative to healthy controls of the same genotype (-12.3%); this effect was exaggerated in males with schizophrenia (-15.6%). For rs3824927, compared to major allele homozygotes, minor allele carriers with schizophrenia had lower Intelligence Quotients (IQ). Examination in hippocampal postmortem tissue showed no difference in mGluR5 protein expression according to genotype for either rs60954128 or rs3824927. While these genetic variants in GRM5 were associated with cognitive impairments and right hippocampal volume reduction in schizophrenia, they did not affect protein expression. Further study of these mechanisms may help to delineate new targets for the treatment of cognitive deficits in schizophrenia, and may be relevant to other disorders.

Common variation in ZNF804A (rs1344706) is not associated with brain morphometry in schizophrenia or healthy participants.

BACKGROUND: The single nucleotide polymorphism (SNP) rs1344706 [A>C] within intron 2 of the zinc finger protein 804A gene (ZNF804A) is associated with schizophrenia at the genome-wide level, but its function in relation to the development of psychotic disorders, including its influence on brain morphology remains unclear. METHODS: Using both univariate (voxel-based morphometry, VBM; cortical thickness) and multivariate (source-based morphometry, SBM) approaches, we examined the effects of variation of the rs1344706 polymorphism on grey matter integrity in 214 Caucasian schizophrenia cases and 94 Caucasian healthy individuals selected from the Australian Schizophrenia Research Bank. RESULTS: Neither univariate nor multivariate analyses showed any associations between indices of grey matter and rs1344706 variation in schizophrenia or healthy participants. This was revealed in the context of the typical pattern of decreased grey matter integrity in schizophrenia compared to healthy individuals, including: (1) large grey matter volume reductions in the orbitofrontal and anterior cingulate cortices and the left fusiform/inferior temporal gyri; (2) decreased cortical thickness in the left inferior temporal and fusiform gyri, the left orbitofrontal gyrus, as well as in the right pars opercularis/precentral gyrus; and (3) decreased covariation of grey matter concentration in frontal and limbic brain regions emerging from the SBM analyses. CONCLUSIONS: Contrary to some - but not all - previous findings, this study of a large sample of schizophrenia cases and healthy controls reveals no evidence for association between grey matter alterations and variation in rs1344706 (ZNF804A). Differences in sample sizes and ethnicities may account for discrepant findings between the present and previous studies.

Alterations of ubiquitin related proteins in the pathology and development of schizophrenia: Evidence from human and animal studies.

Gene expression analyses in post-mortem schizophrenia brains suggest that a number of ubiquitin proteasome system (UPS) genes are associated with schizophrenia; however the status of UPS proteins in the schizophrenia brain is largely unknown. Ubiquitin related proteins are inherently involved in memory, neuronal survival and morphology, which are processes implicated in neurodevelopmental disorders such as schizophrenia. We examined levels of five UPS proteins (Protein Inhibitor of Activated STAT2 [PIAS2], F-Box and Leucine rich repeat protein 21 [FBXL21], Mouse Double Minute 2 homolog [MDM2], Ubiquitin Carboxyl-Terminal Hydrolase-L1 [UCHL1] and Ubiquitin Conjugating Enzyme E2D1 [UBE2D1]) involved in these neuronal processes, within the dorsolateral prefrontal cortex of post-mortem schizophrenia subjects and matched controls (n = 30/group), in addition to across neurodevelopmental time-points (juvenile, adolescent and adult stages of life), utilizing a well-established neurodevelopmental phencyclidine (PCP) animal model of schizophrenia. We observed significant reductions in PIAS2, FBXL21 and MDM2 in schizophrenia subjects compared to controls (p-values ranging from 0.002 to 0.004). In our developmental PCP model, MDM2 protein was significantly reduced in adult PCP-treated rats compared to controls (p = 0.034). Additionally, FBXL21 (p = 0.022) and UCHL1 (p = 0.022) were significantly decreased, whilst UBE2D1 was increased (p = 0.022), in juvenile phencyclidine-treated rats compared to controls. This is the first study reporting alterations of UPS proteins in post-mortem human schizophrenia subjects and in a neurodevelopmental model of schizophrenia. The findings from this study provide strong support for a role of these UPS proteins in the pathology and development of schizophrenia.

Shifting towards a model of mGluR5 dysregulation in schizophrenia: Consequences for future schizophrenia treatment.

Metabotropic glutamate receptor subtype 5 (mGluR5), encoded by the GRM5 gene, represents a compelling novel drug target for the treatment of schizophrenia. mGluR5 is a postsynaptic G-protein coupled glutamate receptor strongly linked with several critical cellular processes that are reported to be disrupted in schizophrenia. Accordingly, mGluR5 positive allosteric modulators show encouraging therapeutic potential in preclinical schizophrenia models, particularly for the treatment of cognitive dysfunctions against which currently available therapeutics are largely ineffective. More work is required to support the progression of mGluR5-targeting drugs into the clinic for schizophrenia treatment, although some obstacles may be overcome by comprehensively understanding how mGluR5 itself is involved in the neurobiology of the disorder. Several processes that are necessary for the regulation of mGluR5 activity have been identified, but not examined, in the context of schizophrenia. These processes include protein-protein interactions, dimerisation, subcellular trafficking, the impact of genetic variability or mutations on protein function, as well as epigenetic, post-transcriptional and post-translational processes. It is essential to understand these aspects of mGluR5 to determine whether they are affected in schizophrenia pathology, and to assess the consequences of mGluR5 dysfunction for the future use of mGluR5-based drugs. Here, we summarise the known processes that regulate mGluR5 and those that have already been studied in schizophrenia, and discuss the consequences of this dysregulation for current mGluR5 pharmacological strategies. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Preclinical and Clinical Evidence of DNA Methylation Changes in Response to Trauma and Chronic Stress.

Exposure to chronic stress, either repeated severe acute or moderate sustained stress, is one of the strongest risk factors for the development of psychopathologies such as post-traumatic stress disorder and depression. Chronic stress is linked with several lasting biological consequences, particularly to the stress endocrine system but also affecting intermediate phenotypes such as brain structure and function, immune function, and behavior. Although genetic predisposition confers a proportion of the risk, the most relevant molecular mechanisms determining those susceptible and resilient to the effects of stress and trauma may be epigenetic. Epigenetics refers to the mechanisms that regulate genomic information by dynamically changing the patterns of transcription and translation of genes. Mounting evidence from preclinical rodent and clinical population studies strongly support that epigenetic modifications can occur in response to traumatic and chronic stress. Here, we discuss this literature examining stress-induced epigenetic changes in preclinical models and clinical cohorts of stress and trauma occurring early in life or in adulthood. We highlight that a complex relationship between the timing of environmental stressors and genetic predispositions likely mediate the response to chronic stress over time, and that a better understanding of epigenetic changes is needed by further investigations in longitudinal and postmortem brain clinical cohorts.

Neurodevelopmental Expression Profile of Dimeric and Monomeric Group 1 mGluRs: Relevance to Schizophrenia Pathogenesis and Treatment.

Group 1 metabotropic glutamate receptors (mGluR1/mGluR5) play an integral role in neurodevelopment and are implicated in psychiatric disorders, such as schizophrenia. mGluR1 and mGluR5 are expressed as homodimers, which is important for their functionality and pharmacology. We examined the protein expression of dimeric and monomeric mGluR1α and mGluR5 in the prefrontal cortex (PFC) and hippocampus throughout development (juvenile/adolescence/adulthood) and in the perinatal phencyclidine (PCP) model of schizophrenia. Under control conditions, mGluR1α dimer expression increased between juvenile and adolescence (209-328%), while monomeric levels remained consistent. Dimeric mGluR5 was steadily expressed across all time points; monomeric mGluR5 was present in juveniles, dramatically declining at adolescence and adulthood (-97-99%). The mGluR regulators, Homer 1b/c and Norbin, significantly increased with age in the PFC and hippocampus. Perinatal PCP treatment significantly increased juvenile dimeric mGluR5 levels in the PFC and hippocampus (37-50%) but decreased hippocampal mGluR1α (-50-56%). Perinatal PCP treatment also reduced mGluR1α dimer levels in the PFC at adulthood (-31%). These results suggest that Group 1 mGluRs have distinct dimeric and monomeric neurodevelopmental patterns, which may impact their pharmacological profiles at specific ages. Perinatal PCP treatment disrupted the early expression of Group 1 mGluRs which may underlie neurodevelopmental alterations observed in this model.