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1.
Int J Mol Sci ; 21(24)2020 Dec 17.
Article in English | MEDLINE | ID: mdl-33348528

ABSTRACT

The astrocytic glutamate transporters excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2) play a key role in nervous system function to maintain extracellular glutamate levels at low levels. In physiology, this is essential for the rapid uptake of synaptically released glutamate, maintaining the temporal fidelity of synaptic transmission. However, EAAT1/2 hypo-expression or hypo-function are implicated in several disorders, including epilepsy and neurodegenerative diseases, as well as being observed naturally with aging. This not only disrupts synaptic information transmission, but in extremis leads to extracellular glutamate accumulation and excitotoxicity. A key facet of EAAT1/2 expression in astrocytes is a requirement for signals from other brain cell types in order to maintain their expression. Recent evidence has shown a prominent role for contact-dependent neuron-to-astrocyte and/or endothelial cell-to-astrocyte Notch signalling for inducing and maintaining the expression of these astrocytic glutamate transporters. The relevance of this non-cell-autonomous dependence to age- and neurodegenerative disease-associated decline in astrocytic EAAT expression is discussed, plus the implications for disease progression and putative therapeutic strategies.


Subject(s)
Aging/metabolism , Astrocytes/metabolism , Excitatory Amino Acid Transporter 1/metabolism , Excitatory Amino Acid Transporter 2/metabolism , Glutamic Acid/metabolism , Animals , Biological Transport , Humans , Neurodegenerative Diseases/metabolism , Neurons/metabolism , Receptors, Notch/metabolism , Signal Transduction
2.
Int J Mol Sci ; 19(4)2018 Mar 21.
Article in English | MEDLINE | ID: mdl-29561757

ABSTRACT

Astrocytes are glial cells that have an intimate physical and functional association with synapses in the brain. One of their main roles is to recycle the neurotransmitters glutamate and gamma-aminobutyric acid (GABA), as a component of the glutamate/GABA-glutamine cycle. They perform this function by sequestering neurotransmitters and releasing glutamine via the neutral amino acid transporter SNAT3. In this way, astrocytes regulate the availability of neurotransmitters and subsequently influence synaptic function. Since many plasma membrane transporters are regulated by protein kinase C (PKC), the aim of this study was to understand how PKC influences SNAT3 glutamine transport in astrocytes located immediately adjacent to synapses. We studied SNAT3 transport by whole-cell patch-clamping and fluorescence pH imaging of single astrocytes in acutely isolated brainstem slices, adjacent to the calyx of the Held synapse. Activation of SNAT3-mediated glutamine transport in these astrocytes was reduced to 77 ± 6% when PKC was activated with phorbol 12-myristate 13-acetate (PMA). This effect was very rapid (within ~20 min) and eliminated by application of bisindolylmaleimide I (Bis I) or 7-hydroxystaurosporine (UCN-01), suggesting that activation of conventional isoforms of PKC reduces SNAT3 function. In addition, cell surface biotinylation experiments in these brain slices show that the amount of SNAT3 in the plasma membrane is reduced by a comparable amount (to 68 ± 5%) upon activation of PKC. This indicates a role for PKC in dynamically controlling the trafficking of SNAT3 transporters in astrocytes in situ. These data demonstrate that PKC rapidly regulates the astrocytic glutamine release mechanism, which would influence the glutamine availability for adjacent synapses and control levels of neurotransmission.


Subject(s)
Amino Acid Transport Systems, Neutral/metabolism , Astrocytes/metabolism , Protein Kinase C/metabolism , Synapses/metabolism , Animals , Brain/metabolism , Endocytosis , Enzyme Activation , Isoenzymes/metabolism , Mice, Inbred C57BL , Rats, Wistar
4.
Nat Commun ; 8: 15132, 2017 05 02.
Article in English | MEDLINE | ID: mdl-28462931

ABSTRACT

The influence that neurons exert on astrocytic function is poorly understood. To investigate this, we first developed a system combining cortical neurons and astrocytes from closely related species, followed by RNA-seq and in silico species separation. This approach uncovers a wide programme of neuron-induced astrocytic gene expression, involving Notch signalling, which drives and maintains astrocytic maturity and neurotransmitter uptake function, is conserved in human development, and is disrupted by neurodegeneration. Separately, hundreds of astrocytic genes are acutely regulated by synaptic activity via mechanisms involving cAMP/PKA-dependent CREB activation. This includes the coordinated activity-dependent upregulation of major astrocytic components of the astrocyte-neuron lactate shuttle, leading to a CREB-dependent increase in astrocytic glucose metabolism and elevated lactate export. Moreover, the groups of astrocytic genes induced by neurons or neuronal activity both show age-dependent decline in humans. Thus, neurons and neuronal activity regulate the astrocytic transcriptome with the potential to shape astrocyte-neuron metabolic cooperation.


Subject(s)
Astrocytes/metabolism , Cerebral Cortex/metabolism , Gene Expression Regulation, Developmental , Neurons/metabolism , Tauopathies/genetics , Animals , Astrocytes/cytology , CREB-Binding Protein/genetics , CREB-Binding Protein/metabolism , Cell Communication , Cerebral Cortex/cytology , Cerebral Cortex/growth & development , Coculture Techniques , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Embryo, Mammalian , Gene Expression Profiling , Glucose/metabolism , High-Throughput Nucleotide Sequencing , Humans , Lactic Acid/metabolism , Membrane Potentials/physiology , Mice, Knockout , Neurons/cytology , Rats, Sprague-Dawley , Receptors, Notch/genetics , Receptors, Notch/metabolism , Signal Transduction , Tauopathies/metabolism , Tauopathies/pathology
5.
Glia ; 65(6): 900-916, 2017 06.
Article in English | MEDLINE | ID: mdl-28272791

ABSTRACT

The release of glutamine from astrocytes adjacent to synapses in the central nervous system is thought to play a vital role in the mechanism of glutamate recycling and is therefore important for maintaining excitatory neurotransmission. Here we investigate the nature of astrocytic membrane transport of glutamine in rat brainstem slices, using electrophysiological recording and fluorescent imaging of pHi and Nai+. Glutamine application to perisynaptic astrocytes induced a membrane current, caused by activation of system A (SA) family transporters. A significant electroneutral component was also observed, which was mediated by the system N (SN) family transporters. This response was stimulated by glutamine (KM of 1.57 mM), histidine, and asparagine, but not by leucine or serine, indicating activation of the SNAT3 isoform of SN. We hypothesized that increasing the [Na+ ]i would alter the SNAT3 transporter equilibrium, thereby stimulating glutamine release. In support of this hypothesis, we show that SNAT3 transport can be driven by changing cation concentration and that manipulations to raise [Na+ ]i (activation of excitatory amino acid transporters (EAATs), SA transporters or AMPA receptors) all directly influence SNAT3 transport rate. A kinetic model of glutamine fluxes is presented, which shows that EAAT activation causes the release of glutamine, driven mainly by the increased [Na+ ]i . These data demonstrate that SNAT3 is functionally active in perisynaptic astrocytes in situ. As a result, astrocytic Nai+ signaling, as would be stimulated by neighboring synaptic activity, has the capacity to stimulate astrocytic glutamine release to support glutamate recycling.


Subject(s)
Amino Acid Transport Systems, Neutral/metabolism , Astrocytes/metabolism , Glutamine/metabolism , Intracellular Space/metabolism , Sodium/metabolism , Synapses/metabolism , Amino Acid Transport System A/metabolism , Animals , Astrocytes/drug effects , Brain Stem/drug effects , Brain Stem/metabolism , Cations, Monovalent/metabolism , Female , Glutamate Plasma Membrane Transport Proteins/metabolism , Hydrogen-Ion Concentration , Intracellular Space/drug effects , Kinetics , Lithium/metabolism , Male , Models, Neurological , Rats, Wistar , Receptors, AMPA/metabolism , Synapses/drug effects , Tissue Culture Techniques
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