Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion

Abstract

Astrocytes, secretory cells of the CNS

The concept of astrocytes as secretory cells is almost as old as the discovery of these glial cells. The secretory potential of astrocytes became known only 15 years after Michael von Lenhossék coined the term “astrocyte” (von Lenhossék, 1895). In 1909, Hans Held observed, using the molybdenum haematoxylin stain, granular inclusions in neuroglial processes, which he interpreted as a sign of active secretion (Held, 1909). A year later, Jean Nageotte reported secretory granules in glial cells of the grey matter (i.e. astrocytes) using the Altmann method of fucsin labelling. Nageotte concluded that he was “able to present evidence of a robust and active secretion phenomenon in the protoplasm of these cells” (Nageotte, 1910). These granules, later called gliosomes by Alois Alzheimer (see (Glees, 1955) for historic narration), were often observed, and the hypothesis of astroglial secretion was also entertained by Wilder Penfield (Penfield, 1932). Of note, this early 20^th century term should not be confused with the recent use of the name gliosomes for describing glial sub‐cellular re‐sealed particles (Nakamura et al, 1993) containing transmitter‐laden vesicles (Stigliani et al, 2006). Be this as it may, both Nageotte and Penfield regarded astrocytes as true endocrine elements that release their secretions into the blood from their endfeet tightly associated with the brain vasculature. This endocrine role of astroglia has not been experimentally confirmed. However, research carried out in recent years has provided a remarkable body of evidence indicating that astrocytes secrete diverse substances that contribute to the regulation of CNS development and homeostasis, synaptogenesis and cognitive function. In that, astrocytes act as a part of a neuroglial secretory network, which, by analogy with the endocrine system, can be defined as the gliocrine system of the CNS (Vardjan & Zorec, 2015). Other known cellular components of the gliocrine system are microglia and oligodendroglia, which all secrete numerous factors important for trophic support, homeostatic control and defence of the nervous tissue. It is highly likely that NG2 cells could be annexed to the gliocrine system, albeit experimental evidence on this account is lacking at present. Astroglia‐derived secretory substances include (Table 1): (i) classical neurotransmitters, (ii) neurotransmitter precursors, (iii) neuromodulators, (iv) hormones and peptides, (v) eicosanoids, (vi) metabolic substrates, (vii) scavengers of ROS, (viii) growth factors, (ix) various factors that can be defined as “plastic” (e.g. factors that regulate synaptogenesis and synaptic connectivity) and, finally, (x) pathologically relevant molecules such as inflammatory factors. These different molecules are released by astrocytes through several pathways (Fig 1 and see Malarkey & Parpura, 2008 for details) represented by: (i) vesicle‐based exocytosis (e.g. that of d‐serine (Martineau et al, 2013) or glutamate (Montana et al, 2004); (ii) diffusion through plasmalemmal pores/channels (e.g. release of ATP and/or glutamate through anion channels, connexin hemichannels or dilated P2X₇ receptors, Cotrina et al, 1998; Suadicani et al, 2006) and (iii) extrusion through plasmalemmal transporters (e.g. the release of GABA via the reversed operation of GAT‐3 transporters, Unichenko et al, 2012). Often, the same molecule can be released through different pathways, which affects the complexity/specificity of its action. The release of these molecules to the extracellular space, along with their subsequent transport by the convective glymphatic system (Thrane et al, 2014), occurs within various brain regions in different time spans and with multiple functional consequences. In this review, we primarily focus on the exocytotic secretory pathway.

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Figure 1

Multiple secretory pathways operating in astrocyte

 

Exocytosis: multiple mechanisms

Exocytotic release, engaging various types of membrane‐bound organelles laden with heterogeneous cargo, emerged early in evolution (Vardjan et al, 2010; Spang et al, 2015) and is present in the majority of eukaryotic cells. Fusion of organelles with the plasma membrane is key for intercellular signalling and for targeting various molecules (e.g. receptors or transporters) to the plasmalemma. Exocytosis is regulated by cytosolic free calcium ions and can occur either without stimulation (constitutive secretion) or in response to exogenous stimulation (regulated secretion, Kasai et al, 2012). In the brain, neurones are an exemplary model to study the exocytotic signalling pathway due to the spatially and temporally precise release of neurotransmitters at chemical synapses. Astrocytes are similarly capable of exocytosis, but this process is different in terms of spatial arrangements, kinetics and molecular mechanisms.

Vesicular release is supported by the evolutionary conserved family of SNARE proteins (Sollner et al, 1993). They are further divided into two categories, R‐SNAREs and Q‐SNAREs (Fasshauer et al, 1998; Jahn & Scheller, 2006). The former are associated with the vesicular membrane (also referred to as VAMPs), while the later are either integral plasma membrane proteins (e.g. syntaxins) or proteins associated with the plasma membrane (e.g. SNAP25 in neurones or SNAP23 in astrocytes). In the presence of supra‐threshold cytosolic Ca²⁺ concentrations, R‐SNARE and Q‐SNARE proteins form the ternary SNARE complex by contributing their SNARE domains (one from each VAMP2 and syntaxin and two from SNAP23/25) to form a 4 α‐helical bundle (SNAREpin). This bundle facilitates the fusion of vesicular and plasma membranes (Sutton et al, 1998; Weber et al, 1998). Kinetics of exocytosis is highly heterogeneous (Table 2). Fusion develops in < 1 ms in fast CNS synapses, whereas in endocrine or in kidney cells exocytosis proceeds over many hundreds of milliseconds or even seconds (Coorssen & Zorec, 2012; Kasai et al, 2012; Neher, 2012). Time course of exocytotic release is determined by several factors. First, it is the sensitivity of secretory apparatus to [Ca²⁺]_i, which is heterogeneous in different cell types. Second, the spatiotemporal progression of local [Ca²⁺]_i signals differs markedly between cells. For instance, in synaptic terminals excitation–secretion coupling is exceedingly fast due to the organisation of Ca²⁺ nanodomains that reflect a close proximity of the Ca²⁺ source and exocytotic machinery (Eggermann et al, 2012). Finally, slow regulated exocytosis may also evince a distinct vesicle nanoarchitecture (e.g. arrangement and density of R‐SNAREs, see Fig 2) and the heterogeneity of Q‐SNAREs (Takamori et al, 2006; Singh et al, 2014). Multiple mechanisms controlling exocytosis may coexist within the confinement of a single cell resulting in complex kinetics of secretion (Rupnik et al, 2000).

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Figure 2

Arrangement of VAMP2 on vesicles in astrocytes

(A) The diagram represents an astrocytic vesicle with the architecture of VAMP2 across the vesicle membrane. VAMP2 is appended at its luminal C‐terminus with yellow phluorin (YpH, shown in green) and can be immunolabelled at its N‐terminus (cytoplasmic site) using primary and secondary antibodies, the latter tagged with Atto594, a red fluorophore. The graph (below) shows the measurements of distance between the two fluorophores obtained from an astrocyte shown in (B), indicating the length of VAMP2 in astrocytes. (B) Structured illumination microscopy (SIM) micrograph of an astrocyte expressing VAMP2 marked fluorescently at luminal and cytoplasmic sites as schematized in (A). There is an incomplete co‐localisation, that is separation, between the red and green puncta, disclosed as the distance in (A); co‐localisation of YpH and Atto594 is disclosed in yellow. An area (box) of an astrocyte is shown in the inset (bottom right). Scale bar: 10 µm (300 nm in inset). (Modified with permission from Singh et al, 2014).

Diversity of astroglial secretory organelles

Eukaryotic cells produce different types of membranous secretory organelles that are classified as intracellular or extracellular. Intracellular vesicles are represented by transport vesicles, lysosomes and various types of secretory vesicles, whereas extracellular vesicles are ectosomes, exosomes, microvesicles (microparticles), membrane particles and apoptotic vesicles (van der Pol et al, 2012; Cocucci & Meldolesi, 2015). Intracellular vesicles are cellular organelles that may completely fuse with cellular membranes, whereas extracellular vesicles are membranous compartments released into the surrounding environment. Generally, vesicles undergoing constitutive or regulated exocytosis derive either from the trans‐Golgi network or from early or recycling endosomes, although multivesicular bodies and lysosomes have been reported to undergo exocytosis under certain conditions.

Several secretory organelles undergo regulated exocytosis in astrocytes (Fig 3). These include clear electron lucent SLMVs that morphologically resemble synaptic vesicles (Bezzi et al, 2004; Crippa et al, 2006; Jourdain et al, 2007; Bergersen & Gundersen, 2009; Martineau et al, 2013), DCVs (Calegari et al, 1999; Parpura & Zorec, 2010) and secretory lysosomes (Zhang et al, 2007; Li et al, 2008; Verderio et al, 2012). All these organelles can store and release low (amino acids) and/or high (peptides and proteins) molecular weight chemical transmitters (Parpura & Zorec, 2010; Gucek et al, 2012; Vardjan & Zorec, 2015). Secretory vesicles can also act as recycling vesicles that take up extracellular molecules (e.g. by endocytosis) and promote their subsequent release (Vardjan et al, 2014b). This function may be essential for defining the composition of the cerebrospinal fluid and for the function of the glymphatic system (Thrane et al, 2014).

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Figure 3

Diversity of astroglial vesicles

Bottom panel shows schematic overview of astroglial secretory organelles. Top panel demonstrates ultrastructure of astroglial secretory organelles. (a) Electron micrograph of small clear vesicles (red arrowheads) in an astrocyte (Ast) from the rat hippocampus in situ. Scale bars: 100 nm (50 nm in inset). Modified with permission from Bergersen et al (2012). (b) Small clear vesicles from cultured astrocytes. Courtesy of Dr. Michela Matteoli. Scale bar: 50 nm. (c) Electron micrograph of a dense‐core vesicle in cultured astrocytes stained by immunogold for secretogranin II. Modified with permission from Calegari et al (1999). Arrow shows DCV stained for secretogranin II. The open arrowhead points to the intermediate filament immunolabelled for GFAP. Scale bar: 100 nm. (d) Electron micrograph showing a dense‐core vesicle in cultured astrocytes. Scale bar: 500 nm. Modified with permission from Prada et al (2011). (e) Electron micrograph of lysosomes (L) in astrocytes. Scale bar: 1 µm. ©2012 by National Academy of Sciences. Modified with permission from Di Malta et al (2012). (f) Electron microscopy images of an ADF glioma cell. Arrows point to multilamellar organelles. Scale bar: 150 nm. Modified with permission from Verderio et al (2012). (g) Electron micrograph showing a multivesicular body‐like structure from a rat cultured astrocyte. Arrowheads indicate vesicles. Scale bars: 200 µm. Modified with permission from Brignone et al (2015). (h) Electron micrograph (negative staining) showing an exosome secreted by cultured astrocytes following stimulation with 100 mM BzATP, a P2X agonist, for 20 min. Scale bar: 300 nm. Modified with permission from Bianco et al (2009). Abbreviations: aSMase, acid sphingomyelinase; BMP, bis(monoacylglycero)phosphate; FGF2, fibroblast growth factor 2; Hsp70, 70 kilodalton heat shock protein; miRNA, microRNA; MMPs, matrix metalloproteinases; mtRNA, mitochondrial RNA; nSMase2, neutral sphingomyelinase 2; NTPDase, nucleoside triphosphate diphosphohydrolases; PS, phosphatidylserine; REST, RE‐1‐silencing transcription factor; tPA, tissue plasminogen activator; VGEF, vascular endothelial growth factor.

Extracellular vesicles

Extracellular vesicles are broadly divided into exosomes and ectosomes. ECVs are typically loaded with a wide spectrum of bioactive substances including cytokines, signalling proteins, mRNA and microRNA (Mause & Weber, 2010). Exosomes are vesicles of 40–100 nm in diameter, produced through the formation of MVBs and their subsequent fusion with the plasma membrane (Mathivanan et al, 2010). Ectosomes, on the other hand, range from 100 to more than 1,000 nm in diameter and are formed and released by shedding off the plasma membrane.

The formation of exosomes follows the typical endocytic route, where transmembrane proteins are endocytosed and trafficked to early endosomes and subsequently to late endosomes. Intraluminal vesicles are generated by neutral sphingomyelinase 2 and ceramide‐dependent process (Trajkovic et al, 2008) that also requires the ESCRT to generate MVBs (van Niel et al, 2006). Fusion of MVBs and release of exosomes involve Rab11, Rab27 and Rab35 (Vanlandingham & Ceresa, 2009; Hsu et al, 2010; Ostrowski et al, 2010; Baietti et al, 2012). During differentiation, MVBs become enriched in lipids such as cholesterol, lysobisphosphatidic acid and sphingomyelins containing ceramide (Kobayashi et al, 1998; Chevallier et al, 2008).

Ectosomes form by direct budding off the plasma membrane (Thery et al, 2009). Similarly to exosomes, ceramide is required for ectosome release (Bianco et al, 2009); ceramide together with ESCRT subunits participate in ectosome assembly and budding. During ectosome shedding from the plasma membrane, ARRDC1 interacts with ESCRT component TSG101 (Nabhan et al, 2012).

Astrocytes release both types of ECVs. Ectosome shedding from astrocytes occurs upon the activation of P2X₇ purinoceptors and involves the rapid activation of acid sphingomyelinase that moves to the plasma membrane outer leaflet. Sphingomyelinase alters membrane structure/fluidity leading to vesicle blebbing and shedding (Bianco et al, 2009). Diameters of ectosomes shed by cultured astrocytes from the 2‐day‐old rat cortex vary between 100 and 1,000 nm (Proia et al, 2008; Bianco et al, 2009). Some ECVs are even larger. For example, in cultured human foetal astrocytes spontaneous shedding of large (~4 μm diameter on average) ECVs has been detected. These large ECVs can contain mitochondria and lipid droplets and are decorated with β‐1 integrin, a shedding marker (Falchi et al, 2013). The physiological relevance of this type of ECVs remains to be resolved; it cannot be excluded that they represent apoptotic bodies. Astrocyte‐derived ectosomes carry numerous factors that regulate the activity of neighbouring cells including fibroblast growth factor 2 and vascular endothelial growth factor (Proia et al, 2008), IL‐1β (Bianco et al, 2009), nucleoside triphosphate diphosphohydrolases (Ceruti et al, 2011) and matrix metalloproteinases (Sbai et al, 2010). Ectosomes also contain acid sphingomyelinase and high levels of phosphatidylserine on their membrane outer leaflet (Bianco et al, 2009). Finally, both exosomes and ectosomes contain nucleic acids, mainly microRNAs, small RNA regulators that have essential roles in different biological processes. Exosomes containing microRNAs can be utilized in communication between astrocytes and neurones. For instance, astrocytes treated with morphine and HIV Tat increase the expression of miR‐29b that is released by exosomes; miR29b in turn is taken up by neurones where it downregulates the expression of PDGF_B receptors (Hu et al, 2012). Of note, astrocyte‐derived exosomes have been reported to contain mitochondrial DNA (Guescini et al, 2010). Whether ECVs also carry neurotransmitters is yet to be elucidated.

Exosomes are released from astrocytes in response to oxidative and heat stress (Taylor et al, 2007) and also in pathological conditions. Secretion of exosomes containing PAR4 and ceramide is increased in astrocytes surrounding amyloid plaques in a mouse model of familial Alzheimer's disease. These PAR4‐ and ceramide‐enriched exosomes are subsequently taken up by astrocytes and induce apoptosis even in the absence of β‐amyloid (Wang et al, 2012). Given the role of PAR4‐ and ceramide‐containing exosomes in apoptotic processes, they are defined as “apoxosomes”. Whether exosomes are discharged by astrocytes through a physiologically regulated process and whether exosomal release in vivo has a physiological function remains unclear. Owing to the lack of methods to specifically block exosome secretion without affecting secretion of other membrane vesicles, the resolution to these issues cannot be reached at the time being.

Molecular machinery of astroglial exocytosis

Twenty years ago, it was demonstrated that SNAREs are present in cultured astrocytes (Parpura et al, 1995). Subsequent studies have revealed that astrocytes express proteins characteristic for neuronal synaptic vesicles such as VAMP2 and proteins that are found in exocytotic trafficking vesicles of non‐neuronal cells such as SCAMP and VAMP3 (Parpura et al, 1995; Maienschein et al, 1999; Wilhelm et al, 2004; Mothet et al, 2005; Crippa et al, 2006; Montana et al, 2006; Martineau et al, 2008). The lysosome‐associated TI‐VAMP/VAMP7 is expressed in astrocytes (Zhang et al, 2007; Martineau et al, 2008; Verderio et al, 2012) along with components of secretory machinery, Q‐SNARE proteins SNAP23, syntaxins 1, 2, 3 and 4 (Hepp et al, 1999; Zhang et al, 2004b; Paco et al, 2009), SNARE‐associated proteins such as synaptotagmin 4 (Zhang et al, 2004a) and isoforms of Munc18 (Paco et al, 2009). Cleavage of SNARE proteins with tetanus or botulinum neurotoxins (Verderio et al, 1999) reduce glutamate and d‐serine and, to a lesser extent, ATP release in cultured astrocytes (Coco et al, 2003). Similarly, the treatment with tetanus toxin suppressed exocytosis measured by monitoring amperometric spikes (Chen et al, 2005) or by recording membrane capacitance (Kreft et al, 2004). The residual, toxin‐insensitive component of Ca²⁺‐evoked exocytosis could be due to the contribution by other secretory organelles, such as lysosomes carrying toxin‐insensitive VAMP7 (Verderio et al, 2012).

Several SNARE proteins, including VAMP2 (Wilhelm et al, 2004), VAMP3 (Bezzi et al, 2004; Zhang et al, 2004a; Jourdain et al, 2007; Bergersen & Gundersen, 2009; Schubert et al, 2011), VAMP7 (Verderio et al, 2012), SNAP23 and syntaxin 1 (Schubert et al, 2011), have been detected in astrocytes in situ. VAMP2 and 3 co‐localise with VGLUT1 and 2 on SLMVs that store glutamate (Bezzi et al, 2004; Zhang et al, 2004a; Jourdain et al, 2007; Bergersen & Gundersen, 2009) and likely d‐serine (Martineau et al, 2013). Several synaptotagmin isoforms including synaptotagmins 4, 5, 7 and 11 are also present in astrocytes (Zhang et al, 2004a; Mittelsteadt et al, 2009). However, typical neuronal SNARE‐associated proteins, such as synaptotagmins 1 and 2, and synaptophysin (Wilhelm et al, 2004) have not been observed in astrocytes in situ.

Inactivation of VAMP2 and/or VAMP3 by tetanus neurotoxin abolished the release of glutamate (Jourdain et al, 2007; Perea & Araque, 2007) and, likely, d‐serine in astrocytes in brain slices (Henneberger et al, 2010). A transgenic mouse model expressing a dominant negative (dn) SNARE (i.e. the cytosolic tail of VAMP2) in astrocytes (Pascual et al, 2005; Halassa et al, 2009) showed changes in behaviour, synaptic transmission and maturation of neurones (Pascual et al, 2005; Hines & Haydon, 2013; Nadjar et al, 2013; Turner et al, 2013; Lalo et al, 2014; Sultan et al, 2015), suggesting a role for astrocytic VAMP2‐dependent exocytosis in vivo. Of note, VAMP2 cytosolic tail is supposed to compete with VAMP2 for binding to other components forming the ternary complexes, leading to the reduced number of complexes formed and hence inhibiting regulated exocytosis. Although these experiments provide strong support for a function of SNARE proteins in astroglial regulated exocytosis, there are indications that neurones might also express dnSNARE in the transgenic mice, thus raising the possibility that the impairment of neuronal, rather than astroglial, exocytosis may account for the phenotype observed (Fujita et al, 2014). The debate that ensued (Sloan & Barres, 2014) highlights technical matters, and particular aspects of astroglial glutamate secretion in the context of synaptic transmission, without questioning the general concept of exocytosis‐mediated astroglial secretion. These technical dissensions nonetheless emphasize the need for refining the existing experimental strategies and developing new approaches directly attacking the various facets of astroglial secretion in physiological and pathophysiological contexts (Jahn et al, 2015).

Astroglial exocytosis is slow

Visualisation of fluorescently labelled VGLUT1/2‐containing vesicles revealed that fusion events in isolated astrocytes occur within hundreds of milliseconds after the increase in cytosolic Ca²⁺ (Bezzi et al, 2004; Cali et al, 2008; Marchaland et al, 2008; Santello et al, 2011). Even slower kinetics of vesicular fusions has been reported by using synapto‐pHluorin (spH), a fluorescently tagged‐VAMP2, (Bowser & Khakh, 2007). Treatment of astrocytes with the Ca²⁺ ionophore ionomycin triggered exocytotic fusion of spH‐labelled SLMVs within seconds (Liu et al, 2011). Similarly, the TIRF microscopy (Malarkey & Parpura, 2011) showed slow exocytotic bursts occurring within seconds after mechanical stimulation of astrocytes. Secretion of NPY from peptidergic vesicles occurred with a > 1‐min delay after stimulation (Ramamoorthy & Whim, 2008; Prada et al, 2011). Exocytotic release from peptidergic vesicles in 8‐Br‐cAMP‐matured astrocytes also began minutes after the stimulation (Paco et al, 2009). Similar observations have been made for secretory lysosomes, which labelled with FM dyes fused with the plasma membrane with an ~1‐min delay after exposure of astrocytes to Ca²⁺ ionophores or ATP (Zhang et al, 2007; Li et al, 2008). Exocytotic fusion of quinacrine‐loaded vesicles that express lysosomal VAMP7 occurred with a > 2‐min delay after exposure to various stimuli including ionomycin, glutamate, ATP or UV‐induced Ca²⁺ uncaging (Kreft et al, 2004; Pangrsic et al, 2007; Pryazhnikov & Khiroug, 2008). Likewise, EGFP‐LAMP1‐ and FITC‐dextran‐labelled lysosomes underwent exocytotic fusion with a > 40‐s delay after administration of ionomycin (Liu et al, 2011) or the group I mGluR agonist DHPG (Jaiswal et al, 2007).

Taken together, these imaging data indicate that in contrast to neurones, where the fusion occurs within < 0.5 ms after the Ca²⁺ entry into the cytosol (Neher, 2012; Sudhof, 2012), exocytotic release of various molecules from astrocytes is a much slower process, occurring with a substantial post‐stimulus delay (Vardjan et al, 2015). Indeed, capacitance measurements on isolated astrocytes confirm that the kinetics of vesicle fusion is at least 2 orders of magnitude slower than in neurones (Fig 4 and Table 2; Kreft et al, 2003, 2004). Incidentally, inhibition of astroglial exocytosis (using astroglia targeted expression of dnSNARE or pharmacological tools) affects only slow electrical oscillations in the cortex (Fellin et al, 2009), while fast neuronal electrical activity seems to be unaffected by corrupted (using a mouse model rendering a reduction of VAMP1‐3 expression in Müller cells, a specialized astroglia of the retina) gliotransmission (Slezak et al, 2012).

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Figure 4

Slowness of astroglial exocytosis

(A) Comparison of kinetics of neuronal and astroglial exocytosis. Time‐dependent changes in membrane capacitance (Cm) recorded in a neuronal cell (trace in red, photoreceptor, modified from Kreft et al, 2003) and an astrocyte (trace in blue, modified from Kreft et al, 2004), elicited by a flash photolysis‐induced increase in cytosolic Ca²⁺. Note that the blue trace recorded in an astrocyte displays a significant delay between the stimulus (asterisk) and the response (trace components above the dotted line). (B) Neuronal vs. astrocytic SNAREs. Neurones and astrocytes alike express SNAREs VAMP2 and syntaxin 1; many astrocytes can also express VAMP3 in lieu of or in addition to VAMP2. Astrocytes express SNAP23, a homologue of neuronal SNAP25. At the plasma membrane, syntaxin 1A can form a binary cis complex with SNAP25B or SNAP23A, which then interacts with vesicular VAMP2 to form a ternary complex. A single ternary complex can tether the vesicle at the plasma membrane for a longer period of time (1.9 s vs. 0.8 s), when it contains SNAP25B rather than SNAP23A, respectively. Of note, truncated syntaxin 1, lacking the N‐terminal Habc domain and the linker region to the SNARE domain, is shown for simplicity. Drawings are not to scale.

The somewhat lethargic kinetics of astroglial vesicular release likely reflects distinct organisation of the exocytotic machinery. First, electron microscopy studies (Bezzi et al, 2004; Jourdain et al, 2007; Bergersen et al, 2012) have shown that astrocytes lack structurally organised vesicle clusters typical of the active zone present in presynaptic terminals, which may make the stimulus–secretion coupling looser. In neurones, SNARE proteins are associated with vesicles clustered at active zones that are essentially release sites. This spatial localisation arguably is linked to the minimisation of the delay between the stimulus and the secretory output (Kasai et al, 2012). Second, the SNARE components and SNARE‐associated proteins of the exocytotic apparatus are not identical in astrocytes and neurones, neither is the stability of SNARE complexes, nor are the numbers of SNARE molecules associated with a single vesicle.

VAMP isoforms with similar structural properties can participate in the formation of several different SNARE complexes (Wilhelm et al, 2004; Montana et al, 2009), which may affect the mechanism of vesicle fusion with the plasma membrane. In neuronal terminals, the ternary fusion complex forms between VAMP2, SNAP25 and syntaxin, whereas in astrocytes the ternary SNARE fusion complex assembles from VAMP2/3 or TI‐VAMP, SNAP23 and syntaxin (Montana et al, 2009; Hamilton & Attwell, 2010). At a single molecule level, the presence of SNAP23A (as opposed to SNAP25B) in the ternary complex decreases the complex stability by half, arguably retarding the tethering/docking/fusion process (Fig 4B). Moreover, the density of R‐SNAREs associated with a single vesicle in astrocytes is lesser than in neurones; in the latter, a single synaptic vesicle contains ~70 VAMP2 molecules (Takamori et al, 2006) vs. ~25 in a single astroglial vesicle (Singh et al, 2014). This paucity of VAMP2 would lead to the reduced density of ternary SNARE complexes, which would contribute to further retardation of docking and fusion process in astrocytes.

Trafficking of astroglial vesicles

Quantitative measurements of secretory vesicles mobility in astrocytes (Potokar et al, 2005, 2013b) revealed two types of vesicle mobility: (i) directional, when vesicles travel along tracks, such as cytoskeletal elements, including intermediate filaments, or (ii) non‐directional, characterised by contorted vesicle trajectories, typical for the Brownian movement of particles. These experiments also unveiled a dichotomy of vesicular traffic: glutamatergic vesicles accelerate with an increase of cytosolic Ca²⁺ (Stenovec et al, 2007), whereas peptidergic vesicles and endolysosomes slow down (Potokar et al, 2008, 2010). Such stimulation‐dependent vesicle mobility regulation has not been observed in neurones and may represent an adaptive mechanism for astrocytes to redistribute vesicles to the correct location.

Astroglial exocytosis in physiology and pathophysiology

Concluding remarks

Astrocytes express a complex exocytotic machinery that is associated with several types of secretory vesicles involved in the secretion of a wide variety of neurotransmitters, neurotransmitter precursors, hormones, trophic and plastic factors, etc. Astroglial secretion contributes to the intrinsic CNS gliocrine network that provides for the regulation of multiple physiological and pathophysiological processes. Likely owing to the difference in secretory machinery, astroglial exocytosis is much slower that the neuronal counterpart. This fundamental difference reflects distinct physiological specialisation of astroglia as a key homeostatic component of the neural network.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

Notes

The EMBO Journal (2016) 35: 239–257 [PMC free article] [PubMed] [Google Scholar]

Notes

See the Glossary for abbreviations used in this article.

References