Exposure to significant levels of stress and trauma throughout life is a leading risk factor for the development of major psychiatric disorders. Despite this, we do not have a comprehensive understanding of the mechanisms that explain how stress raises psychiatric disorder risk. Stress in humans is complex and produces variable molecular outcomes depending on the stress type, timing, and duration. Deciphering how stress increases disorder risk has consequently been challenging to address with the traditional single-target experimental approaches primarily utilized to date. Importantly, the molecular processes that occur following stress are not fully understood but are needed to find novel treatment targets. Sequencing-based omics technologies, allowing for an unbiased investigation of physiological changes induced by stress, are rapidly accelerating our knowledge of the molecular sequelae of stress at a single-cell resolution. Spatial multi-omics technologies are now also emerging, allowing for simultaneous analysis of functional molecular layers, from epigenome to proteome, with anatomical context. The technology has immense potential to transform our understanding of how disorders develop, which we believe will significantly propel our understanding of how specific risk factors, such as stress, contribute to disease course. Here, we provide our perspective of how we believe these technologies will transform our understanding of the neurobiology of stress, and also provided a technical guide to assist molecular psychiatry and stress researchers who wish to implement spatial omics approaches in their own research. Finally, we identify potential future directions using multi-omics technology in stress research.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer’s disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

Genes involved in regulating the hypothalamic-pituitary-adrenal (HPA) axis, including the glucocorticoid receptor (GR), are linked to various stress- related psychopathologies including bipolar disorder as well as other mood and trauma- related disorders. The protein product of the cell cycle gene, SKA2, is a GR interaction partner in peripheral cells. However, the precise roles of SKA2 in stress and GR signaling in the brain, specifically in nonreplicating postmitotic neurons, and its involvement in HPA axis regulation remain unclear. Here, we demonstrate, using diverse in vitro cell assays, a mechanism by which SKA2 promotes GR signaling through enhancing GR-FKBP4 interaction leading to dissociation of FK506bindingprotein 51 (FKBP5) from the complex. FKBP4 and FKBP5 are cochaperones known to regulate GR function in opposite directions. Notably in mice, SKA2 in Crh+ neurons of the paraventricular nucleus of the hypothalamus is crucial for HPA axis responsiveness and for maintaining the negative feedback loop underlying allostasis. Moreover, we show that SKA2 expression is increased in postmortem human hippocampus and amygdala from individuals with BD. Our study highlights a critical role of SKA2 in HPA axis function, adds to the understanding of the molecular basis of stress- related psychiatric disorders, and points to potential targets for intervention.

Severe psychological stress is one of the most potent risk factors for developing a mood or psychotic disorder, yet the underlying molecular mechanisms are poorly understood. Astrocytes are a key brain cell type associated with stress and psychiatric phenotypes in animals, but how this translates to humans is largely unknown. Here, we show that cortical astrocytes are persistently changed both physically and molecularly in humans with psychiatric disorders exposed to profound stress before diagnosis. By profiling the diversity of human astrocytes with single nucleus and spatial transcriptomics, we identified distinct alterations to glutamate-related synaptic functions, supported by histological quantification of >20,000 astrocytes. Alterations were pronounced in females compared to males and in cases exposed to profound stress during childhood. The use of human pluripotent stem cell-derived astrocytes confirmed that glutamate signalling is directly impacted by glucocorticoid activation. Our findings suggest that astrocytes are strategic pharmacological targets for future intervention strategies.

The expression of FKBP5, and its resulting protein FKBP51, is strongly induced by glucocorticoids. Numerous studies have explored their involvement in a plethora of cellular processes and diseases. There is, however, a lack of knowledge on the role of the different RNA splicing variants and the two protein isoforms, one missing functional C-terminal motifs. In this study, we use in vitro models (HeLa and Jurkat cells) as well as peripheral blood cells of a human cohort (N = 26 male healthy controls) to show that the two expressed variants are both dynamically upregulated following dexamethasone, with significantly earlier increases (starting 1-2 h after stimulation) in the short isoform both in vitro and in vivo. Protein degradation assays in vitro showed a reduced half-life (4 h vs. 8 h) of the shorter isoform. Only the shorter isoform showed a subnuclear cellular localization. The two isoforms also differed in their effects on known downstream cellular pathways, including glucocorticoid receptor function, macroautophagy, immune activation, and DNA methylation regulation. The results shed light on the difference between the two variants and highlight the importance of differential analyses in future studies with implications for targeted drug design.

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.

High levels of proinflammatory cytokines induce neurotoxicity and catalyze inflammation-driven neurodegeneration, but the specific release mechanisms from microglia remain elusive. Here we show that secretory autophagy (SA), a non-lytic modality of autophagy for secretion of vesicular cargo, regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling. SKA2 inhibits SA-dependent IL-1β release by counteracting FKBP5 function. Hippocampal Ska2 knockdown in male mice hyperactivates SA resulting in neuroinflammation, subsequent neurodegeneration and complete hippocampal atrophy within six weeks. The hyperactivation of SA increases IL-1β release, contributing to an inflammatory feed-forward vicious cycle including NLRP3-inflammasome activation and Gasdermin D-mediated neurotoxicity, which ultimately drives neurodegeneration. Results from protein expression and co-immunoprecipitation analyses of male and female postmortem human brains demonstrate that SA is hyperactivated in Alzheimer’s disease. Overall, our findings suggest that SKA2-regulated, hyperactive SA facilitates neuroinflammation and is linked to Alzheimer’s disease, providing mechanistic insight into the biology of neuroinflammation.

Learning and memory rely on changes in postsynaptic AMPA-type glutamate receptor (AMPA receptor) number, spatial organization and function. The Hippo pathway component WWC1 regulates AMPA receptor surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPA receptor complexes are largely unknown. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice, and by generating a hippocampus-specific interactome we show that WWC1 is a major regulatory platform in AMPA receptor signalling networks. Interaction of the Hippo pathway members WWC1 and LATS limited the impact of WWC1 on synaptic proteins. Truncation of WWC1/LATS-binding through a point mutation at WWC1 elevated the abundance of WWC1 in AMPA receptor complexes and improved hippocampal-dependent learning and memory. Thus, relocation of WWC1 from the Hippo-pathway to AMPA receptor regulatory complexes provides an innovative strategy for the treatment of cognitive impairments.

The mediobasal hypothalamus (MBH) is the central region in the physiological response to metabolic stress. The FK506-binding protein 51 (FKBP51) is a major modulator of the stress response and has recently emerged as a scaffolder regulating metabolic and autophagy pathways. However, the detailed protein-protein interactions linking FKBP51 to autophagy upon metabolic challenges remain elusive. We performed mass spectrometry-based metabolomics of FKBP51 knockout (KO) cells revealing an increased amino acid and polyamine metabolism. We identified FKBP51 as a central nexus for the recruitment of the LKB1/AMPK complex to WIPI4 and TSC2 to WIPI3, thereby regulating the balance between autophagy and mTOR signaling in response to metabolic challenges. Furthermore, we demonstrated that MBH FKBP51 deletion strongly induces obesity, while its overexpression protects against high-fat diet (HFD) induced obesity. Our study provides an important novel regulatory function of MBH FKBP51 within the stress-adapted autophagy response to metabolic challenges.

Deducing genes capable of classifying biologically-distinct psychiatric subtypes, and their targets for treatment, is a priority approach in psychiatry. FKBP5 is one such gene with strong evidence of utility to delineate a trans-diagnostic psychiatric subtype. Yet how brain-expressed FKBP5 is affected in psychiatric disorders in humans is not fully understood and critical for propelling FKBP5-targeting treatment development. We performed a large-scale postmortem study (n=895) of FKBP5 using dorsolateral prefrontal cortex samples derived from individuals with severe psychiatric disorders with a comprehensive battery of bulk/single-cell omics and histological analyses. We observed consistently heightened FKBP5 mRNA and protein in psychopathology, moderated by genotype and age, and accompanied by DNA methylation changes in key enhancers. These effects were most prominent in superficial-layer pyramidal cells. Heightened FKBP5 was also differentially associated with downstream pathways according to age, being specifically associated with synaptic transmission in early adulthood, and neuroinflammation and neurodegeneration in later life.

SUMMARY Deducing genes capable of classifying biologically-distinct psychiatric subtypes, and their targets for treatment, is a priority approach in psychiatry. FKBP5 is one such gene with strong evidence of utility to delineate a trans-diagnostic psychiatric subtype. Yet how brain-expressed FKBP5 is affected in psychiatric disorders in humans is not fully understood and critical for propelling FKBP5 -targeting treatment development. We performed a large-scale postmortem study (n=895) of FKBP5 using dorsolateral prefrontal cortex samples derived from individuals with severe psychiatric disorders with a comprehensive battery of bulk/single-cell omics and histological analyses. We observed consistently heightened FKBP5 mRNA and protein in psychopathology, moderated by genotype and age, and accompanied by DNA methylation changes in key enhancers. These effects were most prominent in superficial-layer pyramidal cells. Heightened FKBP5 was also differentially associated with downstream pathways according to age, being specifically associated with synaptic transmission in early adulthood, and neuroinflammation and neurodegeneration in later life.

Dementia is a devastating age-related disorder. Its therapy would largely benefit from the identification of susceptible subjects at early, prodromal stages of the disease. To search for such prognostic markers of cognitive impairment, we studied spatial navigation in male BALBc vs. B6N mice in combination with in vivo magnetic resonance spectroscopy (1H-MRS). BALBc mice consistently showed higher escape latencies than B6N mice, both in the Water Cross Maze (WCM) and the Morris water maze (MWM). These performance deficits coincided with higher levels of myo-inositol (mIns) in the dorsal hippocampus before and after training. Subsequent biochemical analyses of hippocampal specimens by capillary immunodetection and liquid chromatography mass spectrometry-based (LC/MS) metabolomics revealed a higher abundance of glial markers (IBA-1, S100B, and GFAP) as well as distinct alterations in metabolites including a decrease in vitamins (pantothenic acid and nicotinamide), neurotransmitters (acetylcholine), their metabolites (glutamine), and acetyl-L-carnitine. Supplementation of low abundant acetyl-L-carnitine via the drinking water, however, failed to revert the behavioral deficits shown by BALBc mice. Based on our data we suggest (i) BALBc mice as an animal model and (ii) hippocampal mIns levels as a prognostic marker of mild cognitive impairment (MCI), due to (iii) local changes in microglia and astrocyte activity, which may (iv) result in decreased concentrations of promnesic molecules.

Disturbed activation or regulation of the stress response through the hypothalamic-pituitary-adrenal (HPA) axis is a fundamental component of multiple stress-related diseases, including psychiatric, metabolic, and immune disorders. The FK506 binding protein 51 (FKBP5) is a negative regulator of the glucocorticoid receptor (GR), the main driver of HPA axis regulation, and FKBP5 polymorphisms have been repeatedly linked to stress-related disorders in humans. However, the specific role of Fkbp5 in the paraventricular nucleus of the hypothalamus (PVN) in shaping HPA axis (re)activity remains to be elucidated. We here demonstrate that the deletion of Fkbp5 in Sim1+ neurons dampens the acute stress response and increases GR sensitivity. In contrast, Fkbp5 overexpression in the PVN results in a chronic HPA axis over-activation, and a PVN-specific rescue of Fkbp5 expression in full Fkbp5 KO mice normalizes the HPA axis phenotype. Single-cell RNA sequencing revealed the cell-type-specific expression pattern of Fkbp5 in the PVN and showed that Fkbp5 expression is specifically upregulated in Crh+ neurons after stress. Finally, Crh-specific Fkbp5 overexpression alters Crh neuron activity, but only partially recapitulates the PVN-specific Fkbp5 overexpression phenotype. Together, the data establish the central and cell-type-specific importance of Fkbp5 in the PVN in shaping HPA axis regulation and the acute stress response.

ABSTRACTIdentification and characterisation of novel targets for treatment is a priority in the field of psychiatry.FKBP5is 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 thatFKBP5/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 propelFKBP5/1-targeted treatment development. Here, we performed an extensive, large-scale postmortem study (n=1024) ofFKBP5/1 examining prefrontal cortex (BA9, BA11, BA24) 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, aging and genotype on corticalFKBP5/1 expression including in a cell-type specific manner. We identified consistently heightenedFKBP5/1 levels in psychopathology and with age, but not genotype, with these effects strongest in schizophrenia. Using single-nucleus RNA sequencing (snRNAseq) and targeted histology, we established that these disease- and aging-effects onFKBP5/1 expression were most pronounced in excitatory supragranular neurons. We then found that this increase inFKBP5levels likely impacts on synaptic plasticity, asFKBP5gex levels strongly and inversely correlated with dendritic mushroom spine density and brain-derived neurotrophic factor (BDNF) levels in supragranular neurons. These findings pinpoint a novel cellular and molecular mechanism that has significant potential to open a new avenue of FKBP51 drug development to treat cognitive symptoms in psychiatric disorders.

The stress response is an essential mechanism for maintaining homeostasis, and its disruption is implicated in several psychiatric disorders. On the cellular level, stress activates, among other mechanisms, autophagy that regulates homeostasis through protein degradation and recycling. Secretory autophagy is a recently described pathway in which autophagosomes fuse with the plasma membrane rather than with lysosomes. Here, we demonstrate that glucocorticoid-mediated stress enhances secretory autophagy via the stress-responsive co-chaperone FK506-binding protein 51. We identify the matrix metalloproteinase 9 (MMP9) as one of the proteins secreted in response to stress. Using cellular assays and in vivo microdialysis, we further find that stress-enhanced MMP9 secretion increases the cleavage of pro-brain-derived neurotrophic factor (proBDNF) to its mature form (mBDNF). BDNF is essential for adult synaptic plasticity and its pathway is associated with major depression and posttraumatic stress disorder. These findings unravel a cellular stress adaptation mechanism that bears the potential of opening avenues for the understanding of the pathophysiology of stress-related disorders.

Raw data associated to the following publication: “Mediobasal hypothalamic FKBP51 acts as a molecular switch linking autophagy to whole-body metabolism". The stress-responsive FKBP51 controls body weight gain by regulating the balance between autophagy and mTOR signaling. We identified FKBP51 as a central nexus for the recruitment of the LKB1/AMPK complex to WIPI4 and TSC2 to WIPI3, thereby regulating the balance between autophagy and mTOR signaling in response to metabolic challenges. MBH FKBP51 dose-dependently regulates autophagy both in the brain as well as in peripheral metabolic tissues. Consequently, deletion of MBH FKBP51 strongly induces obesity, while its overexpression protects against high-fat diet (HFD) induced obesity. IC data in Thermo RAW data format; 2nd of 2 datasets.

The stress response is an essential mechanism that strives to maintain homeostasis, and its disruption is implicated in several psychiatric disorders. As a cellular response to stressors, autophagy is activated to regulate homeostasis through protein degradation and recycling. Secretory autophagy is a recently described pathway where autophagosomes fuse with the plasma membrane rather than lysosomes. In this study, we demonstrate that glucocorticoid-mediated stress enhances secretory autophagy, via the stress-responsive co-chaperone FK506-binding protein 51. We identified the matrix metalloproteinase 9 (MMP9) as one of the stress-induced secreted proteins. Using cellular assays and in vivo microdialysis, we further found that stress-enhanced MMP9 secretion increases the cleavage of pro-brain derived neurotrophic factor (proBDNF) to its mature form. BDNF is essential for adult synaptic plasticity and its pathway is associated with major depression and posttraumatic stress disorder. These findings unravel a novel mechanistic link between stress, stress adaptation and the development of psychiatric disorders, with possible therapeutic implications.

We report here the involvement of the stress-responsive glucocorticoid receptor co-chaperone FKBP51 in the mechanism of in vivo secretion of mature BDNF (mBDNF). We used a novel method combining brain microdialysis with a capillary electrophoresis-based immunoassay, to examine mBDNF secretion in the medial prefrontal cortex (mPFC) in vivo in freely moving mice. By combining optogenetic, neurochemical (KCl-evoked depolarization), and transgenic (conditional BDNF knockout mice) means, we have shown that the increase in extracellular mBDNF in vivo is determined by neuronal activity. Withal, mBDNF secretion in the mPFC of mice was stimulated by a systemic administration of S-ketamine (10 or 50 mg/kg) or S-hydroxynorketamine (10 mg/kg). KCl- and S-ketamine-evoked mBDNF secretion was strongly dependent on the expression of FKBP51. Moreover, the inability of S-ketamine to evoke a transient secretion in mBDNF in the mPFC in FKBP51- knockout mice matched the lack of antidepressant-like effect of S-ketamine in the tail suspension test. Our data reveal a critical role of FKBP51 in mBDNF secretion and suggest the involvement of mBDNF in the realization of immediate stress-coping behavior induced by acute S-ketamine.