71 results on '"Vardjan N"'
Search Results
2. The fabrics of astrocyte vesicle traffic in health and disease: W07–03
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Vardjan, N., Potokar, M., Jorgačevski, J., Singh, P., Trkov, S., Gabrijel, M., Stenovec, M., Jeras, M., Pekny, M., Kreft, M., and Zorec, R.
- Published
- 2013
3. Distinct labelling of fusion events in rat lactotrophs by FM 1–43 and FM 4–64 is associated with conformational differences
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Stenovec, M., Šolmajer, T., Perdih, A., Vardjan, N., Kreft, M., and Zorec, R.
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- 2007
4. Fusion Pore: An Evolutionary Invention of Nucleated Cells
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Vardjan, N., primary, Stenovec, M., additional, Jorgačevski, J., additional, Kreft, M., additional, and Zorec, R., additional
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- 2010
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5. Distinct labelling of fusion events in rat lactotrophs by FM 1?43 and FM 4?64 is associated with conformational differences
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Stenovec, M., primary, Šolmajer, T., additional, Perdih, A., additional, Vardjan, N., additional, Kreft, M., additional, and Zorec, R., additional
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- 2007
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6. IFN-γ-induced increase in the mobility of MHC class II compartments in astrocytes depends on intermediate filaments
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Vardjan Nina, Gabrijel Mateja, Potokar Maja, Švajger Urban, Kreft Marko, Jeras Matjaž, de Pablo Yolanda, Faiz Maryam, Pekny Milos, and Zorec Robert
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Astrocytes ,Vesicle mobility ,Late endosomes/lysosomes ,Major histocompatibility class II compartments ,Interferon-γ ,Dextran labeling ,Immune response ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Abstract Background In immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-γ (IFN-γ) can express major histocompatibility complex (MHC) class II molecules and antigens on their surface. MHC class II molecules are thought to be delivered to the cell surface by membrane-bound vesicles. However, the characteristics and dynamics of this vesicular traffic are unclear, particularly in reactive astrocytes, which overexpress intermediate filament (IF) proteins that may affect trafficking. The aim of this study was to determine the mobility of MHC class II vesicles in wild-type (WT) astrocytes and in astrocytes devoid of IFs. Methods The identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN-γ activation was determined immunocytochemically by using confocal microscopy. Time-lapse confocal imaging and Alexa Fluor546-dextran labeling of late endosomes/lysosomes in IFN-γ treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using ParticleTR software. Results Confocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-γ induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor546-conjugated dextran. Live imaging revealed faster movement of dextran-positive vesicles in IFN-γ-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-γ-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-γ-induced increase in the mobility of MHC class II compartments is IF-dependent. Conclusions Since reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the up-regulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface. In vivo, such regulatory mechanisms may enable antigen-presenting reactive astrocytes to respond rapidly and in a controlled manner to CNS inflammation.
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- 2012
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7. Inhibiting glycolysis rescues memory impairment in an intellectual disability Gdi1-null mouse
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Maja Malnar, Angela Bachi, Helena H. Chowdhury, Anemari Horvat, Patrizia D’Adamo, Antonia Gurgone, Saša Trkov Bobnar, Marko Muhič, Lorenzo Piemonti, Maria Lidia Mignogna, Michela Masetti, Jelena Velebit, Veronica Bianchi, Robert Zorec, Matjaž Stenovec, Alessia Mercalli, Katja Fink, Sara Belloli, Stefano Taverna, Maja Potokar, Marko Kreft, Rosa Maria Moresco, Nina Vardjan, Maddalena Ripamonti, Umberto Restuccia, D'Adamo, Patrizia, Horvat, Anemari, Gurgone, Antonia, Mignogna, Maria Lidia, Bianchi, Veronica, Masetti, Michela, Ripamonti, Maddalena, Taverna, Stefano, Velebit, Jelena, Malnar, Maja, Muhič, Marko, Fink, Katja, Bachi, Angela, Restuccia, Umberto, Belloli, Sara, Moresco, Rosa Maria, Mercalli, Alessia, Piemonti, Lorenzo, Potokar, Maja, Bobnar, Saša Trkov, Kreft, Marko, Chowdhury, Helena H, Stenovec, Matjaž, Vardjan, Nina, Zorec, R, D'Adamo, P, Horvat, A, Gurgone, A, Mignogna, M, Bianchi, V, Masetti, M, Ripamonti, M, Taverna, S, Velebit, J, Malnar, M, Muhic, M, Fink, K, Bachi, A, Restuccia, U, Belloli, S, Moresco, R, Mercalli, A, Piemonti, L, Potokar, M, Bobnar, S, Kreft, M, Chowdhury, H, Stenovec, M, and Vardjan, N
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0301 basic medicine ,CTX, context memory ,Male ,Endocrinology, Diabetes and Metabolism ,Glucose uptake ,Intellectual disability ,FRET, Förster Resonance Energy Transfer ,SV, synaptic vesicle ,XLID, X-linked intellectual disability ,Mice ,0302 clinical medicine ,Endocrinology ,Basic Science ,GDI1 knockout mice ,Aerobic glycolysis ,Astrocytes ,cAMP ,Glycolysis ,Gdi1 KO, full knockout of Gdi1 ,Cells, Cultured ,Guanine Nucleotide Dissociation Inhibitors ,NA, noradrenaline ,Mice, Knockout ,Cultured ,3-Cl-5-OH-BA, 3-chloro-5-hydroxybenzoic acid ,Animals ,Brain ,Deoxyglucose ,Down-Regulation ,Glucose ,Intellectual Disability ,Maze Learning ,Memory ,Memory Disorders ,[18F]-FDG, [18F]-fluoro-2-deoxy-d-glucose ,Aerobic glycolysi ,cAMP, cyclic adenosine monophosphate ,GlastGdi1flox/Y, GLAST:CreERT2+/Gdi1lox/Y inducible astrocyte-specific Gdi1 KO male mice ,medicine.anatomical_structure ,intellectual disability ,Knockout mouse ,Astrocyte ,Gdi1 WT, wild type ,medicine.medical_specialty ,Cells ,Knockout ,030209 endocrinology & metabolism ,Biology ,2-DG, 2-deoxy-d-glucose ,sEPSCs, spontaneous excitatory postsynaptic currents ,CNS, central nervous system ,SEM, standard error of the mean ,03 medical and health sciences ,αGDI, α guanosine dissociation inhibitor protein coded by GDI1 gene ,CFP, cyan fluorescent protein ,Downregulation and upregulation ,Internal medicine ,medicine ,aerobic glycolysis ,GlastGdi1X/Y, male mice (Gdi1X/Y) carrying the GLAST:CreERT2 transgene ,GLUT1, d-glucose transporter ,Wild type ,astrocytes ,GFAP, glial fibrillary acidic protein ,PSD, postsynaptic density ,GDI1, guanosine dissociation inhibitor 1 gene ,YFP, yellow fluorescent protein ,030104 developmental biology ,GPCR, G-protein coupled receptor ,Anaerobic glycolysis ,GPR81, G-protein receptor 81 ,CS, conditional stimulus, tone ,PKA, protein kinase A ,MCTs, monocarboxylate transporters ,Homeostasis - Abstract
Objectives GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. Methods We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. Results Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. Conclusions These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice., Highlights • Mutations in human Gdi1, encoding αGDI, a protein controlling vesicle traffic, are responsible for Intellectual Disability. • Gdi1 knockout revealed significant changes in astrocyte-resident glycolytic enzymes and facilitated D-glucose utilization. • Astrocyte-selective Gdi1 deletion impairs working memory, which can be rescued by administration of 2-deoxy-D-glucose. • Astrocyte-based glycolysis is a new target to treat Intellectual Disability.
- Published
- 2021
8. Hippocampal Noradrenaline Regulates Spatial Working Memory in the Rat
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Rosario Gulino, Domenico Nunziata, Anna Kostenko, Giampiero Leanza, Serena Alexa Emmi, Gioacchino de Leo, Vardjan, N and Zorec, R, Gulino, Rosario, Kostenko, Anna, de Leo, Gioacchino, Emmi, Serena Alexa, Nunziata, Domenico, and Leanza, Giampiero
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Alzheimer’s disease ,noradrenaline ,locus coeruleus ,immunolesion ,transplantation ,water maze ,working memory ,rat ,Working memory ,Water maze ,Hippocampal formation ,Spatial memory ,Lesion ,Transplantation ,medicine ,Locus coeruleus ,medicine.symptom ,Cognitive decline ,Psychology ,locus coeruleu ,Neuroscience - Abstract
Degeneration of noradrenergic neurons in the locus coeruleus and loss of fiber terminals in the neocortical and hippocampal target regions represent prominent and early features of Alzheimer’s disease, however, whether these events are causally linked to cognitive decline in Alzheimer’s disease is still unclear. In the present study, the noradrenergic contribution to the regulation of hippocampus-dependent spatial learning and memory was investigated. Postnatal day 4 rats underwent selective immunolesioning of hippocampal noradrenergic afferents and, 4 days later, the bilateral intrahippocampal implantation of locus coeruleus noradrenergic neuroblasts. Starting from 4 weeks and up to about 9 months postsurgery, sensory-motor and spatial navigation abilities were evaluated, followed by postmortem tissue analyses. All animals in the Control, Lesion, and Lesion+Transplant groups exhibited normal sensory-motor function and were equally efficient in the reference memory version of the water maze task, whereas working memory abilities were seen consistently impaired in the Lesion-only rats. Notably, the noradrenergic reinnervation promoted by the grafted progenitors reinstated a fairly normal working memory performance, suggesting a primary role for coeruleo-hippocampal noradrenergic inputs in the maintenance of specific aspects of cognition.
- Published
- 2017
9. ERNEST COST action overview on the (patho)physiology of GPCRs and orphan GPCRs in the nervous system.
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Birgül Iyison N, Abboud C, Abboud D, Abdulrahman AO, Bondar AN, Dam J, Georgoussi Z, Giraldo J, Horvat A, Karoussiotis C, Paz-Castro A, Scarpa M, Schihada H, Scholz N, Güvenc Tuna B, and Vardjan N
- Abstract
G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. This overview emphasises the GPCRs of the nervous system, which are the research focus of the members of ERNEST COST action (CA18133) working group 'Biological roles of signal transduction'. First, the (patho)physiological role of the nervous system GPCRs in the modulation of synapse function is discussed. We then debate the (patho)physiology and pharmacology of opioid, acetylcholine, chemokine, melatonin and adhesion GPCRs in the nervous system. Finally, we address the orphan GPCRs, their implication in the nervous system function and disease, and the challenges that need to be addressed to deorphanize them., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)
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- 2024
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10. Brain energy metabolism: A roadmap for future research.
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Rae CD, Baur JA, Borges K, Dienel G, Díaz-García CM, Douglass SR, Drew K, Duarte JMN, Duran J, Kann O, Kristian T, Lee-Liu D, Lindquist BE, McNay EC, Robinson MB, Rothman DL, Rowlands BD, Ryan TA, Scafidi J, Scafidi S, Shuttleworth CW, Swanson RA, Uruk G, Vardjan N, Zorec R, and McKenna MC
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- Animals, Humans, Brain metabolism, Energy Metabolism
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Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD
+ , as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research., (© 2024 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)- Published
- 2024
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11. Adrenergic regulation of astroglial aerobic glycolysis and lipid metabolism: Towards a noradrenergic hypothesis of neurodegeneration.
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Zorec R and Vardjan N
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- Humans, Astrocytes metabolism, Lipid Metabolism, Norepinephrine metabolism, Locus Coeruleus metabolism, Glycolysis physiology, Adrenergic Agents, Alzheimer Disease metabolism
- Abstract
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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest in relation to this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2023
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12. The Activation of GPR27 Increases Cytosolic L-Lactate in 3T3 Embryonic Cells and Astrocytes.
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Dolanc D, Zorec TM, Smole Z, Maver A, Horvat A, Pillaiyar T, Trkov Bobnar S, Vardjan N, Kreft M, Chowdhury HH, and Zorec R
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- 3T3 Cells, Animals, Glycogen metabolism, Mice, Rats, Receptors, G-Protein-Coupled metabolism, Astrocytes metabolism, Lactic Acid metabolism
- Abstract
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.- Published
- 2022
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13. Pathophysiology of Lipid Droplets in Neuroglia.
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Smolič T, Zorec R, and Vardjan N
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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.
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- 2021
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14. Astrocyte arborization enhances Ca 2+ but not cAMP signaling plasticity.
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Pirnat S, Božić M, Dolanc D, Horvat A, Tavčar P, Vardjan N, Verkhratsky A, Zorec R, and Stenovec M
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- Calcium Signaling physiology, Cells, Cultured, Astrocytes metabolism, Signal Transduction
- Abstract
The plasticity of astrocytes is fundamental for their principal function, maintaining homeostasis of the central nervous system throughout life, and is associated with diverse exposomal challenges. Here, we used cultured astrocytes to investigate at subcellular level basic cell processes under controlled environmental conditions. We compared astroglial functional and signaling plasticity in standard serum-containing growth medium, a condition mimicking pathologic conditions, and in medium without serum, favoring the acquisition of arborized morphology. Using opto-/electrophysiologic techniques, we examined cell viability, expression of astroglial markers, vesicle dynamics, and cytosolic Ca
2+ and cAMP signaling. The results revealed altered vesicle dynamics in arborized astrocytes that was associated with increased resting [Ca2+ ]i and increased subcellular heterogeneity in [Ca2+ ]i , whereas [cAMP]i subcellular dynamics remained stable in both cultures, indicating that cAMP signaling is less prone to plastic remodeling than Ca2+ signaling, possibly also in in vivo contexts., (© 2021 The Authors. GLIA published by Wiley Periodicals LLC.)- Published
- 2021
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15. Lactate as an Astroglial Signal Augmenting Aerobic Glycolysis and Lipid Metabolism.
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Horvat A, Zorec R, and Vardjan N
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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 via L-lactate-sensitive G
s -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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Horvat, Zorec and Vardjan.)- Published
- 2021
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16. Astrocytes in stress accumulate lipid droplets.
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Smolič T, Tavčar P, Horvat A, Černe U, Halužan Vasle A, Tratnjek L, Kreft ME, Scholz N, Matis M, Petan T, Zorec R, and Vardjan N
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- Animals, Drosophila, Endoplasmic Reticulum metabolism, Lipid Droplets metabolism, Mitochondria, Rats, Astrocytes
- Abstract
When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. We imaged fluorescently labeled LDs by microscopy in isolated and brain tissue rat astrocytes and in glia-like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca
2+ , likely to enhance the LD-organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress-induced lipotoxicity., (© 2021 The Authors. Glia published by Wiley Periodicals LLC.)- Published
- 2021
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17. Ca 2+ as the prime trigger of aerobic glycolysis in astrocytes.
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Horvat A, Muhič M, Smolič T, Begić E, Zorec R, Kreft M, and Vardjan N
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- Animals, Astrocytes drug effects, Cells, Cultured, Cerebral Cortex cytology, Cerebral Cortex drug effects, Cerebral Cortex metabolism, Female, Glycolysis drug effects, Isoproterenol pharmacology, Phenylephrine pharmacology, Rats, Rats, Wistar, Astrocytes metabolism, Calcium metabolism, Glucose metabolism, Glycolysis physiology
- Abstract
Astroglial aerobic glycolysis, a process during which d-glucose is converted to l-lactate, a brain fuel and signal, is regulated by the plasmalemmal receptors, including adrenergic receptors (ARs) and purinergic receptors (PRs), modulating intracellular Ca
2+ and cAMP signals. However, the extent to which the two signals regulate astroglial aerobic glycolysis is poorly understood. By using agonists to stimulate intracellular α1 -/β-AR-mediated Ca2+ /cAMP signals, β-AR-mediated cAMP and P2 R-mediated Ca2+ signals and genetically encoded fluorescence resonance energy transfer-based glucose and lactate nanosensors in combination with real-time microscopy, we show that intracellular Ca2+ , but not cAMP, initiates a robust increase in the concentration of intracellular free d-glucose ([glc]i ) and l-lactate ([lac]i ), both depending on extracellular d-glucose, suggesting Ca2+ -triggered glucose uptake and aerobic glycolysis in astrocytes. When the glycogen shunt, a process of glycogen remodelling, was inhibited, the α1 -/β-AR-mediated increases in [glc]i and [lac]i were reduced by ∼65 % and ∼30 %, respectively, indicating that at least ∼30 % of the utilization of d-glucose is linked to glycogen remodelling and aerobic glycolysis. Additional activation of β-AR/cAMP signals aided to α1 -/β-AR-triggered [lac]i increase, whereas the [glc]i increase was unaltered. Taken together, an increase in intracellular Ca2+ is the prime mechanism of augmented aerobic glycolysis in astrocytes, while cAMP has only a moderate role. The results provide novel information on the signals regulating brain metabolism and open new avenues to explore whether astroglial Ca2+ signals are dysregulated and contribute to neuropathologies with impaired brain metabolism., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)- Published
- 2021
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18. Noradrenaline-induced l-lactate production requires d-glucose entry and transit through the glycogen shunt in single-cultured rat astrocytes.
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Fink K, Velebit J, Vardjan N, Zorec R, and Kreft M
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- Animals, Animals, Newborn, Arabinose pharmacology, Brain metabolism, Citric Acid Cycle drug effects, Deoxyglucose pharmacology, Energy Metabolism, Fluorescence Resonance Energy Transfer, Imino Furanoses pharmacology, Nitro Compounds pharmacology, Oxidative Phosphorylation, Primary Cell Culture, Propionates pharmacology, Rats, Rats, Wistar, Sugar Alcohols pharmacology, Transfection, Astrocytes metabolism, Glucose metabolism, Glycogen metabolism, Lactic Acid biosynthesis, Norepinephrine pharmacology
- Abstract
During cognitive efforts mediated by local neuronal networks, approximately 20% of additional energy is required; this is mediated by chemical messengers such as noradrenaline (NA). NA targets astroglial aerobic glycolysis, the hallmark of which is the end product l-lactate, a fuel for neurons. Biochemical studies have revealed that astrocytes exhibit a prominent glycogen shunt, in which a portion of d-glucose molecules entering the cytoplasm is transiently incorporated into glycogen, a buffer and source of d-glucose during increased energy demand. Here, we studied single astrocytes by measuring cytosolic L-lactate ([lac]
i ) with the FRET nanosensor Laconic. We examined whether NA-induced increase in [lac]i is influenced by: (a) 2-deoxy-d-glucose (2-DG, 3 mM), a molecule that enters the cytosol and inhibits the glycolytic pathway; (b) 1,4-dideoxy-1,4-imino-d-arabinitol (DAB, 300 µM), a potent inhibitor of glycogen phosphorylase and glycogen degradation; and (c) 3-nitropropionic acid (3-NPA, 1 mM), an inhibitor of the Krebs cycle. The results of these pharmacological experiments revealed that d-glucose uptake is essential for the NA-induced increase in [lac]i , and that this exclusively arises from glycogen degradation, indicating that most, if not all, d-glucose molecules in NA-stimulated cells transit the glycogen shunt during glycolysis. Moreover, under the defined transmembrane d-glucose gradient, the glycolytic intermediates were not only used to produce l-lactate, but also to significantly support oxidative phosphorylation, as demonstrated by an elevation in [lac]i when Krebs cycle was inhibited. We conclude that l-lactate production via aerobic glycolysis is an essential energy pathway in NA-stimulated astrocytes; however, oxidative metabolism is important at rest., (© 2021 Wiley Periodicals LLC.)- Published
- 2021
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19. Inhibiting glycolysis rescues memory impairment in an intellectual disability Gdi1-null mouse.
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D'Adamo P, Horvat A, Gurgone A, Mignogna ML, Bianchi V, Masetti M, Ripamonti M, Taverna S, Velebit J, Malnar M, Muhič M, Fink K, Bachi A, Restuccia U, Belloli S, Moresco RM, Mercalli A, Piemonti L, Potokar M, Bobnar ST, Kreft M, Chowdhury HH, Stenovec M, Vardjan N, and Zorec R
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- Animals, Brain drug effects, Brain metabolism, Cells, Cultured, Deoxyglucose therapeutic use, Down-Regulation drug effects, Glucose metabolism, Guanine Nucleotide Dissociation Inhibitors deficiency, Intellectual Disability drug therapy, Intellectual Disability metabolism, Intellectual Disability pathology, Male, Maze Learning drug effects, Memory drug effects, Memory Disorders genetics, Mice, Mice, Knockout, Deoxyglucose pharmacology, Glycolysis drug effects, Guanine Nucleotide Dissociation Inhibitors genetics, Intellectual Disability genetics, Memory Disorders prevention & control
- Abstract
Objectives: GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear., Methods: We carried out proteomic analysis and monitored [
18 F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests., Results: Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18 F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection., Conclusions: These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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20. Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca 2+ signaling and dysregulated cell metabolism.
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Velebit J, Horvat A, Smolič T, Prpar Mihevc S, Rogelj B, Zorec R, and Vardjan N
- Subjects
- Amyotrophic Lateral Sclerosis metabolism, Amyotrophic Lateral Sclerosis pathology, Animals, Astrocytes pathology, Cells, Cultured, Glycolysis, Humans, Inclusion Bodies pathology, Rats, Wistar, Receptors, Adrenergic metabolism, Signal Transduction, Astrocytes metabolism, Calcium metabolism, Cyclic AMP metabolism, DNA-Binding Proteins metabolism, Inclusion Bodies metabolism, Norepinephrine metabolism
- Abstract
Most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons and non-neuronal cells, including astrocytes, which metabolically support neurons with nutrients. Neuronal metabolism largely depends on the activation of the noradrenergic system releasing noradrenaline. Activation of astroglial adrenergic receptors with noradrenaline triggers cAMP and Ca
2+ signaling and augments aerobic glycolysis with production of lactate, an important neuronal energy fuel. Astrocytes with cytoplasmic TDP-43 inclusions can cause motor neuron death, however, whether astroglial metabolism and metabolic support of neurons is altered in astrocytes with TDP-43 inclusions, is unclear. We measured lipid droplet and glucose metabolisms in astrocytes expressing the inclusion-forming C-terminal fragment of TDP-43 or the wild-type TDP-43 using fluorescent dyes or genetically encoded nanosensors. Astrocytes with TDP-43 inclusions exhibited a 3-fold increase in the accumulation of lipid droplets versus astrocytes expressing wild-type TDP-43, indicating altered lipid droplet metabolism. In these cells the noradrenaline-triggered increases in intracellular cAMP and Ca2+ levels were reduced by 35% and 31%, respectively, likely due to the downregulation of β2 -adrenergic receptors. Although noradrenaline triggered a similar increase in intracellular lactate levels in astrocytes with and without TDP-43 inclusions, the probability of activating aerobic glycolysis was facilitated by 1.6-fold in astrocytes with TDP-43 inclusions and lactate MCT1 transporters were downregulated. Thus, while in astrocytes with TDP-43 inclusions noradrenergic signaling is reduced, aerobic glycolysis and lipid droplet accumulation are facilitated, suggesting dysregulated astroglial metabolism and metabolic support of neurons in TDP-43-associated ALS and FTD.- Published
- 2020
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21. The European Research Network on Signal Transduction (ERNEST): Toward a Multidimensional Holistic Understanding of G Protein-Coupled Receptor Signaling.
- Author
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Sommer ME, Selent J, Carlsson J, De Graaf C, Gloriam DE, Keseru GM, Kosloff M, Mordalski S, Rizk A, Rosenkilde MM, Sotelo E, Tiemann JKS, Tobin A, Vardjan N, Waldhoer M, and Kolb P
- Abstract
G protein-coupled receptors (GPCRs) are intensively studied due to their therapeutic potential as drug targets. Members of this large family of transmembrane receptor proteins mediate signal transduction in diverse cell types and play key roles in human physiology and health. In 2013 the research consortium GLISTEN (COST Action CM1207) was founded with the goal of harnessing the substantial growth in knowledge of GPCR structure and dynamics to push forward the development of molecular modulators of GPCR function. The success of GLISTEN, coupled with new findings and paradigm shifts in the field, led in 2019 to the creation of a related consortium called ERNEST (COST Action CA18133). ERNEST broadens focus to entire signaling cascades, based on emerging ideas of how complexity and specificity in signal transduction are not determined by receptor-ligand interactions alone. A holistic approach that unites the diverse data and perspectives of the research community into a single multidimensional map holds great promise for improved drug design and therapeutic targeting., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)
- Published
- 2020
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22. Astrocyte Specific Remodeling of Plasmalemmal Cholesterol Composition by Ketamine Indicates a New Mechanism of Antidepressant Action.
- Author
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Lasič E, Lisjak M, Horvat A, Božić M, Šakanović A, Anderluh G, Verkhratsky A, Vardjan N, Jorgačevski J, Stenovec M, and Zorec R
- Subjects
- Animals, Antidepressive Agents therapeutic use, Astrocytes pathology, Cell Membrane metabolism, Cyclic AMP metabolism, Depressive Disorder, Major drug therapy, Exocytosis drug effects, Female, Ketamine therapeutic use, PC12 Cells, Rats, Rats, Wistar, Antidepressive Agents pharmacology, Astrocytes drug effects, Cholesterol metabolism, Ketamine pharmacology
- 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.- Published
- 2019
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23. Metabolic Plasticity of Astrocytes and Aging of the Brain.
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Morita M, Ikeshima-Kataoka H, Kreft M, Vardjan N, Zorec R, and Noda M
- Subjects
- Animals, Brain growth & development, Humans, Adaptation, Physiological, Aging metabolism, Astrocytes metabolism, Brain metabolism, Energy Metabolism
- Abstract
As part of the blood-brain-barrier, astrocytes are ideally positioned between cerebral vasculature and neuronal synapses to mediate nutrient uptake from the systemic circulation. In addition, astrocytes have a robust enzymatic capacity of glycolysis, glycogenesis and lipid metabolism, managing nutrient support in the brain parenchyma for neuronal consumption. Here, we review the plasticity of astrocyte energy metabolism under physiologic and pathologic conditions, highlighting age-dependent brain dysfunctions. In astrocytes, glycolysis and glycogenesis are regulated by noradrenaline and insulin, respectively, while mitochondrial ATP production and fatty acid oxidation are influenced by the thyroid hormone. These regulations are essential for maintaining normal brain activities, and impairments of these processes may lead to neurodegeneration and cognitive decline. Metabolic plasticity is also associated with (re)activation of astrocytes, a process associated with pathologic events. It is likely that the recently described neurodegenerative and neuroprotective subpopulations of reactive astrocytes metabolize distinct energy substrates, and that this preference is supposed to explain some of their impacts on pathologic processes. Importantly, physiologic and pathologic properties of astrocytic metabolic plasticity bear translational potential in defining new potential diagnostic biomarkers and novel therapeutic targets to mitigate neurodegeneration and age-related brain dysfunctions.
- Published
- 2019
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24. Astroglial cAMP signalling in space and time.
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Horvat A and Vardjan N
- Subjects
- Animals, Calcium Signaling, Humans, Neurons metabolism, Signal Transduction, Astrocytes metabolism, Cyclic AMP metabolism
- Abstract
To maintain a high level of specificity and normal cell function, the cyclic adenosine monophosphate (cAMP) pathway is tightly regulated in space and time. Recent advances in cAMP reporter technology have provided insights into spatio-temporal characteristics of cAMP signalling in individual living cells, including astrocytes. Astrocytes are glial cells in the central nervous system with many homeostatic functions. In contrast to neurons, astrocytes are electrically silent, but, in response to extracellular stimuli through activation of surface receptors, they can increase intracellular levels of secondary messengers, e.g. Ca
2+ and cAMP. This enables them to communicate with neighbouring cells, such as neurons and endothelial cells of blood vessels. The dynamics of receptor-mediated Ca2+ signalling in astrocytes has been extensively studied in the past in contrast to cAMP signalling. Here, we present the first insights into the temporal dynamics of cAMP signalling in living astrocytes, which revealed that cAMP signals in astrocytes exhibit tonic dynamics and are slower than Ca2+ signals with phasic dynamics. We debate on the heterogeneity of basal cAMP levels in astrocytes and how hypotonicity-induced astrocyte swelling affects temporal dynamics of cAMP signalling. Understanding the spatio-temporal characteristics of cAMP signalling in astrocytes is of extreme importance because cAMP governs many important cellular processes and any malfunctions may lead to pathology., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2019
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25. Physiology of Astroglia.
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Verkhratsky A, Parpura V, Vardjan N, and Zorec R
- Subjects
- Calcium, Homeostasis, Humans, Ion Pumps physiology, Neuroglia, Receptors, Neurotransmitter physiology, Sodium, Astrocytes physiology, Brain physiology, Signal Transduction
- Abstract
Astrocytes are principal cells responsible for maintaining the brain homeostasis. Additionally, these glial cells are also involved in homocellular (astrocyte-astrocyte) and heterocellular (astrocyte-other cell types) signalling and metabolism. These astroglial functions require an expression of the assortment of molecules, be that transporters or pumps, to maintain ion concentration gradients across the plasmalemma and the membrane of the endoplasmic reticulum. Astrocytes sense and balance their neurochemical environment via variety of transmitter receptors and transporters. As they are electrically non-excitable, astrocytes display intracellular calcium and sodium fluctuations, which are not only used for operative signalling but can also affect metabolism. In this chapter we discuss the molecules that achieve ionic gradients and underlie astrocyte signalling.
- Published
- 2019
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26. Gliocrine System: Astroglia as Secretory Cells of the CNS.
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Vardjan N, Parpura V, Verkhratsky A, and Zorec R
- Subjects
- Humans, Membrane Fusion, Astrocytes cytology, Central Nervous System cytology, Exocytosis, Secretory Vesicles physiology
- Abstract
Astrocytes are secretory cells, actively participating in cell-to-cell communication in the central nervous system (CNS). They sense signaling molecules in the extracellular space, around the nearby synapses and also those released at much farther locations in the CNS, by their cell surface receptors, get excited to then release their own signaling molecules. This contributes to the brain information processing, based on diffusion within the extracellular space around the synapses and on convection when locales relatively far away from the release sites are involved. These functions resemble secretion from endocrine cells, therefore astrocytes were termed to be a part of the gliocrine system in 2015. An important mechanism, by which astrocytes release signaling molecules is the merger of the vesicle membrane with the plasmalemma, i.e., exocytosis. Signaling molecules stored in astroglial secretory vesicles can be discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This leads to a fusion pore formation, a channel that must widen to allow the exit of the Vesiclal cargo. Upon complete vesicle membrane fusion, this process also integrates other proteins, such as receptors, transporters and channels into the plasma membrane, determining astroglial surface signaling landscape. Vesiclal cargo, together with the whole vesicle can also exit astrocytes by the fusion of multivesicular bodies with the plasma membrane (exosomes) or by budding of vesicles (ectosomes) from the plasma membrane into the extracellular space. These astroglia-derived extracellular vesicles can later interact with various target cells. Here, the characteristics of four types of astroglial secretory vesicles: synaptic-like microvesicles, dense-core vesicles, secretory lysosomes, and extracellular vesicles, are discussed. Then machinery for vesicle-based exocytosis, second messenger regulation and the kinetics of exocytotic vesicle content discharge or release of extracellular vesicles are considered. In comparison to rapidly responsive, electrically excitable neurons, the receptor-mediated cytosolic excitability-mediated astroglial exocytotic vesicle-based transmitter release is a relatively slow process.
- Published
- 2019
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27. General Pathophysiology of Astroglia.
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Verkhratsky A, Ho MS, Vardjan N, Zorec R, and Parpura V
- Subjects
- Alexander Disease physiopathology, Atrophy, Humans, Mental Disorders physiopathology, Astrocytes pathology, Brain physiopathology
- Abstract
Astroglial cells are involved in most if not in all pathologies of the brain. These cells can change the morpho-functional properties in response to pathology or innate changes of these cells can lead to pathologies. Overall pathological changes in astroglia are complex and diverse and often vary with different disease stages. We classify astrogliopathologies into reactive astrogliosis, astrodegeneration with astroglial atrophy and loss of function, and pathological remodelling of astrocytes. Such changes can occur in neurological, neurodevelopmental, metabolic and psychiatric disorders as well as in infection and toxic insults. Mutation in astrocyte-specific genes leads to specific pathologies, such as Alexander disease, which is a leukodystrophy. We discuss changes in astroglia in the pathological context and identify some molecular entities underlying pathology. These entities within astroglia may repent targets for novel therapeutic intervention in the management of brain pathologies.
- Published
- 2019
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28. Enhancement of Astroglial Aerobic Glycolysis by Extracellular Lactate-Mediated Increase in cAMP.
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Vardjan N, Chowdhury HH, Horvat A, Velebit J, Malnar M, Muhič M, Kreft M, Krivec ŠG, Bobnar ST, Miš K, Pirkmajer S, Offermanns S, Henriksen G, Storm-Mathisen J, Bergersen LH, and Zorec R
- Abstract
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 (EC
50 ≈ 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.- Published
- 2018
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29. Impaired αGDI Function in the X-Linked Intellectual Disability: The Impact on Astroglia Vesicle Dynamics.
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Potokar M, Jorgačevski J, Lacovich V, Kreft M, Vardjan N, Bianchi V, D'Adamo P, and Zorec R
- Subjects
- Animals, Astrocytes pathology, Disease Models, Animal, Endosomes metabolism, Gene Silencing, Guanine Nucleotide Dissociation Inhibitors metabolism, Lysosomes metabolism, Male, Mice, Mutation genetics, Rats, Transfection, Astrocytes metabolism, Cytoplasmic Vesicles metabolism, Genes, X-Linked, Guanine Nucleotide Dissociation Inhibitors genetics, Intellectual Disability genetics
- Abstract
X-linked non-syndromic intellectual disability (XLID) is a common mental disorder recognized by cognitive and behavioral deficits. Mutations in the brain-specific αGDI, shown to alter a subset of RAB GTPases redistribution in cells, are linked to XLID, likely via changes in vesicle traffic in neurons. Here, we show directly that isolated XLID mice astrocytes, devoid of pathologic tissue environment, exhibit vesicle mobility deficits. Contrary to previous studies, we show that astrocytes express two GDI proteins. The siRNA-mediated suppression of expression of αGDI especially affected vesicle dynamics. A similar defect was recorded in astrocytes from the Gdi1
-/Y mouse model of XLID and in astrocytes with recombinant mutated human XLID αGDI. Endolysosomal vesicles studied here are involved in the release of gliosignaling molecules as well as in regulating membrane receptor density; thus, the observed changes in astrocytic vesicle mobility may, over the long time-course, profoundly affect signaling capacity of these cells, which optimize neural activity.- Published
- 2017
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30. Astrocytic face of Alzheimer's disease.
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Zorec R, Parpura V, Vardjan N, and Verkhratsky A
- Subjects
- Animals, Humans, Locus Coeruleus physiopathology, Memory physiology, Alzheimer Disease physiopathology, Astrocytes physiology
- Abstract
Ageing of the central nervous system (CNS) is the major risk factor for Alzheimer's disease (AD), a type of neurodegeneration that is associated with deficits in cognition and memory and clinically manifested as severe senile dementia. Numerous mental processes underline cognition, including attention, producing and understanding language, learning, reasoning, problem solving, decision making and memory formation. In the past, neurones or their parts have been considered to be the exclusive cellular sites of memory and cognitive processes. However, it has become evident that astrocytes, the major homeostatic glial cell of the CNS, provide an essential contribution to memory formation, and astroglial failure may promote cognitive decline in AD. In response to the network reset mechanisms mediated by the noradrenergic projections of neurones located in the locus coeruleus, astrocytes get excited and participate in the morphological remodelling associated with synaptic plasticity, otherwise thought to represent a cellular mechanism of learning and memory. Astroglial morphological plasticity is an energy-demanding process requiring mobilisation of glycogen, which, in the CNS, is almost exclusively stored in astrocytes. Astroglia exhibit cytoplasmic excitability that engages ions (such as Ca
2+ and Na+ ) and second messengers (such as cAMP). These ions/molecules contribute to the reception of extracellular signals and coordinate the secretion of glio-signalling molecules, including peptides such as apolipoporotein E, which participates in lipid transport between glia and neurones. In this setting, astrocytes are positioned as spatio-temporal integrators of neural network coordination, which disintegrates during progression of AD., (Copyright © 2016 Elsevier B.V. All rights reserved.)- Published
- 2017
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31. Astrocytic Pathological Calcium Homeostasis and Impaired Vesicle Trafficking in Neurodegeneration.
- Author
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Vardjan N, Verkhratsky A, and Zorec R
- Subjects
- Alzheimer Disease etiology, Alzheimer Disease metabolism, Alzheimer Disease pathology, Animals, Astrocytes pathology, Atrophy, Cell Communication, Cell Membrane metabolism, Cyclic AMP metabolism, Humans, Locus Coeruleus metabolism, Locus Coeruleus pathology, Neocortex metabolism, Neocortex pathology, Neurodegenerative Diseases etiology, Neurodegenerative Diseases pathology, Neuronal Plasticity, Neurons metabolism, Signal Transduction, Astrocytes metabolism, Calcium metabolism, Homeostasis, Neurodegenerative Diseases metabolism, Transport Vesicles metabolism
- Abstract
Although the central nervous system (CNS) consists of highly heterogeneous populations of neurones and glial cells, clustered into diverse anatomical regions with specific functions, there are some conditions, including alertness, awareness and attention that require simultaneous, coordinated and spatially homogeneous activity within a large area of the brain. During such events, the brain, representing only about two percent of body mass, but consuming one fifth of body glucose at rest, needs additional energy to be produced. How simultaneous energy procurement in a relatively extended area of the brain takes place is poorly understood. This mechanism is likely to be impaired in neurodegeneration, for example in Alzheimer's disease, the hallmark of which is brain hypometabolism. Astrocytes, the main neural cell type producing and storing glycogen, a form of energy in the brain, also hold the key to metabolic and homeostatic support in the central nervous system and are impaired in neurodegeneration, contributing to the slow decline of excitation-energy coupling in the brain. Many mechanisms are affected, including cell-to-cell signalling. An important question is how changes in cellular signalling, a process taking place in a rather short time domain, contribute to the neurodegeneration that develops over decades. In this review we focus initially on the slow dynamics of Alzheimer's disease, and on the activity of locus coeruleus, a brainstem nucleus involved in arousal. Subsequently, we overview much faster processes of vesicle traffic and cytosolic calcium dynamics, both of which shape the signalling landscape of astrocyte-neurone communication in health and neurodegeneration.
- Published
- 2017
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32. Targeting Astrocytes for Treating Neurological Disorders: Carbon Monoxide and Noradrenaline-Induced Increase in Lactate.
- Author
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Horvat A, Vardjan N, and Zorec R
- Subjects
- Animals, Astrocytes drug effects, Drug Delivery Systems methods, Drug Delivery Systems trends, Humans, Nervous System Diseases drug therapy, Neuroprotection drug effects, Astrocytes metabolism, Carbon Monoxide administration & dosage, Lactic Acid metabolism, Nervous System Diseases metabolism, Neuroprotection physiology, Norepinephrine administration & dosage
- Abstract
There are at least three reasons why brain astrocytes represent a new target for treating neurological disorders. First, although the human neocortex represents over 80% of brain mass, neurons are outnumbered by non-neuronal cells, including astrocytes, a neuroglial cell type. Second, as in neurons, vesicle-based release of transmitters is present in astrocytes, however with much slower kinetics than in neurons. Third, astrocytes contain glycogen, which can be transformed to L-lactate in glycolysis. L-lactate is considered to be a fuel and a signalling molecule involved in cognition and neuroprotection. The mechanisms of neuroprotection are unclear but may be linked to carbon monoxide, a product of the heme oxygenase, an evolutionarily conserved cellular cytoprotectant. Increased levels of local carbon monoxide arising from heme oxygenase activity may increase L-lactate, but direct measurements of cytosolic L-lactate are lacking. A fluorescence resonance energy transfer-based nanosensor selective for L-lactate was used to monitor cytosolic levels of L-lactate while cultured astrocytes were exposed to carbon monoxide. The results revealed that in astrocytes exposed to carbon monoxide there is no significant increase in L-lactate, however, when noradrenaline, a potent glycogenolytic agent, is applied, cytosolic levels of Llactate are increased, but strongly attenuated in astrocytes pretreated with carbon monoxide. These first measurements of carbon monoxide-modulated L-lactate levels in astrocytes provide evidence that the L-lactate and heme oxygenase neuroprotective systems may interact. In conclusion, not only the abundance of astrocytes but their signalling capacity using vesicles and metabolites, such as L-lactate, are valid targets for neurological disorders., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)
- Published
- 2017
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33. Time-dependent uptake and trafficking of vesicles capturing extracellular S100B in cultured rat astrocytes.
- Author
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Lasič E, Galland F, Vardjan N, Šribar J, Križaj I, Leite MC, Zorec R, and Stenovec M
- Subjects
- Adenosine Triphosphate pharmacology, Animals, Antibodies, Blocking pharmacology, Calcium metabolism, Cells, Cultured, Cyanoacrylates pharmacology, Cytoplasmic Vesicles ultrastructure, Dynamins antagonists & inhibitors, Endocytosis, Female, Indoles pharmacology, Lysosomes metabolism, Rats, Rats, Wistar, Receptor for Advanced Glycation End Products antagonists & inhibitors, Receptor for Advanced Glycation End Products immunology, Astrocytes metabolism, Cytoplasmic Vesicles metabolism, Extracellular Space metabolism, S100 Calcium Binding Protein beta Subunit metabolism
- Abstract
Astrocytes, the most heterogeneous glial cells in the central nervous system, contribute to brain homeostasis, by regulating a myriad of functions, including the clearance of extracellular debris. When cells are damaged, cytoplasmic proteins may exit into the extracellular space. One such protein is S100B, which may exert toxic effects on neighboring cells unless it is removed from the extracellular space, but the mechanisms of this clearance are poorly understood. By using time-lapse confocal microscopy and fluorescently labeled S100B (S100B-Alexa
488 ) and fluorescent dextran (Dextran546 ), a fluid phase uptake marker, we examined the uptake of fluorescently labeled S100B-Alexa488 from extracellular space and monitored trafficking of vesicles that internalized S100B-Alexa488 . Initially, S100B-Alexa488 and Dextran546 internalized with distinct rates into different endocytotic vesicles; S100B-Alexa488 internalized into smaller vesicles than Dextran546 . At a later stage, S100B-Alexa488 -positive vesicles substantially co-localized with Dextran546 -positive endolysosomes and with acidic LysoTracker-positive vesicles. Cell treatment with anti-receptor for advanced glycation end products (RAGE) antibody, which binds to RAGE, a 'scavenger receptor', partially inhibited uptake of S100B-Alexa488 , but not of Dextran546 . The dynamin inhibitor dynole 34-2 inhibited internalization of both fluorescent probes. Directional mobility of S100B-Alexa488 -positive vesicles increased over time and was inhibited by ATP stimulation, an agent that increases cytosolic free calcium concentration ([Ca2+ ]i ). We conclude that astrocytes exhibit RAGE- and dynamin-dependent vesicular mechanism to efficiently remove S100B from the extracellular space. If a similar process occurs in vivo, astroglia may mitigate the toxic effects of extracellular S100B by this process under pathophysiologic conditions. This study reveals the vesicular clearance mechanism of extracellular S100B in astrocytes. Initially, fluorescent S100B internalizes into smaller endocytotic vesicles than dextran molecules. At a later stage, both probes co-localize within endolysosomes. S100B internalization is both dynamin- and RAGE-dependent, whereas dextran internalization is dependent on dynamin. Vesicle internalization likely mitigates the toxic effects of extracellular S100B and other waste products., (© 2016 International Society for Neurochemistry.)- Published
- 2016
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34. Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state.
- Author
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Guček A, Jorgačevski J, Singh P, Geisler C, Lisjak M, Vardjan N, Kreft M, Egner A, and Zorec R
- Subjects
- Adenosine Triphosphate pharmacology, Animals, Astrocytes drug effects, Astrocytes metabolism, Exocytosis drug effects, Extracellular Vesicles drug effects, Extracellular Vesicles metabolism, Female, Microscopy, Models, Biological, Rats, Wistar, Time Factors, Membrane Fusion drug effects, Peptides metabolism, SNARE Proteins metabolism
- Abstract
Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.
- Published
- 2016
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35. Adrenergic activation attenuates astrocyte swelling induced by hypotonicity and neurotrauma.
- Author
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Vardjan N, Horvat A, Anderson JE, Yu D, Croom D, Zeng X, Lužnik Z, Kreft M, Teng YD, Kirov SA, and Zorec R
- Subjects
- Adrenergic Agents pharmacology, Animals, Astrocytes cytology, Brain Injuries complications, Cell Size drug effects, Cells, Cultured, Disease Models, Animal, Rats, Astrocytes drug effects, Brain Edema etiology, Brain Injuries drug therapy, Spinal Cord Injuries drug therapy
- Abstract
Edema in the central nervous system can rapidly result in life-threatening complications. Vasogenic edema is clinically manageable, but there is no established medical treatment for cytotoxic edema, which affects astrocytes and is a primary trigger of acute post-traumatic neuronal death. To test the hypothesis that adrenergic receptor agonists, including the stress stimulus epinephrine protects neural parenchyma from damage, we characterized its effects on hypotonicity-induced cellular edema in cortical astrocytes by in vivo and in vitro imaging. After epinephrine administration, hypotonicity-induced swelling of astrocytes was markedly reduced and cytosolic 3'-5'-cyclic adenosine monophosphate (cAMP) was increased, as shown by a fluorescence resonance energy transfer nanosensor. Although, the kinetics of epinephrine-induced cAMP signaling was slowed in primary cortical astrocytes exposed to hypotonicity, the swelling reduction by epinephrine was associated with an attenuated hypotonicity-induced cytosolic Ca(2+) excitability, which may be the key to prevent astrocyte swelling. Furthermore, in a rat model of spinal cord injury, epinephrine applied locally markedly reduced neural edema around the contusion epicenter. These findings reveal new targets for the treatment of cellular edema in the central nervous system., (© 2016 Wiley Periodicals, Inc.)
- Published
- 2016
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36. Unproductive exocytosis.
- Author
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Kreft M, Jorgačevski J, Vardjan N, and Zorec R
- Subjects
- Animals, Calcium metabolism, Cell Membrane metabolism, Humans, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels physiology, Exocytosis physiology, Membrane Fusion physiology, Secretory Vesicles physiology
- Abstract
Regulated exocytosis is a multistage process involving a merger between the vesicle and the plasma membrane, leading to the formation of a fusion pore, a channel, through which secretions are released from the vesicle to the cell exterior. A stimulus may influence the pore by either dilating it completely (full-fusion exocytosis) or mediating a reversible closure (transient exocytosis). In neurons, these transitions are short-lived and not accessible for experimentation. However, in some neuroendocrine cells and astrocytes, initial fusion pores may reopen several hundred times, indicating their stability. Frequently, these pores are too narrow to pass luminal molecules to the extracellular space (unproductive exocytosis), but their diameter can dilate upon stimulation. To explain the stability of the initial narrow fusion pores, anisotropic membrane constituents with a non-axisymmetric shape were proposed to accumulate in the fusion pore membrane. Although the nature of these is unclear, they may consist of lipids and proteins, including SNAREs, which may facilitate and regulate the pre- and post-fusional stages of exocytosis. This review highlights models and experimental studies revealing mechanisms of fusion pore stabilization in a narrow, release unproductive state. The fusion pore is a channel that forms when the vesicle and the plasma membranes merge, and mediates the release of secretions from the vesicle lumen to the cell exterior. Frequently, these pores are too narrow to pass molecules to the extracellular space. Anisotropic membrane constituents with a non-axisymmetric shape were proposed to accumulate in the fusion pore membrane. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015)., (© 2016 International Society for Neurochemistry.)
- Published
- 2016
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37. Loose excitation-secretion coupling in astrocytes.
- Author
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Vardjan N, Parpura V, and Zorec R
- Subjects
- Animals, Astrocytes cytology, Calcium metabolism, Humans, SNARE Proteins metabolism, Astrocytes physiology, Cell Communication physiology, Cell Membrane physiology, Exocytosis physiology
- Abstract
Astrocytes play an important housekeeping role in the central nervous system. Additionally, as secretory cells, they actively participate in cell-to-cell communication, which can be mediated by membrane-bound vesicles. The gliosignaling molecules stored in these vesicles are discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This process is termed exocytosis, regulated by SNARE proteins, and triggered by elevations in cytosolic calcium levels, which are necessary and sufficient for exocytosis in astrocytes. For astrocytic exocytosis, calcium is sourced from the intracellular endoplasmic reticulum store, although its entry from the extracellular space contributes to cytosolic calcium dynamics in astrocytes. Here, we discuss calcium management in astrocytic exocytosis and the properties of the membrane-bound vesicles that store gliosignaling molecules, including the vesicle fusion machinery and kinetics of vesicle content discharge. In astrocytes, the delay between the increase in cytosolic calcium activity and the discharge of secretions from the vesicular lumen is orders of magnitude longer than that in neurons. This relatively loose excitation-secretion coupling is likely tailored to the participation of astrocytes in modulating neural network processing., (© 2015 Wiley Periodicals, Inc.)
- Published
- 2016
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38. Adrenergic stimulation of single rat astrocytes results in distinct temporal changes in intracellular Ca(2+) and cAMP-dependent PKA responses.
- Author
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Horvat A, Zorec R, and Vardjan N
- Subjects
- Animals, Astrocytes drug effects, Calcium Signaling drug effects, Cells, Cultured, Cytoplasm metabolism, Isoproterenol pharmacology, Neuroglia metabolism, Rats, Adrenergic Agents pharmacology, Astrocytes metabolism, Calcium metabolism, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases metabolism
- Abstract
During the arousal and startle response, locus coeruleus neurons, innervating practically all brain regions, release catecholamine noradrenaline, which reaches neural brain cells, including astrocytes. These glial cells respond to noradrenergic stimulation by simultaneous activation of the α- and β-adrenergic receptors (ARs) in the plasma membrane with increasing cytosolic levels of Ca(2+) and cAMP, respectively. AR-activation controls a myriad of processes in astrocytes including glucose metabolism, gliosignal vesicle homeostasis, gene transcription, cell morphology and antigen-presenting functions, all of which have distinct temporal characteristics. It is known from biochemical studies that Ca(2+) and cAMP signals in astrocytes can interact, however it is presently unclear whether the temporal properties of the two second messengers are time associated upon AR-activation. We used confocal microscopy to study AR agonist-induced intracellular changes in Ca(2+) and cAMP in single cultured cortical rat astrocytes by real-time monitoring of the Ca(2+) indicator Fluo4-AM and the fluorescence resonance energy transfer-based nanosensor A-kinase activity reporter 2 (AKAR2), which reports the activity of cAMP via its downstream effector protein kinase A (PKA). The results revealed that the activation of α1-ARs by phenylephrine triggers periodic (phasic) Ca(2+) oscillations within 10s, while the activation of β-ARs by isoprenaline leads to a ∼10-fold slower tonic rise to a plateau in cAMP/PKA activity devoid of oscillations. Thus the concomitant activation of α- and β-ARs triggers the Ca(2+) and cAMP second messenger systems in astrocytes with distinct temporal properties, which appears to be tailored to regulate downstream effectors in different time domains., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
39. Excitable Astrocytes: Ca(2+)- and cAMP-Regulated Exocytosis.
- Author
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Vardjan N and Zorec R
- Subjects
- Animals, Cytosol metabolism, Humans, Neuroglia physiology, Astrocytes physiology, Calcium Signaling physiology, Cyclic AMP physiology, Exocytosis physiology
- Abstract
During neural activity, neurotransmitters released at synapses reach neighbouring cells, such as astrocytes. These get excited via numerous mechanisms, including the G protein coupled receptors that regulate the cytosolic concentration of second messengers, such as Ca(2+) and cAMP. The stimulation of these pathways leads to feedback modulation of neuronal activity and the activity of other cells by the release of diverse substances, gliosignals that include classical neurotransmitters such as glutamate, ATP, or neuropeptides. Gliosignal molecules are released from astrocytes through several distinct molecular mechanisms, for example, by diffusion through membrane channels, by translocation via plasmalemmal transporters, or by vesicular exocytosis. Vesicular release regulated by a stimulus-mediated increase in cytosolic second messengers involves a SNARE-dependent merger of the vesicle membrane with the plasmalemma. The coupling between the stimulus and vesicular secretion of gliosignals in astrocytes is not as tight as in neurones. This is considered an adaptation to regulate homeostatic processes in a slow time domain as is the case in the endocrine system (slower than the nervous system), hence glial functions constitute the gliocrine system. This article provides an overview of the mechanisms of excitability, involving Ca(2+) and cAMP, where the former mediates phasic signalling and the latter tonic signalling. The molecular, anatomic, and physiologic properties of the vesicular apparatus mediating the release of gliosignals is presented.
- Published
- 2015
- Full Text
- View/download PDF
40. Memory Formation Shaped by Astroglia.
- Author
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Zorec R, Horvat A, Vardjan N, and Verkhratsky A
- Abstract
Astrocytes, the most heterogeneous glial cells in the central nervous system (CNS), execute a multitude of homeostatic functions and contribute to memory formation. Consolidation of synaptic and systemic memory is a prolonged process and hours are required to form long-term memory. In the past, neurons or their parts have been considered to be the exclusive cellular sites of these processes, however, it has now become evident that astrocytes provide an important and essential contribution to memory formation. Astrocytes participate in the morphological remodeling associated with synaptic plasticity, an energy-demanding process that requires mobilization of glycogen, which, in the CNS, is almost exclusively stored in astrocytes. Synaptic remodeling also involves bidirectional astroglial-neuronal communication supported by astroglial receptors and release of gliosignaling molecules. Astroglia exhibit cytoplasmic excitability that engages second messengers, such as Ca(2+), for phasic, and cyclic adenosine monophosphate (cAMP), for tonic signal coordination with neuronal processes. The detection of signals by astrocytes and the release of gliosignaling molecules, in particular by vesicle-based mechanisms, occurs with a significant delay after stimulation, orders of magnitude longer than that present in stimulus-secretion coupling in neurons. These particular arrangements position astrocytes as integrators ideally tuned to support time-dependent memory formation.
- Published
- 2015
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41. Insulin and Insulin-like Growth Factor 1 (IGF-1) Modulate Cytoplasmic Glucose and Glycogen Levels but Not Glucose Transport across the Membrane in Astrocytes.
- Author
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Muhič M, Vardjan N, Chowdhury HH, Zorec R, and Kreft M
- Subjects
- Animals, Astrocytes cytology, Biological Transport, Active drug effects, Biological Transport, Active physiology, Cell Membrane metabolism, Cells, Cultured, Fluorescence Resonance Energy Transfer, Glucose Transporter Type 4 metabolism, Hypoglycemic Agents metabolism, Insulin metabolism, Insulin-Like Growth Factor I metabolism, Rats, Astrocytes metabolism, Glucose metabolism, Glycogen metabolism, Hypoglycemic Agents pharmacology, Insulin pharmacology, Insulin-Like Growth Factor I pharmacology
- Abstract
Astrocytes contain glycogen, an energy buffer, which can bridge local short term energy requirements in the brain. Glycogen levels reflect a dynamic equilibrium between glycogen synthesis and glycogenolysis. Many factors that include hormones and neuropeptides, such as insulin and insulin-like growth factor 1 (IGF-1) likely modulate glycogen stores in astrocytes, but detailed mechanisms at the cellular level are sparse. We used a glucose nanosensor based on Förster resonance energy transfer to monitor cytosolic glucose concentration with high temporal resolution and a cytochemical approach to determine glycogen stores in single cells. The results show that after glucose depletion, glycogen stores are replenished. Insulin and IGF-1 boost the process of glycogen formation. Although astrocytes appear to express glucose transporter GLUT4, glucose entry across the astrocyte plasma membrane is not affected by insulin. Stimulation of cells with insulin and IGF-1 decreased cytosolic glucose concentration, likely because of elevated glucose utilization for glycogen synthesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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- View/download PDF
42. Pathologic potential of astrocytic vesicle traffic: new targets to treat neurologic diseases?
- Author
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Vardjan N, Verkhratsky A, and Zorec R
- Subjects
- Aquaporins metabolism, Histocompatibility Antigens Class II metabolism, Humans, Nervous System Diseases metabolism, Receptors, G-Protein-Coupled metabolism, Vesicular Glutamate Transport Protein 1 metabolism, Astrocytes metabolism, Nervous System Diseases pathology, Secretory Vesicles physiology
- 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.
- Published
- 2015
- Full Text
- View/download PDF
43. Hyperpolarization-activated cyclic nucleotide-gated channels and cAMP-dependent modulation of exocytosis in cultured rat lactotrophs.
- Author
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Calejo AI, Jorgačevski J, Rituper B, Guček A, Pereira PM, Santos MA, Potokar M, Vardjan N, Kreft M, Gonçalves PP, and Zorec R
- Subjects
- Animals, Calcium Signaling drug effects, Calcium Signaling physiology, Cells, Cultured, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels antagonists & inhibitors, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels genetics, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels physiology, Male, Patch-Clamp Techniques, Potassium Channels genetics, Potassium Channels physiology, Rats, Rats, Wistar, Cyclic AMP physiology, Exocytosis physiology, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels drug effects, Lactotrophs physiology
- Abstract
Hormone and neurotransmitter release from vesicles is mediated by regulated exocytosis, where an aqueous channel-like structure, termed a fusion pore, is formed. It was recently shown that second messenger cAMP modulates the fusion pore, but the detailed mechanisms remain elusive. In this study, we asked whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are activated by cAMP, are involved in the regulation of unitary exocytic events. By using the Western blot technique, a real-time PCR, immunocytochemistry in combination with confocal microscopy, and voltage-clamp measurements of hyperpolarizing currents, we show that HCN channels are present in the plasma membrane and in the membrane of secretory vesicles of isolated rat lactotrophs. Single vesicle membrane capacitance measurements of lactotrophs, where HCN channels were either augmented by transfection or blocked with an HCN channel blocker (ZD7288), show modulated fusion pore properties. We suggest that the changes in local cation concentration, mediated through HCN channels, which are located on or near secretory vesicles, have an important role in modulating exocytosis., (Copyright © 2014 the authors 0270-6474/14/3315638-10$15.00/0.)
- Published
- 2014
- Full Text
- View/download PDF
44. Dynamics of β-adrenergic/cAMP signaling and morphological changes in cultured astrocytes.
- Author
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Vardjan N, Kreft M, and Zorec R
- Subjects
- 1-Methyl-3-isobutylxanthine pharmacology, Adrenergic Agents pharmacology, Animals, Animals, Newborn, Cells, Cultured, Cerebral Cortex cytology, Colforsin pharmacology, Dose-Response Relationship, Drug, Flow Cytometry, Glial Fibrillary Acidic Protein metabolism, Guanine Nucleotide Exchange Factors metabolism, Luminescent Proteins genetics, Luminescent Proteins metabolism, Phosphodiesterase Inhibitors pharmacology, Rats, Rats, Wistar, Signal Transduction drug effects, Time Factors, Astrocytes cytology, Astrocytes metabolism, Cyclic AMP metabolism, Receptors, Adrenergic, beta metabolism, Signal Transduction physiology
- Abstract
The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β-Adrenergic receptors (β-AR), which increase cytosolic cAMP concentration ([cAMP]i ), may affect cell morphology. However, the real-time dynamics of β-AR-mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)-based cAMP biosensor Epac1-camps to study time-dependent changes in [cAMP]i ; morphological changes in primary rat astrocytes were monitored by real-time confocal microscopy. Stimulation of β-AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β-AR, rapidly increased [cAMP]i (∼15 s). The FRET signal response, mediated via β-AR, was faster than in the presence of forskolin (twofold) and dibutyryl-cAMP (>35-fold), which directly activate adenylyl cyclase and Epac1-camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP-dependent processes operate in a slow time domain. Most Epac1-camps expressing astrocytes revealed a morphological change upon β-AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell-shaped dependency on [cAMP]i . The 5-10% decrease in cell cross-sectional area and the 30-50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, β-AR/cAMP-mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease., (Copyright © 2014 Wiley Periodicals, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
45. Regulated Exocytosis in Astrocytes is as Slow as the Metabolic Availability of Gliotransmitters: Focus on Glutamate and ATP.
- Author
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Vardjan N, Kreft M, and Zorec R
- Abstract
It is becoming clear that astrocytes, the most abundant type of glial cells in the mammalian brain, share many properties with neurons. One such property involves vesicles, which play a key role in cell-to-cell signaling. On the one hand, vesicles determine the signaling potential by delivering various receptors and transporters to the plasma membrane by vesicular exocytosis. On the other hand, vesicles are used in astrocytes for the release of vesicle-laden chemical messengers. This chapter compares the properties of Ca(2+)-dependent fusion of the vesicle membrane with the plasma membrane in astrocytes and in neurons, monitored by membrane capacitance techniques. Moreover, we focus on membrane-bound vesicles that store gliotransmitters, glutamate, and adenosine 5'-triphosphate (ATP), to learn why regulated exocytosis in astrocytes is orders of magnitude slower than in neurons and the fact that these signaling molecules are also metabolites. The relatively slow kinetics of regulated exocytosis in astrocytes likely involves vesicle dynamics regulation and mechanisms governing the merger of the vesicle membrane with the plasma membrane, but may also depend on the availability of ATP and glutamate in metabolic pathways for packaging into vesicles via specific vesicle transporters.
- Published
- 2014
- Full Text
- View/download PDF
46. Astrocytic vesicle mobility in health and disease.
- Author
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Potokar M, Vardjan N, Stenovec M, Gabrijel M, Trkov S, Jorgačevski J, Kreft M, and Zorec R
- Subjects
- Animals, Astrocytes cytology, Endocytosis, Humans, Membrane Transport Proteins metabolism, Astrocytes metabolism, Disease, Health, Secretory Vesicles metabolism
- Abstract
Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.
- Published
- 2013
- Full Text
- View/download PDF
47. Neurotoxic phospholipase A2 toxicity model: An insight from mammalian cells.
- Author
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Vardjan N, Mattiazzi M, Rowan EG, Križaj I, Petrovič U, and Petan T
- Abstract
The molecular mechanism of action of presynaptically neurotoxic secreted phospholipases A2 (sPLA2s) has not been fully elucidated. We have recently proposed a model to explain one of the hallmarks of their action - the reduction in endocytosis leading to synaptic vesicle depletion in nerve terminals. Our results speak strongly in favor of a mechanism in which both specific protein-protein interactions and enzymatic activity of the neurotoxic sPLA2 ammodytoxin A (AtxA) are necessary for impairment of clathrin-dependent endocytosis in yeast cells. The reduction of endocytosis was strictly dependent on the enzymatic activity of sPLA2s expressed ectopically in our yeast model cells and was not observed with the catalytically inactive, non-neurotoxic AtxA-homolog, ammodytin L (AtnL). Here we confirm the validity of the model in mammalian cells also, by demonstrating that the enzymatically active mutant of AtnL, shown to inhibit endocytosis in yeast, acts as a presynaptically neurotoxic sPLA2 at the mammalian neuromuscular junction.
- Published
- 2013
- Full Text
- View/download PDF
48. Fusion pores, SNAREs, and exocytosis.
- Author
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Vardjan N, Jorgacevski J, and Zorec R
- Subjects
- Animals, Humans, Secretory Vesicles metabolism, Cell Membrane metabolism, Exocytosis physiology, Membrane Fusion physiology, SNARE Proteins metabolism
- Abstract
Exocytosis is a multistage process involving a merger between the vesicle and the plasma membranes, leading to the formation of a fusion pore, a channel, through which secretions are released from the vesicle to the cell exterior. A stimulus may influence the pore by either dilating it completely (full-fusion exocytosis) or mediating a reversible closure (transient exocytosis). In neurons, these transitions are short-lived and not accessible for experimentation. However, in some neuroendocrine cells, initial fusion pores may reopen several hundred times, indicating their stability. Moreover, these pores are too narrow to pass luminal molecules to the extracellular space, termed release-unproductive. However, on stimulation, their diameter dilates, initiating the release of cargo without de novo fusion pore formation. To explain the stability of the initial narrow fusion pores, anisotropic membrane constituents with non-axisymmetrical shape were proposed to accumulate in the fusion pore membrane. Although the nature of these is unclear, they may consist of lipids and proteins, including SNAREs, which may facilitate and regulate the pre- and post-fusional stages of exocytosis. In the future, a more detailed insight into the molecular control of fusion pore stabilization and regulation will generate a better understanding of fusion pore physiology in health and disease.
- Published
- 2013
- Full Text
- View/download PDF
49. Exocytosis in astrocytes: transmitter release and membrane signal regulation.
- Author
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Guček A, Vardjan N, and Zorec R
- Subjects
- Animals, Calcium metabolism, Cell Membrane metabolism, Humans, Astrocytes metabolism, Exocytosis, Neurotransmitter Agents metabolism, Signal Transduction
- Abstract
Astrocytes, a type of glial cells in the brain, are eukaryotic cells, and a hallmark of these are subcellular organelles, such as secretory vesicles. In neurons vesicles play a key role in signaling. Upon a stimulus-an increase in cytosolic concentration of free Ca(2+) ([Ca(2+)](i))-the membrane of vesicle fuses with the presynaptic plasma membrane, allowing the exit of neurotransmitters into the extracellular space and their diffusion to the postsynaptic receptors. For decades it was thought that such vesicle-based mechanisms of gliotransmitter release were not present in astrocytes. However, in the last 30 years experimental evidence showed that astrocytes are endowed with mechanisms for vesicle- and non-vesicle-based gliotransmitter release mechanisms. The aim of this review is to focus on exocytosis, which may play a role in gliotransmission and also in other forms of cell-to-cell communication, such as the delivery of transporters, ion channels and antigen presenting molecules to the cell surface.
- Published
- 2012
- Full Text
- View/download PDF
50. Fusion pore regulation in peptidergic vesicles.
- Author
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Jorgačevski J, Kreft M, Vardjan N, and Zorec R
- Subjects
- Cell Membrane metabolism, Cholesterol metabolism, Exocytosis physiology, Munc18 Proteins metabolism, Protein Binding, SNARE Proteins metabolism, Secretory Vesicles metabolism, Sphingosine metabolism, Membrane Fusion physiology, Peptides metabolism
- Abstract
Regulated exocytosis, which involves fusion of secretory vesicles with the plasma membrane, is an important mode of communication between cells. In this process, signalling molecules that are stored in secretory vesicles are released into the extracellular space. During the initial stage of fusion, the interior of the vesicle is connected to the exterior of the cell with a narrow, channel-like structure: the fusion pore. It was long believed that the fusion pore is a short-lived intermediate state leading irreversibly to fusion pore dilation. However, recent results show that the diameter of the fusion pore can fluctuate, suggesting that the fusion pore is a subject of stabilization. A possible mechanism is addressed in this article, involving the local anisotropicity of membrane constituents that can stabilize the fusion pore. The molecular nature of such a stable fusion pore to predict how interacting molecules (proteins and/or lipids) mediate changes that affect the stability of the fusion pore and exocytosis is also considered. The fusion pore likely attains stability via multiple mechanisms, which include the shape of the lipid and protein membrane constituents and the interactions between them., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
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