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Abstract Background Regulated protein-protein interactions (PPIs) are pivotal molecular switches that are important for the regulation of signaling processes within eukaryotic cells. Cellular signaling is altered in various disease conditions and offers interesting options for pharmacological interventions. Constitutive PPIs are usually mediated by large interaction domains. In contrast, stimulus-regulated PPIs often depend on small post-translational modifications and are thus better suited targets for drug development. However, the detection of modification-dependent PPIs with biochemical methods still remains a labour- and material-intensive task, and many pivotal PPIs that are potentially suited for pharmacological intervention most likely remain to be identified. The availability of methods to easily identify and quantify stimulus-dependent, potentially also transient interaction events, is therefore essential. The assays should be applicable to intact mammalian cells, optimally also to primary cells in culture. Results In this study, we adapted the split-TEV system to quantify phosphorylation-dependent and transient PPIs that occur at the membrane and in the cytosol of living mammalian cells. Split-TEV is based on a PPI-induced functional complementation of two inactive TEV protease fragments fused to interaction partners of choice. Genetically encoded transcription-coupled and proteolysis-only TEV reporter systems were used to convert the TEV activity into an easily quantifiable readout. We measured the phosphorylation-dependent interaction between the pro-apoptotic protein Bad and the adapter proteins 14-3-3ε and ζ in NIH-3T3 fibroblasts and in primary cultured neurons. Using split-TEV assays, we show that Bad specifically interacts with 14-3-3 isoforms when phosphorylated by protein kinase Akt-1/PKB at Ser136. We also measured the phosphorylation-dependent Bad/14-3-3 interactions mediated by endogenous and transient Akt-1 activity. We furthermore applied split-TEV assays to measure the phosphorylation-dependent interactions of Neuregulin-1-stimulated ErbB4 receptors with several adapter proteins. Conclusion Split-TEV assays are well suited to measure phosphorylation-dependent and transient PPIs that occur specifically at the membrane and in the cytosol of heterologous and primary cultured mammalian cells. Given the high sensitivity of the split-TEV system, all assays were performed in multi-plate formats and could be adapted for higher throughput to screen for pharmacologically active substances.

Abstract Background The different physiological repertoire of CA3 and CA1 neurons in the hippocampus, as well as their differing behaviour after noxious stimuli are ultimately based upon differences in the expressed genome. We have compared CA3 and CA1 gene expression in the uninjured brain, and after cerebral ischemia using laser microdissection (LMD), RNA amplification, and array hybridization. Results Profiling in CA1 vs. CA3 under normoxic conditions detected more than 1000 differentially expressed genes that belong to different, physiologically relevant gene ontology groups in both cell types. The comparison of each region under normoxic and ischemic conditions revealed more than 5000 ischemia-regulated genes for each individual cell type. Surprisingly, there was a high co-regulation in both regions. In the ischemic state, only about 100 genes were found to be differentially expressed in CA3 and CA1. The majority of these genes were also different in the native state. A minority of interesting genes (e.g. inhibinbetaA) displayed divergent expression preference under native and ischemic conditions with partially opposing directions of regulation in both cell types. Conclusion The differences found in two morphologically very similar cell types situated next to each other in the CNS are large providing a rational basis for physiological differences. Unexpectedly, the genomic response to ischemia is highly similar in these two neuron types, leading to a substantial attenuation of functional genomic differences in these two cell types. Also, the majority of changes that exist in the ischemic state are not generated de novo by the ischemic stimulus, but are preexistant from the genomic repertoire in the native situation. This unexpected influence of a strong noxious stimulus on cell-specific gene expression differences can be explained by the activation of a cell-type independent conserved gene-expression program. Our data generate both novel insights into the relation of the quiescent and stimulus-induced transcriptome in different cells, and provide a large dataset to the research community, both for mapping purposes, as well as for physiological and pathophysiological research.

MicroRNAs (miRNAs) have the potential to affect drug metabolism, and some drugs affect cellular miRNA expression. miRNAs are found inside extracellular vesicles (EVs), and the profile of these EV-miRNAs can change across different diseases and disease states. Consequently, in recent years EV-miRNAs have attracted increasing attention as possible non-invasive biomarkers. For example, analyzing the miRNA expression profile of brain-derived EVs in blood may allow us to non-invasively assess miRNA dysregulation and thus to gain knowledge about the pathophysiology of psychiatric disorders and identify potential new predictive targets. We searched PubMed for all studies related to the effects of psychiatric medications on EV-miRNAs and identified 14 relevant articles. Taken together, findings indicate that certain EV-miRNAs may be targets for psychiatric medications and that antipsychotics such as olanzapine and antidepressants such as fluoxetine may alter the expression levels of particular EV-miRNAs. If confirmed and replicated, these findings may lead to the suggested miRNA profiles being used as pharmacodynamic biomarkers. However, heterogeneities and uncertainties remain regarding the role of EV-miRNAs in psychiatric disorders and their interaction with neuronal gene expression and drugs. This minireview summarizes some of the findings on the effects of psychiatric medications on EV-miRNAs and describes the potential role of EV-miRNAs as pharmacodynamic biomarkers for psychiatric disorders., Competing Interests: Declaration of competing interest The authors declare that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Astrocytes regulate brain-wide functions and also show region-specific differences, but little is known about how general and region-specific functions are aligned at the single-cell level. To explore this, we isolated adult mouse diencephalic astrocytes by ACSA-2-mediated magnetic-activated cell sorting (MACS). Single-cell RNA-seq revealed 7 gene expression clusters of astrocytes, with 4 forming a supercluster. Within the supercluster, cells differed by gene expression related to ion homeostasis or metabolism, with the former sharing gene expression with other regions and the latter being restricted to specific regions. All clusters showed expression of proliferation-related genes, and proliferation of diencephalic astrocytes was confirmed by immunostaining. Clonal analysis demonstrated low level of astrogenesis in the adult diencephalon, but not in cerebral cortex grey matter. This led to the identification of Smad4 as a key regulator of diencephalic astrocyte in vivo proliferation and in vitro neurosphere formation. Thus, astrocytes show diverse gene expression states related to distinct functions with some subsets being more widespread while others are more regionally restricted. However, all share low-level proliferation revealing the novel concept of adult astrogenesis in the diencephalon., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Astrocyte-derived cholesterol supports brain cells under physiological conditions. However, in demyelinating lesions, astrocytes downregulate cholesterol synthesis, and the cholesterol that is essential for remyelination has to originate from other cellular sources. Here, we show that repair following acute versus chronic demyelination involves distinct processes. In particular, in chronic myelin disease, when recycling of lipids is often defective, de novo neuronal cholesterol synthesis is critical for regeneration. By gene expression profiling, genetic loss-of-function experiments, and comprehensive phenotyping, we provide evidence that neurons increase cholesterol synthesis in chronic myelin disease models and in patients with multiple sclerosis (MS). In mouse models, neuronal cholesterol facilitates remyelination specifically by triggering oligodendrocyte precursor cell proliferation. Our data contribute to the understanding of disease progression and have implications for therapeutic strategies in patients with MS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many psychiatric disorders, for example, anxiety, are accompanied by disturbances of circadian rhythms, including disturbed sleep/wake cycles, changes in locomotor activity, and abnormal endocrine function. Conversely, alternations of circadian rhythms are a risk factor for the development of psychiatric disorders. This assumption is supported by animals with clock gene mutations which often display behaviors that resemble human psychiatric disorders. In this study, we performed an in-depth behavioral analysis with male mice lacking the central clock genes Cryptochrome 1 and 2 (Cry1/2 -/- ), which are thus unable to express endogenous circadian rhythms. With wild-type and Cry1/2 -/- mice, we performed an extensive behavioral analysis to study their cognitive abilities, social behavior, and their expression of depression-like and anxiety-like behavior. While Cry1/2 -/- mice showed only mild abnormalities at cognitive and social behavioral levels, they were consistently more anxious than wildtype mice. Anxiety-like behavior was particularly evident in reduced mobility in new environments, altered ability to habituate, compensatory behavior, and consistent restless behavior across many behavioral tests. In line with their anxiety-like behavioral phenotype, Cry1/2 -/- mice have higher c-Fos activity in the amygdala after exposure to an anxiogenic stressor than wild-type mice. In our study, we identified Cry1/2 -/- mice as animals that qualify as a translational mouse model for anxiety disorder in humans because of its consistent behavior of restlessness, increased immobility, and dysfunctional habituation in new environments., (© 2020 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

An evolutionarily conserved function of glia is to provide metabolic and structural support for neurons. To identify molecules generated by glia and with vital functions for neurons, we used Drosophila melanogaster as a screening tool, and subsequently translated the findings to mice. We found that a cargo receptor operating in the secretory pathway of glia was essential to maintain axonal integrity by regulating iron buffering. Ferritin heavy chain was identified as the critical secretory cargo, required for the protection against iron-mediated ferroptotic axonal damage. In mice, ferritin heavy chain is highly expressed by oligodendrocytes and secreted by employing an unconventional secretion pathway involving extracellular vesicles. Disrupting the release of extracellular vesicles or the expression of ferritin heavy chain in oligodendrocytes causes neuronal loss and oxidative damage in mice. Our data point to a role of oligodendrocytes in providing an antioxidant defense system to support neurons against iron-mediated cytotoxicity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cognitive deficits are increasingly recognized as a core dimension rather than a consequence of schizophrenia (SCZ). The previous evidence supports the hypothesis of shared genetic factors between SCZ and cognitive ability. The objective of this study was to test whether and to what extent the variation of disease-relevant neurocognitive function in a sample of SCZ patients from the previous clinical interventional studies can be explained by SCZ polygenic risk scores (PRSs) or by hypothesis-driven and biomedical PRSs. The previous studies have described associations of the SNAP25 gene with cognition in SCZ. Likewise, the enrichment of several calcium signaling-related gene sets has been reported by genome-wide association studies (GWAS) in SCZ. Hypothesis-driven PRSs were calculated on the basis of the SNAP-25 interactome and also for genes regulated by phorbol myristate acetate (PMA), an activator of the signal transduction of protein kinase C (PKC) enzymes. In a cohort of 127 SCZ patients who had completed a comprehensive neurocognitive test battery as part of the previous antipsychotic intervention studies, we investigated the association between neurocognitive dimensions and PRSs. The PRS for SCZ and SNAP-25-associated genes could not explain the variance of neurocognition in this cohort. At a p value threshold of 0.05, the PRS for PMA was able to explain 2% of the variance in executive function (p = 0.05, uncorrected). The correlation between the PRS for PMA-regulated genes and cognition can give hints for further patient-derived cellular assays. In conclusion, incorporating biological information into PRSs and other en masse genetic analyses may help to close the gap between genetic vulnerability and the biological processes underlying neuropsychiatric diseases such as SCZ.

Competing Interests: Interessenkonflikte: Mit-Gründer der SYSTASY Bioscience GmbH, die die Patente zur split-Sensor Technology hält; Berater der Boehringer Ingelheim AG & Co. KG.

Objectives: Despite progress in identifying molecular pathophysiological processes in schizophrenia, valid biomarkers are lacking for both the disease and treatment response., Methods: This comprehensive review summarises recent efforts to identify molecular mechanisms on the level of protein and gene expression and epigenetics, including DNA methylation, histone modifications and micro RNA expression. Furthermore, it summarises recent findings of alterations in lipid mediators and highlights inflammatory processes. The potential that this research will identify biomarkers of schizophrenia is discussed., Results: Recent studies have not identified clear biomarkers for schizophrenia. Although several molecular pathways have emerged as potential candidates for future research, a complete understanding of these metabolic pathways is required to reveal better treatment modalities for this disabling condition., Conclusions: Large longitudinal cohort studies are essential that pair a thorough phenotypic and clinical evaluation for example with gene expression and proteome analysis in blood at multiple time points. This approach might identify biomarkers that allow patients to be stratified according to treatment response and ideally also allow treatment response to be predicted. Improved knowledge of molecular pathways and epigenetic mechanisms, including their potential association with environmental influences, will facilitate the discovery of biomarkers that could ultimately be effective tools in clinical practice.

Chronic psychosocial stress is an important environmental risk factor for the development of psychiatric diseases. However, studying the impact of chronic psychosocial stress in mice is time consuming and thus not optimally suited to 'screen' increasing numbers of genetically manipulated mouse models for psychiatric endophenotypes. Moreover, many studies focus on restraint stress, a strong physical stressor with limited relevance for psychiatric disorders. Here, we describe a simple and a rapid method based on the resident-intruder paradigm to examine acute effects of mild psychosocial stress in mice. The OSO paradigm (open field--social defeat--open field) compares behavioral consequences on locomotor activity, anxiety and curiosity before and after exposure to acute social defeat stress. We first evaluated OSO in male C57Bl/6 wildtype mice where a single episode of social defeat reduced locomotor activity, increased anxiety and diminished exploratory behavior. Subsequently, we applied the OSO paradigm to mouse models of two schizophrenia (SZ) risk genes. Transgenic mice with neuronal overexpression of Neuregulin-1 (Nrg1) type III showed increased risk-taking behavior after acute stress exposure suggesting that NRG1 dysfunction is associated with altered affective behavior. In contrast, Tcf4 transgenic mice displayed a normal stress response which is in line with the postulated predominant contribution of TCF4 to cognitive deficits of SZ. In conclusion, the OSO paradigm allows for rapid screening of selected psychosocial stress-induced behavioral endophenotypes in mouse models of psychiatric diseases., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Sharp-1 is a basic helix-loop-helix (bHLH) transcriptional repressor that is involved in a number of cellular processes. Our previous studies have demonstrated that Sharp-1 is a negative regulator of skeletal myogenesis and it blocks differentiation of muscle precursor cells by modulating the activity of MyoD. In order to understand its role in pre- and post-natal myogenesis, we assessed skeletal muscle development and freeze-injury-induced regeneration in Sharp-1-deficient mice. We show that embryonic skeletal muscle development is not impaired in the absence of Sharp-1; however, post-natally, the regenerative capacity is compromised. Although the initial phases of injury-induced regeneration proceed normally in Sharp-1(-/-) mice, during late stages, the mutant muscle exhibits necrotic fibers, calcium deposits and fibrosis. TGF-β expression, as well as levels of phosphorylated Smad2 and Smad3, are sustained in the mutant tissue and treatment with decorin, which blocks TGF-β signaling, improves the histopathology of Sharp-1(-/-) injured muscles. In vitro, Sharp-1 associates with Smad3, and its overexpression inhibits TGF-β- and Smad3-mediated expression of extracellular matrix genes in myofibroblasts. These results demonstrate that Sharp-1 regulates muscle regenerative capacity, at least in part, by modulation of TGF-β signaling.

Hypothalamic functions, including feeding behavior, show a high degree of plasticity throughout life. Doublecortin (DCX) is a marker of plasticity and neuronal migration expressed in the hypothalamus. Therefore, we wanted to map the fate of DCX(+) cells in the arcuate nucleus (ARC) of the hypothalamus. For this purpose, we generated a BAC transgenic mouse line that expresses the inducible recombinase CreER(T2) under control of the DCX locus. Crossing this line with the Rosa26 or Ai14 reporter mouse lines, we found reporter(+) cells in the ARC upon tamoxifen treatment. They were born prenatally and expressed both DCX and the plasticity marker TUC-4. Immediately after labeling, reporter(+) cells had an enlarged soma that normalized over time, suggesting morphological remodeling. Reporter(+) cells expressed β-endorphin and BSX, neuronal markers of the feeding circuit. Furthermore, leptin treatment led to phosphorylation of STAT3 in reporter(+) cells in accordance with the concept that they are part of the feeding circuits. Indeed, we found a negative correlation between the number of reporter(+) cells and body weight and epididymal fat pads. Our data suggest that DCX(+) cells in the ARC represent a cellular correlate of plasticity that is involved in controlling energy balance in adult mice.

PATHOWIKI (http://www.pathowiki.org) is a new specialized information system in the form of a web-based wiki with content from all sub-disciplines of human pathology. Essential components are articles and specimens which are located thematically in dermatopathology. The project is presented on the basis of impressive examples and possibilities. The ability to link all kinds of content and integrate pattern analysis theories creates an effective tool for teaching and training in dermatopathology. Collaborative work ensures the effective usage of available resources and a continually growing amount of content, the quality of which depends on the number of users and should be as high as possible. Therefore, all interested colleagues are invited to support the project., (© The Authors • Journal compilation © Blackwell Verlag GmbH, Berlin.)

The project Pathowiki (www.pathowiki.org) is a free expert database for texts, images, virtual slides and links to all subject areas of pathology in the internet. The aim of this project is to integrate all available information and media, in particular virtual microscopy, to achieve a fast overview of a relevant subject area. Here we present the project’s basic functions and applications and evaluate the project with respect to the ongoing digital developments in pathology.

Data sharing is important in the biological sciences to prevent duplication of effort, to promote scientific integrity, and to facilitate and disseminate scientific discovery. Sharing requires centralized repositories, and submission to and utility of these resources require common data formats. This is particularly challenging for multidimensional microscopy image data, which are acquired from a variety of platforms with a myriad of proprietary file formats (PFFs). In this paper, we describe an open standard format that we have developed for microscopy image data. We call on the community to use open image data standards and to insist that all imaging platforms support these file formats. This will build the foundation for an open image data repository.

Background: Injectable fillers are widely used in aesthetic medicine. Polylactic acid (PLA) is a semipermanent filler that needs to be diluted with sterile water before injection. PLA has been associated with an increased risk of adverse reactions, specifically nodule formation., Objective: To describe adverse reactions to PLA and potential risk factors based on a partly population-based registry over an 8-year period., Methods: The Berlin registry is a partially population-based registry where dermatologists, plastic surgeons, and other specialists are contacted regularly and asked to report patients with adverse events to injectable fillers substances. Additional patients were derived from private practices outside of Berlin. The patients were assessed with a standardized questionnaire. The results were mainly analyzed by descriptive measures., Results: Twenty-two patients (age, 47.82 +/- 12.65 years) with adverse reactions to PLA were included. The most frequent adverse reaction was nodule formation found in all patients. In 13 (59.1%) of the cases, nodule formation was considered to be severe. Nodules appeared after a mean latency period of 6.00 +/- 5.84 months. The duration of the adverse reactions until the interview was 14.32 +/- 10.13 months. The frequency of patients with adverse events to PLA was found to decrease after new recommendations concerning the dilution of the product were launched., Conclusion: PLA is an injectable filler substance that may cause subcutaneous nodules in treated patients. Our data support a decreased risk of adverse reactions with an increased dilution. However, nodule formation still appears to be a characteristic feature of PLA.

Background: Hydroxyethylmethacrylate and ethylmethacrylate in a fixed combination with hyaluronic acid has been used as an injectable filler for nearly a decade. Severe adverse reactions have been associated with this filler., Objective: To characterize the adverse reactions to this filler., Methods: Data from the Berlin registry for adverse reactions to injectable fillers were analyzed. The registry is a partially population-based registry with the aim of collecting adverse reactions to injectable fillers. Patients were interviewed based on a standardized questionnaire., Results: Thirty-four of 118 (28.8%) registered patients were treated with this filler. Of 95 treated areas, 87 responded with a reaction (91.6%). The most frequently observed adverse events were the development of nodules (n=85) in 87 affected areas, discoloration (n=39), erythema or inflammation (n=32), and swelling (n=24). Most nodular reactions were rated as severe. The mean time after the last treatment until appearance of an adverse reaction was 23.1+/-22.8 months., Conclusion: Adverse reactions to this methacrylate filler are common. The mean latency period for these mostly severe rated reactions was nearly 2 years. Based on the frequency and severity of these reactions, the use of this filler does not seem to be advisable.

SHARP1, a basic helix-loop-helix transcription factor, is expressed in many cell types; however, the mechanisms by which it regulates cellular differentiation remain largely unknown. Here, we show that SHARP1 negatively regulates adipogenesis. Although expression of the early marker CCAAT/enhancer binding protein beta (C/EBPbeta) is not altered, its crucial downstream targets C/EBPalpha and peroxisome proliferator-activated receptor gamma (PPARgamma) are downregulated by SHARP1. Protein interaction studies confirm that SHARP1 interacts with and inhibits the transcriptional activity of both C/EBPbeta and C/EBPalpha, and enhances the association of C/EBPbeta with histone deacetylase 1 (HDAC1). Consistently, in SHARP1-expressing cells, HDAC1 and the histone methyltransferase G9a are retained at the C/EBP regulatory sites on the C/EBPalpha and PPARgamma2 promoters during differentiation, resulting in inhibition of their expression. Interestingly, treatment with troglitazone results in displacement of HDAC1 and G9a, and rescues the differentiation defect of SHARP1-overexpressing cells. Our data indicate that SHARP1 inhibits adipogenesis through the regulation of C/EBP activity, which is essential for PPARgamma-ligand-dependent displacement of co-repressors from adipogenic promoters.

Background: The different physiological repertoire of CA3 and CA1 neurons in the hippocampus, as well as their differing behaviour after noxious stimuli are ultimately based upon differences in the expressed genome. We have compared CA3 and CA1 gene expression in the uninjured brain, and after cerebral ischemia using laser microdissection (LMD), RNA amplification, and array hybridization., Results: Profiling in CA1 vs. CA3 under normoxic conditions detected more than 1000 differentially expressed genes that belong to different, physiologically relevant gene ontology groups in both cell types. The comparison of each region under normoxic and ischemic conditions revealed more than 5000 ischemia-regulated genes for each individual cell type. Surprisingly, there was a high co-regulation in both regions. In the ischemic state, only about 100 genes were found to be differentially expressed in CA3 and CA1. The majority of these genes were also different in the native state. A minority of interesting genes (e.g. inhibinbetaA) displayed divergent expression preference under native and ischemic conditions with partially opposing directions of regulation in both cell types., Conclusion: The differences found in two morphologically very similar cell types situated next to each other in the CNS are large providing a rational basis for physiological differences. Unexpectedly, the genomic response to ischemia is highly similar in these two neuron types, leading to a substantial attenuation of functional genomic differences in these two cell types. Also, the majority of changes that exist in the ischemic state are not generated de novo by the ischemic stimulus, but are preexistant from the genomic repertoire in the native situation. This unexpected influence of a strong noxious stimulus on cell-specific gene expression differences can be explained by the activation of a cell-type independent conserved gene-expression program. Our data generate both novel insights into the relation of the quiescent and stimulus-induced transcriptome in different cells, and provide a large dataset to the research community, both for mapping purposes, as well as for physiological and pathophysiological research.