TGFβ signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke

<p>Abstract</p> <p>Background</p> <p>TGFβ is both neuroprotective and a key immune system modulator and is likely to be an important target for future stroke therapy. The precise function of increased TGF-β1 after stroke is unknown and its pleiotropic nature means that it may convey a neuroprotective signal, orchestrate glial scarring or function as an important immune system regulator. We therefore investigated the time course and cell-specificity of TGFβ signaling after stroke, and whether its signaling pattern is altered by gender and aging.</p> <p>Methods</p> <p>We performed distal middle cerebral artery occlusion strokes on 5 and 18 month old TGFβ reporter mice to get a readout of TGFβ responses after stroke in real time. To determine which cell type is the source of increased TGFβ production after stroke, brain sections were stained with an anti-TGFβ antibody, colocalized with markers for reactive astrocytes, neurons, and activated microglia. To determine which cells are responding to TGFβ after stroke, brain sections were double-labelled with anti-pSmad2, a marker of TGFβ signaling, and markers of neurons, oligodendrocytes, endothelial cells, astrocytes and microglia.</p> <p>Results</p> <p>TGFβ signaling increased 2 fold after stroke, beginning on day 1 and peaking on day 7. This pattern of increase was preserved in old animals and absolute TGFβ signaling in the brain increased with age. Activated microglia and macrophages were the predominant source of increased TGFβ after stroke and astrocytes and activated microglia and macrophages demonstrated dramatic upregulation of TGFβ signaling after stroke. TGFβ signaling in neurons and oligodendrocytes did not undergo marked changes.</p> <p>Conclusions</p> <p>We found that TGFβ signaling increases with age and that astrocytes and activated microglia and macrophages are the main cell types that undergo increased TGFβ signaling in response to post-stroke increases in TGFβ. Therefore increased TGFβ after stroke likely regulates glial scar formation and the immune response to stroke.</p

Synaptojanin and Endophilin vermitteln Hals-Bildung während der ultraschnellen Endozytose

Neurons use and recycle synaptic vesicles continuously during synaptic transmission. The mechanisms of synaptic vesicle release and recycling have been a subject of much study. There are several known mechanisms of vesicle endocytosis, and recent work suggests that ultrafast endocytosis (UFE) occurs under physiologic conditions at a much faster timescale than previously thought. In this process, endocytosis of membrane occurs in a clathrin-independent manner on the order of 100ms in both C. elegans and mammalian mouse neurons. Following the retrieval of membrane, endocytosed vesicles fuse to an endosome, from which clathrin coated pits bud off, and regenerate synaptic vesicles within 30-300ms. In spite of its rapid time-scale, separation from the plasma membrane in UFE is dynamin-dependent and requires actin. The other proteins that are required to orchestrate the steps of UFE at its characteristic rapid speed are not yet known. Here we show that synaptojanin and endophilin play critical roles in allowing ultrafast endocytosis to achieve its rapid time-scale. Specifically, we show that endophilin and synaptojanin are each necessary for UFE to occur. In this manuscript, we go one step further to address these proteins’ specific roles in this process. We show that UFE requires a functional 5-phosphatase domain but not Sac-1 domain of synaptojanin to occur at its characteristic rapid timescale and that endophilin is required for neck formation in UFE. We also show that endophilin A1 and A2 can both fill the endophilin requirement for UFE and reacidification. By using “flash-and- freeze” electron microscopy, we examine the trafficking of membranes in the absence or modification of endophilin and synaptojanin. Our fluorescence imaging experiments allow us to observe corresponding effects on protein trafficking and vesicle acidification. Taken together, our data are the first to suggest a specific role for synaptojanin and endophilin in UFE, and allow us to better understand this mechanism of vesicle recycling.Während der synaptischen Übertragung werden kontinuierlich synaptische Vesikel verwendet und recycelt. Die dabei involvierten Mechanismen sind der Gegenstand vieler Untersuchungen und mehrere Mechanismen sind bereits bekannt. Jüngste Ergebnisse legen nahe, dass eine ultraschnelle Endozytose (UFE) unter physiologischen Bedingungen viel schneller auftritt als bisher angenommen. Bei diesem Prozess läuft die Endozytose in einer Clathrin-unabhängigen Weise innerhalb von grob 100ms ab, dies wurde sowohl in C. elegans und Säugetier-Mäuse-Neuronen gezeigt. Nach der Rückholung der Membran, fusionieren endozytierte Vesikel zu einem Endosom, vom dem sich Clathrin-beschichtete Pits ablösen und synaptische Vesikel innerhalb von 30-300ms regenerieren. Trotz des raschen Ablaufs dieses Prozesses ist die Trennung der Plasmamembran im Rahmen der UFE Dynamin-abhängig, und benötigt Aktin. Die anderen Proteine, die erforderlich sind, um die Schritte der UFE mit ihrer charakteristischen schnellen Geschwindigkeit zu orchestrieren, sind noch nicht bekannt. Wir zeigen, dass Synaptojanin und Endophilin einen entscheidenden Beitrag zu diesem raschen Ablauf der ultraschnellen Endozytose leisten. Insbesondere zeigen wir, dass sowohl Endophilin als auch Synaptojanin für das Auftreten von UFE notwendig sind. In diesem Manuskript gehen wir auf die spezifischen Rollen dieser Proteine im Prozess der UFE ein. Wir zeigen, dass UFE eine funktionelle 5-Phosphatase-Domäne, aber keine Sac-1-Domäne von Synaptojanin benötigt, um ultraschnelle Endozytose zu erlauben, und dass Endophilin für die Hals-bildung der Vesikel in UFE erforderlich ist. Wir zeigen auch, dass Endophilin A1 und A2 sowohl den Endophilin-Bedarf für UFE als auch für die Widerherstellung eines sauren Milieus innerhalb der Vesikel erfüllt. Mit der "Flash-and-Freeze" -Elektronenmikroskopie untersuchen wir den Austausch der Membranen in Abwesenheit, oder mit einer Modifikation, von Endophilin und Synaptojanin. Unsere Fluoreszenz-Bildgebungs- Experimente erlauben uns, entsprechende Auswirkungen auf den Proteinverkehr und die Ansäuerung der Vesikel zu beobachten. Unsere Daten weisen erstmals auf eine spezifische Rolle von Synaptojanin und Endophilin im Rahmen der UFE hin und erlauben ein besseres Verständnis der Mechanismen des Vesikel-recyclings

Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity

Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric disease like depression and anxiety but may also permeate from the periphery through blood-brain barrier openings seen in neurodegenerative disease. First, we tested the impact of serotonin on the microglial response to an insult caused by a laser lesion in the cortex of acute slices from Cx3Cr1-GFP mice. In the presence of serotonin the microglial processes moved more rapidly towards the laser lesion which is considered to be a chemotactic response to ATP. Similarly, the chemotactic response of cultured microglia to ATP was also enhanced by serotonin. Quantification of phagocytic activity by determining the uptake of microspheres showed that the amoeboid microglia in slices from early postnatal animals or microglia in culture respond to serotonin application with a decreased phagocytic activity whereas we could not detect any significant change in ramified microglia in situ. The presence of microglial serotonin receptors was confirmed by patch-clamp experiments in culture and amoeboid microglia and by qPCR analysis of RNA isolated from primary cultured and acutely isolated adult microglia. These data suggest that microglia express functional serotonin receptors linked to distinct microglial properties

LSP5-2157 a new inhibitor of vesicular glutamate transport

International audienc

LSP5-2157 a new inhibitor of vesicular glutamate transporters

International audienc