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MRNA for the EAAC1 subtype of glutamate transporter is present in neuronal dendrites in vitro and dramatically increases in vivo after a seizure.

ABSTRACT: The neuronal Na(+)-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is not known if mRNA for EAAC1 may also be targeted to dendrites. Sorting of mRNA to specific cellular domains provides a mechanism by which signals can rapidly increase translation in a local environment; this form of regulated translation has been linked to diverse biological phenomena ranging from establishment of polarity during embryogenesis to synapse development and synaptic plasticity. In the present study, EAAC1 mRNA sequences were amplified from dendritic samples that were mechanically harvested from low-density hippocampal neuronal cultures. In parallel analyses, mRNA for histone deacetylase 2 (HDAC-2) and glial fibrillary acidic protein (GFAP) was not detected, suggesting that these samples are not contaminated with cell body or glial mRNAs. EAAC1 mRNA also co-localized with Map2a (a marker of dendrites) but not Tau1 (a marker of axons) in hippocampal neuronal cultures by in situ hybridization. In control rats, EAAC1 mRNA was observed in soma and proximal dendrites of hippocampal pyramidal neurons. Following pilocarpine- or kainate-induced seizures, EAAC1 mRNA was present in CA1 pyramidal cell dendrites up to 200?m from the soma. These studies provide the first evidence that EAAC1 mRNA localizes to dendrites and suggest that dendritic targeting of EAAC1 mRNA is increased by seizure activity and may be regulated by neuronal activity/depolarization.

SUBMITTER: Ross JR

PROVIDER: S-EPMC3040252 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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mRNA for the EAAC1 subtype of glutamate transporter is present in neuronal dendrites in vitro and dramatically increases in vivo after a seizure.

Ross John R JR Porter Brenda E BE Buckley Peter T PT Eberwine James H JH Robinson Michael B MB

Neurochemistry international 20101224 3

The neuronal Na(+)-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is ...[more]

PMID: 21185901

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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.

Project description:Posttraumatic seizures (PTS) commonly occur following severe traumatic brain injury (sTBI). Risk factors for PTS have been identified, but variability in who develops PTS remains. Excitotoxicity may influence epileptogenesis following sTBI. Glutamate transporters manage glutamate levels and excitatory neurotransmission, and they have been associated with both epilepsy and TBI. Therefore, we aimed to determine if genetic variation in neuronal glutamate transporter genes is associated with accelerated epileptogenesis and increased PTS risk after sTBI.Individuals (N = 253) 18-75 years of age with sTBI were assessed for genetic relationships with PTS. Single nucleotide polymorphisms (SNPs) within SLC1A1 and SLC1A6 were assayed. Kaplan-Meier estimates and log-rank statistics were used to compare seizure rates from injury to 3 years postinjury for SNPs by genotype. Hazard ratios (HRs) were estimated using Cox proportional hazards regression for SNPs significant in Kaplan-Meier analyses adjusting for known PTS risk factors.Thirty-two tagging SNPs were examined (SLC1A1: n = 28, SLC1A6: n = 4). Forty-nine subjects (19.37%) had PTS. Of these, 18 (36.7%) seized within 7 days, and 31 (63.3%) seized between 8 days and 3 years post-TBI. With correction for multiple comparisons, genotypes at SNP rs10974620 (SLC1A1) were significantly associated with time to first seizure across the full 3-year follow-up (seizure rates: 77.1% minor allele homozygotes, 24.8% heterozygotes, 16.6% major allele homozygotes; p = 0.001). When seizure follow-up began day 2 postinjury, genotypes at SNP rs7858819 (SLC1A1) were significantly associated with PTS risk (seizure rates: 52.7% minor allele homozygotes, 11.8% heterozygotes, 21.1% major allele homozygotes; p = 0.002). After adjusting for covariates, we found that rs10974620 remained significant (p = 0.017, minor allele versus major allele homozygotes HR 3.4, 95% confidence interval [CI] 1.3-9.3). rs7858819 also remained significant in adjusted models (p = 0.023, minor allele versus major allele homozygotes HR 3.4, 95%CI 1.1-10.5).Variations within SLC1A1 are associated with risk of epileptogenesis following sTBI. Future studies need to confirm findings, but variation within neuronal glutamate transporter genes may represent a possible pharmaceutical target for PTS prevention and treatment.

Dataset Information

MRNA for the EAAC1 subtype of glutamate transporter is present in neuronal dendrites in vitro and dramatically increases in vivo after a seizure.

Publications

mRNA for the EAAC1 subtype of glutamate transporter is present in neuronal dendrites in vitro and dramatically increases in vivo after a seizure.

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