Astrocytes may represent an important target in AIDS neuropathogenesis. Previous studies (Project 1; Volsky) demonstrated that HIV-1 infection of astrocytes, exposure to gp120 or expression of Tat inhibit uptake of the neurotransmitter glutamate. This revised application proposes to elucidate the repertoire of gene expression changes induced as a consequence of infection of astrocytes with HIV-1 or exposure to purified gp120 or Tat and to identify the regulatory sequences governing expression of the GLAST glutamate transporter (GT). This information will provide important insights into the mechanism by which HIV-1 induces AIDS encephalopathy in children and dementia in adults. Moreover, once regulatory molecules and pathways mediating HIV-1-induced astrocyte dysfunction are identified, it may be possible to exploit this information to develop improved intervention strategies to treat AIDS dementia. A sensitive and efficient subtraction hybridization protocol (Jiang & Fisher, 1993) will be used to identify and clone cDNAs displaying altered expression, either induction of suppression, as a function of infection of astrocytes with astrocytotropic HIV-1 strains (A-HIV-1), i.e., viruses that preferentially infect astrocytes rather than T cells or macrophages (Project 1; Volsky). The functional role of specific full-length cDNAs in modulating HIV-1 toxicity in astrocytes will be investigated using transient and stable sense and antisense expression constructs and a novel retroviral-based antisense functional gene knock-out strategy, Rapid Antisense Gene Extinction (RAGE). To fully respond to Reviewer's comments, our research plan has been extensively revised, including addition of a new aim to study regulation of GLAST expression. Specifically, we propose to: (1) construct temporarily spaced subtracted (TSS) cDNA libraries from astrocytes infected with A-HIV-1; (2) use reverse Northern hybridization procedures to identify cDNAs displaying enhanced and reduced expression as a function of infection of astrocytes with A-HIV-1 and treatment with purified gp120 or Tat; (3) isolate full-length cDNAs of genes displaying altered expressions as a function of infection of astrocytes with A-HIV-1 and treatment with purified gp120 or Tat; (4) test the functionality of specific A-HIV-1 modulated genes in regulating astrocyte physiology as a consequence of infection with A-HIV-1 or treatment with purified gp120 or Tat; and (5) identify and characterize the GLAST GT promoter and cis- acting and trans-acting regulatory elements that control expression of this gene. This studies will result in the identification of potentially important genes that correlate with HIV-1 induced neurodegenerative diseases. These genetic reagents and the information obtained will prove useful to other investigators in this program project studying specific aspects of HIV-1 associated encephalopathy.
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