Unraveling the functional contribution of Fkbp5 to stress vulnerability

Trotz jahrzehntelanger Bemühungen gibt es noch immer eine Vielzahl psychiatrischer Erkrankungen, für die uns keine effektiven Behandlungsmöglichkeiten zur Verfügung stehen. Da wir den Einfluss unserer Umwelt auf die psychische Gesundheit nur zu einem gewissen Grad beeinflussen können, ist die Identifikation von Risikogenen für eben solche Krankheiten besonders wichtig. Einer der Kandidaten, der in den letzten Jahren große Beachtung gefunden hat ist dabei das FKBP5-Gen, welches beim Menschen in starker Verbindung zu Depression oder Posttraumatischer Belastungsstörung steht. FKBP5 ist ein wichtiger Regulator für die Ausschüttung von Stress-Hormonen und an essenzieller Stelle in den molekularen Signalweg der körpereigenen Stressantwort eingebunden. Um unser Verständnis des Gens zu erweitern, habe ich im Rahmen dieser Arbeit eine weitreichende Charakterisierung verschiedener Mausmodelle durchgeführt. Dabei konnte ich zwei monoaminerge Regionen im Gehirn identifizieren, die eine sehr hohe Grundexpression des Gens aufweisen und in denen Fkbp5 stressabhängig reguliert wird. Sowohl virale Überexpression, als auch das genetische Ausschalten des Gens hatten Veränderungen im Verhalten und der hormonellen Stressantwort der Tiere zur Folge. Im nächsten Schritt habe ich SAFit2, einen spezifischen Antagonisten für Fkbp5, eingesetzt und konnte dabei wieder Veränderungen im Verhalten und der endokrinen Reaktion auf einen externen Stressor beobachten. Auch die Kombination mit einem etablierten Antidepressivum hatte verhaltensbasierte Auswirkungen und sollte in zukünftigen Untersuchungen noch erweitert werden. Im Rahmen meiner Versuche konnte ich zudem einen erheblichen Beitrag zur Entwicklung und Erprobung neuer Applikationsmöglichkeiten von chemischen Substanzen leisten. Dabei habe ich eine Reihe von in vivo Tests mit einem neu entwickelten, Gel-basierten Trägerstoff für SAFit2 durchgeführt. Insgesamt bilden die Ergebnisse dieser Arbeit eine hervorragende Basis für die weitere Erprobung von Fkbp5-basierten Behandlungsmethoden. Die Manipulation von FKBP5 ist immer noch eine vielversprechende Alternative zu etablierten Behandlungsmethoden für psychiatrische Erkrankungen

A system for production of defective interfering particles in the absence of infectious influenza A virus

<div><p>Influenza A virus (IAV) infection poses a serious health threat and novel antiviral strategies are needed. Defective interfering particles (DIPs) can be generated in IAV infected cells due to errors of the viral polymerase and may suppress spread of wild type (wt) virus. The antiviral activity of DIPs is exerted by a DI genomic RNA segment that usually contains a large deletion and suppresses amplification of wt segments, potentially by competing for cellular and viral resources. DI-244 is a naturally occurring prototypic segment 1-derived DI RNA in which most of the PB2 open reading frame has been deleted and which is currently developed for antiviral therapy. At present, coinfection with wt virus is required for production of DI-244 particles which raises concerns regarding biosafety and may complicate interpretation of research results. Here, we show that cocultures of 293T and MDCK cell lines stably expressing codon optimized PB2 allow production of DI-244 particles solely from plasmids and in the absence of helper virus. Moreover, we demonstrate that infectivity of these particles can be quantified using MDCK-PB2 cells. Finally, we report that the DI-244 particles produced in this novel system exert potent antiviral activity against H1N1 and H3N2 IAV but not against the unrelated vesicular stomatitis virus. This is the first report of DIP production in the absence of infectious IAV and may spur efforts to develop DIPs for antiviral therapy.</p></div

Mutagenic analysis of the HIV restriction factor shiftless

The interferon-induced host cell protein shiftless (SFL) was reported to inhibit human immunodeficiency virus (HIV) infection by blocking the –1 programmed ribosomal frameshifting (–1PRF) required for expression of the Gag-Pol polyprotein. However, it is not clear how SFL inhibits –1PRF. To address this question, we focused on a 36 amino acids comprising region (termed required for antiviral activity (RAA)) that is essential for suppression of –1PRF and HIV infection and is missing from SFL short (SFLS), a splice variant of SFL with unknown function. Here, we confirm that SFL, but not SFLS, inhibits HIV –1PRF and show that inhibition is cell-type-independent. Mutagenic and biochemical analyses demonstrated that the RAA region is required for SFL self-interactions and confirmed that it is necessary for ribosome association and binding to the HIV RNA. Analysis of SFL mutants with six consecutive amino-acids-comprising deletions in the RAA region suggests effects on binding to the HIV RNA, complete inhibition of –1PRF, inhibition of Gag-Pol expression, and antiviral activity. In contrast, these amino acids did not affect SFL expression and were partially dispensable for SFL self-interactions and binding to the ribosome. Collectively, our results support the notion that SFL binds to the ribosome and the HIV RNA in order to block –1PRF and HIV infection, and suggest that the multimerization of SFL may be functionally important

First-principles molecular-dynamics simulations of a hydrous silica melt: Structural properties and hydrogen diffusion mechanism

We use {\it ab initio} molecular dynamics simulations to study a sample of liquid silica containing 3.84 wt.% H$_2$O.We find that, for temperatures of 3000 K and 3500 K,water is almost exclusively dissolved as hydroxyl groups, the silica network is partially broken and static and dynamical properties of the silica network change considerably upon the addition of water.Water molecules or free O-H groups occur only at the highest temperature but are not stable and disintegrate rapidly.Structural properties of this system are compared to those of pure silica and sodium tetrasilicate melts at equivalent temperatures. These comparisons confirm the picture of a partially broken tetrahedral network in the hydrous liquid and suggest that the structure of the matrix is as much changed by the addition of water than it is by the addition of the same amount (in mole %) of sodium oxide. On larger length scales, correlations are qualitatively similar but seem to be more pronounced in the hydrous silica liquid. Finally, we study the diffusion mechanisms of the hydrogen atoms in the melt. It turns out that HOSi$_2$ triclusters and SiO dangling bonds play a decisive role as intermediate states for the hydrogen diffusion.Comment: 25 pages, 18 figures. submitte

Hippocampal Homer1 Levels Influence Motivational Behavior in an Operant Conditioning Task

Loss of motivation and learning impairments are commonly accepted core symptoms of psychiatric disorders such as depression and schizophrenia. Reward-motivated learning is dependent on the hippocampal formation but the molecular mechanisms that lead to functional incentive motivation in this brain region are still largely unknown. Recent evidence implicates neurotransmission via metabotropic glutamate receptors and Homer1, their interaction partner in the postsynaptic density, in drug addiction and motivational learning. As previous reports mainly focused on the prefrontal cortex and the nucleus accumbens, we now investigated the role of hippocampal Homer1 in operant reward learning in the present study. We therefore tested either Homer1 knockout mice or mice that overexpress Homer1 in the hippocampus in an operant conditioning paradigm. Our results show that deletion of Homer1 leads to a diverging phenotype that either displays an inability to perform the task or outstanding hyperactivity in both learning and motivational sessions. Due to the apparent bimodal distribution of this phenotype, the overall effect of Homer1 deletion in this paradigm is not significantly altered. Overexpression of hippocampal Homer1 did not lead to a significantly altered learning performance in any stage of the testing paradigm, yet may subtly contribute to emerging motivational deficits. Our results indicate an involvement of Homer1-mediated signaling in the hippocampus in motivation-based learning tasks and encourage further investigations regarding the specific molecular underpinnings of the phenotypes observed in this study. We also suggest to cautiously interpret the results of this and other studies regarding the phenotype following Homer1 manipulations in animals, since their behavioral phenotype appears to be highly diverse. Future studies would benefit from larger group sizes that would allow splitting the experimental groups in responders and non-responders

The stress regulator FKBP51: a novel and promising druggable target for the treatment of persistent pain states across sexes

It is well established that FKBP51 regulates the stress system by modulating the sensitivity of the glucocorticoid receptor to stress hormones. Recently, we have demonstrated that FKBP51 also drives long-term inflammatory pain states in male mice by modulating glucocorticoid signalling at spinal cord level. Here, we explored the potential of FKBP51 as a new pharmacological target for the treatment of persistent pain across the sexes. First, we demonstrated that FKBP51 regulates long-term pain states of different aetiologies independently of sex. Deletion of FKBP51 reduced the mechanical hypersensitivity seen in joint inflammatory and neuropathic pain states in female and male mice. Furthermore, FKBP51 deletion also reduced the hypersensitivity seen in a translational model of chemotherapy-induced pain. Interestingly, these 3 pain states were associated with changes in glucocorticoid signalling, as indicated by the increased expression, at spinal cord level, of the glucocorticoid receptor isoform associated with glucocorticoid resistance, GRβ, and increased levels of plasma corticosterone. These pain states were also accompanied by an upregulation of interleukin-6 in the spinal cord. Crucially, we were able to pharmacologically reduce the severity of the mechanical hypersensitivity seen in these 3 models of persistent pain with the unique FKBP51 ligand SAFit2. When SAFit2 was combined with a state-of-the-art vesicular phospholipid gel formulation for slow release, a single injection of SAFit2 offered pain relief for at least 7 days. We therefore propose the pharmacological blockade of FKBP51 as a new approach for the treatment of persistent pain across sexes, likely in humans as well as rodents

Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts.

The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host