Glutamate transport is critical for the synaptic inactivation of glutamate, and failure of this process can be neurotoxic, and may participate in neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Three transporter subtypes have been identified, GLT-1, EAAC1, and GLAST. We intend to combine immunohistochemical, and physiological approaches to study the L-glutamate sodium-coupled neurotransmitter transporter family. We shall prepare specific antibodies to each cloned transporter, and will then use these antibodies to better understand the defect in glutamate transport in ALS. Finally, under controlled culture conditions, we will selectively study each transporter to better understand their role in excitotoxicity.
Aim 1. Localization of Glutamate Transporters - Preparation of selective oligopeptide antibodies to the three rat and human glutamate transporter subtypes. These antibodies will be used as powerful tools to localize glutamate transporter immunoreactive protein in brain and spinal cord at the cellular and ultrastructural level. We hypothesize that GLT-l and GLAST are localized to astroglia, whereas EAAC1 is uniquely located in neurons. Significance: In a selective, yet systematic fashion, we will produce and characterize monospecific antisera to oligopeptides derived from each transporter subtype including EAAC1, GLAST and GLT-1. These reagents will be used to determine the cellular and ultrastructural localization of the various glutamate transporter proteins in rat and human tissue.
Aim 2. Glutamate transporters and ALS - To understand the nature of the glutamate transporter defect in ALS. We hypothesize that the loss of glutamate transport in ALS selective for transport subtypes. Significance: We will use monospecific antibodies to each transporter-subtype to identify the specific transporter defect in AL? These studies will determine the subtype specificity of the disease, as well as localize the defect regionally and ultrastructurally.
Aim 3. Glutamate transporters and chronic neural toxicity - To explore the relationship between individual glutamate transporters and chronic neurotoxicity by selectively blocking the synthesis of individual transporter subtypes, in vitro and in vivo, using antisense oligodeoxynucleotides. In addition, the susceptibility of glutamate transport to oxygen radical mediated insults will be studied in cultured neurons or cells expressing glutamate transporter subtypes. Significance: Chronic neuronal toxicity may involve both direct glutamate injury and oxidative stress. These studies will help dissect the role of neuronal and glia glutamate transporters and determine if a link between oxidative stress and glutamate toxicity could account for neuronal degeneration in ALS.
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