## Results: 6

2.

gp120-induced synapse loss required sequential activation of CXCR4, IL-1β, and NMDA receptors. A, inhibition of CXCR4, IL-1β, or NMDA receptors prevents gp120-induced synapse loss. Bar graph summarizes changes in PSD95-GFP puncta (PSDs) after 24-h treatment under control (□) or gp120-treated (■) conditions in the absence (untreated,

*n*= 24) or presence of the indicated inhibitors. Cultures were treated with either 50 μM TKP (*n*= 13), 1 μM AMD3100 (*n*= 6), or 10 μM MK801 (*n*= 7) for 30 min before the addition of gp120 or cotreated with 1 μg/ml IL-1-ra (*n*= 6) and gp120. Data are expressed as mean ± S.E.M. ***,*p*< 0.001 relative to control, Student's*t*test; ††,*p*< 0.01 relative to gp120 alone (untreated), ANOVA with Bonferroni post-test. B, IL-1β-induced synapse loss is mediated by a Src family tyrosine kinase and NMDA receptors. Bar graph summarizes changes in PSD95-GFP puncta (PSDs) after 24-h treatment under control conditions (□) or after treatment with 3 ng/ml IL-1β (■) in the absence (untreated;*n*= 12) or presence of the indicated inhibitors. Cultures were treated with 1 μg/ml IL-1-ra (*n*= 9), 10 μM MK801 (*n*= 11), 1 μM AMD3100 (*n*= 9), or 10 μM pyrazolopyrimidine 2 (*n*= 7) as indicated. Data are expressed as mean ± S.E.M. ***,*p*< 0.001 relative to control Student's*t*test; †,*p*< 0.05 relative to IL-1β alone (untreated), ANOVA with Bonferroni post test.3.

gp120 induced IL-1β production in hippocampal cultures. A, incubation with 600 pM gp120 for 24 h induced the release of IL-1β in rat hippocampal cultures. IL-1β expression was measured by ELISA as described under

*Materials and Methods*. gp120-evoked IL-1β levels in hippocampal cultures were near the limit of detection with commercially available ELISAs. Data are expressed as mean ± S.E.M. (*n*= 9). **,*p*< 0.01 relative to control, Student's*t*test. B, time course for IL-1β mRNA induction in hippocampal cultures. IL-1β mRNA expression peaked 12 h after treatment with 600 pM gp120 (*n*≥ 3 for each data point). IL-1β mRNA expression was measured by qRT-PCR as described under*Materials and Methods*. C, induction of IL-1β mRNA was attenuated by inhibition of microglia activation or blocking CXCR4. Bar graph summarizes the effects of inhibitors on changes in the expression of IL-1β mRNA induced by 12-h treatment with 600 pM gp120 (gp120,*n*= 12). TKP (50 μM) (*n*= 6) or 1 μM AMD3100 (*n*= 8) was applied 15 min before and during treatment with gp120. Data are expressed as mean ± S.E.M. **,*p*< 0.01 relative to gp120 alone (gp120), ANOVA with Bonferroni post test.4.

Cannabinoid receptor agonist, Win55,212-2, prevented PSD loss induced by gp120, but not that induced by Tat. A, confocal images show PSD95-GFP puncta before and 24-h after exposure to 600 pM gp120 in the absence (untreated) or presence of Win55,212-2 (Win-2). Scale bars, 10 μm. B, bar graph summarizes changes in PSD95-GFP puncta (PSDs) after 24-h treatment under control conditions (control, □), after treatment with 600 pM gp120 (■), or after treatment with 50 ng/ml Tat (▩). Experiments were performed in the absence (

*n*= 18 for gp120;*n*= 6 for Tat) or presence of Win-2 (*n*= 15 for gp120;*n*= 7 for Tat). The cannabinoid receptor antagonists rimonabant (100 nM;*n*= 15) or AM630 (100 nM;*n*= 16) were applied 5 min before and during exposure to Win-2 and gp120. Data are mean ± S.E.M. **,*p*< 0.01 relative to control; ††,*p*< 0.01 relative to gp120, ANOVA with Bonferroni post test. C, gp120-evoked IL-1β expression is inhibited by cannabinoids. qRT-PCR was performed as described under*Materials and Methods*. Bar graph summarizes the effects of cannabinoid receptor ligands on gp120-induced IL-1β mRNA expression. Cultures were treated with 600 pM gp120 in the absence (gp120,*n*= 12) or presence of 100 nM Win-2. Cultures were treated with Win-2 alone (Win-2 + gp120,*n*= 7) or pretreated with 100 nM rimonabant (rim + Win-2 + gp120,*n*= 7) or 100 nM AM630 (AM630 + Win-2 + gp120,*n*= 8) 15 min before and during exposure to Win-2 in the presence of gp120. Data are expressed as mean ± S.E.M. ***,*p*< 0.001 relative to gp120 alone (gp120), Student's*t*test; †,*p*< 0.05 relative to Win-2 pretreatment in the presence of gp120 (Win + gp120), ANOVA with Bonferroni post test.5.

HIV-1 gp120 IIIB induced PSD loss and cell death in a time and concentration-dependent manner. A, confocal fluorescent images display maximum

*z*-projections of neurons expressing PSD95-GFP and DsRed2 before and 24 h after treatment with 600 pM gp120. Processing of PSD95-GFP images identified PSDs as fluorescent puncta meeting intensity and size criteria and in contact with a mask derived from the DsRed2 image. Labeled PSDs were dilated and overlaid on the DsRed2 image for visualization purposes (processed). Insets, enlarged images of the boxed region. Scale bars, 10 μm. B, graph shows time-dependent changes in the number of PSD95-GFP puncta for untreated cells (control, ■) and cells treated with 600 pM gp120 (●). Data are expressed as mean ± S.E.M. (*n*≥ 4 for each data point). **,*p*< 0.01 relative to PSDs counted before the addition of gp120 (0 h), repeated-measures ANOVA with Bonferroni post test. C, plot shows concentration-dependent changes in the number of PSD95-GFP puncta for cells treated with the indicated concentration of gp120. The mean ± S.E.M. of the net change in PSD95-GFP puncta 24 h after treatment with gp120 is plotted for the concentrations indicated (*n*≥ 4 for each data point). The curve was fit by a logistic equation of the form % PSD change = [(A_{1}− A_{2})/(1 + (X/EC_{50})^{p})] + A_{2}, where X = gp120 concentration, A_{1}= 31 ± 8% PSD change without gp120, A_{2}= −22 ± 5% PSD change at a maximally effective gp120 concentration, and p = slope factor. EC_{50}was calculated using a nonlinear, least-squares curve-fitting program. EC_{50}and p were 153 ± 50 pM and 2 ± 2 respectively. D, gp120-induced cell death. Cell death was measured using the PI fluorescence assay detailed under*Materials and Methods*. Cell death was measured after 24 h (squares) and 48 h (circles) treatment with the indicated concentrations of gp120. PI fluorescence was normalized to that measured from cells treated with 1 mM glutamate (100%) with PI fluorescence from untreated wells subtracted (0%). The 24- and 48-h curves were fit to logistic equations resulting in EC_{50}and p values of 68 ± 77 pM and 3 ± 8 (24 h), and 85 ± 44 pM and 0.6 ± 0.1 (48 h).6.

gp120 induced PSD loss via the ubiquitin-proteasome pathway and induced cell death by activating NOS. A, processed images display labeled PSDs superimposed on DsRed2 fluorescence acquired before and after 24-h treatment with 600 pM gp120 in control cultures (untreated) and cultures pretreated with 100 nM nutlin-3. Scale bars, 10 μm. B, bar graph summarizes changes in PSD-GFP puncta (PSDs) after 24-h treatment under control conditions (□) or after treatment with 600 pM gp120 (■) in the absence (untreated,

*n*= 24) or presence of inhibitors. Cells were treated with 100 nM nutlin-3 (*n*= 8) or 100 μM l-NAME (*n*= 7) 15 min before and during treatment with gp120 as indicated. Cells expressing p14 ARF (ARF;*n*= 12) or PSD95ΔPEST-GFP (ΔPEST; in lieu of PSD95-GFP;*n*= 7) are indicated. Data are expressed as mean ± S.E.M. ***,*p*< 0.001 relative to control, Student's*t*test; †,*p*< 0.05 relative to gp120 alone (untreated), ANOVA with Bonferroni post test. C, gp120-induced cell death. Cell death was measured using the PI fluorescence assay detailed under*Materials and Methods*after 48-h treatment with the indicated concentrations of gp120 in the absence (untreated, circles,*n*≥ 11) or presence of 100 nM nutlin-3 (▵,*n*≥ 11) or 100 μM l-NAME (■, n ≥6). PI fluorescence from untreated wells was subtracted from each curve (0%) and normalized to that measured from cells treated for 48 h with 1 mM glutamate (100%). Concentration-response curves were generated by fitting a logistic equation to the data using a nonlinear, least-squares curve fitting program (Origin 6.0; OriginLab Corporation, Northampton, MA) and EC_{50}values calculated. A logistic equation of the form ΔPI Fluorescence = [(A_{1}− A_{2})/(1 + (X/EC_{50})^{p})] + A_{2}where X is the gp120 concentration, A_{1}is the percentage of change in PI fluorescence without gp120, A_{2}is the percentage of change in PI fluorescence at maximal gp120 concentration, and p is the slope factor. Values for A_{1}, A_{2}, EC_{50}, and p for untreated and nutlin-3-treated curves were, respectively, −2 ± 5%, 62 ± 12%, 11 ± 9 pM, and 0.5 ± 0.2 (untreated) and 13 ± 1%, 60 ± 2%, 11 ± 2 pM, and 1 ± 0.2 (nutlin-3). All data are presented as mean ± S.E.M. (*n*≥ 6). A set of triplicate wells from a single plating of cells was defined as a single experiment (*n*= 1).