Proteome Analysis and Conditional Deletion of the EAAT2 Glutamate Transporter Provide Evidence against a Role of EAAT2 in Pancreatic Insulin Secretion in Mice

Zhou, Yun; Waanders, Leonie F.; Holmseth, Silvia; Guo, Caiying; Berger, Urs V.; Li, Yuchuan; Lehre, Anne-Catherine; Lehre, Knut P.; Danbolt, Niels Christian

doi:10.1074/jbc.m113.529065

Cited by 46 publications

(53 citation statements)

References 97 publications

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“…In agreement, deletion of the EAAT2 gene in mice caused an almost complete loss (about 95 %) of glutamate uptake activity (Tanaka et al, 1997;Otis and Kavanaugh, 2000;Matsugami et al, 2006;Kiryk et al, 2008;Holmseth et al, 2012b;Zhou et al, 2014b).…”

Section: Eaat2mentioning

confidence: 66%

“…A selective deletion of EAAT2 in the brain is sufficient to reproduce this phenotype confirming that EAAT2 plays its most important role in the brain (Zhou et al, 2014b). The heterozygote EAAT2 knockout mice (+/-) have only half the EAAT2-concentrations as wildtype mice, but do not show any apparent morphological brain abnormalities (Kiryk et al, 2008).…”

Section: Eaat2mentioning

confidence: 91%

“…Antibodies became readily available and a large number of papers were published. Unfortunately, not all antibodies and procedures were validated well enough (for discussion see: Holmseth et al, 2005;Holmseth et al, 2006;Rhodes and Trimmer, 2006;Lorincz and Nusser, 2008;Couchman, 2009;Kalyuzhny, 2009;Holmseth et al, 2012a;Zhou et al, 2014b).…”

Section: Cellular Distribution Of Glutamate Transportersmentioning

confidence: 99%

“…Three different research teams generated, independently of each other, their own flox-EAAT2 mice (Zhou et al, 2014b;Petr et al, 2015;Aida et al, 2015). The first of these three mouse lines (Zhou et al, 2014b) is already available from Jackson Laboratory (JAX Stock No.…”

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

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Neuronal vs glial glutamate uptake: Resolving the conundrum

Danbolt

Furness

Zhou

2016

Neurochemistry International

186

152

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Neither normal brain function nor the pathological processes involved in neurological diseases can be adequately understood without knowledge of the release, uptake and metabolism of glutamate. The reason for this is that glutamate (a) is the most abundant amino acid in the brain, (b) is at the cross-roads between several metabolic pathways, and (c) serves as the major excitatory neurotransmitter. In fact most brain cells express glutamate receptors and are thereby influenced by extracellular glutamate. In agreement, brain cells have powerful uptake systems that constantly remove glutamate from the extracellular fluid and thereby limit receptor activation. It has been clear since the 1970s that both astrocytes and neurons express glutamate transporters. However, the relative contribution of neuronal and glial transporters to the total glutamate uptake activity, however, as well as their functional importance, has been hotly debated ever since. The present short review provides (a) an overview of what we know about neuronal glutamate uptake as well as an historical description of how we got there, and (b) a hypothesis reconciling apparently contradicting observations thereby possibly resolving the paradox. ABBREVIATIONSCre, Cyclization recombinase (Le and Sauer, 2000); EAAC1, glutamate transporter (EAAT3; slc1a1; Kanai and Hediger, 1992; Bjørås et al., 1996); EAAT, excitatory amino acid transporter (synonym to glutamate transporter); GABA, γ-aminobutyric acid; GLAST, rat glutamate transporter (EAAT1; slc1a3; Storck et al., 1992;Tanaka, 1993a); GLT-1, rat glutamate transporter (EAAT2; slc1a2; Pines et al., 1992); GLUL, glutamine synthetase; TBOA, DL-threo-β-benzyloxyaspartate AbstractNeither normal brain function nor the pathological processes involved in neurological diseases can be adequately understood without knowledge of the release, uptake and metabolism of glutamate. The reason for this is that glutamate (a) is the most abundant amino acid in the brain, (b) is at the cross-roads between several metabolic pathways, and (c) serves as the major excitatory neurotransmitter. In fact most brain cells express glutamate receptors and are thereby influenced by extracellular glutamate. In agreement, brain cells have powerful uptake systems that constantly remove glutamate from the extracellular fluid and thereby limit receptor activation. It has been clear since the 1970s that astrocytes and neurons express glutamate transporters. The relative contribution of neuronal and glial transporters to the total glutamate uptake activity, however, as well as their functional importance, has been hotly debated ever since. The present short review provides (a) an overview of what we know about neuronal glutamate uptake as well as an historical description of how we got there, and (b) an hypothesis reconciling apparently contradicting observations thereby possibly resolving the paradox.

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Section: Eaat2mentioning

confidence: 66%

Section: Eaat2mentioning

confidence: 91%

Section: Cellular Distribution Of Glutamate Transportersmentioning

confidence: 99%

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

See 2 more Smart Citations

Neuronal vs glial glutamate uptake: Resolving the conundrum

Danbolt

Furness

Zhou

2016

Neurochemistry International

186

152

View full text Add to dashboard Cite

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“…This cross-reactive protein is present in both wildtype mice and EAAT2 knockout mice. EAAT2 protein itself is not detectable in young adult mouse pancreas (Zhou et al, 2014b). The identity of the cross-reacting antigens is unknown, but it is interesting that islet cells contain several proteins that often give rise to autoantibodies (Arvan et al, 2012).…”

Section: Specificity Testing By Immunoblottingmentioning

confidence: 99%

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

Self Cite

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Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control. GLIA 2016;64:2045–2064

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Maintaining the presynaptic glutamate supply for excitatory neurotransmission

Marx

Billups

2015

J of Neuroscience Research

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Glutamate released from synapses during excitatory neurotransmission must be rapidly recycled to maintain neuronal communication. This review evaluates data from physiological experiments at hippocampal CA3 to CA1 synapses and the calyx of Held synapse in the brainstem to analyze quantitatively the rates of release and resupply of glutamate required to sustain neurotransmission. We calculate that, without efficient recycling, the presynaptic glutamate supply will be exhausted within about a minute of normal synaptic activity. We also discuss replenishment of the presynaptic pool by diffusion from the soma, direct uptake of glutamate back into the presynaptic terminal, and uptake of glutamate precursor molecules. Diffusion of glutamate from the soma is calculated to be fast enough to resupply presynaptic glutamate in the hippocampus but not at the calyx of Held. However, because the somatic cytoplasm will also quickly run out of glutamate and synapses can function continually even if the presynaptic axon is severed, mechanisms other than diffusion must be present to resupply glutamate for release. Direct presynaptic uptake of glutamate is not present at the calyx of Held but may play a role in glutamate recycling in the hippocampus. Alternatively, glutamine or tricarboxylic acid cycle intermediates released from glia can serve as a precursor for glutamate in synaptic terminals, and we calculate that the magnitude of presynaptic glutamine uptake is sufficient to supply enough glutamate to sustain neurotransmission. The nature of these mechanisms, their relative abundance, and the co-ordination between them remain areas of intensive investigation.

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Proteome Analysis and Conditional Deletion of the EAAT2 Glutamate Transporter Provide Evidence against a Role of EAAT2 in Pancreatic Insulin Secretion in Mice

Cited by 46 publications

References 97 publications

Neuronal vs glial glutamate uptake: Resolving the conundrum

Neuronal vs glial glutamate uptake: Resolving the conundrum

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Maintaining the presynaptic glutamate supply for excitatory neurotransmission

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