Die Bedeutung der β1-Integrine in einem neuartigen in vitro Blut-Hirn- Schranken-Modell während der Entzündung

The blood-brain barrier (BBB) is a specialised layer consisting of endothelial cells, pericytes and astrocytes as well as a basement membrane that restricts the diffusion for small hydrophilic solutes and macromolecules as well as the transmigration of leukocytes into the central nervous system. During inflammation, pro-inflammatory cytokines induce changes in the expression of adhesion proteins and matrix metalloproteinases as well as the composition of tight junctions resulting in BBB leakage and an increase in infiltrating leukocytes. The process of transendothelial migration is characterised by expression and function of leukocyte integrins. But less is known about the impact of integrins expressed on the endothelium during these processes. To investigate the role of endothelial β1 integrins at the BBB during inflammation a novel THBMEC-based in vitro BBB model was established which fulfils the morphological, physical and functional requirements of the BBB and presents a more restrictive barrier than the models that have been published so far for THBMEC and other endothelial cell lines. It was demonstrated that this system can be modified by pro-inflammatory cytokines. It was shown that IL-1β and not TNFα induce the most striking inflammatory effects in these cells. Therefore, this system represents a useful tool to investigate the role of β1 integrins in the IL-1 β-induced inflammation at the BBB. Moreover, it has been found that endothelial β1 integrins are involved in inflammatory processes at the BBB including integrin-mediated adhesion and production of proteins of the extracellular matrix, such as collagen IV and fibronectin. It was shown that β1 integrins are up-regulated and activated on endothelial cells under inflammatory conditions. Furthermore, immunofluorescence studies suggest that β1 integrins accumulate in cell-cell contacts during inflammation. Additionally, the application of function-blocking antibodies directed against β1 integrins revealed that the proper function of the α5β1 integrin on endothelial cells is important for maintaining the integrity of the BBB. Moreover, inhibition of α5β1 integrin and α6β1 integrin on endothelial cells reduce the transmigration of leukocytes - especially monocytes - across the in vitro model. Since this effect is enhanced under inflammatory conditions the data indicates that these receptors participate in the transendothelial migration of leukocytes during inflammation.Die Blut-Hirn-Schranke (BHS) ist eine spezialisierte Barriere, die aus Endothelzellen, Astrozyten und Perizyten sowie einer Basalmembran besteht. Sie beschränkt die Diffusion kleiner hydrophiler Stoffe und Makromoleküle sowie die Transmigration von Leukozyten in das zentrale Nervensystem. Während einer Entzündung induzieren pro-inflammatorische Zytokine Veränderungen in der Expression von Adhäsionsmolekülen und Matrixmetalloproteinasen sowie in der Zusammensetzung von Tight Junctions. Das bewirkt das Durchlässigwerden der BHS und ermöglicht so das Eindringen von Leukozyten in das Hirnparenchym. Der Prozess der transendothelialen Migration geht einher mit der vermehrten Expression und Aktivierung von Leukozyten-Integrinen. Wenig ist allerdings darüber bekannt, ob auch Integrine, die auf dem Endothel exprimiert werden, an diesem Prozess beteiligt sind. Um die Rolle der endothelialen β1 Integrine an der BHS während der Entzündung zu untersuchen, wurde mit Hilfe der Endothelzelllinie THBMEC ein neuartiges in vitro BHS-Modell etabliert, welches die morphologischen, physikalischen und funktionellen Anforderungen einer BHS erfüllt und darüber hinaus eine stärkere Barriere darstellt, als bisher für diese und andere Endothelzelllinien publiziert ist. Weiterhin lässt sich dieses System durch die Zugabe von pro-inflammatorischen Zytokinen beeinflussen. Es konnte gezeigt werden, dass die stärksten entzündlichen Effekte in dieser Endothelzelllinie durch IL-1β und nicht durch TNFα ausgelöst werden. Diese Ergebnisse demonstrieren, dass dieses in vitro Modell ein nützliches Hilfsmittel für die Untersuchung der Bedeutung der β1 Integrine während der IL-1β-induzierten Entzündung an der BHS ist. Die in dieser Arbeit vorgestellten Ergebnisse zeigen, dass endotheliale β1 Integrine an entzündlichen Prozessen an der BHS beteiligt sind. Diese Prozesse beinhalten die Integrin-abhängige Zelladhäsion sowie die Synthese von Proteinen der Extrazellularen Matrix, wie Kollagen IV und Fibronektin. Darüber hinaus geht die Entzündung mit einer vermehrten Expression und Aktivierung von β1 Integrinen auf Endothelzellen einher. Außerdem gaben Immunfluoreszenz-Studien Hinweise darauf, dass β1 Integrine unter entzündlichen Bedingungen in den Zell-Zell-Kontakten akkumulieren. In Studien mit funktionsblockierenden Antikörpern gegen β1 Integrine konnte demonstriert werden, dass die korrekte Funktion des α5β1 Integrins wichtig für die Aufrechterhaltung der Integrität der BHS ist. Zusätzlich konnte gezeigt werden, dass die Blockade der Integrine α5β1 und α6β1 auf Endothelzellen die Transmigration von Leukozyten, besonders die von Monozyten, über das BHS-Modell verringert. Da dieser Effekt unter entzündlichen Bedingungen verstärkt ist, lässt sich schlussfolgern, dass diese Rezeptoren an der transendothelialen Migration von Leukozyten während der Entzündung beteiligt sind

Serotonin 5-HT7 receptor increases the density of dendritic spines and facilitates synaptogenesis in forebrain neurons

Precise control of dendritic spine density and synapse formation is critical for normal and pathological brain functions. Therefore, signaling pathways influencing dendrite outgrowth and remodeling remain a subject of extensive investigations. Here, we report that prolonged activation of the serotonin 5-HT7 receptor (5-HT7R) with selective agonist LP-211 promotes formation of dendritic spines and facilitates synaptogenesis in postnatal cortical and striatal neurons. Critical role of 5-HT7R in neuronal morphogenesis was confirmed by analysis of neurons isolated from 5-HT7R-deficient mice and by pharmacological inactivation of the receptor. Acute activation of 5-HT7R results in pronounced neurite elongation in postnatal striatal and cortical neurons, thus extending previous data on the morphogenic role of 5-HT7R in embryonic and hippocampal neurons. We also observed decreased number of spines in neurons with either genetically (i.e. 5-HT7R-knock-out) or pharmacologically (i.e. antagonist treatment) blocked 5-HT7R, suggesting that constitutive 5-HT7R activity is critically involved in the spinogenesis. Moreover, cyclin-dependent kinase 5 and small GTPase Cdc42 were identified as important downstream effectors mediating morphogenic effects of 5-HT7R in neurons. Altogether, our data suggest that the 5-HT7R-mediated structural reorganization during the postnatal development might have a crucial role for the development and plasticity of forebrain areas such as cortex and striatum, and thereby can be implicated in regulation of the higher cognitive functions. Read the Editorial Highlight for this article on doi: 10.1111/jnc.13981

Synaptic Remodeling Depends on Signaling between Serotonin Receptors and the Extracellular Matrix

Rewiring of synaptic circuitry pertinent to memory formation has been associated with morphological changes in dendritic spines and with extracellular matrix (ECM) remodeling. Here, we mechanistically link these processes by uncovering a signaling pathway involving the serotonin 5-HT7 receptor (5-HT7R), matrix metalloproteinase 9 (MMP-9), the hyaluronan receptor CD44, and the small GTPase Cdc42. We highlight a physical interaction between 5-HT7R and CD44 (identified as an MMP-9 substrate in neurons) and find that 5-HT7R stimulation increases local MMP-9 activity, triggering dendritic spine remodeling, synaptic pruning, and impairment of long-term potentiation (LTP). The underlying molecular machinery involves 5-HT7R-mediated activation of MMP-9, which leads to CD44 cleavage followed by Cdc42 activation. One important physiological consequence of this interaction includes an increase in neuronal outgrowth and elongation of dendritic spines, which might have a positive effect on complex neuronal processes (e.g., reversal learning and neuronal regeneration)

Amelioration of Tau pathology and memory deficits by targeting 5-HT7 receptor

International audienceTauopathies comprise a heterogeneous family of neurodegenerative diseases characterized by pathological accumulation of hyperphosphorylated Tau protein. Pathological changes in serotonergic signaling have been associated with tauopathy etiology, but the underlying mechanisms remain poorly understood. Here, we studied the role of the serotonin receptor 7 (5-HT7R), in a mouse model of tauopathy induced by overexpressing the human Tau[R406W] mutant associated with inherited forms of frontotemporal dementia. We showed that the constitutive 5-HT7R activity is required for Tau hyperphosphorylation and formation of highly bundled Tau structures (HBTS) through G-protein-independent, CDK5-dependent mechanism. We also showed that 5-HT7R physically interacts with CDK5. At the systemic level, 5-HT7R-mediated CDK5 activation induces HBTS leading to neuronal death, reduced long-term potentiation (LTP), and impaired memory in mice. Specific blockade of constitutive 5-HT7R activity in neurons that overexpressed Tau[R406W] prevents Tau hyperphosphorylation, aggregation, and neurotoxicity. Moreover, 5-HT7R knockdown in the prefrontal cortex fully abrogates Tau[R406W]-induced LTP deficits and memory impairments. Thus, 5-HT7R/CDK5 signaling emerged as a new, promising target for tauopathy treatments