Primary cell culture and drug treatments
Primary cultures of astrocytes from the rat cortex were established and maintained as previously published protocol[20]. Briefly, cortical astrocytes were dissected from Sprague-Dawley (SD) neonatal rats (1-3 days) with the aseptic operation and digested with 0.25% trypsin (Gibco, USA) at 37℃ for 20 min. The cell suspension was dispersed through the cell strainer (75 μm pore size, Bioland, China) and seeded into the poly-D-lysine coated T-75 flasks (Corning, USA) with DMEM/F12 (Gibco, USA) supplemented with 10% FBS, 1% penicillin/streptomycin. After 10 days of incubation, the cells were purified in a shaking incubator (250 r/min) at 37℃ for 24 h, and astrocytes were cultured at desired densities in 6-well plates. The cells were identified as >98% pure astrocytes by Cy3-conjugated GFAP immunostaining (at 1:400 dilution, Abcam, UK). Cultured astrocytes were treated with 150 pM HIV-1 gp120 CM (ProSpec-Tany TechnoGene Ltd., Israel) for 12 h before application of 5 or 25 μM KYNA (Sigma-Aldrich, USA) for a further 12 h. In addition, gp120-induced (150 pM, 11 h) astrocytes were pretreated for 1 h with media in the presence or absence of 10 nM methyllycacontitine (MLA, MCE, USA) or 50 μM AG490 (Selleckchem, USA) followed by a 12 h co-incubation period with KYNA.
Total RNA isolation and quantitative reverse transcription PCR (RT-qPCR) analysis
According to the manufacturer’s instructions, total RNA was extracted from samples using RNAiso Plus Reagent. Reverse transcription was performed following the protocol of the PrimeScript™ RT Master Mix kit. Then, cDNA was run for quantitative PCR using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) kit on QuantStudio 6 and 7 Flex real-time PCR systems (Thermo Fisher, USA). The reagents mentioned above were obtained from Takara (Japan). Primers for RT-qPCR were provided by Sangon Co. (Shanghai, China) and attached to Supplement Table 1-2. The extracellular or plasma levels of Il-1β, Il-6, and Tnf-α were confirmed by ELISA using commercially available kits.
Western Blotting
Clarified cell and tissue lysates were fractionated by SDS-PAGE using 8-15% gradient polyacrylamide gels and transferred onto a PVDF membrane. After blocking with TBST containing 5% non-fat milk (Bio-Rad, USA) for 1 h, the membranes were stained with primary antibodies in the diluted solution overnight at 4 ℃. Primary antibodies used were: anti-MAP2 (1:1000), anti-NeuN (1:1000), anti-Synaptophysin (1:5000), anti-GFAP (1:5000), anti-C3/C3b/C3c (1:1000), anti-CHRNA7 (1:2500), anti-JAK2 (1:500), anti-STAT3 (1:2000), rabbit monoclonal anti-phosphorylated-JAK2 (1:5000, phospho Y1007+Y1008, Abcam, UK), rabbit monoclonal anti-phosphorylated-STAT3 (1:10000, phospho Y705, Abcam, UK), and mouse monoclonal anti-GAPDH (1:10000). Except for the indicated antibodies, other antibodies mentioned above were rabbit polyclonal antibodies purchased from Proteintech (USA). Following incubation with appropriate HRP-conjugated secondary antibodies (1:5000, Bioss, USA) for 1 h at room temperature, the membrane was treated with Clarity Western ECL blotting substrate (Bio-Rad, 1705060, USA). Bands were quantified using Fuji ImageJ software.
Immunolabeling assay for primary astrocytes
Cells were fixed with 100% methanol for 20 min at -20 ℃ and permeabilized with 0.25% Triton X-100 prepared in PBS for 10 min, followed by blockage with mixture buffer (1% w/v BSA, Sigma-Aldrich, USA; 22.52 mg/ml glycine in PBST). The cells were incubated with mouse monoclonal Cy3-conjugated anti-GFAP (1:400, not requiring secondary antibody, Abcam, UK), anti-C3/C3b/C3c (1:500), Alexa Fluor 488-conjugated α-bungarotoxin (5 μg/ml, Thermo Fisher Invitrogen, USA), anti-JAK2 (1:200), anti-STAT3 (1:100) for overnight at 4 ℃, followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500), goat anti-rabbit IgG H&L (Cy5, 1:2500, Abcam, UK) for 2 h in the dark at room temperature. DAPI (4 μg/mL, Thermo Fisher, USA) was used as nuclei stain. Stained cells were examined using a fluorescent microscope (E800 Nikon, Japan) connected to a color digital camera.
Animals and treatment
The HIV-1 gp120 transgenic mice (gp120tg mice, 3 months old, 6 months old, 12 months old, 24 months old; n = 20 per group; 10 females and 10 males) on SJL/BL6/129 background (cross between C57BL/6J female x Sv129 male) expressed gp120 in astrocytes under the control of a modified murine Gfap gene. The α7nAChR knockout mice (α7-/- mice, B6.129S7-Chrna7tm1Bay/J) were purchased from the Jackson Laboratory. The gp120tg mice intercrossed with α7-/- mice to generate α7-/-gp120tg mice. C57BL/6J black mice (12 months old) were used as wild-type controls. Genotypes of all mice were confirmed by PCR analysis of tail DNA with the following primer sequences: gp120 forward, 5’-GCGGGAGAATGATAATGGAG-3’; gp120 reverse, 5’-TATGGGAATTGGCTCAAAGG-3’; α7nAChR forward, 5’-TTCCTGGTCCTGCTGTGTTA-3’; α7nAChR wild-type reverse, 5’-ATCAGATGTTGCTGGCATGA-3’; α7nAChR knockout reverse, 5’-CCCTTTATAGATTCGCCCTTG-3’. All mice were bred in groups of 5 per cage and maintained in a pathogen-free animal facility with free access to autoclaved distilled water and standard chow under a constant temperature of 22 ± 1 ℃ on a 12 h light/dark cycle. For tryptophan (Trp) supplement, 12-month-old mice were randomly divided into 4 groups using the random number generator (GraphPad), including WT+Vehicle, WT+Trp, gp120tg+Vehicle, gp120tg+Trp (n = 20 mice per group; 10 females and 10 males). Mice were fed the control diet supplemented with 0.1% tryptophan (Sigma-Aldrich, USA) dissolved in sterile saline (0.9 % NaCl) for 1 month.
Morris water maze test and open field test
For the MWM test, mice used 4 unique geometric figures providing landmarks in the testing room to locate a submerged platform (1 cm below the white-opaque water surface) in a circular pool (1.2 m in diameter and 76 cm in height). Mice were allowed 60 s to locate the hidden platform in one maze quadrant under a randomized starting position for 4 trails per day over 5 consecutive days. Upon completion of training, mice were allowed to locate the removed platform for 60 s in the retrieval test. Performances were recorded with a camera suspended 250 cm above the center and analyzed by the image tracking system. Mice were evaluated in the open field test of anxiety and exploratory behavior. Briefly, mice were allowed to move unfetteredly in an enclosure and brightly lit square arena (40×40 cm) for 15 minutes on 2 consecutive days. Distance moved and time spent in the central area were videotaped with a near-infrared camera positioned above the center of the arena. The central zone was 32 cm in diameter with 4 cm from the peripheral walls. An effective center entry was not deemed to have occurred until 90% or over of the mouse’s body had entered the designated center.
Histopathology assessment
Upon anesthesia of mice by sodium pentobarbital (60 mg/kg, Sigma-Aldrich, USA), brains were dissected and fixed by immersion in 10% formalin solution overnight at room temperature. For viewing cellular and tissue structure detail, deparaffinized and rehydrated slides were counterstained with Mayers Hematoxylin and alcoholic-Eosin (H&E). In addition, Nissl-stained sections were labeled with 0.1% cresyl violet solution at 37 ℃ for 10 min after deparaffinization and hydration. Next, the slides were differentiated in 95% ethyl alcohol for 5 to 10 min, followed by incubation with 100% alcohol and xylene.
Immunofluorescence and immunohistochemistry
For immunofluorescence, deparaffinized and rehydrated brain sections were stained with anti-MAP2 (1:500), anti-NeuN (1:500), Cy3-conjugated anti-GFAP (1:400), and anti-C3/C3b/C3c (1:500). Following PBS washes, slides were reacted with Alexa Fluor 488-labeled goat anti-rabbit antibody (1:1000) at room temperature for 1 h. The slides were washed and subsequently counterstained with DAPI (1.5 μg/mL, Thermo Fisher, USA). Five sections from the hippocampus were randomly selected, and the numbers of C3+/GFAP+ cells were counted. For immunohistochemistry, slides were reacted with anti-phosphorylated-JAK2 (1:100) and anti-phosphorylated-STAT3 (1:100) antibodies overnight at 4 ℃ in a humidity chamber. After rinsing with PBS, sections were stained with a biotinylated antibody for 60 min and then incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution for 5 min. Subsequently, sections were counterstained with hematoxylin.
Statistical analysis
Graphs and statistical significance are generated by GraphPad Prism 8 (version 8.3.0). The normality and homogeneity of variance were assessed by the Shapiro-Wilk test and Levene’s test, respectively. Data were analyzed using an unpaired Student’s t test (two-tailed) for a two-group comparison. One-way ANOVA followed by post hoc Dunnett’s multiple comparisons was used to compare more than three groups when compared with the one indicated group.