Mobilnost sekrecijskih mešičkov in homeostaza kalcija v astrocitih (Slovene)

Brainscience has entered a new era in which glial cells are considered as active participants in the control of brain circuit function and behaviour in health and disease. Astrocytes, the most abundant glial cell type in the human brain, are regarded as multifunctional cells modulating synaptic plasticity, learning, memory and sleep. Astrocytes signal to neighboring cells via distinct gateways, like channels, transporters, membrane receptors, however the key mechanism of chemical signaling is likely mediated by regulated exocytosis, which is still debated. Despite our understanding of critical factors involved in vesicles fusion, we know surprisingly little about patho-/physiological regulation of astroglial vesicle trafficking affecting release of gliotransmitters. To verify the hypothesis of patho-/physiological regulation of vesicle trafficking, one has to be able to measure spatio-temporal patterns of calcium activity, translocations of individual vesicles and exocytotic release from astrocytes. By deploying two optophysiological techniques we will investigate alterations in i) calcium homeostasis, ii) vesicles mobility and iii) cargo exocytosis triggered by physiological regulators of astrocytic activity (L-glutamate, ATP), which act via metabotropic and ionotropic glutamate receptors, and via purinergic receptors to, at least in part, regulate the release of calcium from intracellular stores. Astrocytes will be preloaded with permeable fluorescent calcium indicator Fluo-4-AM (Kd ~345 nM) and elevations in calcium activity measured in real time as increases in Fluo-4 fluorescence. We will quantify the peak fluorescence response, the half-life, the frequency of calcium oscillations and estimate the total time of elevated calcium activity. The impact onto mobility will be examined in two distinct vesicle types – the peptidergic vesicles preloaded by secretory peptide tagged with mutant green fluorescenct protein (ANP.emd) and in lysotracker-loaded acidic vesicles comprising endosomes and lysososmes. We will measure changes in the average vesicles speed, the total pathway vesicles traveled, the maximal displacement and the directionality of vesicle motions. The efficacy of exocytosis will be determined in whole cells as relative diminishment in the number of fluorescing vesicles and in individual vesicles by measuring the rate of ANP.emd and Lysotracker fluorescence decrease indicating discharge of cargo. Then, we will explore the causal relationship between the vesicle mobility and the probability for release-productive fusion. We will attempt to identify which vesicles - either mobile or stationary - are the likely candidates to undergo release-productive fusion upon stimulation. We will also examine the impact of lipid compounds that were reported to interfere with SNARE complex assembly and regulate vesicle exocytosis in neurons and neuroendocrine cells, but were not examined whether they act in astrocytes. For this, we will collaborate with Dr. Bazbek Davletov (Cambridge, UK) and screen for lipids – in particular for sphingosine that activates Synaptobrevin-2 in neurons and in neuroendocrine cells and fingolimod (FTY720), a pharmacologically relevant structural analogue of sphingosine - with potential regulatory role in activating Synaptobrevin-2 to form the SNARE complex implicated in astrocytic membrane fusion. We will test the impact of lipids onto vesicle trafficking and/or exocytosis from cultured astrocytes by deploying time-laps confocal imaging and Total Internal Reflection Fluorescence (TIRF) imagining to reveal potential differences in the two dissimilar cellular locations – in the thin layer adjacent to the basal plasma membrane and in the cell interior. Finally, we will estimate the time characterizing intermolecular interactions in the end phase of regulated exocytosis by measuring the latency between arrestment of the mobile vesicles at the site of release and the actual onset of cargo release.

Sphingosine, a natural sphingolipid metabolite, and fingolimod (FTY720), a sphingosine-mimetic drug, attenuate mobility of astrocyte vesicles and inhibit astroglial secretion by increasing intracellular calcium concentration ([Ca2+]i). Astrocytes, the most abundant glial cells in the mammalian brain, provide metabolic support to neurons, actively tune synaptic activity of neurons and co-regulate brain microcirculation. Insults to neurons in the central nervous system (CNS) caused by infections, trauma, autoimmune responses and neurodegenerative diseases activate astrocytes, which initiate the process of astrogliosis. Proliferating reactive astrocytes reduce the spread of inflammatory cells, repair the blood-brain barrier, decrease tissue damage and lesion size, and decrease neuronal loss and demyelination. Disturbances of astrocyte homeostatic function thus have the potential to underlie neuronal dysfunction and various disease states including multiple sclerosis. A key process mediating chemical communication between astrocytes and neighboring cells is regulated exocytosis that consists of multistage merger between the membrane of the secretory vesicle and the plasma membrane; cargo stored inside vesicle lumen is subsequently released into extracellular space by diffusion. Astrocytes release various chemicals like L-glutamate, D-serine, ATP, secretory peptides, tumor necrosis factor ?, interleukins and prostaglandins, which may play a crucial role in the progression of inflammatory diseases of the CNS. Here we have shown that sphingosine and FTY720 (the non-phosphorylated forms), inhibit regulated exocytosis of gliotransmitters by attenuating mobility of astrocyte veiscles (peptidergic, glutamatergic and endosomes/lyzosomes) and prevent effective vesicle access/interaction with the plasma membrane docking/release sites. We have further addressed the specific intracellular signaling pathways that lead to inhibited astroglial secretion, and revealed positive relationship between the altered calcium homeostasis and attenuated mobility of astrocyte vesicles upon exposure to non-phosphorilated bioactive sphingolipids. Sphingolipids-evoked increases in [Ca2+]i coincided with diminished mobility of peptidergic vesicles, which was independently confirmed in astrocytes exposed to transient Ca2+ signaling triggered by stimulation of plasmalemmal purine and glutamate receptors. FTY720 and sphingosine evoked long-lasting increases in [Ca2+]i in the presence and absence of extracellular Ca2+. By using pharmacological blockers, we have found that the activation of phospholipase C and inositol-1,4,5-triphosphate receptors was necessary and sufficient to evoke the lipid-mediated increases in [Ca2+]i. Exogenous, sphingosine-like lipids exert complex, Ca2+-dependent effects on astrocyte cytoskeleton and vesicle mobility by disrupting microtubule-dependent movements of secretory vesicles. The selective inhibition of exocytosis in astrocytes may prevent astrocyte-mediated toxicity to neurons and delay disease onset. An important question is whether the FTY720 effects observed in this study occur in patients taking this drug on longer terms. While the therapeutic dose of the drug is expected to be in nanomolar range in the blood plasma, it is becoming apparent that micromolar concentrations of FTY720 can be attained in the brain parenchyma, especially at the astrocyte-vascular interface, due to the lipophilic nature of the drug. Thus, local concentration of FTY720 in the CNS may reach concentration that affect astrocyte vesicle mobility and inhibit their secretory activity, raising the possibility that some beneficial effects of the drug in treatment of multiple sclerosis may be in part due to changes in astrocytes physiology.

The new platform for survey of biologically active molecules suitable for assessment of cell quality and control of production of recombinant proteins on the basis of intracellular organelles mobility analysis in relation to disease states. The new method enables survey of biological effects in serum and cerebrospinal fluid constituents acting to eukaryotic cells with specifically labelled mobile intracellular organelles. Cultured eukaryotic cells are exposed to either hydrophilic ligands that acts on the plasmalemmal receptor(s) and activate intracellular signaling pathway(s), or to lipophilic molecules that permeate through the plasmalemma and (in)directly affect organelle mobility by affecting cytoskeleton structure and/or the probability for spontaneous or stimulated fusion of vesicles and subsequent discharge of luminal cargo into the extracellular space. During the course of the project, we have prepared this patent proposal. After obtaining the patent rights at the national patent office (The Slovenian Intellectual Property Office) in July 11-th 2011 we have aimed to expand this patent to the international patent in 2012. The later request was submitted to World Intellectual Property Organization - WIPO in Geneva, Switzerland (PCT/EP2012/001759) on April 24-th 2012. The patent was published along with the patent report on January 17-th 2013 (WO/2013/007325). The validity of the patent at WIPO was prolonged in 2013. In February, 2014, we have submitted the patent to the US Patent and Trademark Office (USPTO).