The process of neurotransmission is critically dependent upon a constant supply of glucose. This vital requirement is satisfied, in large measure, by a group of glucose transporters, perhaps the most important of which is Glut 3. Glut 3 is the cell-specific facilitative transporter that is localized to axons and is subject to developmental- and neuronal cell differentiation- linked transcriptional regulation. We hypothesize that Glut 3 is essential in normal central nervous system development and neuronal function. Our long term goal is to understand the mechanisms which regulate the fetal and postnatal expression and site-specific functional activity of this transporter. To this end, we will pursue the following specific aims: First, in the mouse we will establish the site- specific expression of Glut 3 (in comparison with Glut 1, the blood-brain barrier and glial cell glucose transporter isoform) as a function of perinatal and postnatal brain development and correlate this expression with an analysis of localized glucose transport. Next, we will quantitate Glut 3 (in comparison with Glut 1) mRNA abundance, transcriptional rate and protein levels in brain as a function of perinatal and postnatal development. This will be accomplished by a combination of in-situ hybridization, using cloned mouse Glut 3 (and Glut 1) cDNAs, and immunohistochemistry using antibodies generated against Glut 3 (and Glut 1) peptides, followed by Northern, Western and nuclear run-on analyses. Second, we will clone, size, and precisely define the 5'-regulatory regions of the Glut 3 gene. The cloning will be accomplished using a Glut 3 cDNA to screen a mouse genomic library for genomic Glut 3. This will be followed by (1) sequencing, (2) transfection into an SV-T immortalized hippocampal neuronal cell line to determine the minimal sequence necessary to regulate transcription in-vitro and (3) DNA footprinting using brain extracts taken from different stages of development, and neuronal cell extracts from different stages of differentiation, to identify sequences involved in DNA-protein interactions. Our proposed studies will characterize the developmentally-induced transcriptional regulation of neuronal cell- and region-specific Glut 3 expression. These investigations will provide the essential tools necessary for producing Glut 3 overexpressing and Glut 3 deficient mice in the future. The phenotype of these animals will fully test the hypothesis that Glut 3 is essential in normal central nervous system development and neuronal function.
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