ABSTRACT: The neurodegenerative process in HIV encephalitis (HIVE) is associated with extensive damage to the dendritic and synaptic structure that often leads to cognitive impairment. Several mechanisms might be at play, including release of neurotoxins, oxidative stress and decreased activity of neurotrophic factors. Furthermore, HIV-mediated dysregulation of genes involved in neuronal maintenance might play an important role. For this purpose, cRNA was prepared from the brains of 17 AIDS patients for analysis with the Affymetrix Human U95Av2 GeneChip and analyzed with the GeneSpring Expression Analysis Software. Out of 12,625 genes analyzed, 74 were downregulated and 59 were upregulated compared to controls. Initial alternative analysis of RNA was performed by ribonuclease protection assay (RPA). In cases with HIVE, downregulated genes included neuronal molecules involved in synaptic plasticity and transmission (ion channels, synaptogyrin, synapsin II), cell cycle (p35, p39, CDC-L2, CDC42, PAK1) and signaling molecules (PI3K, Ras-Raf-MEK1), transcription factors and cytoskeletal components (MAP-1B, MAP-2, tubulin, adducin-2). Upregulated genes included those involved in neuroimmune (IgG, MHC, ?2microglobulin) and anti-viral responses (interferon-inducible molecules), transcription (STAT1, OLIG2, Pax-6) and signaling modulation (MEK3, EphB1) of the cytoskeleton (myosin, aduccin-3, radixin, dystrobrevin). Taken together, this study suggests that HIV proteins released from infected macrophages might not only induce a neuroinflammatory response, but also may promote neurodegeneration by interfering with neuronal transcription of genes involved in regulating signaling and cytoskeletal molecules important in maintaining synapto-dendritic functioning and integrity. RNA was obtained from brain slabs that are rapidly frozen after removal and stored at ?80 °C for subsequent molecular and biochemical analysis. From each case, a frontal cortex (Broadmann area 46) block (1 mm3) was dissected from the frozen brain slabs. The underlying white matter was dissected out with a blade, thus most of the RNA obtained is isolated from a sample of gray matter. Total RNA (approximately 50 ?g) was purified from 250 mg of this frozen tissue block with TRIzol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol, with an additional centrifugation step to remove unwanted cellular debris. RNA was then further purified using the RNeasy mini kit (Qiagen, Valencia, CA) and the quantity of total RNA was assessed by 260-nm UV absorption, with quality analyzed by gel electrophoresis and analysis of the ribosomal RNA bands. This analysis confirmed that in the samples included for this study, the 18S and 28S bands were clearly identified, indicating that the RNA was well preserved (Fig. 1). For the protocol, 10 ?g of total RNA was utilized per experiment. This was prepared to synthesize cDNA that was then used as a template to produce biotinylated cRNA. Affymetrix Test 2 chips were used to assess RNA quality by examining the 3? to 5? ratios for actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A total of 15 ?g cRNA was prepared in a hybridization cocktail of 300 ml total volume, of which 200 ?l was placed on an Affymetrix Human U95Av2 GeneChip, containing 12,625 independent probe sets to analyze gene expression. After hybridization, this sample was returned to the original aliquot, which was then frozen. For the frontal lobe samples, a further 200 ?l was sampled from the original cocktail and hybridized to a separate duplicate GeneChip within 30 days. Hybridization and subsequent washing and staining were carried out using the Affymetrix Fluidics Station 400 following supplied protocols. Chips were then double stained and scanned according to basic Affymetrix protocol to create GeneChip image files that were converted to text-bases files using Microarray Suite 5.0 software (Affymetrix, Santa Clara, CA). Experiments were repeated at least once to verify the specificity of the results.
