Indeed, a set of elegant experiments by Lipton et al. activity and localization are also affected by treatment with the HIV proteins transactivator of transcription (tat) and glycoprotein 120 (gp120) (Chen et al., 2013; Kim et al., 2013; Bae et al., 2014). However, it remains unclear what role BACE1 plays in HIV-associated neurotoxicity and neuropathogenesis. Macrophages sustain productive viral contamination in HIV patient brains (Koenig et al., 1986; Petito et al., 1986) and infected macrophages may mediate HIV-associated neurotoxicity by secreting factors that include viral proteins, chemokines, and glutamate (Kaul, 2008). Glutamate release in particular has been linked to neuronal damage and cognitive dysfunction in HIV both and (Jiang et al., 2001; Zink et al., 2002). Similarly to AD pathology (Mehta et al., 2013), evidence suggests that glutamate may cause neuronal damage in HIV through NMDAR-dependent mechanisms of excitotoxicity (Giulian et al., 1990; Chen et al., 2002; O’Donnell et al., 2006). Therefore, we used a previously developed and well characterized model of HIV-associated neurotoxicity (Chen et al., 2002; O’Donnell et al., 2006) in which cultured rat neurons are exposed to supernatants collected from HIV-infected human monocyte-derived macrophages (HIV/MDMs). In this model, neurotoxic injury induced by HIV/MDM supernatants is usually entirely dependent on NMDAR activation (Giulian et al., 1990; Jiang et al., 2001; Chen et al., 2002; O’Donnell et al., 2006). Based on the similarities observed thus far between AD and HANDs in relation to amyloid metabolism (Ortega and Ances, 2014), we hypothesized that neurotoxicity induced by HIV/MDM supernatants is dependent upon NMDAR-mediated upregulation of BACE1 and a resultant increase in amyloidogenic APP processing. To address the potential clinical relevance of this mechanism, we also CMPDA hypothesized that A oligomers and BACE1 protein levels are increased in HANDs individual brains. Materials and Methods Chemicals and reagents. The following antibodies were used in this study: -site amyloid precursor protein cleaving enzyme 1 (BACE1; catalog #5606S RRID:AB_1903900), presenilin 1 (PS1; catalog #5643S RRID:AB_10706356), and -actin (catalog #3700 also 3700P, 3700S RRID:AB_2242334) (all from Cell Signaling Technology); binding Ig protein (BiP; catalog #610978 RRID:AB_398291; BD Transduction Laboratories; APP (catalog #ab32136 RRID:AB_2289606), a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10; catalog #ab1997 RRID:AB_302747), and microtubule-associated protein 2 (MAP2; catalog #ab5392 RRID:AB_2138153) (all from Abcam); actin (catalog #A2066 RRID:AB_476693; Sigma Aldrich); and MAP2 (catalog #801801 RRID:AB_2564643; BioLegend. The mouse monoclonal antibody against BACE1 (3d5) was developed by Dr. Robert Neurod1 Vassar (Feinberg School of Medicine, Northwestern University or college, Chicago, IL). The antibody against A-oligomers (Nab61) was kindly provided by Dr. CMPDA Virginia Lee (The Perelman School of Medicine, University or college of Pennsylvania, Philadelphia, PA). The following chemical reagents were used: DAPI (Citifluor); DMEM, neurobasal medium, and B27 product (all from Invitrogen); Bradford protein assay dye, polyvinylidene fluoride (PVDF) membrane, and prestained broad range molecular excess weight ladder (all from Bio-Rad); Tween 20, Triton X-100, Fast Green FCF, protease inhibitor combination, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and cytosine -d-arabinofuranoside hydrochloride (AraC) (all from Sigma Aldrich); poly-l-lysine (Peptides International); normal antibody diluent (NAD; Scytek Laboratories); HBSS, trypsin, and GlutaMAX (all from Thermo Fisher Scientific); Luminata Classico ECL and -secretase inhibitor (BSI) II and IV (all from Millipore); and amino-5-phosphonovaleric acid (AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), dizocilpine (MK-801), and MRK 560 (MRK) (all from Tocris Bioscience). All HRP-conjugated secondary antibodies were obtained from Thermo Fisher Scientific and all fluorescent dye-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. Preparation of main rat cortical neuron cultures. CMPDA Main rat cortical cultures were prepared from embryonic day 18 Sprague Dawley rat embryos (Charles River Laboratories, RRID:RGD_734476). Brains were isolated and dissected cortices were incubated for 40 min in DMEM and 0.027% trypsin as described previously (Wilcox et al., 1994). Cells were then washed in saline, triturated, resuspended in neurobasal medium supplemented with B27, and plated on poly-l-lysine-coated 6-well (9.4 cm2 growth area) or 24-well (1.9 cm2 growth area) plates (USA Scientific) at a concentration of 750,000 cells/ml. After 48 h, cells were treated.
