Effects of the nerve agent VX on hiPSC-derived motor neurons.

Poisoning with the organophosphorus nerve agent VX can be life-threatening due to limitations of the standard therapy with atropine and oximes. To date, the underlying pathomechanism of VX affecting the neuromuscular junction has not been fully elucidated structurally. Results of recent studies investigating the effects of VX were obtained from cells of animal origin or immortalized cell lines limiting their translation to humans. To overcome this limitation, motor neurons (MN) of this study were differentiated from in-house feeder- and integration-free-derived human-induced pluripotent stem cells (hiPSC) by application of standardized and antibiotic-free differentiation media with the aim to mimic human embryogenesis as closely as possible. For testing VX sensitivity, MN were initially exposed once to 400 µM, 600 µM, 800 µM, or 1000 µM VX and cultured for 5 days followed by analysis of changes in viability and neurite outgrowth as well as at the gene and protein level using µLC-ESI MS/HR MS, XTT, IncuCyte, qRT-PCR, and Western Blot. For the first time, VX was shown to trigger neuronal cell death and decline in neurite outgrowth in hiPSC-derived MN in a time- and concentration-dependent manner involving the activation of the intrinsic as well as the extrinsic pathway of apoptosis. Consistent with this, MN morphology and neurite network were altered time and concentration-dependently. Thus, MN represent a valuable tool for further investigation of the pathomechanism after VX exposure. These findings might set the course for the development of a promising human neuromuscular test model and patient-specific therapies in the future.

Molecular pathways of major depressive disorder converge on the synapse.

Major depressive disorder (MDD) is a psychiatric disease of still poorly understood molecular etiology. Extensive studies at different molecular levels point to a high complexity of numerous interrelated pathways as the underpinnings of depression. Major systems under consideration include monoamines, stress, neurotrophins and neurogenesis, excitatory and inhibitory neurotransmission, mitochondrial dysfunction, (epi)genetics, inflammation, the opioid system, myelination, and the gut-brain axis, among others. This review aims at illustrating how these multiple signaling pathways and systems may interact to provide a more comprehensive view of MDD's neurobiology. In particular, considering the pattern of synaptic activity as the closest physical representation of mood, emotion, and conscience we can conceptualize, each pathway or molecular system will be scrutinized for links to synaptic neurotransmission. Models of the neurobiology of MDD will be discussed as well as future actions to improve the understanding of the disease and treatment options.

The Efficiency of Direct Maturation: the Comparison of Two hiPSC Differentiation Approaches into Motor Neurons.

Motor neurons (MNs) derived from human-induced pluripotent stem cells (hiPSC) hold great potential for the treatment of various motor neurodegenerative diseases as transplantations with a low-risk of rejection are made possible. There are many hiPSC differentiation protocols that pursue to imitate the multistep process of motor neurogenesis in vivo. However, these often apply viral vectors, feeder cells, or antibiotics to generate hiPSC and MNs, limiting their translational potential. In this study, a virus-, feeder-, and antibiotic-free method was used for reprogramming hiPSC, which were maintained in culture medium produced under clinical good manufacturing practice. Differentiation into MNs was performed with standardized, chemically defined, and antibiotic-free culture media. The identity of hiPSC, neuronal progenitors, and mature MNs was continuously verified by the detection of specific markers at the genetic and protein level via qRT-PCR, flow cytometry, Western Blot, and immunofluorescence. MNX1- and ChAT-positive motoneuronal progenitor cells were formed after neural induction via dual-SMAD inhibition and expansion. For maturation, an approach aiming to directly mature these progenitors was compared to an approach that included an additional differentiation step for further specification. Although both approaches generated mature MNs expressing characteristic postmitotic markers, the direct maturation approach appeared to be more efficient. These results provide new insights into the suitability of two standardized differentiation approaches for generating mature MNs, which might pave the way for future clinical applications.

Highly stable peptide adducts from hard keratins as biomarkers to verify local sulfur mustard exposure of hair by high-resolution mass spectrometry.

In the recent past, the blister agent sulfur mustard (SM) deployed by the terroristic group Islamic State has caused a huge number of civilian and military casualties in armed conflicts in the Middle East. The vaporized or aerolized agent might be inhaled and have direct contact to skin and hair. Reaction products of SM with plasma proteins (adducts) represent well-established systemic targets for the bioanalytical verification of exposure. The SM-derived hydroxyethylthioethyl (HETE)-moiety is attached to nucleophilic amino acid side chains and allows unambiguous adduct detection. For shipping of common blood and plasma samples, extensive packaging rules are to be followed as these matrices are considered as potentially infectious material. In contrast, hair is considered as non-infectious thus making its handling and transportation much less complicated. Therefore, we addressed this matrix to develop a procedure for bioanalytical verification. Following optimized lysis of SM-treated human scalp hair and pepsin-catalyzed proteolysis of adducts of keratin type I and II, microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS) was used to detect three alkylated keratin-derived biomarker peptides: AE(-HETE)IRSDL, FKTIE(-HETE)EL, and LE(-HETE)TKLQF simultaneously. All bear the HETE-moiety bound to a glutamic acid residue. Protein adducts were stable for at least 14 weeks at ambient temperature and contact to air, and were not affected by washing the hair with shampoo. The biomarker peptides were also obtained from beard, armpit, abdominal, and pubic hair. This is the first report introducing stable local peptide adduct biomarkers from hair, that is easily accessible by a non-invasive sampling process.

Tricyclic antidepressants target FKBP51 SUMOylation to restore glucocorticoid receptor activity.

FKBP51 is an important inhibitor of the glucocorticoid receptor (GR) signaling. High FKBP51 levels are associated to stress-related disorders, which are linked to GR resistance. SUMO conjugation to FKBP51 is necessary for FKBP51's inhibitory action on GR. The GR/FKBP51 pathway is target of antidepressant action. Thus we investigated if these drugs could inhibit FKBP51 SUMOylation and therefore restore GR activity. Screening cells using Ni2+ affinity and in vitro SUMOylation assays revealed that tricyclic antidepressants- particularly clomipramine- inhibited FKBP51 SUMOylation. Our data show that clomipramine binds to FKBP51 inhibiting its interaction with PIAS4 and therefore hindering its SUMOylation. The inhibition of FKBP51 SUMOylation decreased its binding to Hsp90 and GR facilitating FKBP52 recruitment, and enhancing GR activity. Reduction of PIAS4 expression in rat primary astrocytes impaired FKBP51 interaction with GR, while clomipramine could no longer exert its inhibitory action. This mechanism was verified in vivo in mice treated with clomipramine. These results describe the action of antidepressants as repressors of FKBP51 SUMOylation as a molecular switch for restoring GR sensitivity, thereby providing new potential routes of antidepressant intervention.

Harnessing autophagy to fight SARS-CoV-2: An update in view of recent drug development efforts.

Drug repurposing is an attractive option for identifying new treatment strategies, in particular in extraordinary situations of urgent need such as the current coronavirus disease 2019 (Covid-19) pandemic. Recently, the World Health Organization announced testing of three drugs as potential Covid-19 therapeutics that are known for their dampening effect on the immune system. Thus, the underlying concept of selecting these drugs is to temper the potentially life-threatening overshooting of the immune system reacting to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This viewpoint discusses the possibility that the impact of these and other drugs on autophagy contributes to their therapeutic effect by hampering the SARS-CoV-2 life cycle.

Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: Electrophoretic and mass spectrometric identification and characterization.

For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS). It was found that the Cys10 -residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 µM to 2 mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.

Analysis of the cerebellar molecular stress response led to first evidence of a role for FKBP51 in brain FKBP52 expression in mice and humans.

As the cerebellar molecular stress response is understudied, we assessed protein expression levels of hypothalamic-pituitary-adrenal (HPA) axis regulators and neurostructural markers in the cerebellum of a male PTSD mouse model and of unstressed vs. stressed male FK506 binding protein 51 (Fkbp5) knockout (KO) vs. wildtype mice. We explored the translatability of our findings in the Fkbp5 KO model to the situation in humans by correlating mRNA levels of candidates with those of FKBP5 in two whole transcriptome datasets of post-mortem human cerebellum and in blood of unstressed and stressed humans. Fkbp5 deletion rescued the stress-induced loss in hippocampal, prefrontal cortical, and, possibly, also cerebellar FKBP52 expression and modulated post-stress cerebellar expression levels of the glucocorticoid receptor (GR) and possibly (trend) also of glial fibrillary acidic protein (GFAP). Accordingly, expression levels of genes encoding for these three genes correlated with those of FKBP5 in human post-mortem cerebellum, while other neurostructural markers were not related to Fkbp5 either in mouse or human cerebellum. Also, gene expression levels of the two immunophilins correlated inversely in the blood of unstressed and stressed humans. We found transient changes in FKBP52 and persistent changes in GR and GFAP in the cerebellum of PTSD-like mice. Altogether, upon elucidating the cerebellar stress response we found first evidence for a novel facet of HPA axis regulation, i.e., the ability of FKBP51 to modulate the expression of its antagonist FKBP52 in the mouse and, speculatively, also in the human brain and blood and, moreover, detected long-term single stress-induced changes in expression of cerebellar HPA axis regulators and neurostructural markers of which some might contribute to the role of the cerebellum in fear extinction.

Post-translational modifications and stress adaptation: the paradigm of FKBP51.

Adaptation to stress is a fundamental requirement to cope with changing environmental conditions that pose a threat to the homeostasis of cells and organisms. Post-translational modifications (PTMs) of proteins represent a possibility to quickly produce proteins with new features demanding relatively little cellular resources. FK506 binding protein (FKBP) 51 is a pivotal stress protein that is involved in the regulation of several executers of PTMs. This mini-review discusses the role of FKBP51 in the function of proteins responsible for setting the phosphorylation, ubiquitination and lipidation of other proteins. Examples include the kinases Akt1, CDK5 and GSK3ß, the phosphatases calcineurin, PP2A and PHLPP, and the ubiquitin E3-ligase SKP2. The impact of FKBP51 on PTMs of signal transduction proteins significantly extends the functional versatility of this protein. As a stress-induced protein, FKBP51 uses re-setting of PTMs to relay the effect of stress on various signaling pathways.

Peptidylprolylisomerases, Protein Folders, or Scaffolders? The Example of FKBP51 and FKBP52.

Peptidylprolyl-isomerases (PPIases) comprise of the protein families of FK506 binding proteins (FKBPs), cyclophilins, and parvulins. Their common feature is their ability to expedite the transition of peptidylprolyl bonds between the cis and the trans conformation. Thus, it seemed highly plausible that PPIase enzymatic activity is crucial for protein folding. However, this has been difficult to prove over the decades since their discovery. In parallel, more and more studies have discovered scaffolding functions of PPIases. This essay discusses the hypothesis that PPIase enzymatic activity might be the consequence of binding to peptidylprolyl protein motifs. The main focus of this paper is the large immunophilins FKBP51 and FKBP52, but other PPIases such as cyclophilin A and Pin1 are also described. From the hypothesis, it follows that the PPIase activity of these proteins might be less relevant, if at all, than the organization of protein complexes through versatile protein binding. Also see the video abstract here https://youtu.be/A33la0dx5LE.

Molecular Psychiatry: Trends and Study Examples.

In contrast to about 20-30 years ago, the concept that psychiatric diseases have a molecular basis is now widely accepted [...].

SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection.

Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.

Is There a Role of Autophagy in Depression and Antidepressant Action?

Autophagy has been recognized as evolutionary conserved intracellular pathway that ensures energy, organelle, and protein homeostasis through lysosomal degradation of damaged macromolecules and organelles. It is activated under various stress situations, e.g., food deprivation or proteotoxic conditions. Autophagy has been linked to several diseases, more recently also including stress-related diseases such as depression. A growing number of publications report on the role of autophagy in neurons, also referred to as "neuronal autophagy" on the one hand, and several studies describe effects of antidepressants-or of compounds that exert antidepressant-like actions-on autophagy on the other hand. This minireview highlights the emerging evidence for the involvement of autophagy in the pathology and treatment of depression and discusses current limitations as well as potential avenues for future research.

Epigenetic upregulation of FKBP5 by aging and stress contributes to NF-κB-driven inflammation and cardiovascular risk.

Aging and psychosocial stress are associated with increased inflammation and disease risk, but the underlying molecular mechanisms are unclear. Because both aging and stress are also associated with lasting epigenetic changes, a plausible hypothesis is that stress along the lifespan could confer disease risk through epigenetic effects on molecules involved in inflammatory processes. Here, by combining large-scale analyses in human cohorts with experiments in cells, we report that FKBP5, a protein implicated in stress physiology, contributes to these relations. Across independent human cohorts (total n > 3,000), aging synergized with stress-related phenotypes, measured with childhood trauma and major depression questionnaires, to epigenetically up-regulate FKBP5 expression. These age/stress-related epigenetic effects were recapitulated in a cellular model of replicative senescence, whereby we exposed replicating human fibroblasts to stress (glucocorticoid) hormones. Unbiased genome-wide analyses in human blood linked higher FKBP5 mRNA with a proinflammatory profile and altered NF-κB-related gene networks. Accordingly, experiments in immune cells showed that higher FKBP5 promotes inflammation by strengthening the interactions of NF-κB regulatory kinases, whereas opposing FKBP5 either by genetic deletion (CRISPR/Cas9-mediated) or selective pharmacological inhibition prevented the effects on NF-κB. Further, the age/stress-related epigenetic signature enhanced FKBP5 response to NF-κB through a positive feedback loop and was present in individuals with a history of acute myocardial infarction, a disease state linked to peripheral inflammation. These findings suggest that aging/stress-driven FKBP5-NF-κB signaling mediates inflammation, potentially contributing to cardiovascular risk, and may thus point to novel biomarker and treatment possibilities.

Is Autophagy Involved in the Diverse Effects of Antidepressants?

Autophagy has received increased attention as a conserved process governing cellular energy and protein homeostasis that is thus relevant in a range of physiological and pathophysiological conditions. Recently, autophagy has also been linked to depression, mainly through its involvement in the action of antidepressants. Some antidepressant drugs and psychotropic medication have been reported to exert beneficial effects in other diseases, for example, in cancer and neurodegenerative diseases. This review collates the evidence for the hypothesis that autophagy contributes to the effects of antidepressants beyond depression treatment.

The Stress-Inducible Protein DRR1 Exerts Distinct Effects on Actin Dynamics.

Cytoskeletal dynamics are pivotal to memory, learning, and stress physiology, and thus psychiatric diseases. Downregulated in renal cell carcinoma 1 (DRR1) protein was characterized as the link between stress, actin dynamics, neuronal function, and cognition. To elucidate the underlying molecular mechanisms, we undertook a domain analysis of DRR1 and probed the effects on actin binding, polymerization, and bundling, as well as on actin-dependent cellular processes. METHODS: DRR1 domains were cloned and expressed as recombinant proteins to perform in vitro analysis of actin dynamics (binding, bundling, polymerization, and nucleation). Cellular actin-dependent processes were analyzed in transfected HeLa cells with fluorescence recovery after photobleaching (FRAP) and confocal microscopy. RESULTS: DRR1 features an actin binding site at each terminus, separated by a coiled coil domain. DRR1 enhances actin bundling, the cellular F-actin content, and serum response factor (SRF)-dependent transcription, while it diminishes actin filament elongation, cell spreading, and actin treadmilling. We also provide evidence for a nucleation effect of DRR1. Blocking of pointed end elongation by addition of profilin indicates DRR1 as a novel barbed end capping factor. CONCLUSIONS: DRR1 impacts actin dynamics in several ways with implications for cytoskeletal dynamics in stress physiology and pathophysiology.

Expression and glucocorticoid-dependent regulation of the stress-inducible protein DRR1 in the mouse adult brain.

Identifying molecular targets that are able to buffer the consequences of stress and therefore restore brain homeostasis is essential to develop treatments for stress-related disorders. Down-regulated in renal cell carcinoma 1 (DRR1) is a unique stress-induced protein in the brain and has been recently proposed to modulate stress resilience. Interestingly, DRR1 shows a prominent expression in the limbic system of the adult mouse. Here, we analyzed the neuroanatomical and cellular expression patterns of DRR1 in the adult mouse brain using in situ hybridization, immunofluorescence and Western blot. Abundant expression of DRR1 mRNA and protein was confirmed in the adult mouse brain with pronounced differences between distinct brain regions. The strongest DRR1 signal was detected in the neocortex, the CA3 region of the hippocampus, the lateral septum and the cerebellum. DRR1 was also present in circumventricular organs and its connecting regions. Additionally, DRR1 was present in non-neuronal tissues like the choroid plexus and ependyma. Within cells, DRR1 protein was distributed in a punctate pattern in several subcellular compartments including cytosol, nucleus as well as some pre- and postsynaptic specializations. Glucocorticoid receptor activation (dexamethasone 10 mg/kg s.c.) induced DRR1 expression throughout the brain, with particularly strong induction in white matter and fiber tracts and in membrane-rich structures. This specific expression pattern and stress modulation of DRR1 point to a role of DRR1 in regulating how cells sense and integrate signals from the environment and thus in restoring brain homeostasis after stressful challenges.

Hsp90 and FKBP51: complex regulators of psychiatric diseases.

Mood disorders affect nearly a quarter of the world's population. Therefore, understanding the molecular mechanisms underlying these conditions is of great importance. FK-506 binding protein 5 (FKBP5) encodes the FKBP51 protein, a heat shock protein 90 kDa (Hsp90) co-chaperone, and is a risk factor for several affective disorders. FKBP51, in coordination with Hsp90, regulates glucocorticoid receptor (GR) activity via a short negative feedback loop. This signalling pathway rapidly restores homeostasis in the hypothalamic-pituitary-adrenal (HPA) axis following stress. Expression of FKBP5 increases with age through reduced DNA methylation. High levels of FKBP51 are linked to GR resistance and reduced stress coping behaviour. Moreover, common allelic variants in the FKBP5 gene are associated with increased risk of developing affective disorders like anxiety, depression and post-traumatic stress disorder (PTSD). This review highlights the current understanding of the Hsp90 co-chaperone, FKBP5, in disease from both human and animal studies. In addition, FKBP5 genetic implications in the clinic involving life stress exposure, gender differences and treatment outcomes are discussed.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.