Astrocytes regulate many important functions in the brain, including processes that influence the physiological activity and survival of neurons. Recent studies indicate that astrocytes may also represent a significant target for HIV-1 induced dementia (HAD). The broad aims of this program project grant (PPG) are to mechanistically understand how HIV-1 infection alters astroctye functions thereby contributing to neuropathogenesis. It is hypothesized that HIV-1 infection or exposure to HIV-1 viral glycoprotein (gp120) results in alterations in gene expression resulting in decreased glutamate transport, thereby resulting in accumulation of glutamate adjacent to neurons culminating in excitotoxicity and neuronal toxicity. Using a sensitive and efficient subtraction hybridization approach, rapid subtraction hybridization (RASH), genes displaying enhanced expression in astrocytes, astrocyte elevated genes (AEGs), and decreased expression In astrocytes, astrocyte suppressed genes (ASGs), following HIV-1 infection or treatment with gp120 have been cloned during the prior funding cycle. Based on structure, chromosomal location and preliminary functional studies, one novel astrocyte gone that displays enhanced expression after HIV-1 infection, AEG-1, may play a prominent role in intracellular signal transduction pathways (functioning as a membrane associated G-protein) affecting normal astrocyte physiology and development of malignant gliomas. Using a sequential progressive genemic scanning (SPGS) approach the promoter region of excitatory amino acid transporter 2 (EAAT2), the primary regulator of glutamate transport in astrocytes, was cloned. Infection of primary human fetal astrocytes (PHFA) with HIV-1 or treatment with gp120 or the neurotoxic agent tumor necrosis factor-alpha (TNF-alpha) results in induction of AEG-1 and downregulation of EAAT2 promoter activity, mRNA, protein and glutamate transport. AEG-1 decreases EAAT2 promoter activity when transiently expressed in PHFA and in most malignant gliomas AEG-1 expression is elevated and EAAT2 promoter activity is low. These findings suggest an inverse correlation between EAAT2 promoter activity and AEG-1 expression in PHFA and malignant gliomas. This component of the PPG will dissect the roles of EAAT2 and AEG-1 in astrocyte dysfunction potentially resulting in HAD and malignant glioma pathogenesis. Experiments will: (1) molecularly characterize the EAAT2 promoter and determine its relationship to neuropathogenesis; (2) define the biochemical and functional properties of AEG-1 and its biological rote in normal astrocyte and malignant glioma development and progression; and (3) determine the functional role of AEG-1 on EAAT2 promoter activity in astrocytes and in malignant glioma development and pathogenesis. To provide insights into the role of AEG-1 on EAAT2 activity, response to HIV-1 infection and in malignant glioma development, a transgenic mouse will be constructed in which AEG-1 expression is targeted in brain astrocytes using the GFAP promoter. The current project will provide significant insights into the mechanism of glutamate transport regulation in normal astrocytes and in malignant gliomas, directed through the study of a novel HIV-1/gp120/TNF-alpha inducible gone, AEG-1. These studies offer a unique opportunity to understand how HIV-1 infection of astrocytes contributes to HAD and the role of aberrant signal transduction pathways in mediating changes in glutamate transport and neuropathogenesis. This information could provide the basis for developing rational approaches for inhibiting or preventing pathogenesis associated with HIV-1 infection and neurotoxic agents, such as TNF-alpha.
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