METH increases the generation of ROS in rhNPC.

<p>Neurospheres derived from rhNPC were digested and cells were plated on poly-D-lysine-coated cover slips. Cells were treated with 300 µM METH for 24 hours or pre-treated with 10 mM NAC for 30 min, then treated with 300 µM METH for 24 hours. Cells were then subjected to mitochondrial ROS staining with MitoTracker® CM-H₂ROS (a reduced, nonfluorescent dye that fluoresces upon oxidation) (a2-d2, red), immunostaining with mouse nestin monoclonal antibody (a1-d1, green) and nucleus staining with DAPI (a3-d3, blue) and visualized by Zeiss Axiovert microscope. Cells were treated with 200 µM H₂O₂ for 30 min as a positive control (e1-f4).</p

METH-induced apoptosis of rhNPC is accompanied by mitochondrial fragmentation.

<p>Neurospheres were digested and plated on poly-D-lysine-coated cover slips. After 24 hours rhNPC were treated with METH at indicated concentrations for another 24 hours. (A) NPC were stained with MitoTracker®Red CMXRos dye (red) and fixed with 3.7% formaldehyde. After permeabilization with 0.2% Triton X-100 in PBS at 4°C for 10 min, cells were subjected to immunostaining with monoclonal nestin antibody and nuclei were stained with DAPI (blue), then visualized by Zeiss Axiovert microscope. (B) Apoptotic nuclei, showing highly condensed and fragmented chromatin, as indicated with arrows in A-b2 and A-c2. The results are expressed as average±SD of triplicate samples. # denotes <i>p</i><0.05 in comparison to control; ## denotes <i>p</i><0.001 compared with control. (C) Cells were analyzed for mitochondrial morphology by fluorescence microscope using MitoTracker®Red. Typical mitochondrial phenotypes (A-a1) and fragmented mitochondria (A-b1 and A-c1) were quantified by counting cell number with or without fragmented mitochondria. # denotes <i>p</i><0.01 in comparison to control; ## denotes <i>p</i><0.001 compared with control.</p

Isolation and differentiation of rhNPC.

<p>rhNPC were dissociated from rat hippocampi and cultured with NPBM neural progenitor basal medium containing EGF, bFGF and NSF-1. The neurospheres were formed after culture for 7 days (Fig. 1A). Neurospheres were digested and plated on poly-D-lysine-coated cover slips and cultured with astrocyte differentiation medium or neuron differentiation medium for 7 days and then subjected to immunostaining with nestin (Fig. 1B), GFAP (Fig. 1C) and β-tubulin III (Fig. 1D) antibodies. rhNPC were cultured in six-well plates with differentiation medium for 3, 5 and 7 days, respectively, then subjected to western blotting with GFAP and β-tubulin III antibodies (Fig. 1E, 1F).</p

METH induces mitochondrial fragmentation in rhNPC in a time-dependent manner.

<p>Neurospheres were digested and plated on poly-D-lysine-coated cover slips. After 24 hours NPC were treated with 300 µM METH for the indicated times and then subjected to mitochondrial staining with MitoTracker®Red (upper panel) and immunostaining with nestin monoclonal antibody (green) and nuclear staining with DAPI (blue) and visualized by Zeiss Axiovert microscope.</p

METH has no effect on Ca²⁺ influx or glutamate-mediated Ca²⁺ influx in rhNPC.

<p>rhNPC cultured on poly-D-lysine-coated cover slips (25 mm), were loaded with Fura-2 and monitored for calcium influx by micro-fluorescent imaging. (A) Different concentrations of METH were tested and calcium influx was measured. METH concentrations were 1: 50 µM; 2: 100 µM; 3: 500 µM; 4: 1 mM; 5: 5 mM; 6:100 µM glutamate as a positive control. (B) METH has no effect on glutamate-mediated AMPA receptor responses; a: glutamate (100 µM); b: glutamate (100 µM)+cyclothiazide (10 µM); c: glutamate (100 µM)+cyclothiazide (10 µM)+CNQX (10 µM); d: glutamate (100 µM)+500 µM METH+cyclothiazide (10 µM).</p

Human GLS1 promoter construct and sequence analysis.

<p>(A). Schematic diagram of the human GLS1 promoter-driven luciferase reporter construct. The construct contains the Firefly luciferase coding sequence (black) with regions of the promoter (blank) and the 5' UTR (shaded). The restriction enzymes used for cloning are shown. (B). Sequence analysis of the human GLS1 promoter. The TSS is designated as +1. The 5' UTR is shown in gray. The coding sequence is underlined. Putative transcription factor binding sites along the promoter sequence are highlighted in different colors, with their corresponding transcription factors shown on top of the sequence. Highlighted in red: TATA box, dark green: CAAT box, pink: AP-1, blue: SP-1, bright green: STAT, grey: 5' UTR glutaminase; font color in blue: NF-1.</p

IFN-α specifically activates human GLS1 promoter.

<p>HEK 293T cells were co-transfected with the human GLS1 promoter construct and pRL-SV40. 24 hours later, the cells were treated with either various individual cytokines for another 24 hours (A), IFN-α of varying doses for 24 hours (B), or with 100 U/ml IFN-α for varied time lengths (C). In (A), 100 U/ml IFN-α, 100 ng/ml IFN-γ, 10 ng/ml IL-1β, 10 ng/ml IL-6, 25 ng/ml IL-10, 50 ng/ml TRAIL, or 50 ng/ml TNF-α was used. Luciferase activity in the lysates was measured by luminescence detection. Renilla luciferase was used as internal control to normalize transfection efficiency. The data are representative of three independent experiments and are the means of triplicate samples. #, p<0.05, <i>*</i>, p<0.01, **, p<0.001 in comparison to control.</p

Significant correlation between STAT1 and GAC mRNA levels in brain tissues of HAD patients.

<p>Total RNA from post-mortem brain tissue collected from HAD patients, HIV serum-positive patients without dementia, and HIV serum negative individuals were extracted and subjected to real time RT-PCR for STAT1, GAC, IFN-α2 and IFN-β. GAPDH expression was used as an internal control. (A) Levels of STAT1 are normalized as a ratio to GAPDH and shown as fold change relative to the average of HIV serum negative controls. Results are shown as the average ± SEM, #, p<0.05. Correlation of GAC with STAT1 (B), IFN-α2 (C) and IFN-β (D) mRNA levels was determined by Spearman correlation.</p

STAT1 binds directly with the GLS1 promoter in IFN-α treated MDM.

<p>(A). The predicted STAT1 binding sites in the human GLS1 promoter, TSS is designated as +1. (B). MDM were treated with 100 U/ml IFN-α for 1 hour, then ChIP assay was performed using digested chromatin, p-STAT1 (Tyr 701) and STAT1 antibodies, or IgG antibody as a negative control. Purified DNA was analyzed by quantitative real-time PCR using specific primers. The amount of immunoprecipitated DNA is represented as signal relative to the total amount of input chromatin. The data are representative of three independent experiments using three different donors. #, p<0.05, *, p<0.01 in comparison with control. (C). MDM were pretreated with 1 µM fludarabine or 1:10,000 DMSO for 1 hour, then treated with or without 100 U/ml IFN-α for another hour. ChIP assay was performed using p-STAT1 antibody as in (B). The data are representative of two independent experiments using two different donors. *, p<0.01 in comparison with control that were treated with DMSO only; #, p<0.05 in comparison to cells treated with IFN-α and DMSO.</p

STAT1 binds directly with the GLS1 promoter in HIV-1 infected MDM.

<p>(A-E). MDM were infected with or without HIV-1_ADA for 5 days. (A). ChIP was performed using p-STAT1 (Tyr 701) antibody; IgG was used as a negative control. (B). Supernatants were tested for RTase activity. (C). p-STAT1 and STAT1 were detected by Western blot with β-actin used as a loading control. (D). Glutamate was detected in supernatants by HPLC. (E). Intracellular glutamate was detected using the Amplex^® Red Glutamic Acid/Glutamate Oxidase Assay Kit. The data (A-E) are representative of three independent experiments using three different donors. (F) Human MDM were infected with HIV-1 for 5 days, then total RNA was collected. IFN-α and IFN-β mRNA levels were determined by real-time RT-PCR. Results shown are representative of three independent experiments using three different donors and are means of triplicate samples. (G-I) Human MDM were infected with HIV-1 for 5 days in the presence of IgG or IFN-α/IFN-β neutralizing antibodies. Total RNA and cell lysates were collected and GAC mRNA (G) and protein (H and I) levels were determined by real time RT-PCR and Western blot, respectively. For quantification, GAC expression were normalized as a ratio to β-actin and shown as fold change relative to IgG control . Results (F, I) are shown as the average ± SEM of three independent experiments with three different donors, #, p<0.05, *, p<0.01, **, p<0.001, when compared with uninfected MDM or IgG control.</p