The principal glucose transporter at the brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo, is the Glut1 isoform of the sodium independent glucose transporter gene family. Previous studies have demonstrated an important mode for regulation of BBB Glut1 gene expression is at the post-transcriptional level in pathophysiologic states such as human brain tumors, glucose deprivation/hypoglycemia, development, and cerebral ischemia. Post-transcriptional regulation of mRNA function is mediated via the interaction of cytosolic or polysome proteins with specific cis-elements located within either the 5' - or 3' -untranslated region (UTR) of the Glut1 mRNA. Previous studies have described a polysome protein, designated p44, and a cytosolic protein, designated p88, which interact with two different cis-elements within the 3'-UTR of the glout1 mRNA. The p88 cis-element is associated with an increase in Glut1 gene expression and mediates the stabilization of the Glut1mRNA. The p44 cis-element causes destabilization of the Glut1 transcript, and is associated with an endonuclease/exonuclease activity. The proposed work is comprised of 3 specific aims. Fist, the mechanism of post-transcriptional regulation of Glut1 gene expression will be further defined using a luciferase reporter gene, and cells will also be stably transfected with the Glut1 gene containing selective deletions of th4 putative cis-elements within 3'-UTR. The stably transfected cells will be subjected to hypoxia and glucose deprivation in tissue culture. In parallel, in vivo studies of regional cerebral ischemia using the middle cerebral artery occlusion (MCAO) model as well as global cereal ischemia, using the transient forebrain ischemia (TFI) model, will be performed and correlations will be made between polysome p44, cytosol p88, and Glut1 mRNA levels in whole brain and polysomes, immunoreacitve Glut1 protein, both in brain and at the BBB in vivo. Second, cDNAs encoding the glut1 mRNA UTR-binding proteins will be cloned, sequenced, and analyzed, in parallel with cloning of cDNAs encoding non-glut1 transcripts expressing the cis-acting elements found in the glut1 mRNA 3'-UTR. Third, the secondary structure of the Glut1 mRNA both with and without binding to the p44 and p88 proteins will be determined experimentally using both chemical and enzymatic approaches. These studies will provide insight into molecular mechanisms of regulation of a step crucial to the maintenance of cerebral intermediary metabolism, i.e., the continuous transport of glucose across the blood-brain barrier in vivo.
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