Fluoxetine requires the endfeet protein aquaporin-4 to enhance plasticity of astrocyte processes

Morphological alterations in astrocytes are characteristic for post mortem brains of patients affected by major depressive disorder (MDD). Recently, a significant reduction in the coverage of blood vessels (BVs) by aquaporin-4 (AQP-4)-positive astrocyte endfeet has been shown in the prefrontal cortex (PFC) of MDD patients, suggesting that either alterations in the morphology of endfeet or in AQP-4 distribution might be responsible for the disease phenotype or constitute a consequence of its progress. Antidepressant drugs (ADs) regulate the expression of several proteins, including astrocyte-specific ones. Thus, they may target AQP-4 to induce morphological changes in astrocytes and restore their proper shape or relocate AQP-4 to endfeet. Using an animal model of depression, rats selectively bred for high anxiety-like behavior (HAB), we confirmed a reduced coverage of BVs in the adult PFC by AQP-4-immunoreactive (AQP-4-IR) astrocyte processes with respect to non-selected Wistar rats (NAB), thereby validating it for our study. A further evaluation of the morphology of astrocyte in brain slices (ex vivo) and in vitro using an antibody against the astrocyte-specific cytoskeletal protein glial fibrillary acidic protein (GFAP) revealed that HAB astrocytes extended less processes than NAB cells. Furthermore, short-term drug treatment in vitro with the AD fluoxetine (FLX) was sufficient to increase the plasticity of astrocyte processes, enhancing their number in NAB-derived cells and recovering their basal number in HAB-derived cells. This enhanced FLX-dependent plasticity occurred, however, only in the presence of intact AQP-4, as demonstrated by the lack of effect after the downregulation of AQP-4 with RNAi in both NAB and HAB cells. Nonetheless, a similar short-term treatment did neither modulate the coverage of BVs with AQP-4-positive astrocyte endfeet in NAB nor in HAB rats, although dosage and time of treatment were sufficient to fully recover GFAP expression in HAB brains. Thus, we suggest that longer treatment regimes may be needed to properly restore the coverage of BVs or to relocate AQP-4 to astrocyte endfeet. In conclusion, FLX requires AQP-4 to modulate the plasticity of astrocyte processes and this effect might be essential to re-establish a functional glia-vasculature interface necessary for a physiological communication between bloodstream and brain parenchyma

T Cell-Specific Overexpression of Acid Sphingomyelinase Results in Elevated T Cell Activation and Reduced Parasitemia During Plasmodium yoelii Infection

The enzyme acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and is thereby involved in several cellular processes such as differentiation, proliferation, and apoptosis in different cell types. However, the function of ASM in T cells is still not well characterized. Here, we used T cell-specific ASM overexpressing mice (t-ASM/CD4cre) to clarify the impact of cell-intrinsic ASM activity on T cell function in vitro and in vivo. We showed that t-ASM/CD4cre mice exhibit decreased frequencies of Foxp3+ T regulatory cells (Tregs) within the spleen. Enforced T cell-specific ASM expression resulted in less efficient induction of Tregs and promoted differentiation of CD4+CD25− naïve T cells into IFN-γ producing Th1 cells in vitro. Further analysis revealed that ASM-overexpressing T cells from t-ASM/CD4cre mice show elevated T cell receptor (TCR) signaling activity accompanied with increased proliferation upon stimulation in vitro. Plasmodium yoelii infection of t-ASM/CD4cre mice resulted in enhanced T cell activation and was associated with reduced parasitemia in comparison to infected control mice. Hence, our results provide evidence that ASM activity modulates T cell function in vitro and in vivo

Rolle der sauren Sphingomyelinase bei der Aktivierung von T-Lymphozyten

Die Saure Sphingomyelinase und Ceramid modulieren diverse Aspekte der T-Zellaktivierung. T-Lymphozyten kommt bei der adaptiven Immunabwehr von Pathogenen eine zentrale Rolle zu. Tuberkulose ist eine der häufigsten Infektionskrankheiten (weltweit), die jährlich noch immer zu einer Vielzahl an Todesfällen führt. In diesem Projekt wird die Rolle der Sauren Sphingomyelinase in der Signaltransduktion von T-Zellen näher beleuchtet. Dies wird anhand dreier T-Zellmodelle vollzogen: 1) transgene CD4-positive T-Zellen, die einen T-Zellrezeptor exprimieren, welcher das tuberkulosespezifische Peptid25 erkennt, sowie 2) primäre Mauslymphozyten und 3) Jurkat-Zellen, eine humane Leukämiezelllinie, die von T-Zellen abstammt. Die pharmakologische Inhibition der sauren Sphingomyelinase durch Imipramin beeinträchtigt die Aktivierung verschiedener Kinasen signifikant, während die genetische Defizienz der Sauren Sphingomyelinase nur zu geringfügigen Effekten führt. Darüber hinaus wurde die Bedeutung der Sauren Sphingomyelinase für die Aktivierung von T-Zellrezeptor-transgenen CD4-positiven T-Zellen durch das tuberkulosespezifische Peptid25 untersucht. Hierbei wurde gezeigt, dass unter Imipramineinfluss die initiale Aktivierung der Signalwege durch die Stimulation mit Peptid25 weniger effektiv erfolgt, welches wiederum zu einer deutlichen Reduktion der Proliferation, Differenzierung und Zytokinproduktion führt und zudem zur Induktion des Zelltodes beiträgt. Im Gegensatz dazu zeigen T-Zellrezeptor-transgene CD4-positive T-Zellen, die defizient für die Saure Sphingomyelinase sind, sehr ähnliche Reaktionen in Bezug auf späte Aktivierungsereignisse wie solche, die die Saure Sphingomyelinase exprimieren. Das Bacillus Calmette-Guerin ist eine attenuierte Form des Mycobacterium bovis mit einem Antigen-Profil, welches dem des Mycobacterium tuberculosis sehr ähnlich ist. In Analogie zu den Befunden in vitro zeigt die systemische Infektion mit dem Bacillus Calmette-Guerin von Saure Sphingomyelinase-defizienten im Vergleich mit genetisch unveränderten Mäusen ebenfalls, dass die Saure Sphingomyelinase im Kontext der Aktivierung von CD4- und CD8-positiven T-Zellen in vivo nur eine untergeordnete Rolle spielt. Weiterhin hat die massenspektrometrische Analyse der Lipidzusammensetzung von Jurkatzellen gezeigt, dass die Behandlung mit Imipramin zu verringerten Sphingosin- und Sphingosin-1-phosphat-Spiegeln führt. Dies deutet darauf hin, dass die Effekte der Gabe von Imipramin nicht allein auf die Inhibition der Sauren Sphingomyelinase, sondern auch auf die Beeinträchtigung der Funktion der Sauren Ceramidase zurückzuführen sein könnten. Diese Arbeit gibt somit neue Einblicke in die Bedeutung der Sauren Sphingomyelinase in Bezug auf die T-Zellaktivierung.Acid sphingomyelinase and ceramide modulate several aspects of T lymphocyte activation. T lymphocytes are the vital players in adaptive immune response against invading pathogens. Tuberculosis is one of the most common infectious diseases, still imposing a huge death toll every year. This project investigates the role of acid sphingomyelinase in tuberculosis-specific peptide25 T cell receptor (TCR) transgenic CD4+ T cells, murine primary lymphocytes and human Jurkat cells. Data reveals that pharmacological inhibition of acid sphingomyelinase by Imipramine significantly impairs the activation of several TCR signaling kinases, however genetic deficiency of acid sphingomyelinase only shows limited effects. Moreover, examination of peptide25-induced activation of TCR transgenic CD4+ T cells demonstrates that Imipramine significantly inhibits the late activation events, i.e., proliferation, differentiation, cytokine production and induces cell death following stimulation due to ineffective initial activation. In contrast, acid sphingomyelinase-deficient transgenic (P25/Asm-/-) CD4+ T cells present very similar responses regarding the late activation events, compared to wild-type control cells (P25/Asm+/+). In parallel, systemic infection of wild-type and acid sphingomyelinase-deficient mice with Bacillus Calmette-Guerin, which is a live attenuated form of Mycobacterium bovis with a similar antigenic profile to Mycobacterium tuberculosis, reveals the insignificant function of acid sphingomyelinase in both CD4+ and CD8+ T cell activation in vivo. In addition, mass spectrometry analysis of lipid composition of Jurkat cells following Imipramine treatment reveals a diminished level of sphingosine and sphingosine-1-phosphate. This indicates that the inhibitory effects of Imipramine in T cell signaling and late activation events might not be entirely due to the inhibition of acid sphingomyelinase but also acid ceramidase. Thus, this project gives some new insights into the role of acid sphingomyelinase in T lymphocyte activation