Glutamate and aspartate are the predominant rapid excitatory neurotransmitters in the mammalian central nervous system (CNS), and are involved in several forms of plasticity in the developing and adult nervous system. Excessive activation of EAA receptors contributes to brain damage observed in several acute insults to the CNS, including stroke and head trauma. The extracellular concentrations of these excitatory amino acids (EAAs) are controlled by a family of NA+-dependent high-affinity transporters. We have recently developed evidence that platelet-derived growth factor (PDGF) increases cell surface expression of EAAC1, one of the neuronal glutamate transporters. We have also found that wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreases cell surface expression of this same transporter. The overall goal of this project is to study the mechanisms that control this regulation, and to define the functional consequences of this altered cell surface expression. Based on our preliminary data, these alterations in cell surface expression are due to trafficking between intracellular vesicles and the plasma membrane. We hypothesize that PDGF increases cell surface expression through PI3-K and a serine-threonine kinase called Akt (also known as protein kinase B). We hypothesize that SNAREs are involved in the trafficking to the cell surface, and that dynamin, a GTPase, is important for endocytosis of the transporter. We hypothesize that this regulation is specific for the EAAC1 subtype of transporter, and that chimeras will help to delineate both the mechanisms involved in this regulation and the portions of EAAC1 that govern this regulated trafficking. Finally, we hypothesize that this PDGF-mediated increase in cell surface expression may contribute to its previously documented neuroprotective activity. These hypotheses will be tested using C6 glioma, a model system that only expresses the EAAC1 subtype of transporter and primary neuron-enriched cultures. We propose using a variety of complimentary biochemical, molecular biological, pharmacological, and cell biologic approaches to address these hypotheses. Since EAAC1 is enriched in cortex and hippocampus, two areas that are particularly vulnerable to excitotoxic insults, the proposed studies may provide opportunities to develop new strategies to limit excitotoxic brain damage.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS039011-02
Application #
6188296
Study Section
Special Emphasis Panel (ZRG1-MDCN-4 (01))
Program Officer
Chiu, Arlene Y
Project Start
1999-05-15
Project End
2003-04-30
Budget Start
2000-05-01
Budget End
2001-04-30
Support Year
2
Fiscal Year
2000
Total Cost
$312,345
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Gonzalez, Marco I; Krizman-Genda, Elizabeth; Robinson, Michael B (2007) Caveolin-1 regulates the delivery and endocytosis of the glutamate transporter, excitatory amino acid carrier 1. J Biol Chem 282:29855-65
Sheldon, Amanda L; Robinson, Michael B (2007) The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention. Neurochem Int 51:333-55
Waxman, Elisa A; Baconguis, Isabelle; Lynch, David R et al. (2007) N-methyl-D-aspartate receptor-dependent regulation of the glutamate transporter excitatory amino acid carrier 1. J Biol Chem 282:17594-607
Fournier, Keith M; Robinson, Michael B (2006) A dominant-negative variant of SNAP-23 decreases the cell surface expression of the neuronal glutamate transporter EAAC1 by slowing constitutive delivery. Neurochem Int 48:596-603
Sheldon, Amanda L; Gonzalez, Marco I; Robinson, Michael B (2006) A carboxyl-terminal determinant of the neuronal glutamate transporter, EAAC1, is required for platelet-derived growth factor-dependent trafficking. J Biol Chem 281:4876-86
Krizman-Genda, Elizabeth; Gonzalez, Marco I; Zelenaia, Olga et al. (2005) Evidence that Akt mediates platelet-derived growth factor-dependent increases in activity and surface expression of the neuronal glutamate transporter, EAAC1. Neuropharmacology 49:872-82
Gonzalez, Marco I; Robinson, Michael B (2004) Protein kinase C-dependent remodeling of glutamate transporter function. Mol Interv 4:48-58
Kalandadze, Avtandil; Wu, Ying; Fournier, Keith et al. (2004) Identification of motifs involved in endoplasmic reticulum retention-forward trafficking of the GLT-1 subtype of glutamate transporter. J Neurosci 24:5183-92
Gonzalez, Marco I; Robinson, Michael B (2004) Neurotransmitter transporters: why dance with so many partners? Curr Opin Pharmacol 4:30-5
Fournier, Keith M; Gonzalez, Marco I; Robinson, Michael B (2004) Rapid trafficking of the neuronal glutamate transporter, EAAC1: evidence for distinct trafficking pathways differentially regulated by protein kinase C and platelet-derived growth factor. J Biol Chem 279:34505-13

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