Fig. 4

PPARγ agonists reverse HIV-1_ADA gp120-mediated downregulation of GLT-1 in vitro and in vivo. Primary cultures of rat astrocytes were treated with PPARγ ligands 1 h prior to gp120 (5 nM) exposure for 6 h, and protein expression of GLT-1 was analyzed through immunoblotting (a). Adult Wistar rats were administered IP, 30 min prior to ICV bilateral injection of 4 μg/ventricle HIV-1_ADA gp120 with rosiglitazone (10 mg/kg) or co-administration of rosiglitazone with GW9662 (5 mg/kg). Saline (control) and gp120 (vehicle) animals received the same volume of DMSO/saline 1:10 IP. Hippocampus brain regions were isolated 24 h post ICV; protein expression of GLT-1 was analyzed through immunoblotting (b), and GLT-1 mRNA levels were measured using qPCR (c). For immunoblotting, cell or tissue protein lysate (50 μg) was resolved on a 10% SDS-polyacrylamide gel, transferred to a PVDF membrane. GLT-1 was detected using a rabbit polyclonal antibody (1:1000, dilution). Actin was detected using a mouse monoclonal antibody (1:5000, dilution). Data generated from densitometric analysis is presented as a ratio of GLT-1 expression normalized to actin (loading control). Cyclophilin B was used as the housekeeping gene for qPCR. Results are expressed as mean ± SEM relative to DMSO (control, in vitro) or saline (control, in vivo) of at least 3 separate experiments in vitro and n = 5–7 animals/group in vivo. Asterisks and pound symbol represent data points significantly different from DMSO or saline (control) and gp120 (vehicle) respectively (**p < 0.01, *p < 0.05, #p < 0.05)