Deep phenotyping reveals CRH and FKBP51-dependent behavioral profiles following chronic social stress exposure in male mice.

The co-chaperone FKBP51, encoded by FKBP5 gene, is recognized as a psychiatric risk factor for anxiety and depressive disorders due to its crucial role in the stress response. Another key modulator in stress response regulation is the corticotropin releasing hormone (CRH), which is co-expressed with FKBP51 in many stress-relevant brain-regions and cell-types. Together, they intricately influence the balance of the hypothalamic-pituitary-adrenal (HPA) axis, one of the primary stress response systems. Previous research underscores the potential moderating effects these genes have on the regulation of the stressful life events towards the vulnerability of major depressive disorder (MDD). However, the specific function of FKBP51 in CRH-expressing neurons remains largely unexplored. Here, through deep behavioral phenotyping, we reveal heightened stress effects in mice lacking FKBP51 in CRH co-expressing neurons (CRHFKBP5-/-), particularly evident in social contexts. Our findings highlight the importance of considering cell-type specificity and context in comprehending stress responses and advocate for the utilization of machine-learning-driven phenotyping of mouse models. By elucidating these intricacies, we lay down the groundwork for personalized interventions aimed at enhancing stress resilience and individual well-being.

Hypothalamic protein profiling from mice subjected to social defeat stress.

The Hypothalmic-Pituitary-Adrenal axis also known as the HPA axis is central to stress response. It also acts as the relay center between the body and the brain. We analysed hypothalamic proteome from mice subjected to chronic social defeat paradigm using iTRAQ based quantitative proteomics to identify changes associated with stress response. We identified greater than 2000 proteins after processing our samples analysed through Q-Exactive (Thermo) and Orbitrap Velos (Thermo) at 5% FDR. Analysis of data procured from the runs showed that the proteins whose levels were affected belonged primarily to mitochondrial and metabolic processes, translation, complement pathway among others. We also found increased levels of fibrinogen, myelin basic protein (MBP) and neurofilaments (NEFL, NEFM, NEFH) in the hypothalamus from socially defeated mice. Interestingly, research indicates that these proteins are upregulated in blood and CSF of subjects exposed to trauma and stress. Since hypothalamus secreted proteins can be found in blood and CSF, their utility as biomarkers in depression holds an impressive probability and should be validated in clinical samples.

Automatically annotated motion tracking identifies a distinct social behavioral profile following chronic social defeat stress.

Severe stress exposure increases the risk of stress-related disorders such as major depressive disorder (MDD). An essential characteristic of MDD is the impairment of social functioning and lack of social motivation. Chronic social defeat stress is an established animal model for MDD research, which induces a cascade of physiological and behavioral changes. Current markerless pose estimation tools allow for more complex and naturalistic behavioral tests. Here, we introduce the open-source tool DeepOF to investigate the individual and social behavioral profile in mice by providing supervised and unsupervised pipelines using DeepLabCut-annotated pose estimation data. Applying this tool to chronic social defeat in male mice, the DeepOF supervised and unsupervised pipelines detect a distinct stress-induced social behavioral pattern, which was particularly observed at the beginning of a novel social encounter and fades with time due to habituation. In addition, while the classical social avoidance task does identify the stress-induced social behavioral differences, both DeepOF behavioral pipelines provide a clearer and more detailed profile. Moreover, DeepOF aims to facilitate reproducibility and unification of behavioral classification by providing an open-source tool, which can advance the study of rodent individual and social behavior, thereby enabling biological insights and, for example, subsequent drug development for psychiatric disorders.

Sex-specific and opposed effects of FKBP51 in glutamatergic and GABAergic neurons: Implications for stress susceptibility and resilience.

Mental health disorders often arise as a combination of environmental and genetic factors. The FKBP5 gene, encoding the GR co-chaperone FKBP51, has been uncovered as a key genetic risk factor for stress-related illness. However, the exact cell type and region-specific mechanisms by which FKBP51 contributes to stress resilience or susceptibility processes remain to be unravelled. FKBP51 functionality is known to interact with the environmental risk factors age and sex, but so far data on behavioral, structural, and molecular consequences of these interactions are still largely unknown. Here we report the cell type- and sex-specific contribution of FKBP51 to stress susceptibility and resilience mechanisms under the high-risk environmental conditions of an older age, by using two conditional knockout models within glutamatergic (Fkbp5Nex) and GABAergic (Fkbp5Dlx) neurons of the forebrain. Specific manipulation of Fkbp51 in these two cell types led to opposing effects on behavior, brain structure and gene expression profiles in a highly sex-dependent fashion. The results emphasize the role of FKBP51 as a key player in stress-related illness and the need for more targeted and sex-specific treatment strategies.

Metabolic effects of early life stress and pre-pregnancy obesity are long lasting and sex specific in mice.

Early life stress (ELS) is associated with metabolic, cognitive, and psychiatric diseases and has a very high prevalence, highlighting the urgent need for a better understanding of the versatile physiological changes and identification of predictive biomarkers. In addition to programming the hypothalamic-pituitary-adrenal (HPA) axis, ELS may also affect the gut microbiota and metabolome, opening up a promising research direction for identifying early biomarkers of ELS-induced (mal)adaptation. Other factors affecting these parameters include maternal metabolic status and diet, with maternal obesity shown to predispose offspring to later metabolic disease. The aim of the present study was to investigate the long-term effects of ELS and maternal obesity on the metabolic and stress phenotype of rodent offspring. To this end, offspring of both sexes were subjected to an adverse early-life experience, and their metabolic and stress phenotypes were examined. In addition, we assessed whether a prenatal maternal and an adult high-fat diet (HFD) stressor further shape observed ELS-induced phenotypes. We show that ELS has long-term effects on male body weight (BW) across the lifespan, whereas females more successfully counteract ELS-induced weight loss, possibly by adapting their microbiota, thereby stabilizing a balanced metabolome. Furthermore, the metabolic effects of a maternal HFD on BW are exclusively triggered by a dietary challenge in adult offspring and are more pronounced in males than in females. Overall, our study suggests that the female microbiota protects against an ELS challenge, rendering them more resilient to additional maternal- and adult nutritional stressors than males.

Psychosocial Stress Induces Schizophrenia-Like Behavior in Mice With Reduced MMP-9 Activity.

Understanding gene-environment interactions in the pathogenesis of schizophrenia remains a major research challenge. Matrix metalloproteinase-9 (MMP-9) has been previously implicated in the pathophysiology of schizophrenia. In the present study, adolescent Mmp-9 heterozygous mice, with a genetically lower level of MMP-9, were subjected to resident-intruder psychosocial stress for 3 weeks and then examined in behavioral tests that evaluated cognitive deficits and positive- and negative-like symptoms of schizophrenia. Cognitive and positive symptoms in unstressed Mmp-9 heterozygous mice were unaffected by stress exposure, whereas negative symptoms were manifested only after stress exposure. Interestingly, negative symptoms were ameliorated by treatment with the antipsychotic drug clozapine. We describe a novel gene × environment interaction mouse model of schizophrenia. Lower MMP-9 levels in the brain might be a risk factor for schizophrenia that, in combination with environmental factors (e.g., psychosocial stress), may evoke schizophrenia-like symptoms that are sensitive to antipsychotic treatment.

MMPs in learning and memory and neuropsychiatric disorders.

Matrix metalloproteinases (MMPs) are a group of over twenty proteases, operating chiefly extracellularly to cleave components of the extracellular matrix, cell adhesion molecules as well as cytokines and growth factors. By virtue of their expression and activity patterns in animal models and clinical investigations, as well as functional studies with gene knockouts and enzyme inhibitors, MMPs have been demonstrated to play a paramount role in many physiological and pathological processes in the brain. In particular, they have been shown to influence learning and memory processes, as well as major neuropsychiatric disorders such as schizophrenia, various kinds of addiction, epilepsy, fragile X syndrome, and depression. A possible link connecting all those conditions is either physiological or aberrant synaptic plasticity where some MMPs, e.g., MMP-9, have been demonstrated to contribute to the structural and functional reorganization of excitatory synapses that are located on dendritic spines. Another common theme linking the aforementioned pathological conditions is neuroinflammation and MMPs have also been shown to be important mediators of immune responses.

Absence of Wdr13 Gene Predisposes Mice to Mild Social Isolation - Chronic Stress, Leading to Depression-Like Phenotype Associated With Differential Expression of Synaptic Proteins.

We earlier reported that the male mice lacking the Wdr13 gene (Wdr13-/0) showed mild anxiety, better memory retention, and up-regulation of synaptic proteins in the hippocampus. With increasing evidences from parallel studies in our laboratory about the possible role of Wdr13 in stress response, we investigated its role in brain. We observed that Wdr13 transcript gets up-regulated in the hippocampus of the wild-type mice exposed to stress. To further dissect its function, we analyzed the behavioral and molecular phenotypes of Wdr13-/0 mice when subjected to mild chronic psychological stress, namely; mild (attenuated) social isolation. We employed iTRAQ based quantitative proteomics, real time PCR and western blotting to investigate molecular changes. Three weeks of social isolation predisposed Wdr13-/0 mice to anhedonia, heightened anxiety-measured by Open field test (OFT), increased behavior despair- measured by Forced swim test (FST) and reduced dendritic branching along with decreased spine density of hippocampal CA1 neurons as compared to wild-type counterparts. This depression-like-phenotype was however ameliorated when treated with anti-depressant imipramine. Molecular analysis revealed that out of 1002 quantified proteins [1% False discovery rate (FDR), at-least two unique peptides], strikingly, a significant proportion of synaptic proteins including, SYN1, CAMK2A, and RAB3A were down-regulated in the socially isolated Wdr13-/0 mice as compared to its wild-type counterparts. This was in contrast to the elevated levels of these proteins in non-stressed mutants as compared to the controls. We hypothesized that a de-regulated transcription factor upstream of the synaptic genes might be responsible for the observed phenotype. Indeed, in the socially isolated Wdr13-/0 mice, there was an up-regulation of GATA1 - a transcription factor that negatively regulates synaptic genes and has been associated with Major Depression (MD) in humans. The present study demonstrates significant genotype × enviornment interaction for Wdr13 gene as shown by the reversal in the expression levels of several synaptic proteins in the mutant vis-à-vis wild-type mouse when exposed to social isolation stress.

Implication of Genetic Deletion of Wdr13 in Mice: Mild Anxiety, Better Performance in Spatial Memory Task, with Upregulation of Multiple Synaptic Proteins.

WDR13 expresses from the X chromosome and has a highly conserved coding sequence. There have been multiple associations of WDR13 with memory. However, its detailed function in context of brain and behavior remains unknown. We characterized the behavioral phenotype of 2 month old male mice lacking the homolog of WDR13 gene (Wdr13 (-/0)). Taking cue from analysis of its expression in the brain, we chose hippocampus for molecular studies to delineate its function. Wdr13 (-/0) mice spent less time in the central area of the open field test (OFT) and with the novel object in novel object recognition test (NOR) as compared to the wild-type. However, these mice didn't show any significant changes in total time spent in arms or in frequency of arm entries in elevated plus maze (EPM). In the absence of Wdr13, there was a significant upregulation of synaptic proteins, viz., SYN1, RAB3A, CAMK2A etc. accompanied with increased spine density of hippocampal CA1 neurons and better spatial memory in mice as measured by increased time spent in the target quadrant of Morris water maze (MWM) during probe test. Parallel study from our lab has established c-JUN, ER α/ß, and HDAC 1,3,7 as interacting partners of WDR13. WDR13 represses transcription from AP1 (c-JUN responsive) and Estrogen Receptor Element (ERE) promoters. We hypothesized that absence of Wdr13 would result in de-regulated expression of a number of genes including multiple synaptic genes leading to the observed phenotype. Knocking down Wdr13 in Neuro2a cell lines led to increased transcripts of Camk2a and Nrxn2 consistent with in-vivo results. Summarily, our data provides functional evidence for the role of Wdr13 in brain.