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Abstract Background The expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes. Methods In human GBM, the expression of glial fibrillary acidic protein (GFAP), AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively. Results A positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin’s abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines. Conclusions Plectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs.

Abstract Astroglia are a broad class of neural parenchymal cells primarily dedicated to homoeostasis and defence of the central nervous system (CNS). Astroglia contribute to the pathophysiology of all neurological and neuropsychiatric disorders in ways that can be either beneficial or detrimental to disorder outcome. Pathophysiological changes in astroglia can be primary or secondary and can result in gain or loss of functions. Astroglia respond to external, non-cell autonomous signals associated with any form of CNS pathology by undergoing complex and variable changes in their structure, molecular expression, and function. In addition, internally driven, cell autonomous changes of astroglial innate properties can lead to CNS pathologies. Astroglial pathophysiology is complex, with different pathophysiological cell states and cell phenotypes that are context-specific and vary with disorder, disorder-stage, comorbidities, age, and sex. Here, we classify astroglial pathophysiology into (i) reactive astrogliosis, (ii) astroglial atrophy with loss of function, (iii) astroglial degeneration and death, and (iv) astrocytopathies characterised by aberrant forms that drive disease. We review astroglial pathophysiology across the spectrum of human CNS diseases and disorders, including neurotrauma, stroke, neuroinfection, autoimmune attack and epilepsy, as well as neurodevelopmental, neurodegenerative, metabolic and neuropsychiatric disorders. Characterising cellular and molecular mechanisms of astroglial pathophysiology represents a new frontier to identify novel therapeutic strategies.

Ageing is a key factor in the development of cognitive decline and dementia, an increasing and challenging problem of the modern world. The most commonly diagnosed cognitive decline is related to Alzheimer's disease (AD), the pathophysiology of which is poorly understood. Several hypotheses have been proposed. The cholinergic hypothesis is the oldest, however, recently the noradrenergic system has been considered to have a role as well. The aim of this review is to provide evidence that supports the view that an impaired noradrenergic system is causally linked to AD. Although dementia is associated with neurodegeneration and loss of neurons, this likely develops due to a primary failure of homeostatic cells, astrocytes, abundant and heterogeneous neuroglial cells in the central nervous system (CNS). The many functions that astrocytes provide to maintain the viability of neural networks include the control of ionic balance, neurotransmitter turnover, synaptic connectivity and energy balance. This latter function is regulated by noradrenaline, released from the axon varicosities of neurons arising from the locus coeruleus (LC), the primary site of noradrenaline release in the CNS. The demise of the LC is linked to AD, whereby a hypometabolic CNS state is observed clinically. This is likely due to impaired release of noradrenaline in the AD brain during states of arousal, attention and awareness. These functions controlled by the LC are needed for learning and memory formation and require activation of the energy metabolism. In this review, we address first the process of neurodegeneration and cognitive decline, highlighting the function of astrocytes. Cholinergic and/or noradrenergic deficits lead to impaired astroglial function. Then, we focus on adrenergic control of astroglial aerobic glycolysis and lipid droplet metabolism, which play a protective role but also promote neurodegeneration under some circumstances, supporting the noradrenergic hypothesis of cognitive decline. We conclude that targeting astroglial metabolism, glycolysis and/or mitochondrial processes may lead to important new developments in the future when searching for medicines to prevent or even halt cognitive decline.

Abstract In 2009, new EU legislation regulating advanced therapy medicinal products (ATMPs), consisting of gene therapy, tissue engineering and cell-based medicines, was introduced. Although less than 20 ATMPs were authorized since that time, the awarding of the Nobel Prize for Physiology or Medicine in 2018 revived interest in developing new cancer immunotherapies involving significant manipulation of the patient's own immune cells, including lymphocytes and dendritic cells. The lymphocytes are mainly thought to directly affect tumour cells, dendritic cells are involved in indirect mechanisms by antigen presentation to other leukocytes orchestrating the immune response. It is the latter cells that are the focus of this brief review. Based on the recent results of our study treating patients with castration-resistant prostate cancer (CRPC) with an immunohybridoma cell construct (termed aHyC), produced by electrofusion of autologous tumour and dendritic cells, we compare their effectiveness with a matched documented control group of patients. The results revealed that cancer-specific survival and the time to next in-line therapy (TTNT) were both significantly prolonged versus controls. When patients were observed for longer periods since the time of diagnosis of CRPC, 20% of patients had not yet progressed to the next in-line therapy even though the time under observation was ~ 80 months. Interestingly, analysis of survival of patients revealed that the effectiveness of treatment was independent of the number of cells in the vaccine used for treatment. It is concluded that autologous dendritic cell-based immunotherapy is a new possibility to treat not only CRPC but also other solid tumours.

Astrocytes are increasingly recognized as important viral host cells in the central nervous system. These cells can produce relatively high quantities of new virions. In part, this can be attributed to the characteristics of astrocyte metabolism and its abundant and dynamic cytoskeleton network. Astrocytes are anatomically localized adjacent to interfaces between blood capillaries and brain parenchyma and between blood capillaries and brain ventricles. Moreover, astrocytes exhibit a larger membrane interface with the extracellular space than neurons. These properties, together with the expression of various and numerous viral entry receptors, a relatively high rate of endocytosis, and morphological plasticity of intracellular organelles, render astrocytes important target cells in neurotropic infections. In this review, we describe factors that mediate the high susceptibility of astrocytes to viral infection and replication, including the anatomic localization of astrocytes, morphology, expression of viral entry receptors, and various forms of autophagy.

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second most common cause of death due to cancer. About 30% of patients with PCa who have been castrated develop a castration-resistant form of the disease (CRPC), which is incurable. In the last decade, new treatments that control the disease have emerged, slowing progression and spread and prolonging survival while maintaining the quality of life. These include immunotherapies; however, we do not yet know the optimal combination and sequence of these therapies with the standard ones. All therapies are not always suitable for every patient due to co-morbidities or adverse effects of therapies or both, so there is an urgent need for further work on new therapeutic options. Advances in cancer immunotherapy with an immune checkpoint inhibition mechanism (e.g., ipilimumab, an anti-CTLA-4 inhibitor) have not shown a survival benefit in patients with CRPC. Other immunological approaches have also not given clear results, which has indirectly prevented breakthrough for this type of therapeutic strategy into clinical use. Currently, the only approved form of immunotherapy for patients with CRPC is a cell-based medicine, but it is only available to patients in some parts of the world. Based on what was gained from recently completed clinical research on immunotherapy with dendritic cell-based immunohybridomas, the aHyC dendritic cell vaccine for patients with CRPC, we highlight the current status and possible alternatives that should be considered in the future.

Abstract 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.

A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K+ concentration ([K+]o) alters neuronal excitability, thus contributing to depression. We examined how ketamine affects inwardly rectifying K+ channel Kir4.1, the principal regulator of K+ buffering and neuronal excitability in the brain. Cultured rat cortical astrocytes were transfected with plasmid-encoding fluorescently tagged Kir4.1 (Kir4.1-EGFP) to monitor the mobility of Kir4.1-EGFP vesicles at rest and after ketamine treatment (2.5 or 25 µM). Short-term (30 min) ketamine treatment reduced the mobility of Kir4.1-EGFP vesicles compared with the vehicle-treated controls (p < 0.05). Astrocyte treatment (24 h) with dbcAMP (dibutyryl cyclic adenosine 5′-monophosphate, 1 mM) or [K+]o (15 mM), which increases intracellular cAMP, mimicked the ketamine-evoked reduction of mobility. Live cell immunolabelling and patch-clamp measurements in cultured mouse astrocytes revealed that short-term ketamine treatment reduced the surface density of Kir4.1 and inhibited voltage-activated currents similar to Ba2+ (300 µM), a Kir4.1 blocker. Thus, ketamine attenuates Kir4.1 vesicle mobility, likely via a cAMP-dependent mechanism, reduces Kir4.1 surface density, and inhibits voltage-activated currents similar to Ba2+, known to block Kir4.1 channels.

Exocytosis is a universal process of eukaryotic cells, consisting of fusion between the vesicle and the plasma membranes, leading to the formation of a fusion pore, a channel through which vesicle cargo exits into the extracellular space. In 1986, Rand and Parsegian proposed several stages to explain the nature of membrane fusion. Following stimulation, it starts with focused stress destabilization of membranes in contact, followed by the coalescence of two membrane surfaces. In the next fraction of a millisecond, restabilization of fused membranes is considered to occur to maintain the cell’s integrity. This view predicted that once a fusion pore is formed, it must widen abruptly, irreversibly and fully, whereby the vesicle membrane completely integrates with and collapses into the plasma membrane (full fusion exocytosis). However, recent experimental evidence has revealed that once the fusion pore opens, it may also reversibly close (transient or kiss-and-run exocytosis). Here, we present a historical perspective on understanding the mechanisms that initiate the membrane merger and fusion pore formation. Next, post-fusion mechanisms that regulate fusion pore stability are considered, reflecting the state in which the forces of widening and constriction of fusion pores are balanced. Although the mechanisms generating these forces are unclear, they may involve lipids and proteins, including SNAREs, which play a role not only in the pre-fusion but also post-fusion stages of exocytosis. How molecules stabilize the fusion pore in the open state is key for a better understanding of fusion pore physiology in health and disease.

Glycogen synthase kinase 3 (GSK-3) plays an important role in metabolic regulation in skeletal muscles, and both insulin and adrenaline stimulate GSK-3 phosphorylation. The aim of the present study was to study the effect of insulin and adrenaline on GSK-3 localisation in skeletal muscles. We characterized subcellular localization of (GSK-3) signal protein in fully differentiated muscle fibre by immunofluorescence and confocal microscopy. We stimulated muscle fibres with insulin and/or adrenaline. Images were analysed by segmentation of single central optical section of the muscle. We found GSK-3 to be localised in clusters. The number of GSK-3 clusters and their average size were increased after stimulation with insulin and/or adrenaline. Average GSK-3 particle size is linearly related to their quantity. We conclude that subcellular GSK-3 in isolated skeletal muscle fibres is localized in clusters and clustering increased after stimulation with insulin and/or adrenaline.

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.

Abstract 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.

At the end of 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was discovered in China, causing a new coronavirus disease, termed COVID-19 by the WHO on February 11, 2020. At the time of this paper (January 31, 2021), more than 100 million cases have been recorded, which have claimed over 2 million lives worldwide. The most important clinical presentation of COVID-19 is severe pneumonia; however, many patients present various neurological symptoms, ranging from loss of olfaction, nausea, dizziness, and headache to encephalopathy and stroke, with a high prevalence of inflammatory central nervous system (CNS) syndromes. SARS-CoV-2 may also target the respiratory center in the brainstem and cause silent hypoxemia. However, the neurotropic mechanism(s) by which SARS-CoV-2 affects the CNS remain(s) unclear. In this paper, we first address the involvement of astrocytes in COVID-19 and then elucidate the present knowledge on SARS-CoV-2 as a neurotropic virus as well as several other neurotropic flaviviruses (with a particular emphasis on the West Nile virus, tick-borne encephalitis virus, and Zika virus) to highlight the neurotropic mechanisms that target astroglial cells in the CNS. These key homeostasis-providing cells in the CNS exhibit many functions that act as a favorable milieu for virus replication and possibly a favorable environment for SARS-CoV-2 as well. The role of astrocytes in COVID-19 pathology, related to aging and neurodegenerative disorders, and environmental factors, is discussed. Understanding these mechanisms is key to better understanding the pathophysiology of COVID-19 and for developing new strategies to mitigate the neurotropic manifestations of COVID-19.

G-protein-coupled receptors (GPCRs) represent a family with over 800 members in humans, and one-third of these are targets for approved drugs. A large number of GPCRs have unknown physiologic roles. Here, we investigated GPR27, an orphan GPCR belonging to the family of super conserved receptor expressed in the brain, with unknown functions. Cytosolic levels of L-lactate ([lactate]i), the end product of aerobic glycolysis, were measured with the Laconic fluorescence resonance energy transfer nanosensor. In single 3T3 wild-type (WT) embryonic cells, the application of 8535 (1 µM), a surrogate agonist known to activate GPR27, resulted in an increase in [lactate]i. Similarly, an increase was recorded in primary rat astrocytes, a type of neuroglial cell abundant in the brain, which contain glycogen and express enzymes of aerobic glycolysis. In CRISPR-Cas9 GPR27 knocked out 3T3 cells, the 8535-induced increase in [lactate]i was reduced compared with WT controls. Transfection of the GPR27-carrying plasmid into the 3T3KOGPR27 cells rescued the 8535-induced increase in [lactate]i. These results indicate that stimulation of GPR27 enhances aerobic glycolysis and L-lactate production in 3T3 cells and astrocytes. Interestingly, in the absence of GPR27 in 3T3 cells, resting [lactate]i was increased in comparison with controls, further supporting the view that GPR27 regulates L-lactate homeostasis.

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.

Abstract Neurotransmission and secretion of hormones involve a sequence of protein/lipid interactions with lipid turnover impacting on vesicle trafficking and ultimately fusion of secretory vesicles with the plasma membrane. We previously demonstrated that sphingosine, a sphingolipid metabolite, promotes formation of the SNARE complex required for membrane fusion and also increases the rate of exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and in hippocampal neurons. Recently a fungi-derived sphingosine homologue, FTY720, has been approved for treatment of multiple sclerosis. In its non-phosphorylated form FTY720 accumulates in the central nervous system, reaching high levels which could affect neuronal function. Considering close structural similarity of sphingosine and FTY720 we investigated whether FTY720 has an effect on regulated exocytosis. Our data demonstrate that FTY720 can activate vesicular synaptobrevin for SNARE complex formation and enhance exocytosis in neuroendocrine cells and neurons.

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.

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−brain and blood−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.

In the past, manipulation of the cholinergic system was seen as the most likely therapeutic for neurodegeneration-based cognitive decline in Alzheimer's disease (AD) (Whitehouse et al., 1982). However, targeting the noradrenergic system also seems a promising strategy, since more recent studies revealed that in post-mortem tissue from patients with AD and other neurodegenerative disorders there is a robust correlation between cognitive decline and loss of neurons from the Locus coeruleus (LC), a system with diffuse noradrenaline (NA) innervation in the central nervous system (CNS). Therefore, the hypothesis has been considered that increasing NA signaling in the CNS will prevent, or at least halt the progression of neurodegeneration and cognitive decline. A hallmark of the age- and neurodegeneration-related cognitive decline is reduced neurogenesis. We here discuss noradrenergic dysfunction in AD-related cognitive decline in humans and its potential involvement in AD pathology and disease progression. We also focus on animal models to allow the validation of the noradrenergic hypothesis of AD, including those based upon the immunotoxin-mediated ablation of LC based on saporin, a protein synthesis interfering agent, which offers selective and graded demise of LC neurons, Finally, we address how astrocytes, an abundant and functionally heterogeneous cell type of neuroglia maintaining homeostasis, may participate in the regulation of neurogenesis, a new strategy for preventing LC neuron loss.

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.

Dipeptidyl peptidase 4 (DPP4), a transmembrane protein, has been identified in human adipose tissue and is considered to be associated with obesity-related type 2 diabetes. Since adipose tissue is relatively hypoxic in obese participants, we investigated the expression of DPP4 in human preadipocytes (hPA) and adipocytes in hypoxia, during differentiation and upon insulin stimulation. The results show that DPP4 is abundantly expressed in hPA but very sparsely in adipocytes. During differentiation in vitro, the expression of DPP4 in hPA is reduced on the addition of differentiation medium, indicating that this protein can be hPA marker. Long term hypoxia altered the expression of DPP4 in hPA. In in vitro hypoxic conditions the protease activity of shed DPP4 is reduced; however, in the presence of insulin, the increase in DPP4 expression is potentiated by hypoxia.

Using electron microscopy, we studied the morphology of secretory granules in rat pars intermedia cells. We found figures of apparent intergranule fusion, characterized by a tight association of two granules. The fusion was detected in around 2% of all measured granules, indicating a low occurrence of intergranule fusion. To study whether intergranule fusion affects the distribution of granule diameters a simple probabilistic model was developed. It is based on the theory that larger granules are formed by fusion of two or more spherical granules of fixed size, and that the surface of a newly formed granule is equal to the sum of fused granule membranes. The model accounts for the bias on granule diameter measurements due to sectioning of granules. Although the electron microscopy data strongly indicates the existence of intergranule fusion in rat melanotrophs, this process as modelled in the present work does not contribute to the granule diameter distribution significantly. It is likely that in addition to the fusion of larger granules, other processes, such as fusion of microvesicles, may affect the distribution of granule diameters.

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.

Tick-borne encephalitis virus (TBEV) causes one of the most dangerous human neuroinfections in Europe and Asia. To infect neurons it must cross the blood-brain-barrier (BBB), and presumably also cells adjacent to the BBB, such as astrocytes, the most abundant glial cell type. However, the knowledge about the viral infection of glial cells is fragmental. Here we studied whether TBEV infects rat astrocytes. Rats belong to an animal group serving as a TBEV amplifying host. We employed high resolution quantitative fluorescence microscopy to investigate cell entry and cytoplasmic mobility of TBEV particles along with the effect on the cell cytoskeleton and cell survival. We report that infection of astrocytes with TBEV increases with time of exposure to TBEV and that with post-infection time TBEV particles gained higher mobility. After several days of infection actin cytoskeleton was affected, but cell survival was unchanged, indicating that rat astrocytes resist TBEV-mediated cell death, as reported for other mammalian cells. Therefore, astrocytes may present an important pool of dormant TBEV infections and a new target for therapeutic intervention.

Astroglial cells, due to their passive electrical properties, were long considered subservient to neurons and to merely provide the framework and metabolic support of the brain. Although astrocytes do play such structural and housekeeping roles in the brain, these glial cells also contribute to the brain's computational power and behavioural output. These more active functions are endowed by the Ca 2+ -based excitability displayed by astrocytes. An increase in cytosolic Ca 2+ levels in astrocytes can lead to the release of signalling molecules, a process termed gliotransmission, via the process of regulated exocytosis. Dynamic components of astrocytic exocytosis include the vesicular-plasma membrane secretory machinery, as well as the vesicular traffic, which is governed not only by general cytoskeletal elements but also by astrocyte-specific IFs (intermediate filaments). Gliotransmitters released into the ECS (extracellular space) can exert their actions on neighbouring neurons, to modulate synaptic transmission and plasticity, and to affect behaviour by modulating the sleep homoeostat. Besides these novel physiological roles, astrocytic Ca 2+ dynamics, Ca 2+ -dependent gliotransmission and astrocyte–neuron signalling have been also implicated in brain disorders, such as epilepsy. The aim of this review is to highlight the newer findings concerning Ca 2+ signalling in astrocytes and exocytotic gliotransmission. For this we report on Ca 2+ sources and sinks that are necessary and sufficient for regulating the exocytotic release of gliotransmitters and discuss secretory machinery, secretory vesicles and vesicle mobility regulation. Finally, we consider the exocytotic gliotransmission in the modulation of synaptic transmission and plasticity, as well as the astrocytic contribution to sleep behaviour and epilepsy.

The fusion pore is an aqueous channel that is formed upon the fusion of the vesicle membrane with the plasma membrane. Once the pore is open, it may close again (transient fusion) or widen completely (full fusion) to permit vesicle cargo discharge. While repetitive transient fusion pore openings of the vesicle with the plasma membrane have been observed in the absence of stimulation, their frequency can be further increased using a cAMP-increasing agent that drives the opening of nonspecific cation channels. Our model hypothesis is that the openings and closings of the fusion pore are driven by changes in the local concentration of cations in the connected vesicle. The proposed mechanism of fusion pore dynamics is considered as follows: when the fusion pore is closed or is extremely narrow, the accumulation of cations in the vesicle (increased cation concentration) likely leads to lipid demixing at the fusion pore. This process may affect local membrane anisotropy, which reduces the spontaneous curvature and thus leads to the opening of the fusion pore. Based on the theory of membrane elasticity, we used a continuum model to explain the rhythmic opening and closing of the fusion pore.

Vesicles are cellular organelles, in which signaling molecules (neurotransmitters or hormones) are stored and are essential for the function of neurons and endocrine cells in supporting the communication between tissues and organs. Upon stimulation the signaling molecules stored inside vesicles are released from cells by exocytosis. This fundamental biological process consists of membrane fusion between the vesicles and the plasma membrane, leading to the formation of an aqueous channel – the fusion pore – through which signaling molecules exit into the extracellular space or blood stream. The vesicle cargo discharge initially requires the delivery of vesicles to the plasma membrane, where vesicles dock and get primed for fusion with the plasma membrane. Classical view holds that stimulation initiates the fusion pore formation and vesicle cargo discharge in an all-or-none fashion. Once formed the fusion pore may close (transient, “kiss-and-run” exocytosis) or expand, leading to the full collapse of the vesicle membrane into the plasma membrane (full fusion exocytosis). However, recent studies indicate that exocytosis may not be as simple. Here we highlight the novel findings which indicate that transient fusion pore is subject to regulations, which affect the release competence of a single vesicle. Our recent studies have shown that in pituitary lactotrophs vesicle release of peptide signaling molecules involves modulation of fusion pore kinetics and fusion pore conductance.