Exploring the underlying mechanisms mediated by FKBP51, GR and Tac2 in anxiety-related behaviour

Excessive or chronic stress can lead to maladaptive anxiety. Anxiety disorders are highly prevalent and there is a pressing demand for a more comprehensive understanding of the molecular mechanisms that underlie such disorders. Dysregulation of the stress response and genetic risk factors can contribute towards an increased susceptibility to maladaptive anxiety. The co-chaperone FKBP51 is an immunophilin protein best known as a regulator of the glucocorticoid receptor (GR) and the stress response system. Human and animal studies have shown that aberrant GR signalling as well as genetic variants within the gene encoding FKBP51, FKBP5, can precede stress-related pathology. Notably, the effects of FKBP51 are highly region- and cell type specific. The central extended amygdala, which comprises the central amygdala (CeA) and the bed nucleus of the stria terminalis (BNST), more specifically the oval BNST (ovBNST), is a promising limbic structure of the forebrain that has been repeatedly associated with the modulation of anxiety states. However, the region-specific function of FKBP51 in this nucleus has not been explored yet. Moreover, Tachykinin 2 (Tac2) is an emerging neuropeptide that is also expressed in the BNST and highly likely involved in mediating anxiety-like behaviour. In this thesis, we aimed to elucidate molecular mechanisms mediated by the GR, FKBP51 and Tac2 that underlie stress-induced anxiety states. Initially we addressed whether GR signalling in forebrain neurons contributes to fear and anxiety-related behaviour. Interestingly, we found that forebrain glutamatergic, but not GABAergic, neurons mediate the anxiogenic effects of the GR. Moreover, virally-mediated GR deletion revealed that fear-related behaviour is regulated exclusively by GRs in the glutamatergic neurons of the basolateral amygdala (BLA). We then proceeded to explore whether the lack of FKBP51 would lead to alterations in brain architecture and connectivity in mice. Using in vivo structural magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) we reported two clusters of significantly larger volumes in the hypothalamus, periaqueductal grey (PAG), and dorsal raphe (DR) region of wildtype (WT) animals. DTI measurements, however, highlighted statistically higher fractional anisotropy (FA) values for FKBP51 knockout (51KO) animals in locations including the anterior commissure, fornix, and posterior commissure/superior colliculus region. In order to dissect the role of FKBP51 in anxiety, we characterised the function of FKBP51 in the ovBNST and assessed its impact on HPA axis function and anxiety-related behaviour. Notably, our data suggests that stress-induced increase of FKBP5 in the ovBNST may in fact have a protective role, leading to decreased anxiety and suppression of a future stress-induced HPA axis activation. Finally, we convey a first impression of the function of Tac2 within the ovBNST and its implication in anxiety-like behaviour. Here we show that Tac2 is upregulated by acute stress in the ovBNST and that ovBNST Tac2 positive neurons are involved in anxiety circuitry and behaviour. The collective findings of the current thesis provide novel evidence for the contribution of GR, Tac2 and FKBP51 towards the underlying molecular mechanisms of stress-induced anxiety-like states

Genomic organization of eukaryotic tRNAs

BACKGROUND: Surprisingly little is known about the organization and distribution of tRNA genes and tRNA-related sequences on a genome-wide scale. While tRNA gene complements are usually reported in passing as part of genome annotation efforts, and peculiar features such as the tandem arrangements of tRNA gene in Entamoeba histolytica have been described in some detail, systematic comparative studies are rare and mostly restricted to bacteria. We therefore set out to survey the genomic arrangement of tRNA genes and pseudogenes in a wide range of eukaryotes to identify common patterns and taxon-specific peculiarities. RESULTS: In line with previous reports, we find that tRNA complements evolve rapidly and tRNA gene and pseudogene locations are subject to rapid turnover. At phylum level, the distributions of the number of tRNA genes and pseudogenes numbers are very broad, with standard deviations on the order of the mean. Even among closely related species we observe dramatic changes in local organization. For instance, 65% and 87% of the tRNA genes and pseudogenes are located in genomic clusters in zebrafish and stickleback, resp., while such arrangements are relatively rare in the other three sequenced teleost fish genomes. Among basal metazoa, Trichoplax adherens has hardly any duplicated tRNA gene, while the sea anemone Nematostella vectensis boasts more than 17000 tRNA genes and pseudogenes. Dramatic variations are observed even within the eutherian mammals. Higher primates, for instance, have 616 +/- 120 tRNA genes and pseudogenes of which 17% to 36% are arranged in clusters, while the genome of the bushbaby Otolemur garnetti has 45225 tRNA genes and pseudogenes of which only 5.6% appear in clusters. In contrast, the distribution is surprisingly uniform across plant genomes. Consistent with this variability, syntenic conservation of tRNA genes and pseudogenes is also poor in general, with turn-over rates comparable to those of unconstrained sequence elements. Despite this large variation in abundance in Eukarya we observe a significant correlation between the number of tRNA genes, tRNA pseudogenes, and genome size. CONCLUSIONS: The genomic organization of tRNA genes and pseudogenes shows complex lineage-specific patterns characterized by an extensive variability that is in striking contrast to the extreme levels of sequence-conservation of the tRNAs themselves. The comprehensive analysis of the genomic organization of tRNA genes and pseudogenes in Eukarya provides a basis for further studies into the interplay of tRNA gene arrangements and genome organization in general

Pharmacological Modulation of the Psychiatric Risk Factor FKBP51 Alters Efficiency of Common Antidepressant Drugs

Despite a growing body of research over the last few decades, mental disorders, including anxiety disorders or depression, are still one of the most prevalent and hardest to treat health burdens worldwide. Since pharmacological treatment with a single drug is often rather ineffective, approaches such as co-medication with functionally diverse antidepressants (ADs) have been discussed and tried more recently. Besides classical ADs, there is a growing number of candidate targets identified as potential starting points for new treatment methods. One of these candidates, the FK506 binding protein 51 (FKBP51) is linked to a number of psychiatric disorders in humans. In this study, we used SAFit2-a newly developed modulator of FKBP51, which has shown promising results in rodent models for stress-related disorders delivered in a depot formulation. We combined SAFit2 with the commonly prescribed selective serotonin reuptake inhibitor (SSRI) escitalopram and performed basic behavioral characterization in a mouse model. Remarkably, co-application of SAFit2 lowered the efficacy of escitalopram in anxiety-related tests but improved stress coping behavior. Given the fact that mental diseases such as anxiety disorders or depression can be divided into different sub-categories, some of which more or less prone to stress, SAFit2 could indeed be a highly beneficial co-medication in very specific cases. This study could be a first, promising step towards the use of FKBP51 modulators as potent and specific enhancers of AD efficiency for subclasses of patients in the future

The co-chaperone Fkbp5 shapes the acute stress response in the paraventricular nucleus of the hypothalamus of male mice

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

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.This work was supported by the “GUTMOM” grant of the ERA-Net Cofund HDHL-INTIMIC (INtesTInal MIcrobiomics) under the JPI HDHL (Joint Programming Initiative – A healthy diet for a healthy life) umbrella (01EA1805; MVS), the SCHM2360-5-1 grant (MVS) from the German Research Foundation (DFG), the ZonMw grant from the Netherlands Organisation for Health Research and Development (project number 529051019), the DIM-ELI-2 grant of La Fundación La Marató-TV3 (ref. 2018-27/30-31), the PID2019-108973RB-C22 and PCIN2017-117 grants from the Ministerio de Ciencia e Innovación of Spain and the grants GV/2020/048 and IDIFEDER/2021/072 from the Generalitat Valenciana of Spain. Open Access funding enabled and organized by Projekt DEAL.Peer reviewe

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells

© 2018 Sutanto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAEC CF ) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508-del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAEC CF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAEC CF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAEC CF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations

Introducing evolutionary biologists to the analysis of big data: guidelines to organize extended bioinformatics training courses

Research in evolutionary biology has been progressively influenced by big data such as massive genome and transcriptome sequencing data, scalar measurements of several phenotypes on tens to thousands of individuals, as well as from collecting worldwide environmental data at an increasingly detailed scale. The handling and analysis of such data require computational skills that usually exceed the abilities of most traditionally trained evolutionary biologists. Here we discuss the advantages, challenges and considerations for organizing and running bioinformatics training courses of 2–3 weeks in length to introduce evolutionary biologists to the computational analysis of big data. Extended courses have the advantage of offering trainees the opportunity to learn a more comprehensive set of complementary topics and skills and allowing for more time to practice newly acquired competences. Many organizational aspects are common to any course, as the need to define precise learning objectives and the selection of appropriate and highly motivated instructors and trainees, among others. However, other features assume particular importance in extended bioinformatics training courses. To successfully implement a learning-by-doing philosophy, sufficient and enthusiastic teaching assistants (TAs) are necessary to offer prompt help to trainees. Further, a good balance between theoretical background and practice time needs to be provided and assured that the schedule includes enough flexibility for extra review sessions or further discussions if desired. A final project enables trainees to apply their newly learned skills to real data or case studies of their interest. To promote a friendly atmosphere throughout the course and to build a close-knit community after the course, allow time for some scientific discussions and social activities. In addition, to not exhaust trainees and TAs, some leisure time needs to be organized. Finally, all organization should be done while keeping the budget within fair limits. In order to create a sustainable course that constantly improves and adapts to the trainees’ needs, gathering short- and long-term feedback after the end of the course is important. Based on our experience we have collected a set of recommendations to effectively organize and run extended bioinformatics training courses for evolutionary biologists, which we here want to share with the community. They offer a complementary way for the practical teaching of modern evolutionary biology and reaching out to the biological community.Peer reviewe