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Abstract

Vesicles are small intracellular organelles that are fundamental for constitutive housekeeping of the plasmalemma, intercellular transport, and cell-to-cell communications. In astroglial cells, traffic of vesicles is associated with cell morphology, which determines the signaling potential and metabolic support for neighboring cells, including when these cells are considered to be used for cell transplantations or for regulating neurogenesis. Moreover, vesicles are used in astrocytes for the release of vesicle-laden chemical messengers. Here we review the properties of membrane-bound vesicles that store gliotransmitters, endolysosomes that are involved in the traffic of plasma membrane receptors, and membrane transporters. These vesicles are all linked to pathological states, including amyotrophic lateral sclerosis, multiple sclerosis, neuroinflammation, trauma, edema, and states in which astrocytes contribute to developmental disorders. In multiple sclerosis, for example, fingolimod, a recently introduced drug, apparently affects vesicle traffic and gliotransmitter release from astrocytes, indicating that this process may well be used as a new pathophysiologic target for the development of new therapies.

Keywords

Astrocytes Glia Vesicles Trafficking Reactive astrogliosis Morphology Gliotransmitter Antigen presentation Neurogenesis Neuroinflammation Amyotrophic lateral sclerosis Multiple sclerosis

The Neurology in Crisis

Neurological diseases, represented by more than 600 nosological forms, remain the least understood and the most resilient forms of human pathology. Most somatic diseases became fundamentally curable during the last century; the neurological diseases, from neurotrauma to neurodegeneration and from autism to major psychiatric diseases, do not have pathogenetic therapeutic strategies, simply because the mechanisms behind neuropathological developments remain virtually unknown. Thus, deficiencies in fundamental knowledge stipulate medicinal failure.

The fundamentals of modern neurophysiology and neuropathophysiology are revolving around a neuronocentric doctrine, which, after being firmly established in the first half of the 20th century, postulates that neurons and neurons only are the cellular cornerstones of nervous function, and their malfunction ultimately means neurological disease.

The supposition of neurons being the only site of a disease has never been unequivocally corroborated by experimentation. Conceptually, any disease is primarily a homeostatic failure, and logically, the neuroglia that provides for homeostasis and defense of the nervous system may play a fundamental, although long time underappreciated, role in neuropathology.

New directions in how the brain should be investigated are in line with the recognized global need to investigate these complex systems in large-scale projects [i.e., Human Brain Project (38) in the EU and BRAIN in the US], which generally ignore nonexcitable cells (35) but nevertheless are central for keeping the nervous system in healthy balance.

Astroglia and Secretory Vesicles

The concept of neuroglia as a connective tissue of the nervous system into which nerves and nerve cells are embedded was introduced by Rudolf Virchow in the mid-19th century (57, 142). In the central nervous system (CNS), neuroglia is represented by astroglia, oligodendroglia, and NG2 glia, which are all of neural origin, and microglia, which are myeloid cells that invade the CNS early in development. In this article, we shall focus on astroglial cells. Astrocytes populate gray and white matter of the CNS and are, arguably, the most heterogeneous (in form and function) type of glia (99, 137). Astrocytes can be broadly defined as primary homeostatic cells of the CNS responsible for a wide variety of functions that include, for example, the regulation of synaptogenesis, synaptic maturation, neurotransmitter homeostasis, brain microcirculation, brain metabolism, and control over formation and maintenance of the blood-brain barrier (BBB) (1, 3, 26, 30, 41, 48, 56, 77, 79, 80, 110, 121, 138, 154). All these processes depend, to a large extent, on the mechanisms by which astrocytes communicate with the surrounding cells. These include plasma membrane channels, receptors, transporters, and mechanisms that mediate the exchange of molecules by exo- and endocytotic processes (43, 58, 83, 93 -95, 155). Exo- and endocytotic processes require vesicles, from which signaling molecules are released or extracellular material is internalized into vesicles. In the cytoplasm, both exo- and endocytotic vesicles are mobile in the cytoplasm, which involves the cytoskeleton (101, 102).

The cargo contained and carried by astrocytic vesicles is exceptionally diverse and may include classical neurotransmitters [glutamate or adenosine 5′-triphosphate (ATP)], neuromodulators (d-serin or taurine), amino acids, nucleotides, and peptides; in addition, vesicles contribute to the trafficking of large molecules such as transporters, water channels, and receptors (9, 21, 54, 60, 67, 68, 86, 91, 95, 105, 133). A singularly complex system regulates vesicle mobility and hence their delivery to the destination. Vesicular mobility is controlled by numerous signaling pathways and by cytoskeleton dynamics. Under pathologic conditions (ischemia, trauma, edema, neuroinflammation), different molecular cues alter vesicle mobility, as shown by several studies utilizing single-vesicle trafficking (101, 103 -105, 107, 108, 119, 120, 128, 133). We shall provide an overview of the mobility properties of exocytotic vesicles that transport signaling molecules such as glutamate, ATP, atrial natriuretic peptide (ANP), and brain-derived neurotrophic factor (BDNF), plasmalemmal transporters for glutamate also known as an excitatory amino acid transporter 2 (EAAT2); aquaporins 4 (AQP4), and antigen-presenting receptors represented by the major histocompatibility complex class (MHC-II) in physiology and in pathologic conditions.

Alterations in Vesicle Dynamics Contribute to Neurological Diseases

Diseases of the CNS result from homeostatic insufficiency and, in particular, follow the disruption of the balance between cell damage and repair. Astrocytes provide homeostatic support, and hence, these cells, arguably, are involved in every kind of neuropathology (40, 114, 140). Notably, astrocytes contribute to various aspects of CNS defense through (i) existing homeostatic mechanisms [which, for example, provide for regulation of glutamate and ion movements and hence contain excitotoxicity (111) or protect against reactive oxygen species by astrogliaderived glutathione (31)] or (ii) by mounting an evolutionary conserved defensive response known as reactive astrogliosis. Reactive astrogliosis aims at protecting the CNS, isolating the damaged area, reconstructing the BBB, and promote remodeling of the neural circuitry after the resolution of pathology (99, 145). Astrocytes, however, may also contribute to neuronal damage through failure or reversal of various homeostatic cascades that assume neurotoxic proportions (40, 78).

Vesicles contribute profoundly to the pathologic potential of astrocytes. In contrast to neurons, where vesicular release usually occurs in the submillisecond temporal domain (113), regulated exocytosis in astrocytes is at least one or two orders of magnitude slower (43). In addition, the surface area of cultured astrocytes is turned over by constitutive vesicle traffic in 5-6 h (59). If such slow constitutive plasma membrane turnover occurs in astrocytes in vivo, even the slightest imbalances in the rate of vesicle delivery to the plasma membrane versus the rate of retrieval from the plasma membrane may, over time, affect the properties of astrocytes and their responsiveness to physiological and pathological stimuli. For example, tissue remodeling in Alzheimer's disease (AD), where Aβ protein can be internalized into the astrocytes (75, 76), likely involves altered vesicle dynamics, which may contribute to the progression of AD.

Another example of impaired vesicle traffic may occur in the intellectual deficiency (ID), formerly known as human mental retardation (MR), a common disorder characterized by an IQ lower than 85. Poor cognitive ability can be the only visible sign in nonspecific ID, whereas behavioral deficits accompanied by other clinical signs may compose a syndrome (e.g., Down syndrome) or may be associated with metabolic, mitochondrial, or developmental disorders (63). Symptoms appear early in life and affect between 2% and 3% of the population. Family studies have demonstrated a relatively large number of X chromosome-linked forms of ID (XLID) with an incidence of about 0.9-1.4 in 1,000 males (129). One of the first genes discovered to be mutated in patients with XLID is GDI1 (23). GDI1 encodes for guanine nucleotide dissociation inhibitor (αGDI), a protein physiologically involved in retrieving inactive GDP-bound Rab proteins from the membrane. The identification of GDI1 as one of the genes causing human ID suggested that vesicular traffic in neuronal cells is an important pathway for development of cognitive functions (10, 24). Although the importance of αGDI in neuronal function has been demonstrated, it is unclear whether its role in glia vesicle trafficking contributes to the etiology of disease. The αGDI protein regulates the function of Rab GTPases, such as Rab 4 and Rab 5, which have recently been shown to regulate the traffic of endolysosomes in astrocytes (103), and it is likely that impaired vesicle traffic in astrocytes contributes to the ID.

Brain Plasticity is Associated with Changes in Astrocyte Morphology

The extracellular space in the rodent brain is changing diurnally. During the night, the “glymphatic ” tunnels, associated with astrocytic endfeet, get enlarged, which increases the flux of cerebrospinal fluid, which helps in removing the extracellular debris during sleep (149). Changes in the flux of cerebrospinal fluid are regulated by adrenergic receptors (149), and it is possible that astroglial morphological plasticity is regulated by norepinephrine (134).

This mechanism seems to involve G-protein-coupled receptors (GPCRs). Astroglial β-adrenergic receptors (β-ARs) functionally regulate astrocyte morphology (47). An increase in intracellular cAMP production following β-AR stimulation induces astrocyte stellation in vitro (Fig. 1), that is, transformation from a flattened irregular morphology to a stellate process-bearing morphology (11, 115). The β-ARs are abundantly expressed by astrocytes in both white and gray matter (4, 18, 29, 123, 151), and these receptors are likely to be involved in various forms of pathology.

Figure 1.

Morphological changes in astrocytes (stellation) induced by β-AR activation, which increases cAMP. (A, B) Fluorescing astrocytes transfected with the cAMP nanosensor Epac1-camps (top) and their corresponding differential interference contrast images (bottom) before (left) and within 30 min after (right) the addition of (A) extracellular solution as control (CTRL) and (B) 1 μM β-AR agonist isoprenaline (Iso), which increases intracellular cAMP levels through activation of β-ARs in astrocytes. (B) Rapid change in the cross-sectional area and perimeter of astrocytes, where thinning of processes indicates astrocyte stellation. The perimeter of individual cells expressing Epac1-cAMPs was traced using LSM510 META software, which also outlines the cross-sectional area of the cell. Scale bar: 20 μm. Astrocytes were cultured from rat cortex. Modified from Vardjan, N., et al. (2014) Glia 62(4): 566-579; with permission.

Synaptic transmission may also be directly affected by changes in shape and volume of astroglial processes that tightly enwrap most of the CNS synapses (5, 48, 109). Local retractions or expansions of astrocytic processes modify the geometry of the extracellular space, affecting neuron-glia interactions (126). The apposition of astrocyte membrane to the synaptic cleft is an important determinant for the efficient glutamate removal, which defines the properties of synaptic signals (66). Removal of glutamate from the synaptic cleft consists of diffusion of glutamate in the synaptic cleft and flux into the astrocyte via membrane glutamate transporters; glutamate then diffuses in the cytoplasm to sites where it is metabolized (109). Thus, morphological changes in astrocytes play an important role in pathologic states. Astrocytes may swell and contribute to the development of brain edema, and here vesicles carrying AQP4 may be important (105). Hypertrophic astrocytes are associated with reactive gliosis (145).

At the early stages of AD, reduced adrenergic innervation of the brain due to degeneration of locus coeruleus occurs (44), which may likely result in decreased cAMP signaling and facilitate astrocyte atrophy, as observed in the triple transgenic animal model of AD (82). Such astrocytic atrophy may lead to synaptic loss due to insufficient metabolic support for synapses by astrocytes.

Pathologic Potential of Astroglial Transmitter Vesicles

Astrocytes are endocranial secretory cells, which release multiple signaling molecules synthesized and/or stored in glia (95). Storage of gliotransmitters in membrane-bound vesicles as opposed to having them dissolved in the cytoplasm provides several advantages for cell-to-cell communication (43). In astrocytes, several types of vesicles have been observed; these vesicles reportedly contain glutamate and d-serine (9, 58, 67, 68, 71, 72, 92), ATP (21, 86), and peptides such as ANP (58, 60), BDNF (8), and tissue plasminogen activator (tPA) (17). These vesicles can discharge their content after fusing with the plasma membrane, which leads to the formation of the fusion pore, a channel that provides for the exit of secretions into the extracellular space. Prior to the merger with the plasma membrane, vesicles have to be delivered to the plasmalemma. Therefore, there are many stages at which the complex system of vesicle-based secretion of astrocytes may go wrong in pathological conditions. One of these possibilities is vesicle traffic itself.

Spontaneous mobility of membrane-bound vesicles in astrocytes was reported ~10 years ago (102) and was subsequently confirmed (22). Vesicle mobility is determined by monitoring vesicle movements and recording parameters such as total track length, the path a vesicle travels in a given period of time, the average velocity, the displacement, and the directionality index (ratio between the maximal displacement/total track length). Maximal displacement represents a measure of the maximal net translocation of vesicles (144). Directionality index (DI) values equaling 1 indicate that vesicles move ideally linearly along cytoskeletal elements. If DI is less than 1, this indicates that the coupling between the vesicle and the cytoskeleton is loose; the lower the DI value is, the looser are the interactions with the cytoskeleton. In physiological conditions, two distinct modes of vesicle mobility have been described in astrocytes, similarly to other cell types (13, 32, 102, 130). These modes were described as directional (vesicle tracks being a straight line) and nondirectional (vesicle tracks being a contorted line), and these modes of mobility were able to switch while a vesicle was being observed (102) (Fig. 2A).

Figure 2.

Single-vesicle mobility tracks revealing directional and nondirectional vesicle mobility. (A) Displacement from the origin versus time for a directional vesicle. The first quarter of the vesicle's mobility tracking time from the origin consists of almost constant displacements (a to b) and, during this time, the vesicle remains close to the origin of tracking [see inset (A)]. In the next third of the tracking time (b-d1), vesicle displacements increase rapidly in a preferential direction with a brief pause (c). After a short period with equal displacements (d1-d2), the vesicle seems to move backward, however, apparently not on the same track (d2-e1 and inset). (B) Displacement from the origin for a nondirectional vesicle. Minor mobility was observed, and the vesicle did not translocate far from the origin of tracking (see inset). The mean square displacement (MSD) shown in (C, D) was calculated according to the equation MSD = [d(t) - d(t + Δt)]². (C) The MSD of directional vesicles. The dashed line represents a linear function fitted to the data using an equation with the form MSD (μm²) = (0.4702 ± 0.0099) × time (s). The upwardly curving line represents a quadratic function fitted to the data following the equation MSD (μm²) = (0.2189 ± 0.0148) × time (s) + (0.0221 ± 0.0013) × time² (s²). (D) The MSD of nondirectional vesicles. The linear function was fitted to the data following the equation MSD (μm²) = (0.0038 ± 0.0001) × time (s). Scale bar: 2.5 μm. Astrocytes were cultured from rat cortex. Modified from Potokar, M., et al. (2005) Biochem Biophys Res Commun 329:678-683; with permission.

Motility of Different Types of Astroglial Vesicles

In this section, we will discuss mobility of vesicles containing specific cargo. Mobility of different vesicles is regulated distinctly by stimulation (some of the vesicles accelerate and some slow down), which may in pathological conditions lead to vesicle traffic imbalances and potential traffic deficits. Distinct mobility properties of amino acid- and peptide-loaded vesicles are shown in Figure 3.

Figure 3.

The maximal displacement (MD) plotted versus the track length (TL, abscissa in μm) in vesicles tracked in nonstimulated (black) and ATP-stimulated conditions (gray) in astrocytes isolated from WT (left) and from GFAP^-/-Vim^-/- mice (right): (A) amino acid-loaded vesicles, marked by VGLUT1; (B) peptide-loaded vesicles, marked for atrial natriuretic peptide (ANP). Cells were either left untreated (nonstimulated) or stimulated with 1 mM ATP to increase cytosolic level of free Ca²+. A linear function with the form: [MD = MD₀ + a*(TL)] was fitted to the data. The slope values (a) for VGLUT1 and ANP experiments in WT and in GFAP^-/-Vim^-/-mice are for WT VGLUT1 vesicles in nonstimulated conditions 0.48 ± 0.02 (black) and for stimulated conditions 0.65 ± 0.02 (gray, statistically increased). In GFAP^-/-Vim^-/- astrocytes, the slope was in nonstimulated conditions 0.62 ± 0.02 (black) and 0.68 ± 0.02 in stimulated conditions (gray). For WT ANP vesicles, the slopes were in nonstimulated conditions 0.67 ± 0.01 (black) and for stimulated conditions 0.57 ± 0.01 (gray), statistically reduced (ANOVA). However in GFAP^-/-Vim^-/- astrocytes, the slopes were in nonstimulated conditions 0.61 ± 0.01 (black) and in stimulated conditions 0.61 ± 0.01 (gray), not statistically different. Modified from Potokar, M., et al. (2010) Glia 58(10):1208-19; with permission.

Amino Acid-Loaded Vesicles

In astrocytes, glutamate is accumulated into vesicles by the vesicular glutamate transporters (VGLUTs) VGLUT1, VGLUT2, and VGLUT3 (25, 95). Although the existence of VGLUT1 in mouse astrocytes has been questioned (61), VGLUT1-containing vesicles in rat astrocytes have been described as small and electron lucent, with an estimated diameter of ~30 nm (9) and ~50 nm when they recycle (119), although larger sizes have also been reported (19, 64).

Vesicular mobility is regulated by cytosolic Ca²⁺ concentration ([Ca²⁺]_i). This regulation was directly studied in glutamatergic vesicles labeled by extracellular antibodies, which, after Ca²⁺-dependent exocytosis, are internalized by endocytosis (119). At higher intracellular [Ca²⁺]_i in cells exposed to 4 μM ionomycin or stimulated by 1 mM ATP, vesicle mobility increased significantly. This positive modulation was absent in the cells preloaded with high-affinity Ca²⁺ buffer (BAPTA-AM), indicating that Ca²⁺ is essential for enhanced vesicle mobility. Microtubules, actin, and intermediate filament vimentin all contribute to the mobility of VGLUT1 vesicles because the disruption of actin attenuated their mobility (119); vesicle mobility was suppressed when astrocytes were depleted of intermediate filaments GFAP and vimentin (Fig. 3). Expression of both GFAP and vimentin is upregulated in pathological conditions being a part of the astrogliotic response (99, 139). This, in turn, may affect the function of astrocytes through alterations in vesicle traffic.

ATP- and Peptide-Containing Vesicles

Stimulation of astrocytes induced a reduction in the mobility of peptidergic vesicles and endosomal structures (104, 106, 107), which contain also ATP. ATP is a universal chemical transmitter ubiquitously utilized for purinergic signaling in virtually all tissues, including the CNS (14, 15). In the brain and in the spinal cord, ATP acts through P2X ionotropic and P2Y metabotropic receptors widely expressed in neurons and all types of neuroglia (2, 141). In the CNS, ATP is released from neurons during synaptic transmission; ATP can be stored on its own in specific purinergic vesicles or share vesicles with other neurotransmitters such as glutamate or GABA (87, 88) and peptides (84). In astrocytes, ATP can be released by diffusion through plasmalemmal channels [e.g., connexin hemichannels (118, 122) or volume-sensitive organic osmolyte and anion channels, abbreviated as VSOAC (12)]. The Ca²⁺-dependent exocytotic ATP release from astrocytes has also been described and characterized in detail (95). In microglia, ATP is stored in nonlysosomal compartments (50). In cultured astrocytes, the vesicular presence of ATP has been shown to overlap with the marker of dense-core granules in the hippocampus, the secretogranin II (SgII) (16, 21); a high concentration of ATP was also found in lysosomes (53, 62, 136, 153). In neonatal cortical rat astrocytes, ATP-containing vesicles also store ANP (86). Elevation of [Ca²⁺]_i completely abolished the directional mobility of ATP-containing vesicles. After induction of the cytosolic Ca²⁺ signal, the number of ATP vesicles in astrocytes decreased probably due to Ca²⁺-activated discharge of the fluorescent cargo by regulated exocytosis. This effect was suppressed in the presence of the dominant-negative soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) domain peptide, which interferes with the formation of the SNARE complex (86, 152).

ANP, which is stored in membrane-bound vesicles that also contain ATP (84), is released from astrocytes by Ca²⁺-dependent exocytosis (60). The vesicular mobility in the secretory pathway was monitored using ANP.emd recombinant protein (45), whereas the mobility in the recycling pathway was monitored using an immunolabeling approach with extracellular antibodies against the vesicle cargo in living cells (107). ANP vesicle mobility decreased significantly after depolymerization of micro-tubules, actin filaments, and intermediate filaments (101). These observations support the hypothesis that intermediate filaments are required for long-range directional vesicle mobility by acting as a three-dimensional conduit. The importance of astrocytic intermediate filaments in vesicle mobility under pathologic conditions has also been confirmed in more recent studies (133).

The mobility of ANP vesicles in the recycling pathway of secretory vesicles, which are released by stimulated exocytosis (125), has been monitored in rat astrocytes. During recycling, the vesicles remain almost intact, except for the loss of the content and some membrane proteins. Recycling occurs when the fusion pore is rapidly resealed in the exocytotic process, and the vesicle is retrieved into the cytoplasm without intermixing of membranes and without collapse of the vesicle membrane into the surface membrane (51, 125, 132). Recycling ANP vesicles (those that take up external antibodies) exhibited 10 times slower mobility than secretory ANP vesicles (108). What is the physiologic significance of these results remains to be studied in the future, especially in relation to neuropathology.

A specialized form of neuronal-glial bidirectional communication involves signaling peptides. The BDNF secreted from neurons in its precursor form (pro-BDNF) is accumulated into neighboring astrocytes, which internalize it by formation of a complex with the pan-neurotrophin receptor p75 and subsequent clathrin-dependent endocytosis. Endocytosed pro-BDNF is then routed into a fast recycling pathway for successive SNARE-dependent secretion triggered by glutamate (8). The very similar pathway operates for tPA, which after being released by neurons is constitutively endocytosed by astrocytes through the low-density lipoprotein-related protein receptor and is then exocytosed in a regulated manner. Extracellular glutamate, however, inhibits vesicular recycling of tPA by astrocytes. Astroglial kainate receptors act as sensors of extracellular glutamate and, by employing a protein kinase C signaling pathway, modulate the exocytosis of tPA (17).

Pathophysiology of Endolysosomal Vesicles in Astrocytes

Endolysosomes are a heterogeneous group of vesicles with multiple roles in eukaryotic cells. Apart from being secretory organelles (storing ATP and perhaps peptides, too), they contain degrading enzymes, contribute to antigen presentation and to the uptake of debris and pathogens including viruses (100). The mobility properties of endolysosomes have been described in detail in mouse (104, 133) and rat astrocytes (120). The endolysosomal vesicles are conveniently labeled by LysoTracker dye (Ly) and exhibit relatively slow mobility compared with other vesicle types (108). The direction and speed of Ly vesicles is affected in the absence of astrocytic intermediate filaments. The trafficking of Ly-labeled vesicles is regulated differently from glutamate-containing (VGLUT1-positive) and ATP or peptide-containing (ANP-positive) vesicles under different physiologic conditions. Increase in [Ca²⁺]_i significantly reduced the mobility of Ly-labeled vesicles in normal astrocytes but had no effect in astrocytes in which GFAP and vimentin were genetically deleted (the GFAP^-/-Vim^-/- mice) (34, 98, 147) (Fig. 3).

Pathology can markedly change the regulation of endolysosomal mobility. The mobility of Ly-labeled vesicles was increased in a Ca²⁺-dependent manner following application of purified immunoglobulin G (IgG) antibodies from patients with sporadic amyotrophic lateral sclerosis (ALS). Since endolysosomes typically reduce their mobility upon stimulation (104), these results indicate that acidic compartments may not represent a functionally homogeneous subcellular compartment, since under some pathologic conditions, they may exhibit enhanced mobility upon stimulation (120). The altered mobility is possibly associated with deregulated Ca²⁺ homeostasis caused by ALS IgGs (69). The hallmark of ALS is selective death of motor neurons, although glial cells are also affected. In ALS, astrocytic function is compromised, and that impairs neuronal survival. Astroglial deficits include (i) reduced glutamate uptake due to loss of EAAT2 (112) and (ii) deficient release of neurotrophic factors (33), nerve growth factor (NGF) or extracellular mutant superoxide dismutase 1 known as SOD1 (97, 131). Motor neurons survived less when cocultured on astrocytes expressing the ALS-associated mutant form of Cu-Zn SOD1, when compared with normal astrocytes (28). The application of conditioned medium from mutant SOD1-expressing astrocytes also decreased the survival of motor neurons, suggesting the presence of astrocyte-secreting neurotoxic molecules (74). Alterations in vesicle dynamics may thus reflect changes associated with the progression of the disease and may offer possibilities for the development of new diagnostic tests.

Peptidergic vesicles exhibit similar mobility properties to endolysosomes, which may also change in pathologic conditions. BDNF is the most prevalent growth factor in the CNS, and it is widely implicated in psychiatric diseases, such as major depressive disorder (MDD), schizophrenia, addiction, and Rett syndrome (6). Notably, N-methyl-d-aspartate receptor (NMDAR) antagonists may produce fast-acting behavioral antidepressant effects in patients with depression, and studies in mouse models indicate that these effects depend on the rapid synthesis of BDNF. However, in addition to the regulation of protein synthesis as a therapeutic target in the treatment of MDD, one cannot rule out the possibility that fast-acting antidepressants affect trafficking and/or release of pre-synthesized BDNF from brain cells. Patients with depression report alleviation of MDD symptoms within 2 h of a single, low-dose, intravenous infusion of the antidepressant drug (6, 55, 150).

In neurotrauma, astrocytes undergo reactive remodeling and upregulate functions limiting the extent of tissue damage. In part, this is related to Notch signaling that depends on vesicle traffic of endolysosomes (146).

Cellular Edema and Aquaporin-Transporting Vesicles

Vasogenic edema results mainly from an increased permeability of the BBB (46), whereas cytotoxic edema results from cellular swelling mainly caused by failure of energy metabolism. Astrocytes are the only cells in the CNS that undergo rapid changes in volume (85, 110, 127) caused by ionic deregulation (114). As a result, Na⁺ accumulates within the cells, leading to uptake of water mediated by aquaporin AQP4 water channels to maintain osmotic homeostasis (65, 90, 105, 127). Water transport through the cell membrane is, in part, regulated by the changes in permeability properties of AQP4 (42, 81), by the heterogeneity of AQP4 crystalline-like orthogonal arrays (49), and, as recently suggested, by the mobility of the AQP4 vesicles that are delivered to and from the plasma membrane (105). The AQP4e is one of the newly described AQP4 isoforms (70). In unstimulated conditions, the mobility of AQP4e vesicles resembled the mobility of slow recycling and endosomal vesicles (104, 107, 119).

At the early stages of brain edema, astrocytes swell (77, 89, 110). A reduction in osmolarity triggers an increase in the somatic volume; this has been quantified in situ and in cultured rat astrocytes (85, 124, 127). The increase in cell volume may be accompanied by an increased rate of insertion of exocytotic vesicles in the membrane (96). Hypo-osmotic conditions affected plasma membrane localization of AQP4 in rat astrocytes; in particular, hypo-osmotic stimulation triggered a transient increase in AQP4 plasma membrane localization (105). These changes were related to modified AQP4e vesicle traffic; an increase in AQP4 plasma membrane localization overlapped with the observed decrease in mobility of AQP4e vesicles, whereas the subsequent decrease in AQP4 plasma membrane localization overlapped with increased AQP4e vesicle mobility. The changes in mobility occurred predominantly in vesicles with directional mobility (see Fig. 2). These studies have shown that vesicle dynamics is playing a role in the cell swelling response, possibly by regulating the density of AQP4 channels in the plasma membrane, and this process may represent a new target for pharmacological intervention.

Vesicle Traffic of MHC Proteins may Regulate Neuroinflammation

Intracellular traffic of astrocytic vesicles, including endolysosomes, may also serve to deliver MHC proteins to the plasma membrane (117, 133).

After exposure to the proinflammatory cytokine, interferon-γ (IFN-γ) astrocytes (that are otherwise immunologically silent) start to express MHC-II molecules and antigens on their surface and act as nonprofessional antigen-presenting cells (APCs). It has been suggested that IFN-γ-activated astrocytes participate in antigen presentation and activation of CD4 helper T-cells in immune-mediated disorders of the CNS including multiple sclerosis (36, 117) and experimental autoimmune encephalomyelitis (116).

In general, the delivery of MHC-II molecules from MHC-II compartments to the cell surface of APCs is mediated via a cytoskeletal network and is most likely accomplished by the fusion of MHC-II-carrying late endosomes/lysosomes with the plasma membrane. Actin microfilaments (7), microtubules (143, 148), and their motor proteins (135, 148) all have been shown to mediate trafficking of MHC-II. Recently, the role of intermediate filaments in MHC-II trafficking was investigated in IFN-γ-activated reactive (i.e., overexpressing GFAP) astrocytes (133).

Exposure to IFN-γ induces expression of MHC-II molecules on the astrocytic plasma membrane and late endosomes/lysosomes (133). The latter can be specifically labeled with Alexa Fluor 546-conjugated dextran (39, 52, 133) (Fig. 4A). Time-lapse confocal imaging and dextran labeling of late endolysosomes in normal astrocytes and in GFAP^-/-Vim^-/- astrocytes revealed faster and more directional movement of late endolysosomes in IFN-γ-treated astrocytes than in untreated astrocytes (108). However, vesicle mobility was lower and less directional in IFN-γ-treated GFAP^-/-Vim^-/- astrocytes than in control astrocytes (Fig. 4B, C), indicating that the IFN-γ-induced increase in the mobility of MHC-II-carrying late endolysosomes is intermediate filament dependent. The ATP-induced [Ca²⁺]_i elevation attenuated the mobility of late endolysosomes, which was more apparent in the control cells expressing GFAP and vimentin (Fig. 4B, C).

Figure 4.

The IFN-γ-induced increase in the mobility of MHC-II compartments in astrocytes is intermediate filament (IF) dependent. (A) Alexa Fluor 546-dextran labels MHC-II-positive compartments in IFN-γ-treated WT and GFAP^-/-Vim^-/- (IF deficient) primary mouse astrocytes. Fluorescence images of astrocytes labeled with dextran, fixed, and immunostained with antibodies against MHC-II molecules. White pixels (Mask) represent the colocalization mask of green (MHC-II) and red fluorescence pixels (Dextran). Scale bars: 10 μ. (B) Histogram of average vesicle track lengths in control (Ctrl.) and IFN-γ-treated (+IFN-γ) WT and GFAP^-/-Vim^-/- cells. (C) Histogram of the mean maximal displacements of vesicles in control (Ctrl.) and IFN-γ-treated (+IFN-γ) WT and GFAP^-/-Vim^-/-cells. The numbers on the bars are the numbers of vesicles analyzed. Values are mean ± SEM. *p < 0.05 (ANOVA). Modified from Vardjan, N., et al. (2012) J Neuroinflammation 9:144; with permission.

In conclusion, upregulation of intermediate filaments allows faster and therefore more efficient delivery of MHC-II molecules to the cell surface. Reduced mobility of late endolysosomes at high [Ca²⁺]_i may increase their probability of docking and fusion (104), which, in astrocytes acting as APCs, may serve as an additional regulatory mechanism that controls the onset of late endolysosomal fusion and final delivery of MHC-II molecules to the cell surface (133). Besides IFN-γ, endogenous suppressors, including norepinephrine, have been shown to regulate the expression of MHC-II molecules in astrocytes (27, 37). The effects of norepinephrine on astroglial cells are mediated through activation of β-ARs with subsequent stimulation of the cAMP signaling pathway. Our recent unpublished data suggest that the mobility and fusion of late endolysosomes involved in antigen presentation are also regulated by astrocytic β-ARs. Although these studies were performed in vitro, all these regulatory mechanisms may enable antigen-presenting reactive astrocytes in vivo to respond rapidly and in a controlled manner during CNS inflammation.

New Drugs to Treat Neurologic Diseases Target Vesicle Dynamics in Astrocytes

With advances in understanding the nature of vesicle mobility in astrocytes, in health and in disease, a question has emerged as to whether there are any drugs that affect vesicle traffic and could be used to treat neurologic diseases. Drugs that affect vesicle traffic have many consequences on astrocyte function. These endocranial secreting cells are affected in terms of release by vesicular mechanisms, by alterations in the plasma membrane surface signaling landscape (altered densities of transporters, receptors, and associated signaling mechanisms) and changes in vesicle dynamics associated with morphology of astrocytes. All these factors may contribute to changes in communication between astrocytes and neighboring cells, especially neurons. However, one may consider that attenuation of vesicle dynamics may affect astrocyte functions in physiological and pathophysiological contexts. Vesicle mobility was decreased by fingolimod or FTY720 (128). This drug has been recently introduced for the treatment of multiple sclerosis (20). It was shown that FTY720 accumulates in the white matter in the CNS, where it can reach concentrations that affect astrocytic vesicle mobility and consequently their ability to participate in regulated exocytosis (128). This action may be part of its therapeutic efficacy in patients with multiple sclerosis. The mechanism of reduction of vesicle mobility by fingolimod likely involves fingolimod-induced changes in [Ca²⁺]_i homeostasis, which impair all types of vesicles tested (128). Moreover, astrocytes were considered to be the major source of other molecules, including eicosanoids (prostaglandins, prostacyclins, thromboxanes, and leukotrienes), and these proinflammatory signaling molecules in the CNS are released via an ATP-dependent mechanism (73). In astrocytes, ATP itself is released via regulated exocytosis, which participates in the neuroinflammatory and other pathologic states in the CNS. Thus, new therapeutics, such as FTY720 (128), that affect vesicle mobility represent a novel possibility for the treatment of neurologic diseases.

Future Perspectives

Vesicle traffic in astrocytes changes in neurologic diseases. The vesicle dynamics may also contribute to shape changes in astrocytes. These changes occur when astrocytes swell in brain edema or become hypertrophic or atrophic in disease states. Second messenger cAMP is involved in shape changes in astrocytes and also regulates other processes such as energy provision and inhibition of inflammation in the brain. It will be important to understand how these functions are regulated in view of vesicle dynamics and excitability of astrocytes. Therefore, understanding how excitation-energy coupling relates to vesicle traffic, together with measurements of shape changes will help us to understand the spatiotemporal coupling and interactions between neurons and glia.

Footnotes

Acknowledgments

The authors' work is supported by grants from the Slovenian Research Agency (P3 310, J3 4051, J3 4146, L3 3654, J3 3236), CIPKEBIP, COST Nanonet. The authors declare no conflict of interest.

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Pathologic Potential of Astrocytic Vesicle Traffic: New Targets to Treat Neurologic Diseases?

Abstract

Keywords

The Neurology in Crisis

Astroglia and Secretory Vesicles

Alterations in Vesicle Dynamics Contribute to Neurological Diseases

Brain Plasticity is Associated with Changes in Astrocyte Morphology

Pathologic Potential of Astroglial Transmitter Vesicles

Motility of Different Types of Astroglial Vesicles

Amino Acid-Loaded Vesicles

ATP- and Peptide-Containing Vesicles

Pathophysiology of Endolysosomal Vesicles in Astrocytes

Cellular Edema and Aquaporin-Transporting Vesicles

Vesicle Traffic of MHC Proteins may Regulate Neuroinflammation

New Drugs to Treat Neurologic Diseases Target Vesicle Dynamics in Astrocytes

Future Perspectives

Footnotes

Acknowledgments

References