Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.
COBISS.SI-ID: 32590041
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.
COBISS.SI-ID: 33324505
Astrocytes are excitable neural cells that contribute to brain information processing via bidirectional communication with neurons. This involves the release of gliosignaling molecules that affect synapses patterning and activity. Mechanisms mediating the release of these molecules likely consist of non-vesicular and vesicular based mechanisms. It is the vesicle-based regulated exocytosis that is an evolutionary more complex process. It is well established that the release of gliosignaling molecules has profound effects on information processing in different brain regions (e.g., hippocampal astrocytes contribute to long-term potentiation [LTP]), which has traditionally been considered as one of the cellular mechanisms underlying learning and memory. However, the paradigm of vesicle-based regulated release of gliosignaling molecules from astrocytes is still far from being unanimously accepted. One of the most important questions is to what extent can the conclusions obtained from cultured astrocytes be translated to in vivo conditions. Here, we overview the properties of vesicle mobility and their fusion with the plasma membrane in cultured astrocytes and compare these parameters to those recorded in astrocytes from acute brain hippocampal slices. The results from both experimental models are similar, which validates experiments on isolated astrocytes and further supports arguments in favor of in vivo vesicle-based exocytotic release of gliosignaling molecules.
COBISS.SI-ID: 33194969
The finding that ketamine, an anaesthetic, can elicit a rapid antidepressant effect at low doses that lasts for weeks in patients with depression is arguably a major achievement in psychiatry in the last decades. However, the mechanisms of action are unclear. The glutamatergic hypothesis of ketamine action posits that ketamine is a NMDAR antagonist modulating downstream cytoplasmic events in neurons. In addition to targeting NMDARs in synaptic transmission, ketamine may modulate the function of astroglia, key homeostasis-providing cells in the central nervous system, also playing a role in many neurologic diseases including depression, which affects to 20% of the population globally. We first review studies on astroglia revealing that (sub)anaesthetic doses of ketamine attenuate stimulus-evoked calcium signalling, a process of astroglial cytoplasmic excitability, regulating the exocytotic release of gliosignalling molecules. Then we address how ketamine alters the fusion pore activity of secretory vesicles, and how ketamine affects extracellular glutamate and K+ homeostasis, both considered pivotal in depression. Finally, we also provide evidence indicating reduced cytoplasmic mobility of astroglial vesicles carrying the inward rectifying potassium channel (Kir4.1), which may regulate the density of Kir4.1 at the plasma membrane.
COBISS.SI-ID: 34206937
Astroglia, the primary homeostatic cells of the central nervous system, play an important role in neuroinflammation. They act as facultative immunocompetent antigen-presenting cells (APCs), expressing major histocompatibility complex (MHC) class II antigens upon activation with interferon (IFN)-[gamma] and possibly other proinflammatory cytokines that are upregulated in disease states, including multiple sclerosis (MS).We characterized the anti-inflammatory effects of fingolimod (FTY720), an established drug for MS, and its phosphorylated metabolite (FTY720-P) in IFN-[gamma]-activated cultured rat astrocytes. The expression of MHC class II compartments, [beta]2 adrenergic receptor (ADR-[beta]2), and nuclear factor kappa-light-chain enhancer of activated B cells subunit p65 (NF-KB p65) was quantified in immunofluorescence images acquired by laser scanning confocal microscopy. In addition, MHC class II-enriched endocytotic vesicles were labeled by fluorescent dextran and their mobility analyzed in astrocytes subjected to different treatments. FTY720 and FTY720-P treatment significantly reduced the number of IFN-K-induced MHC class II compartments and substantially increased ADR-[beta]2 expression, which is otherwise small or absent in astrocytes in MS. These effects could be partially attributed to the observed decrease in NF-KB p65 expression, because the NF-KB signaling cascade is activated in inflammatory processes.
COBISS.SI-ID: 34175449