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When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. We imaged fluorescently labeled LDs by microscopy in isolated and brain tissue rat astrocytes and in glia‐like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca2+, likely to enhance the LD–organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress‐induced lipotoxicity., Main points Astroglial lipid droplets are dynamic organelles with confined mobility.Lipid droplet turnover is important for astrocyte survival/proliferation.Stressed astrocytes accumulate lipid droplets likely to provide energy and reduce lipotoxicity.

Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined with high resilience to virus-induced cell death, astrocytes are now considered one of the principal types of cells, responsible for virus retention and dissemination within the brain. Autophagy plays an important role in elimination of intracellular components and in maintaining cellular homeostasis and is also intertwined with the life cycle of viruses. The physiological significance of autophagy in astrocytes, in connection with the life cycle and transmission of viruses, remains poorly investigated. In the present study, we investigated flavivirus-induced modulation of autophagy in human astrocytes by monitoring a tandem fluorescent-tagged LC3 probe (mRFP-EGFP-LC3) with confocal and super-resolution fluorescence microscopy. Astrocytes were infected with tick-borne encephalitis virus (TBEV) or West Nile virus (WNV), both pathogenic flaviviruses, and with mosquito-only flavivirus (MOF), which is considered non-pathogenic. The results revealed that human astrocytes are susceptible to infection with TBEV, WNV and to a much lower extent also to MOF. Infection and replication rates of TBEV and WNV are paralleled by increased rate of autophagy, whereas autophagosome maturation and the size of autophagic compartments are not affected. Modulation of autophagy by rapamycin and wortmannin does not influence TBEV and WNV replication rate, whereas bafilomycin A1 attenuates their replication and infectivity. In human astrocytes infected with MOF, the low infectivity and the lack of efficient replication of this flavivirus are mirrored by the absence of an autophagic response.

The plasticity of astrocytes is fundamental for their principal function, maintaining homeostasis of the central nervous system throughout life, and is associated with diverse exposomal challenges. Here, we used cultured astrocytes to investigate at subcellular level basic cell processes under controlled environmental conditions. We compared astroglial functional and signaling plasticity in standard serum-containing growth medium, a condition mimicking pathologic conditions, and in medium without serum, favoring the acquisition of arborized morphology. Using opto−/electrophysiologic techniques, we examined cell viability, expression of astroglial markers, vesicle dynamics, and cytosolic Ca$^{2+}$ and cAMP signaling. The results revealed altered vesicle dynamics in arborized astrocytes that was associated with increased resting [Ca$^{2+}$]$_i$ and increased subcellular heterogeneity in [Ca$^{2+}$]$_i$, whereas [cAMP]$_i$ subcellular dynamics remained stable in both cultures, indicating that cAMP signaling is less prone to plastic remodeling than Ca$^{2+}$ signaling, possibly also in in vivo contexts.

Astroglial aerobic glycolysis, a process during which D-glucose is converted to L-lactate, a brain fuel and signal, is regulated by the plasmalemmal receptors, including adrenergic receptors (ARs) and purinergic receptors (PRs), modulating intracellular Ca$^{2+}$ and cAMP signals. However, the extent to which the two signals regulate astroglial aerobic glycolysis is poorly understood. By using agonists to stimulate intracellular α$_1$-/β-AR-mediated Ca$^{2+}$/cAMP signals, β-AR-mediated cAMP and P$_2$R-mediated Ca$^{2+}$ signals and genetically encoded fluorescence resonance energy transfer-based glucose and lactate nanosensors in combination with real-time microscopy, we show that intracellular Ca$^{2+}$, but not cAMP, initiates a robust increase in the concentration of intracellular free D-glucose ([glc]$_i$) and L-lactate ([lac]$_i$), both depending on extracellular D-glucose, suggesting Ca$^{2+}$-triggered glucose uptake and aerobic glycolysis in astrocytes. When the glycogen shunt, a process of glycogen remodelling, was inhibited, the α$_1$-/β-AR-mediated increases in [glc]$_i$ and [lac]$_i$ were reduced by ∼65 % and ∼30 %, respectively, indicating that at least ∼30 % of the utilization of D-glucose is linked to glycogen remodelling and aerobic glycolysis. Additional activation of β-AR/cAMP signals aided to α$_1$-/β-AR-triggered [lac]$_i$ increase, whereas the [glc]$_i$ increase was unaltered. Taken together, an increase in intracellular Ca$^{2+}$ is the prime mechanism of augmented aerobic glycolysis in astrocytes, while cAMP has only a moderate role. The results provide novel information on the signals regulating brain metabolism and open new avenues to explore whether astroglial Ca$^{2+}$ signals are dysregulated and contribute to neuropathologies with impaired brain metabolism.

Aims Plectin, a universally expressed multi-functional cytolinker protein, is crucial for intermediate filament networking, including crosstalk with actomyosin and microtubules. In addition to its involvement in a number of diseases affecting skin, skeletal muscle, heart, and other stress-exposed tissues, indications for a neuropathological role of plectin have emerged. Having identified P1c as the major isoform expressed in neural tissues in previous studies, our aim for the present work was to investigate whether, and by which mechanism(s), the targeted deletion of this isoform affects neuritogenesis and proper nerve cell functioning. Methods For ex vivo phenotyping, we used dorsal root ganglion and hippocampal neurons derived from isoform P1c-deficient and plectin-null mice, complemented by in vitro experiments using purified proteins and cell fractions. To assess the physiological significance of the phenotypic alterations observed in P1c-deficient neurons, P1c-deficient and wild-type littermate mice were subjected to standard behavioural tests. Results We demonstrate that P1c affects axonal microtubule dynamics by isoform-specific interaction with tubulin. P1c deficiency in neurons leads to altered dynamics of microtubules and excessive association with tau protein, affecting neuritogenesis, neurite branching, growth cone morphology, and translocation and directionality of movement of vesicles and mitochondria. On the organismal level, we found P1c deficiency manifesting as impaired pain sensitivity, diminished learning capabilities and reduced long-term memory of mice. Conclusions Revealing a regulatory role of plectin scaffolds in microtubule-dependent nerve cell functions, our results have potential implications for cytoskeleton-related neuropathies.

Most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons and non-neuronal cells, including astrocytes, which metabolically support neurons with nutrients. Neuronal metabolism largely depends on the activation of the noradrenergic system releasing noradrenaline. Activation of astroglial adrenergic receptors with noradrenaline triggers cAMP and Ca2+ signaling and augments aerobic glycolysis with production of lactate, an important neuronal energy fuel. Astrocytes with cytoplasmic TDP-43 inclusions can cause motor neuron death, however, whether astroglial metabolism and metabolic support of neurons is altered in astrocytes with TDP-43 inclusions, is unclear. We measured lipid droplet and glucose metabolisms in astrocytes expressing the inclusion-forming C-terminal fragment of TDP-43 or the wild-type TDP-43 using fluorescent dyes or genetically encoded nanosensors. Astrocytes with TDP-43 inclusions exhibited a 3-fold increase in the accumulation of lipid droplets versus astrocytes expressing wild-type TDP-43, indicating altered lipid droplet metabolism. In these cells the noradrenaline-triggered increases in intracellular cAMP and Ca2+ levels were reduced by 35% and 31%, respectively, likely due to the downregulation of β2-adrenergic receptors. Although noradrenaline triggered a similar increase in intracellular lactate levels in astrocytes with and without TDP-43 inclusions, the probability of activating aerobic glycolysis was facilitated by 1.6-fold in astrocytes with TDP-43 inclusions and lactate MCT1 transporters were downregulated. Thus, while in astrocytes with TDP-43 inclusions noradrenergic signaling is reduced, aerobic glycolysis and lipid droplet accumulation are facilitated, suggesting dysregulated astroglial metabolism and metabolic support of neurons in TDP-43-associated ALS and FTD.

In recent years, increasing evidence regarding the functional importance of lipid droplets (LDs), cytoplasmic storage organelles in the central nervous system (CNS), has emerged. Although not abundantly present in the CNS under normal conditions in adulthood, LDs accumulate in the CNS during development and aging, as well as in some neurologic disorders. LDs are actively involved in cellular lipid turnover and stress response. By regulating the storage of excess fatty acids, cholesterol, and ceramides in addition to their subsequent release in response to cell needs and/or environmental stressors, LDs are involved in energy production, in the synthesis of membranes and signaling molecules, and in the protection of cells against lipotoxicity and free radicals. Accumulation of LDs in the CNS appears predominantly in neuroglia (astrocytes, microglia, oligodendrocytes, ependymal cells), which provide trophic, metabolic, and immune support to neuronal networks. Here we review the most recent findings on the characteristics and functions of LDs in neuroglia, focusing on astrocytes, the key homeostasis-providing cells in the CNS. We discuss the molecular mechanisms affecting LD turnover in neuroglia under stress and how this may protect neural cell function. We also highlight the role (and potential contribution) of neuroglial LDs in aging and in neurologic disorders.

Interactive mechanical forces between pairs of individual SNARE proteins synaptobrevin 2 (Sb2) and syntaxin 1A (Sx1A) may be sufficient to mediate vesicle docking. This notion, based on force spectroscopy single molecule measurements probing recombinant Sx1A an Sb2 in silico, questioned a predominant view of docking via the ternary SNARE complex formation, which includes an assembly of the intermediate cis binary complex between Sx1A and SNAP25 on the plasma membrane to engage Sb2 on the vesicle. However, whether a trans binary Sx1A-Sb2 complex alone could mediate vesicle docking in a cellular environment remains unclear. To address this issue, we used atomic force microscopy (AFM) in the force spectroscopy mode combined with fluorescence imaging. Using AFM tips functionalized with the full Sx1A cytosolic domain, we probed native Sb2 studding the membrane of secretory vesicles docked at the plasma membrane patches, referred to as "inside-out lawns", identified based on fluorescence stains and prepared from primary culture of lactotrophs. We recorded single molecule Sx1A-Sb2 mechanical interactions and obtained measurements of force (∼183 pN) and extension (∼21.6 nm) necessary to take apart Sx1A-Sb2 binding interactions formed at tip-vesicle contact. Measured interactive force between a single pair of Sx1A-Sb2 molecules is sufficient to hold a single secretory vesicle docked at the plasma membrane within distances up to that of the measured extension. This finding further advances a notion that native vesicle docking can be mediated by a single trans binary Sx1A-Sb2 complex in the absence of SNAP25.

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca$^{2+}$ ([Ca$^{2+}$]$_i$) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca$^{2+}$] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca$^{2+}$ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca$^{2+}$], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca$^{2+}$-dependent exocytosis regulated by distinct Ca$^{2+}$ sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca$^{2+}$ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

Astrocytes, heterogeneous neuroglial cells, contribute to metabolic homeostasis in the brain by providing energy substrates to neurons. In contrast to predominantly oxidative neurons, astrocytes are considered primarily as glycolytic cells. They take up glucose from the circulation and in the process of aerobic glycolysis (despite the normal oxygen levels) produce L-lactate, which is then released into the extracellular space via lactate transporters and possibly channels. Astroglial L-lactate can enter neurons, where it is used as a metabolic substrate, or exit the brain via the circulation. Recently, L-lactate has also been considered to be a signaling molecule in the brain, but the mechanisms of L-lactate signaling and how it contributes to the brain function remain to be fully elucidated. Here, we provide an overview of L-lactate signaling mechanisms in the brain and present novel insights into the mechanisms of L-lactate signaling via G-protein coupled receptors (GPCRs) with the focus on astrocytes. We discuss how increased extracellular L-lactate upregulates cAMP production in astrocytes, most likely viaL-lactate-sensitive Gs-protein coupled GPCRs. This activates aerobic glycolysis, enhancing L-lactate production and accumulation of lipid droplets, suggesting that L-lactate augments its own production in astrocytes (i.e., metabolic excitability) to provide more L-lactate for neurons and that astrocytes in conditions of increased extracellular L-lactate switch to lipid metabolism.

Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.

Aquaporin 4 (AQP4) is the most abundant water channel in the central nervous system (CNS). Its expression is confined to non-neuronal glial cells, predominantly to astrocytes that represent a heterogeneous glial cell type in the CNS. The membrane of astrocyte processes, which align brain capillaries and pia, is particularly rich in AQP4. Several isoforms of AQP4 have been described; however, only some (AQP4a (M1), AQP4 c (M23), AQP4e, and AQP4ex) have been identified in the plasma membrane assemblies of astrocytes termed orthogonal arrays of particles (OAPs). Intracellular splicing isoforms (AQP4b, AQP4d, AQP4f, AQP4-Δ4) have been documented, and most of them are postulated to have a role in the cell surface distribution of the plasma membrane isoforms and in the formation of OAPs in murine and human astrocytes. Although OAPs have been proposed to play various roles in the functioning of astrocytes and CNS tissue as a whole, many of these still need to be described. OAPs are studied primarily from the perspective of understanding water permeability regulation through the plasma membrane and of their involvement in cell adhesion and in the dynamics of astrocytic processes. This review describes the cellular distribution of various AQP4 isoforms and their implications in OAP assembly, which is regulated by several intracellular and extracellular proteins.

Endocytosis is a vesicle-based mechanism by which eukaryotic cells internalize extracellular material. There are several types of this universal mechanism linked to different types of endocytosed cargo, including pathogens; therefore, several approaches can be applied. Here, we describe techniques that are applicable to study the internalization of flaviviruses; dextrans; transporters, such as, glutamate transporter vGlut1; and peptidergic signaling molecules, including atrial natriuretic peptide into astrocytes, the most heterogeneous neuroglial cells, which play a key homeostatic role in the central nervous system.

During cognitive efforts mediated by local neuronal networks, approximately 20% of additional energy is required; this is mediated by chemical messengers such as noradrenaline (NA). NA targets astroglial aerobic glycolysis, the hallmark of which is the end product l-lactate, a fuel for neurons. Biochemical studies have revealed that astrocytes exhibit a prominent glycogen shunt, in which a portion of d-glucose molecules entering the cytoplasm is transiently incorporated into glycogen, a buffer and source of d-glucose during increased energy demand. Here, we studied single astrocytes by measuring cytosolic L-lactate ([lac]i ) with the FRET nanosensor Laconic. We examined whether NA-induced increase in [lac]i is influenced by: (a) 2-deoxy-d-glucose (2-DG, 3 mM), a molecule that enters the cytosol and inhibits the glycolytic pathway; (b) 1,4-dideoxy-1,4-imino-d-arabinitol (DAB, 300 µM), a potent inhibitor of glycogen phosphorylase and glycogen degradation; and (c) 3-nitropropionic acid (3-NPA, 1 mM), an inhibitor of the Krebs cycle. The results of these pharmacological experiments revealed that d-glucose uptake is essential for the NA-induced increase in [lac]i , and that this exclusively arises from glycogen degradation, indicating that most, if not all, d-glucose molecules in NA-stimulated cells transit the glycogen shunt during glycolysis. Moreover, under the defined transmembrane d-glucose gradient, the glycolytic intermediates were not only used to produce l-lactate, but also to significantly support oxidative phosphorylation, as demonstrated by an elevation in [lac]i when Krebs cycle was inhibited. We conclude that l-lactate production via aerobic glycolysis is an essential energy pathway in NA-stimulated astrocytes; however, oxidative metabolism is important at rest.

Ketamine, a general anaesthetic and psychotomimetic drug, exerts rapid, potent and long-lasting antidepressant effect, albeit the cellular and molecular mechanisms of this action are yet to be discovered. Besides targeting neuronal NMDARs fundamental for synaptic transmission, ketamine affects the function of astroglia the key homeostatic cells of the central nervous system that contribute to pathophysiology of psychiatric diseases including depression. Here we review studies revealing that (sub)anaesthetic doses of ketamine elevate intracellular cAMP concentration ([cAMP]$_i$) in astrocytes, attenuate stimulus-evoked astrocyte calcium signalling, which regulates exocytotic secretion of gliosignalling molecules, and stabilize the vesicle fusion pore in a narrow configuration possibly hindering cargo discharge or vesicle recycling. Next we discuss how ketamine affects astroglial capacity to control extracellular K$^+$ by reducing cytoplasmic mobility of vesicles delivering the inward rectifying potassium channel (Kir4.1) to the plasmalemma. Modified astroglial K$^+$ buffering impacts upon neuronal excitability as demonstrated in the lateral habenula rat model of depression. Finally, we highlight the recent discovery that ketamine rapidly redistributes cholesterol in the plasmalemma of astrocytes, but not in fibroblasts nor in neuronal cells. This alteration of membrane structure may modulate a host of processes that synergistically contribute to ketamine's rapid and prominent antidepressant action.

Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the central nervous system (CNS). It is predominantly expressed in astrocytes lining blood&ndash, brain and blood&ndash, liquor boundaries. AQP4a (M1), AQP4c (M23), and AQP4e, present in the plasma membrane, participate in the cell volume regulation of astrocytes. The function of their splicing variants, AQP4b and AQP4d, predicted to be present in the cytoplasm, is unknown. We examined the cellular distribution of AQP4b and AQP4d in primary rat astrocytes and their role in cell volume regulation. The AQP4b and AQP4d isoforms exhibited extensive cytoplasmic localization in early and late endosomes/lysosomes and in the Golgi apparatus. Neither isoform localized to orthogonal arrays of particles (OAPs) in the plasma membrane. The overexpression of AQP4b and AQP4d isoforms in isoosmotic conditions reduced the density of OAPs, in hypoosmotic conditions, they remained absent from OAPs. In hypoosmotic conditions, the AQP4d isoform was significantly redistributed to early endosomes, which correlated with the increased trafficking of AQP4-laden vesicles. The overexpression of AQP4d facilitated the kinetics of cell swelling, without affecting the regulatory volume decrease. Therefore, although they reside in the cytoplasm, AQP4b and AQP4d isoforms may play an indirect role in astrocyte volume changes.

Astrocytes are the key homeostatic cells in the central nervous system; initiation of reactive astrogliosis contributes to neuroinflammation. Pro-inflammatory cytokine interferon γ (IFNγ) induces the expression of the major histocompatibility complex class II (MHCII) molecules, involved in antigen presentation in reactive astrocytes. The pathway for MHCII delivery to the astrocyte plasma membrane, where MHCII present antigens, is unknown. Rat astrocytes in culture and in organotypic slices were exposed to IFNγ to induce reactive astrogliosis. Astrocytes were probed with optophysiologic tools to investigate subcellular localization of immunolabeled MHCII, and with electrophysiology to characterize interactions of single vesicles with the plasmalemma. In culture and in organotypic slices, IFNγ augmented the astrocytic expression of MHCII, which prominently co-localized with lysosomal marker LAMP1-EGFP, modestly co-localized with Rab7, and did not co-localize with endosomal markers Rab4A, EEA1, and TPC1. MHCII lysosomal localization was corroborated by treatment with the lysosomolytic agent glycyl-l-phenylalanine-β-naphthylamide, which reduced the number of MHCII-positive vesicles. The surface presence of MHCII was revealed by immunolabeling of live non-permeabilized cells. In IFNγ-treated astrocytes, an increased fraction of large-diameter exocytotic vesicles (lysosome-like vesicles) with prolonged fusion pore dwell time and larger pore conductance was recorded, whereas the rate of endocytosis was decreased. Stimulation with ATP, which triggers cytosolic calcium signaling, increased the frequency of exocytotic events, whereas the frequency of full endocytosis was further reduced. In IFNγ-treated astrocytes, MHCII-linked antigen surface presentation is mediated by increased lysosomal exocytosis, whereas surface retention of antigens is prolonged by concomitant inhibition of endocytosis. Electronic supplementary material The online version of this article (10.1007/s00018-019-03350-8) contains supplementary material, which is available to authorized users.

Aim Astrocytes play a homeostatic role in the central nervous system and influence numerous aspects of neurophysiology via intracellular trafficking of vesicles. Intermediate filaments (IFs), also known as nanofilaments, regulate a number of cellular processes including organelle trafficking and adult hippocampal neurogenesis. We have recently demonstrated that the IF protein nestin, a marker of neural stem cells and immature and reactive astrocytes, is also expressed in some astrocytes in the unchallenged hippocampus and regulates neurogenesis through Notch signalling from astrocytes to neural stem cells, possibly via altered trafficking of vesicles containing the Notch ligand Jagged-1. Methods We thus investigated whether nestin affects vesicle dynamics in astrocytes by examining single vesicle interactions with the plasmalemma and vesicle trafficking with high-resolution cell-attached membrane capacitance measurements and confocal microscopy. We used cell cultures of astrocytes from nestin-deficient (Nes-/- ) and wild-type (wt) mice, and fluorescent dextran and Fluo-2 to examine vesicle mobility and intracellular Ca2+ concentration respectively. Results Nes-/- astrocytes exhibited altered sizes of vesicles undergoing full fission and transient fusion, altered vesicle fusion pore geometry and kinetics, decreased spontaneous vesicle mobility and altered ATP-evoked mobility. Purinergic stimulation evoked Ca2+ signalling that was slightly attenuated in Nes-/- astrocytes, which exhibited more oscillatory Ca2+ responses than wt astrocytes. Conclusion These results demonstrate at the single vesicle level that nestin regulates vesicle interactions with the plasmalemma and vesicle trafficking, indicating its potential role in astrocyte vesicle-based communication.

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. © 2017 Wiley Periodicals, Inc.

The neocortex represents one of the largest estates of the human brain. This structure comprises ~30-40 billions of neurones and even more of non-neuronal cells. Astrocytes, highly heterogeneous homoeostatic glial cells, are fundamental for housekeeping of the brain and contribute to information processing in neuronal networks. Gray matter astrocytes tightly enwrap synapses, contact blood vessels and, naturally, are also in contact with the extracellular space, where convection of fluid takes place. Thus astrocytes receive signals from several distinct extracellular domains and can get excited by numerous mechanisms, which regulate cytosolic concentration of second messengers, such as Ca2+ and cAMP. Excited astrocytes often secrete diverse substances (generally referred to as gliosignalling molecules) that include classical neurotransmitters such as glutamate and ATP or neuromodulators such as D-serine or neuropeptides. Astrocytic secretion occurs through several mechanisms: by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release of gliosignalling molecules appears fundamentally similar to that operating in neurones, since it depends on the SNARE proteins-dependent merger of the vesicle membrane with the plasmalemma. However, the coupling between the stimulus and astroglial vesicular secretion is at least one order of magnitude slower than that in neurones. Here we review mechanisms of astrocytic excitability and the molecular, anatomical and physiological properties of vesicular apparatus mediating the release of gliosignalling molecules in health and in the neurodegenerative pathology.

Exocytosis is the process by which stored neurotransmitters and hormones are released via the fusion of secretory vesicles with the plasma membrane. It is a dynamic, rapid and spatially restricted process involving multiple steps including vesicle trafficking, tethering, docking, priming and fusion. For many years great steps have been undertaken in our understanding of how exocytosis occurs in different cell types, with significant focus being placed on synaptic release and neurotransmission. However, this process of exocytosis is an essential component of cell signalling throughout the body and underpins a diverse array of essential physiological pathways. Many similarities exist between different cell types with regard to key aspects of the exocytosis pathway, such as the need for Ca(2+) to trigger it or the involvement of members of the N-ethyl maleimide-sensitive fusion protein attachment protein receptor protein families. However, it is also equally clear that non-neuronal cells have acquired highly specialized mechanisms to control the release of their own unique chemical messengers. This review will focus on several important non-neuronal cell types and discuss what we know about the mechanisms they use to control exocytosis and how their specialized output is relevant to the physiological role of each individual cell type. These include enteroendocrine cells, pancreatic β cells, astrocytes, lactotrophs and cytotoxic T lymphocytes. Non-neuronal cells have acquired highly specialized mechanisms to control the release of unique chemical messengers, such as polarised fusion of insulin granules in pancreatic β cells targeted towards the vasculature (top). This review discusses mechanisms used in several important non-neuronal cell types to control exocytosis, and the relevance of intermediate vesicle fusion pore states (bottom) and their specialized output to the physiological role of each cell type. These include enteroendocrine cells, pancreatic β cells, astrocytes, lactotrophs and cytotoxic T lymphocytes. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

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.

Ketamine is an antidepressant with rapid therapeutic onset and long-lasting effect, although the underlying mechanism(s) remain unknown. Using FRET-based nanosensors we found that ketamine increases [cAMP]i in astrocytes. Membrane capacitance recordings, however, reveal fundamentally distinct mechanisms of effects of ketamine and [cAMP]i on vesicular secretion: a rise in [cAMP]i facilitated, whereas ketamine inhibited exocytosis. By directly monitoring cholesterol-rich membrane domains with a fluorescently tagged cholesterol-specific membrane binding domain (D4) of toxin perfringolysin O, we demonstrated that ketamine induced cholesterol redistribution in the plasmalemma in astrocytes, but neither in fibroblasts nor in PC 12 cells. This novel mechanism posits that ketamine affects density and distribution of cholesterol in the astrocytic plasmalemma, consequently modulating a host of processes that may contribute to ketamine’s rapid antidepressant action.

Astrocytes are principal cells responsible for maintaining the brain homeostasis. Additionally, these glial cells are also involved in homocellular (astrocyte-astrocyte) and heterocellular (astrocyte-other cell types) signalling and metabolism. These astroglial functions require an expression of the assortment of molecules, be that transporters or pumps, to maintain ion concentration gradients across the plasmalemma and the membrane of the endoplasmic reticulum. Astrocytes sense and balance their neurochemical environment via variety of transmitter receptors and transporters. As they are electrically non-excitable, astrocytes display intracellular calcium and sodium fluctuations, which are not only used for operative signalling but can also affect metabolism. In this chapter we discuss the molecules that achieve ionic gradients and underlie astrocyte signalling.

Astrocytes are secretory cells, actively participating in cell-to-cell communication in the central nervous system (CNS). They sense signaling molecules in the extracellular space, around the nearby synapses and also those released at much farther locations in the CNS, by their cell surface receptors, get excited to then release their own signaling molecules. This contributes to the brain information processing, based on diffusion within the extracellular space around the synapses and on convection when locales relatively far away from the release sites are involved. These functions resemble secretion from endocrine cells, therefore astrocytes were termed to be a part of the gliocrine system in 2015. An important mechanism, by which astrocytes release signaling molecules is the merger of the vesicle membrane with the plasmalemma, i.e., exocytosis. Signaling molecules stored in astroglial secretory vesicles can be discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This leads to a fusion pore formation, a channel that must widen to allow the exit of the Vesiclal cargo. Upon complete vesicle membrane fusion, this process also integrates other proteins, such as receptors, transporters and channels into the plasma membrane, determining astroglial surface signaling landscape. Vesiclal cargo, together with the whole vesicle can also exit astrocytes by the fusion of multivesicular bodies with the plasma membrane (exosomes) or by budding of vesicles (ectosomes) from the plasma membrane into the extracellular space. These astroglia-derived extracellular vesicles can later interact with various target cells. Here, the characteristics of four types of astroglial secretory vesicles: synaptic-like microvesicles, dense-core vesicles, secretory lysosomes, and extracellular vesicles, are discussed. Then machinery for vesicle-based exocytosis, second messenger regulation and the kinetics of exocytotic vesicle content discharge or release of extracellular vesicles are considered. In comparison to rapidly responsive, electrically excitable neurons, the receptor-mediated cytosolic excitability-mediated astroglial exocytotic vesicle-based transmitter release is a relatively slow process.

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)-γ 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-γ-activated cultured rat astrocytes. The expression of MHC class II compartments, β2 adrenergic receptor (ADR-β2), and nuclear factor kappa-light-chain enhancer of activated B cells subunit p65 (NF-κB 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-γ-induced MHC class II compartments and substantially increased ADR-β2 expression, which is otherwise small or absent in astrocytes in MS. These effects could be partially attributed to the observed decrease in NF-κB p65 expression, because the NF-κB signaling cascade is activated in inflammatory processes. We also found attenuated trafficking and secretion from dextran-labeled endo-/lysosomes that may hinder efficient delivery of MHC class II molecules to the plasma membrane. Our data suggest that FTY720 and FTY720-P at submicromolar concentrations mediate anti-inflammatory effects on astrocytes by suppressing their action as APCs, which may further downregulate the inflammatory process in the brain, constituting the therapeutic effect of fingolimod in MS.

The intermediate filament (nanofilament) protein nestin is a marker of neural stem cells, but its role in neurogenesis, including adult neurogenesis, remains unclear. Here, we investigated the role of nestin in neurogenesis in adult nestin-deficient (Nes–/–) mice. We found that the proliferation of Nes–/– neural stem cells was not altered, but neurogenesis in the hippocampal dentate gyrus of Nes–/– mice was increased. Surprisingly, the proneurogenic effect of nestin deficiency was mediated by its function in the astrocyte niche. Through its role in Notch signaling from astrocytes to neural stem cells, nestin negatively regulates neuronal differentiation and survival; however, its expression in neural stem cells is not required for normal neurogenesis. In behavioral studies, nestin deficiency in mice did not affect associative learning but was associated with impaired long-term memory.

Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC50 ≈ 5 mM) that is in fat cells part of an autocrine loop, in which the Gi-protein mediates reduction of cytosolic cyclic adenosine monophosphate (cAMP). To study whether a similar signaling loop is present in astrocytes, affecting aerobic glycolysis, we measured the cytosolic levels of cAMP, D-glucose and L-lactate in single astrocytes using fluorescence resonance energy transfer (FRET)-based nanosensors. In contrast to the situation in fat cells, stimulation by extracellular L-lactate and the selective GPR81 agonists, 3-chloro-5-hydroxybenzoic acid (3Cl-5OH-BA) or 4-methyl-N-(5-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4-(2-thienyl)-1,3-thiazol-2-yl)cyclohexanecarboxamide (Compound 2), like adrenergic stimulation, elevated intracellular cAMP and L-lactate in astrocytes, which was reduced by the inhibition of adenylate cyclase. Surprisingly, 3Cl-5OH-BA and Compound 2 increased cytosolic cAMP also in GPR81-knock out astrocytes, indicating that the effect is GPR81-independent and mediated by a novel, yet unidentified, excitatory L-lactate receptor-like mechanism in astrocytes that enhances aerobic glycolysis and L-lactate production via a positive feedback mechanism.

In the brain, astrocytes signal to neighboring cells via regulated exocytotic release of gliosignaling molecules, such as brain-derived neurotrophic factor (BDNF). Recent studies uncovered a role of ketamine, an anesthetic and antidepressant, in the regulation of BDNF expression and in the disruption of astrocytic Ca2+ signaling, but it is unclear whether it affects astroglial BDNF release. We investigated whether ketamine affects ATP-evoked Ca2+ signaling and exocytotic release of BDNF at the single-vesicle level in cultured rat astrocytes. Cells were transfected with a plasmid encoding preproBDNF tagged with the pH-sensitive fluorescent protein superecliptic pHluorin, (BDNF-pHse) to load vesicles and measure the release of BDNF-pHse when the exocytotic fusion pore opens and alkalinizes the luminal pH. In addition, cell-attached membrane capacitance changes were recorded to monitor unitary vesicle interaction with the plasma membrane. Intracellular Ca2+ activity was monitored with Fluo-4 and confocal microscopy, which was also used to immunocytochemically characterize BDNF-pHse-laden vesicles. As revealed by double-fluorescent micrographs, BDNF-pHse localized to vesicles positive for the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, vesicle-associated membrane protein 2 (VAMP2), VAMP3, and synaptotagmin IV. Ketamine treatment decreased the number of ATP-evoked BDNF-pHse fusion/secretion events (P

In the brain, astrocytes provide metabolic and trophic support to neurones. Failure in executing astroglial homeostatic functions may contribute to the initiation and propagation of diseases, including Alzheimer disease (AD), characterized by a progressive loss of neurones over years. Here, we examined whether astrocytes from a mice model of AD isolated in the presymptomatic phase of the disease exhibit alterations in vesicle traffic, vesicular peptide release and purinergic calcium signaling. In cultured astrocytes isolated from a newborn wild-type (wt) and 3xTg-AD mouse, secretory vesicles and acidic endosomes/lysosomes were labeled by transfection with plasmid encoding atrial natriuretic peptide tagged with mutant green fluorescent protein (ANP.emd) and by LysoTracker, respectively. The intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored with Fluo-2 and visualized by confocal microscopy. In comparison with controls, spontaneous mobility of ANP- and LysoTracker-labeled vesicles was diminished in 3xTg-AD astrocytes; the track length (TL), maximal displacement (MD) and directionality index (DI) were all reduced in peptidergic vesicles and in endosomes/lysosomes (P < 0.001), as was the ATP-evoked attenuation of vesicle mobility. Similar impairment of peptidergic vesicle trafficking was observed in wt rat astrocytes transfected to express mutated presenilin 1 (PS1M146V). The ATP-evoked ANP discharge from single vesicles was less efficient in 3xTg-AD and PS1M146V-expressing astrocytes than in respective wt controls (P < 0.05). Purinergic stimulation evoked biphasic and oscillatory [Ca(2+)]i responses; the latter were less frequent (P < 0.001) in 3xTg-AD astrocytes. Expression of PS1M146V in astrocytes impairs vesicle dynamics and reduces evoked secretion of the signaling molecule ANP; both may contribute to the development of AD.

Astrocytes play an important housekeeping role in the central nervous system. Additionally, as secretory cells, they actively participate in cell-to-cell communication, which can be mediated by membrane-bound vesicles. The gliosignaling molecules stored in these vesicles are discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This process is termed exocytosis, regulated by SNARE proteins, and triggered by elevations in cytosolic calcium levels, which are necessary and sufficient for exocytosis in astrocytes. For astrocytic exocytosis, calcium is sourced from the intracellular endoplasmic reticulum store, although its entry from the extracellular space contributes to cytosolic calcium dynamics in astrocytes. Here, we discuss calcium management in astrocytic exocytosis and the properties of the membrane-bound vesicles that store gliosignaling molecules, including the vesicle fusion machinery and kinetics of vesicle content discharge. In astrocytes, the delay between the increase in cytosolic calcium activity and the discharge of secretions from the vesicular lumen is orders of magnitude longer than that in neurons. This relatively loose excitation-secretion coupling is likely tailored to the participation of astrocytes in modulating neural network processing.

Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca2+ concentration ([Ca2+]i) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P

AIM Alzheimer's disease (AD) is largely considered a neuron-derived insult, but also involves failure of astroglia. A recent study indicated that mutated presenilin 1 (PS1M146V), a putative endoplasmic reticulum (ER) Ca2+ channel with decreased Ca2+ conductance, impairs the traffic of astroglial peptidergic vesicles. Whether other pathogenically relevant PS1 mutants, such as PS1ΔE9, which code for ER channel with putative increased Ca2+ conductance, similarly affect vesicle traffic, is unknown. METHODS Here, we cotransfected rat astrocytes with plasmids encoding mutant PS1ΔE9 and atrial natriuretic peptide or vesicular glutamate transporter 1 tagged with fluorescent proteins (pANP.emd or pVGLUT1-EGFP respectively), to microscopically examine whether alterations in vesicle mobility and Ca2+ -regulated release of gliosignalling molecules manifest as a general vesicle-based defect; control cells were transfected to co-express exogenous or native wild-type PS1 and pANP.emd or pVGLUT1-EGFP. The vesicle mobility was analysed at rest and after ATP stimulation that increased intracellular calcium activity. RESULTS In PS1ΔE9 astrocytes, spontaneous mobility of both vesicle types was reduced (P

There are at least three reasons why brain astrocytes represent a new target for treating neurological disorders. First, although the human neocortex represents over 80% of brain mass, neurons are outnumbered by non-neuronal cells, including astrocytes, a neuroglial cell type. Second, as in neurons, vesicle-based release of transmitters is present in astrocytes, however with much slower kinetics than in neurons. Third, astrocytes contain glycogen, which can be transformed to L-lactate in glycolysis. L-lactate is considered to be a fuel and a signalling molecule involved in cognition and neuroprotection. The mechanisms of neuroprotection are unclear but may be linked to carbon monoxide, a product of the heme oxygenase, an evolutionarily conserved cellular cytoprotectant. Increased levels of local carbon monoxide arising from heme oxygenase activity may increase L-lactate, but direct measurements of cytosolic L-lactate are lacking. A fluorescence resonance energy transfer-based nanosensor selective for L-lactate was used to monitor cytosolic levels of L-lactate while cultured astrocytes were exposed to carbon monoxide. The results revealed that in astrocytes exposed to carbon monoxide there is no significant increase in L-lactate, however, when noradrenaline, a potent glycogenolytic agent, is applied, cytosolic levels of Llactate are increased, but strongly attenuated in astrocytes pretreated with carbon monoxide. These first measurements of carbon monoxide-modulated L-lactate levels in astrocytes provide evidence that the L-lactate and heme oxygenase neuroprotective systems may interact. In conclusion, not only the abundance of astrocytes but their signalling capacity using vesicles and metabolites, such as L-lactate, are valid targets for neurological disorders.

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.

Regulated exocytosis can be split into a sequence of steps ending with the formation and the dilation of a fusion pore, a neck-like connection between the vesicle and the plasma membrane. Each of these steps is precisely controlled to achieve the optimal spatial and temporal profile of the release of signalling molecules. At the level of the fusion pore, tuning of the exocytosis can be achieved by preventing its formation, by stabilizing the unproductive narrow fusion pore, by altering the speed of fusion pore expansion and by completely closing the fusion pore. The molecular structure and dynamics of fusion pores have become a major focus of cell research, especially as a promising target for therapeutic strategies. Electrophysiological, optical and electrochemical methods have been used extensively to illuminate how cells regulate secretion at the level of a single fusion pore. Here, we describe recent advances in the structure and mechanisms of the initial fusion pore formation and the progress in therapeutic strategies with the focus on exocytosis.

Intracellular organelles, including secretory vesicles, emerged when eukaryotic cells evolved some 3 billion years ago. The primordial organelles that evolved in Archaea were similar to endolysosomes, which developed, arguably, for specific metabolic tasks, including uptake, metabolic processing, storage and disposal of molecules. In comparison with prokaryotes, cell volume of eukaryotes increased by several orders of magnitude and vesicle traffic emerged to allow for communication between distant intracellular locations. Lysosomes, first described in 1955, a prominent intermediate of endo- and exocytotic pathways, operate virtually in all eukaryotic cells including astroglia, the most heterogeneous type of homeostatic glia in the central nervous system. Astrocytes support neuronal network activity in particular through elaborated secretion, based on a complex intracellular vesicle network dynamics. Deranged homeostasis underlies disease and astroglial vesicle traffic contributes to the pathophysiology of neurodegenerative (Alzheimer's disease, Huntington's disease), neurodevelopmental diseases (intellectual deficiency, Rett's disease) and neuroinfectious (Zika virus) disorders. This review addresses astroglial cell-autonomous vesicular traffic network, as well as its into primary and secondary vesicular network defects in diseases, and considers this network as a target for developing new therapies for neurological conditions.

The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β-Adrenergic receptors (β-AR), which increase cytosolic cAMP concentration ([cAMP]i ), may affect cell morphology. However, the real-time dynamics of β-AR-mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)-based cAMP biosensor Epac1-camps to study time-dependent changes in [cAMP]i ; morphological changes in primary rat astrocytes were monitored by real-time confocal microscopy. Stimulation of β-AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β-AR, rapidly increased [cAMP]i (∼15 s). The FRET signal response, mediated via β-AR, was faster than in the presence of forskolin (twofold) and dibutyryl-cAMP (>35-fold), which directly activate adenylyl cyclase and Epac1-camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP-dependent processes operate in a slow time domain. Most Epac1-camps expressing astrocytes revealed a morphological change upon β-AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell-shaped dependency on [cAMP]i . The 5-10% decrease in cell cross-sectional area and the 30-50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, β-AR/cAMP-mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease.

Neuroendocrine cells contain small and large vesicles, but the functional significance of vesicle diameter is unclear. We studied unitary exocytic events of prolactin-containing vesicles in lactotrophs by monitoring discrete steps in membrane capacitance. In the presence of sphingosine, which recruits VAMP2 for SNARE complex formation, the frequency of transient and full fusion events increased. Vesicles with larger diameters proceeded to full fusion, but smaller vesicles remained entrapped in transient exocytosis. The diameter of vesicle dense cores released by full fusion exocytosis into the extracellular space was larger than the diameter of the remaining intracellular vesicles beneath the plasma membrane. Labeling with prolactin- and VAMP2-antibodies revealed a correlation between the diameters of colocalized prolactin- and VAMP2-positive structures. It is proposed that sphingosine-mediated facilitation of regulated exocytosis is not only related to the number of SNARE complexes per vesicle but also depends on the vesicle size, which may determine the transition between transient and full fusion exocytosis.

Astrocytes are considered essential in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). We have demonstrated previously that immunoglobulins G (IgG) isolated from patients with ALS enhance the mobility of acidic vesicles in cultured astrocytes in a Ca2+-dependent manner. Here we directly examined the impact of purified sporadic ALS IgG on cytosolic [Ca2+] ([Ca2+](i)) in astrocytes. Confocal time-lapse images were acquired and fluorescence of a non-ratiometric Ca2+ indicator was recorded before and after the application of IgG. ALS IgG (0.1 mg/ml) from 7 patients evoked transient increases in [Ca2+](i) in similar to 50% of tested astrocytes. The probability of observing a response was independent of extracellular Ca2+. The peak increase in [Ca2+](i) developed similar to 3 times faster and the time integral of evoked transients was similar to 2-fold larger; the peak amplitude itself was not affected by extracellular Ca2+. Application of pharmacological inhibitors revealed that activation of inositol-1,4,5-triphosphate receptors is necessary and sufficient to initiate transients in [Ca2+](i) the Ca2+ influx through store-operated calcium entry prolongs the transient increase in [Ca2+](i). Thus, ALS IgG acutely affect [Ca2+](i) by mobilizing both, intra- and extracellular Ca2+ into the cytosol of cultured astrocytes. (C) 2013 Elsevier Ltd. All rights reserved.

How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-beta-cyclodextrin (MbetaCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (C(m)), fluctuations of which reflect exocytosis and endocytosis. Treatment with MbetaCD reduced cellular cholesterol and caused a dose-dependent attenuation of the Ca(2+)-evoked increase in C(m) (IC50 = 5.3 mM) vs. untreated cells. Cytosol dialysis of MbetaCD enhanced the attenuation of C(m) increase (IC50 = 3.3 mM), suggesting cholesterol depletion at intracellular membrane sites was involved in attenuating exocytosis. Acute extracellular application of MbetaCD resulted in an immediate C(m) decline, which correlated well with the cellular surface area decrease, indicating the involvement of cholesterol in the regulation of membrane surface area dynamics. This decline in C(m) was three-fold slower than MbetaCD-mediated fluorescent cholesterol decay, implying that exocytosis is the likely physiological means for plasma membrane cholesterol replenishment. MbetaCD had no effect on the specific C(m) and the blockade of endocytosis by Dyngo 4a, confirmed by inhibition of dextran uptake, also had no effect on the time-course of MbetaCD-induced C(m) decline. Thus acute exposure to MbetaCD evokes a C(m) decline linked to the removal of membrane cholesterol, which cannot be compensated for by exocytosis. We propose that the primary contribution of cholesterol to surface area dynamics is via its role in regulated exocytosis.

Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

Background Dynamin is a multidomain GTPase exhibiting mechanochemical and catalytic properties involved in vesicle scission from the plasmalemma during endocytosis. New evidence indicates that dynamin is also involved in exocytotic release of catecholamines, suggesting the existence of a dynamin-regulated structure that couples endo- to exocytosis. Methods Thus we here employed high-resolution cell-attached capacitance measurements and super-resolution structured illumination microscopy to directly examine single vesicle interactions with the plasmalemma in cultured rat astrocytes treated with distinct pharmacological modulators of dynamin activity. Fluorescent dextrans and the lipophilic plasmalemmal marker DiD were utilized to monitor uptake and distribution of vesicles in the peri-plasmalemmal space and in the cell cytosol. Results Dynamin inhibition with Dynole™-34-2 and Dyngo™-4a prevented vesicle internalization into the cytosol and decreased fusion pore conductance of vesicles that remained attached to the plasmalemma via a narrow fusion pore that lapsed into a state of repetitive opening and closing - flickering. In contrast, the dynamin activator Ryngo™-1-23 promoted vesicle internalization and favored fusion pore closure by prolonging closed and shortening open fusion pore dwell times. Immunocytochemical staining revealed dextran uptake into dynamin-positive vesicles and increased dextran uptake into Syt4- and VAMP2-positive vesicles after dynamin inhibition, indicating prolonged retention of these vesicles at the plasmalemma. Conclusions Our results have provided direct evidence for a role of dynamin in regulation of fusion pore geometry and kinetics of endo- and exocytotic vesicles, indicating that both share a common dynamin-regulated structural intermediate, the fusion pore.

Neuroglial contribution to Alzheimer's disease (AD) is pathologically relevant and highly heterogeneous. Reactive astrogliosis and activation of microglia contribute to neuroinflammation, whereas astroglial and oligodendroglial atrophy affect synaptic transmission and underlie the overall disruption of the central nervous system (CNS) connectome. Astroglial function is tightly integrated with the intracellular ionic signalling mediated by complex dynamics of cytosolic concentrations of free Ca2+ and Na+. Astroglial ionic signalling is mediated by plasmalemmal ion channels, mainly associated with ionotropic receptors, pumps and solute carrier transporters, and by intracellular organelles comprised of the endoplasmic reticulum and mitochondria. The relative contribution of these molecular cascades/organelles can be plastically remodelled in development and under environmental stress. In AD astroglial Ca2+ signalling undergoes substantial reorganisation due to an abnormal regulation of expression of Ca2+ handling molecular cascades.

Since the 1970s, much effort was been expended researching mechanisms of regulated exocytosis. Early work focused mainly on the role of proteins. Most notably the discovery of SNARE proteins in the 1980s and the zippering hypothesis brought us much closer to understanding the complex interactions in membrane fusion between vesicle and plasma membranes, a pivotal component of regulated exocytosis. However, most likely due to the predictions of the Singer–Nicholson fluid mosaic membrane model, the lipid components of the exocytotic machinery remained largely overlooked. Lipids were considered passive constituents of cellular membranes, not contributing much, if anything, to the process of exocytosis and membrane fusion. Since the 1990s, this so-called proteocentric view has been gradually giving way to the new perspective best described with the term proteolipidic. Many lipids were found to be of great importance in the regulation of exocytosis. Here we highlight the role of cholesterol. Furthermore, by using high-resolution cell-attached membrane capacitance measurements, we have monitored unitary exocytotic events in cholesterol-depleted membranes. We show that the frequency of these events is attenuated, providing evidence at the single vesicle level that cholesterol directly influences the merger of the vesicle and the plasma membranes.

This Special Issue (SI) of Cell Calcium focuses on regulated exocytosis, a recent evolutionary invention of eukaryotic cells. This essential cellular process consists of several stages: (i) the delivery of membrane bound vesicles to specific plasma membrane sites, (ii) where the merger between the vesicle and the plasma membranes occurs, (iii) leading to the formation of an aqueous channel through which vesicle content starts to be discharged to the cell exterior, (iv) after the full incorporation of the vesicle membrane into the plasma membrane, the added vesicle membrane is retrieved back into the cytoplasm by endocytosis. (v) When a fusion pore opens it may close again, a process known as transient fusion pore opening (also kiss-and-run exocytosis). In some cell types these stages are extremely shortlived, as in some neurons, and thus relatively inaccessible to experimentation. In other cell types the transition between these stages is orders of magnitude slower and can be studied in more detail. However, despite the intense investigations of this critical biological process over the last decades, the molecular mechanisms underlying regulated exocytosis have yet to be fully resolved. We thus still lack a comprehensive physiological insight into the nature of the progressive and coupled stages of exocytosis. Such a molecular-level understanding would help to fully reconstruct this process in vitro, as well as identify potential therapeutic targets for a range of diseases and dysfunctions. There are 18 papers in this SI which have been organized into three sections: Rapid regulated exocytosis and calcium homeostasis with an introduction by Erwin Neher, Molecular mechanisms of regulated exocytosis, and Cell models for regulated exocytosis. Here we briefly outline and integrate the messages of these sections.

In the brain, astrocytes signal to the neighboring cells by the release of chemical messengers (gliotransmitters) via regulated exocytosis. Recent studies uncovered a potential role of signaling lipids in modulation of exocytosis. Hence, we investigated whether sphingosine and the structural analog fingolimod/FTY720, a recently introduced therapeutic for multiple sclerosis, affect (i) intracellular vesicle mobility and (ii) vesicle cargo discharge from cultured rat astrocytes. Distinct types of vesicles, peptidergic, glutamatergic, and endosomes/lysosomes, were fluorescently prelabeled by cell transfection with plasmids encoding atrial natriuretic peptide tagged with mutant green fluorescent protein and vesicular glutamate transporter tagged with enhanced green fluorescent protein or by LysoTracker staining, respectively. The confocal and total internal reflection fluorescence microscopies were used to monitor vesicle mobility in the cytoplasm and near the basal plasma membrane, respectively. Sphingosine and FTY720, but not the membrane impermeable lipid analogs, dose-dependently attenuated vesicle mobility in the subcellular regions studied, and significantly inhibited stimulated exocytotic peptide and glutamate release. We conclude that in astrocytes, cell permeable sphingosine-like lipids affect regulated exocytosis by attenuating vesicle mobility, thereby preventing effective vesicle access/interaction with the plasma membrane docking/release sites. © 2012 Wiley Periodicals, Inc.

Rab4 and Rab5 GTPases are key players in the regulation of endocytosis. Although their role has been studied intensively in the past, it is still unclear how they regulate vesicle mobility. In particular, in astrocytes, the most abundant glial cells in the brain, vesicles have been shown to exhibit nondirectional and directional mobility, which can be intermittent, but the underlying switching mechanisms are not known. By using quantitative imaging, we studied the dynamics of single vesicle movements in astrocytes in real time, by transfecting them with different GDP- and GTP-locked mutants of Rab4 and Rab5. Along with the localization of Rab4 and Rab5 on early and late endocytic compartments, we measured the apparent vesicle size by monitoring the area of fluorescent puncta and determined the patterns of vesicle mobility in the presence of wild-type and Rab mutants. Dominant-negative and dominant-positive mutants, Rab4 S22N, Rab5 S34N and Rab4 Q67L, Rab5 Q79L, induced an increase in the apparent vesicle size, especially Rab5 mutants. These mutants also significantly reduced vesicle mobility in terms of vesicle track length, maximal displacement, and speed. In addition, significant reductions in the fraction of vesicles exhibiting directional mobility were observed in cells expressing Rab4 S22N, Rab4 Q67L, Rab5 S34N, and Rab5 Q79L. Our data indicate that changes in the GDP-GTP switch apparently not only affect fusion events in endocytosis and recycling, as already proposed, but also affect the molecular interactions determining directional vesicle mobility, likely involving motor proteins and the cytoskeleton.