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Neuronal activity regulates glutamate transporter dynamics in developing astrocytes.

ABSTRACT: Glutamate transporters (GluTs) maintain a low ambient level of glutamate in the central nervous system (CNS) and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here, we examined the subcellular distribution and dynamic remodeling of the predominant GluT GLT-1 (excitatory amino acid transporter 2, EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. Green fluorescent protein (GFP)-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse three-dimensional confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of GluTs in developing astrocyte membranes and their position relative to synapses.

SUBMITTER: Benediktsson AM

PROVIDER: S-EPMC3232333 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Neuronal activity regulates glutamate transporter dynamics in developing astrocytes.

Benediktsson Adrienne M AM Marrs Glen S GS Tu Jian Cheng JC Worley Paul F PF Rothstein Jeffrey D JD Bergles Dwight E DE Dailey Michael E ME

Glia 20111102 2

Glutamate transporters (GluTs) maintain a low ambient level of glutamate in the central nervous system (CNS) and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here, we examined the subcellular distribution and dynamic remodeling of the predominant GluT GLT-1 (excitatory amino acid transporter 2, EAAT2) in developing hippocampal astrocytes. ...[more]

PMID: 22052455

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Project description:BackgroundChanges in glutamatergic neurotransmission via decreased glutamate transporter (GLT) activity or expression contributes to multiple neurological disorders. Chemokines and their receptors are involved in neurological diseases but the role of chemokines in the expression of glutamate transporters is unclear.MethodsPrimary astrocytes were prepared from neonatal (<24 hours old) SJL/J mouse brains and incubated with 5 μg/ml lipopolysaccharide (LPS) or 50 ng/ml tumor necrosis factor α (TNF-α) for 24 hours. Soluble macrophage inflammatory protein-2γ (MIP-2γ) in culture supernatants was determined using a sandwich ELISA. The MIP-2γ effect on the expression of GLT-1 was measured by quantitative RT-PCR, flow cytometric analysis or western blot assay. Detergent-resistant membranes from astrocytes were isolated on the basis of their ability to float in density gradients. Raft-containing fractions were tracked by the enrichment of caveolin-1 and the dendritic lipid raft marker, flotillin-1. Cell viability was determined by measuring either the leakage of lactate dehydrogenase or the reduction of 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide by viable cells and confirmed by visual inspection.ResultsThe production of the chemokine MIP-2γ by mouse cortical astrocytes increased significantly after stimulation with LPS or TNF-α in vitro. Astrocytes over-expressing MIP-2γ down-regulated the expression of GLT-1 at the mRNA and protein level and caused redistribution of GLT-1 out of the lipid rafts that mediate glutamate uptake. We used pharmacological inhibitors to identify the downstream signaling pathways underlying MIP-2γ activity. We also found complementary results by knocking down MIP-2γ activity in astrocytes with MIP-2γ small interfering RNA (siRNA). MIP-2γ overexpression in astrocytes enhanced the neuronal toxicity of glutamate by decreasing GLT-1 activity, but MIP-2γ itself was not toxic to neurons.ConclusionsThese results suggest that MIP-2γ mediates the pathogenesis of central nervous system disorders associated with neutrophil infiltration in the brain and decreased GLT-1 activity.

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Neuronal activity regulates glutamate transporter dynamics in developing astrocytes.

Publications

Neuronal activity regulates glutamate transporter dynamics in developing astrocytes.

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