Differential cargo mobilisation within Weibel-Palade bodies after transient fusion with the plasma membrane.

Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼ 2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis

Sphingomimetic multiple sclerosis drug FTY720 activates vesicular synaptobrevin and augments neuroendocrine secretion

Neurotransmission and secretion of hormones involve a sequence of protein/lipid interactions with lipid turnover impacting on vesicle trafficking and ultimately fusion of secretory vesicles with the plasma membrane. We previously demonstrated that sphingosine, a sphingolipid metabolite, promotes formation of the SNARE complex required for membrane fusion and also increases the rate of exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and in hippocampal neurons. Recently a fungi-derived sphingosine homologue, FTY720, has been approved for treatment of multiple sclerosis. In its non-phosphorylated form FTY720 accumulates in the central nervous system, reaching high levels which could affect neuronal function. Considering close structural similarity of sphingosine and FTY720 we investigated whether FTY720 has an effect on regulated exocytosis. Our data demonstrate that FTY720 can activate vesicular synaptobrevin for SNARE complex formation and enhance exocytosis in neuroendocrine cells and neurons

Brain energy rescue:an emerging therapeutic concept for neurodegenerative disorders of ageing

The brain requires a continuous supply of energy in the form of ATP, most of which is produced from glucose by oxidative phosphorylation in mitochondria, complemented by aerobic glycolysis in the cytoplasm. When glucose levels are limited, ketone bodies generated in the liver and lactate derived from exercising skeletal muscle can also become important energy substrates for the brain. In neurodegenerative disorders of ageing, brain glucose metabolism deteriorates in a progressive, region-specific and disease-specific manner — a problem that is best characterized in Alzheimer disease, where it begins presymptomatically. This Review discusses the status and prospects of therapeutic strategies for countering neurodegenerative disorders of ageing by improving, preserving or rescuing brain energetics. The approaches described include restoring oxidative phosphorylation and glycolysis, increasing insulin sensitivity, correcting mitochondrial dysfunction, ketone-based interventions, acting via hormones that modulate cerebral energetics, RNA therapeutics and complementary multimodal lifestyle changes

ATP-Evoked Intracellular Ca Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea

Hearing and its protection is regulated by ATP-evoked Ca2+ signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca2+ imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca2+ signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca2+]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca2+ transients in all three cell types, showing desensitization. Inhibiting the Ca2+ signaling of the ionotropic P2X (omission of extracellular Ca2+) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca2+ stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca2+-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca2+ leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca2+ signaling in these cells. Differences in Ca2+ homeostasis and ATP-induced Ca2+ signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology

The European research network on signal transduction (ERNEST): toward a multidimensional holistic understanding of G protein-coupled receptor signaling

G protein-coupled receptors (GPCRs) are intensively studied due to their therapeutic potential as drug targets. Members of this large family of transmembrane receptor proteins mediate signal transduction in diverse cell types and play key roles in human physiology and health. In 2013 the research consortium GLISTEN (COST Action CM1207) was founded with the goal of harnessing the substantial growth in knowledge of GPCR structure and dynamics to push forward the development of molecular modulators of GPCR function. The success of GLISTEN, coupled with new findings and paradigm shifts in the field, led in 2019 to the creation of a related consortium called ERNEST (COST Action CA18133). ERNEST broadens focus to entire signaling cascades, based on emerging ideas of how complexity and specificity in signal transduction are not determined by receptor-ligand interactions alone. A holistic approach that unites the diverse data and perspectives of the research community into a single multidimensional map holds great promise for improved drug design and therapeutic targeting.The authors are grateful for the continued support of the European Cooperation in Science and Technology (COST) through Actions CM1207 GLISTEN and CA18133 ERNEST. On behalf of ERNEST, M.E.S. thanks the Max Delbrück Center for Molecular Medicine Berlin for support in managing the Action. M.E.S. is supported by the Deutsche Forschungsgemeinschaft (DFG) (SO1037/1-3) and the Berlin Institute of Health (Delbrück Fellowship BIH_PRO_314). J.S. acknowledges support from the Instituto de Salud Carlos III FEDER (PI18/00094) and the ERA-NET NEURON & Ministry of Economy, Industry and Competitiveness (AC18/00030). J.C. receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant Agreement No. 715052). D.E.G. is supported by the Lundbeck Foundation (R313- 2019-526) and Novo Nordisk Foundation (NNF17OC0031226). G.M.K. is funded by the National Brain Research Program (2017-1.2.1-NKP-2017-00002). M.K. acknowledges support from the Israel Science Foundation (Grants 1454/13 and 3512/19) and the DS Research Center at the University of Haifa. S.M. is supported by the Alfred Benzon Foundation (ABF-0-0-312) and Polish National Science Center (HARMONIA 2015/18/M/NZ2/00423). M.M.R. acknowledges support from the European Research Council: VIREX Grant agreement 682549, Call ERC-2105- CoG, the Independent Research Fund Denmark, the NovoNordisk Foundation (NNF17OC0029222:) and the Lundbeck Foundation (R268-2017-409). E.S. thanks the Xunta de Galicia (Centro singular de Investigacion de Galicia ́ acreditacion 2019-2022, ED431G 2019/03 and GI-1597 2017- ́ 2019 ED431B2017/70) and the European Union (European Regional Development Fund - ERDF) for financial support. J.K.S.T. acknowledges support from the DFG (HI1502/1-2) and the Novo Nordisk Foundation (Challenge Grant PRISM). N.V. is funded by grants from the Slovenian Research Agency (P3-310, J3-7605, BI-DE/18-19-015). P.K. is supported by the DFG (KO4095/4-1 and Heisenberg professorship KO4095/5- 1). All coauthors thank the stellar organizers of the eight GLISTEN meetings for their vital contributions and their associated institutes and companies for support, including the University of Warsaw (Poland), Pompeu Fabra University and Autonomous University of Barcelona (Spain), Research Centre for Natural Sciences of the Hungarian Academy of Sciences (Budapest, Hungary), Actelion Pharmaceuticals (Allschwil, Switerland), Vrije Universiteit (Amsterdam, The Netherlands), Friedrich Alexander University Erlangen and Philipps-University Marburg (Germany), University of Chemistry and Technology Prague (Czech Republic), the University of Porto (Portugal), and Sosei Heptares (Cambridge, UK). Parts of this paper are derived from the Memorandum of Understanding for the implementation of the COST Action “European Research Network on Signal Transduction” (ERNEST) CA18133