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Monoacylglycerol Lipase Inhibitor JZL184 Prevents HIV-1 gp120-induced Synapse Loss by Altering Endocannabinoid Signaling

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Monoacylglycerol Lipase Inhibitor JZL184 Prevents HIV-1 gp120-induced Synapse Loss by Altering Endocannabinoid Signaling

Xinwen Zhang et al. Neuropharmacology.

Abstract

Monoacylglycerol lipase (MGL) hydrolyzes 2-arachidonoylglycerol to arachidonic acid and glycerol. Inhibition of MGL may attenuate neuroinflammation by enhancing endocannabinoid signaling and decreasing prostaglandin (PG) production. Almost half of HIV infected individuals are afflicted with HIV-associated neurocognitive disorder (HAND), a neuroinflammatory disease in which cognitive decline correlates with synapse loss. HIV infected cells shed the envelope protein gp120 which is a potent neurotoxin that induces synapse loss. Here, we tested whether inhibition of MGL, using the selective inhibitor JZL184, would prevent synapse loss induced by gp120. The number of synapses between rat hippocampal neurons in culture was quantified by imaging clusters of a GFP-tagged antibody-like protein that selectively binds to the postsynaptic scaffolding protein, PSD95. JZL184 completely blocked gp120-induced synapse loss. Inhibition of MGL decreased gp120-induced interleukin-1β (IL-1β) production and subsequent potentiation of NMDA receptor-mediated calcium influx. JZL184-mediated protection of synapses was reversed by a selective cannabinoid type 2 receptor (CB2R) inverse agonist/antagonist. JZL184 also reduced gp120-induced prostaglandin E2 (PGE2) production; PG signaling was required for gp120-induced IL-1β expression and synapse loss. Inhibition of MGL prevented gp120-induced synapse loss by activating CB2R resulting in decreased production of the inflammatory cytokine IL-1β. Because PG signaling was required for gp120-induced synapse loss, JZL184-induced decreases in PGE2 levels may also protect synapses. MGL presents a promising target for preventing synapse loss in neuroinflammatory conditions such as HAND.

Keywords: Cannabinoid receptor; HIV gp120; JZL184; JZL184, 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (PubChem CID: 25021165); Monoacylglycerol lipase; Prostaglandin E2; Synapse loss.

Conflict of interest statement

Conflict of interest

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
JZL184 inhibits MGL enzyme activity but not FAAH activity. (A) Representative traces show MGL enzymatic activity during incubation with the colorimetric substrate, 4-NPA. Cultures were treated for 24 h with 0, 0.3 or 1 µM JZL184. Cells were lysed and incubated with 4-NPA as described in Materials and Methods. Absorbance was monitored every 5 min for 180 min and presented as relative absorbance units (RAU) normalized to µg protein. (B) Plot shows concentration dependent inhibition of MGL activity by JZL184. Enzyme activity was determined from the linear phase of the reaction (15–55 min in A). The concentration response curve was generated using a nonlinear, least-squares curve fitting program (Origin 6.0, OriginLab Corp.) to fit a logistic equation of the form % of Control = [(A1 – A2)/(1 + (X/IC50)p)]+A2 where X = drug concentration, IC50 = 219 ± 62 nM, A1 = 95 ± 3 % inhibition without drug, A2 = 65 ± 2 % inhibition at a maximally effective drug concentration and p = 3 ± 2 slope factor. Values are expressed as mean ± SEM from 5 separate enzyme assays. The incomplete inhibition produced by a maximal concentration of JZL184 is due to the hydrolysis of 4-NPA by lipid hydrolases other than MGL present in the crude membrane preparation isolated from neuronal cell cultures. (C) Representative traces show FAAH enzymatic activity during incubation with the fluorogenic substrate, arachidonoyl-AMC. Cultures were treated for 24 h with 1 µM JZL184 or 1 µM JZL 195, a potent inhibitor of FAAH and MGL. Cells were lysed and incubated with arachidonoyl-AMC as described below. Fluorescence was monitored every 5 min for 200 min and presented as relative fluorescence units (RFU) normalized to µg protein. (D) Bar graph summarizes FAAH activity relative to untreated cultures (control) during the linear phase of the reaction (0–75 min). FAAH activity in the presence of JZL184 was not significantly different from control. *p<0.05 relative to control (one-way ANOVA with Tukey’s post-test, n=6).
Figure 2
Figure 2
PSD95.FingR-eGFP (Intrabody) labels postsynaptic terminals at excitatory synapses. (A) Representative confocal images of a neuron expressing PSD95.FingR-eGFP and tdTomato were acquired and processed as described in Methods. PSD95.FingR-eGFP puncta were identified by filtering compressed z-stacks (8 µm) of confocal images. All puncta that met size (between 0.13–0.86 µm2) and intensity criteria, and were in contact with a binary mask derived from the tdTomato image were counted as synapses. Puncta were dilated and overlaid on the tdTomato maximum projection (Processed) for display purposes. Insets are enlarged images of the boxed regions. Scale bars represent 10 µm. (B) Representative confocal images of a neuron expressing PSD95.FingR-eGFP (green) and immunolabeled for Bassoon (red). PSD95.FingR-eGFP puncta co-localized with Bassoon immunoreactivity (yellow, Merged). Note that non-transfected cells were also present in the field, and thus not all Bassoon immunoreactive puncta (red) co-localized with PSD95.FingR-eGFP (green) puncta. Insets are enlarged images of the boxed regions. Scale bars represent 10 µm.
Figure 3
Figure 3
HIV gp120-induced loss of synapses and IL-1β expression were blocked by inhibition of MGL. (A) Graph shows time-dependent changes in the number of PSD95.FingR-eGFP puncta for untreated cells (control, circles) and cells treated with 600 pM gp120 (gp120, squares). Data are expressed as mean ± SEM. Repeated measures ANOVA revealed gp120 by time interactions [F3, 35 = 6.25, p<0.01]. *p<0.05, **p<0.01 relative to control at the same time-point (Tukey’s post-test, n = 7). (B) Bar graph summarizes changes in PSD95.FingR-eGFP puncta after 24 h treatment under control conditions (open bars) in the absence (n = 8) or presence (n = 6) of 1 µM JZL184 or treated with 600 pM gp120 (solid bars) in the absence (n = 7) or presence (n = 6) of JZL184. JZL184 was added 15 min prior to gp120 for all experiments. Data are expressed as mean ± SEM. *p<0.05 relative to untreated control, #p<0.05 relative to untreated gp120 (one-way ANOVA with Tukey’s post-test). (C) Representative processed images of neurons with no treatment (control), or treated with 600 pM gp120 in the absence or presence of 1 µM JZL184 for 24 h. The insets are enlarged images of the boxed region. Scale bars represent 10 µm. (D) Bar graph summarizes IL-1β mRNA expression relative to control (n = 12) after treatment with 600 pM gp120 and in the absence (n = 18) or presence of 1 µM JZL184 (n = 16) for 4 h. Data are expressed as mean ± SEM. **p<0.01 relative to control, ##p<0.01 relative to gp120 treated group (one-way ANOVA with Tukey’s post-test).
Figure 4
Figure 4
Inhibition of MGL suppresses gp120-induced potentiation of NMDA receptors by blocking IL-1β production. (A–B) HIV-1 gp120-induced a biphasic change in NMDA-evoked [Ca2+]i responses. (A) representative traces show NMDA-evoked [Ca2+]i (10 µM × 60 s) increases from a control neuron (0 h) or neurons treated with 600 pM gp120 for the times indicated above the traces. (B) plot summarizes NMDA-evoked [Ca2+]i responses after treatment with gp120 for 0 to 48 h. Data are expressed as mean ± SEM. **p<0.01 relative to 0 h time-point, ##p<0.01 relative to 4 h time-point (ANOVA with Tukey’s post-test, n ≥ 6). (C) Representative traces show increase in [Ca2+]i evoked by application of 10 µM NMDA (60s) to control neurons and neurons treated with 600 pM gp120 for 4 h in the absence and presence of 1 µg mL−1 IL-1ra. IL-1ra was added 15 min before gp120. (D) Bar graph summarizes net [Ca2+]i increase (Δ[Ca2+]i) evoked by 10 µM NMDA for control and neurons treated with 600 pM gp120 for 4 h in the absence and presence of 1 µg mL−1 IL-1ra. ***p<0.001 relative to untreated control; ###p<0.001 relative to gp120 only treatment (one-way ANOVA with Tukey’s post-test, n=16). (E) Representative traces show increase in [Ca2+]i evoked by application of 10 µM NMDA (60s) to control neurons and neurons treated with 600 pM gp120 for 4 h in the absence and presence of 1 µM JZL184. JZL184 was added 15 min before gp120. (F) Bar graph summarizes Δ [Ca2+]i evoked by 10 µM NMDA for control neurons in the absence (n=10) and presence (n=6) of 1 µM JZL184 and neurons treated with 600 pM gp120 for 4 h in the absence (n=13) and presence (n=11) of 1 µM JZL184. **p<0.01 relative to untreated control; #p<0.05, relative to the group only treated with gp120 (one-way ANOVA with Tukey’s post-test). (G) Representative traces show NMDA13 evoked increase in [Ca2+]i for control neurons and neurons treated with 3 ng mL−1 IL-1β for 4 h in the absence or presence of 1 µM JZL184. JZL184 was added 15 min before IL-1β. (H) Bar graph shows Δ [Ca2+]i evoked by 10 µM NMDA for control neurons in the absence (n=17) and presence (n=10) of 1 µM JZL184 and neurons treated with 3 ng mL−1 IL-1β for 4 h in the absence (n=20) and presence of 1 µM JZL184 (n=19). ***p<0.001 relative to untreated control, ††p<0.01 relative to JZL184 treated control (one-way ANOVA with Tukey’s post-test).
Figure 5
Figure 5
Activation of CB2R but not CB1R was required for JZL184 inhibition of gp120-induced neurotoxicity. (A) Bar graph shows changes in PSD95.FingR-eGFP puncta number after 24 h treatment with the indicated treatment groups: untreated (n=12), 600 pM gp120 (n=16), gp120 + 1 µM JZL184 (n=9), and gp120 + JZL184 + 100 nM AM630 (n=8). The CB2R inverse agonist/antagonist AM630 was added 5 min prior to JZL184. ***p<0.001 relative to untreated, #p<0.05 relative to the group only treated with gp120, †p<0.05 relative the group treated with gp120 + JZL184 (one-way ANOVA with Tukey’s post-test). (B) Bar graph shows changes in PSD95.FingR-eGFP puncta number after 24 h treatment with the indicated treatment groups: untreated (n=10), 600 pM gp120 (n=10), gp120 + 1 µM JZL184 (n=10), and gp120 + JZL184 + 100 nM SR144528 (n=10). The CB2R inverse agonist SR144528 was added 5 min prior to JZL184. **p<0.01 relative to untreated, ###p<0.001 relative to the group only treated with gp120, ††p<0.01 relative the group treated with gp120 + JZL184 (one-way ANOVA with Tukey’s post-test). (C) Bar graph shows changes in PSD95.FingR-eGFP puncta number after 24 h treatment with the indicated treatment groups: untreated (n=8), 600 pM gp120 (n=8), gp120 + 1 µM JZL184 (n=7), and gp120 + JZL184 + 100 nM rimonabant (n=9). The CB1R inverse agonist rimonabant was added 5 min prior to JZL184. **p<0.01 relative to untreated; #p<0.05, ##p<0.01 relative to the group only treated with gp120 (one-way ANOVA with Tukey’s post-test). (D) Bar graph shows changes in PSD95.FingR-eGFP puncta number after 24 h treatment with the indicated treatment groups: untreated (n=9), 600 pM gp120 (n=8), gp120 + 1 µM JZL184 (n=9), and gp120 + JZL184 + 1 µM LY320135 (n=8). The CB1R inverse agonist LY320135 was added 5 min prior to JZL184. *p<0.05 relative to untreated; #p<0.05, ###p<0.001 relative to the group only treated with gp120 (one-way ANOVA with Tukey’s post-test). (E) Bar graph summarizes IL-1β mRNA expression measured using qRT-PCR assay after 4 h treatment with the indicated treatments: untreated (n=10), 600 pM gp120 (n=13), gp120 + 1 µM JZL184 (n=9), and gp120 + JZL184 + 100 nM AM630 (n=9). AM630 was added 5 min prior to JZL184. ***p<0.001 relative to untreated group, #p<0.05 relative to the group only treated with gp120, †p<0.05 relative to the group treated with gp120 and JZL184 (one-way ANOVA with Tukey’s post-test).
Figure 6
Figure 6
MGL inhibition blocks gp120-induced PGE2 production. EP1–2R activation is required for synapse loss and IL-1β expression. (A) Bar graph shows changes in PGE2 levels in culture media measured using ELISA. PGE2 levels are shown for control conditions in the absence (n=14) or presence of 1 µM JZL184 (n=9) and after treatment with 600 pM gp120 for 4 h in the absence (n=17) or presence (n=14) of 1 µM JZL184. *p<0.05 relative to untreated control, #p<0.05 relative to the group only treated with gp120 (one-way ANOVA with Tukey’s post-test). (B) Bar graph shows changes in the number of PSD95.FingR-eGFP puncta under control conditions in the absence (n=11) or presence (n=6) of 10 µM AH6809, an EP1–2R antagonist, or 24 h following treatment with 600 pM gp120 in the absence (n=9) or presence (n=9) AH6809. **p<0.01 relative to untreated control, #p<0.05 relative to the group treated with gp120 alone (one-way ANOVA with Tukey’s post-test). (C) Bar graph summarizes changes of IL-1β mRNA expression in untreated cultures (n=10) and after 4 h treatment with 600 pM gp120 in the absence (n=11) or presence (n=10) of 10 µM AH6809. Data are expressed as fold induction relative to untreated group. *p<0.05, **p<0.01 relative to untreated group; #p<0.05 relative to the group treated with gp120 alone (one-way ANOVA with Tukey’s post-test). (D) Bar graph shows Δ[Ca2+]i evoked by 10 µM NMDA for control cells in the absence (n=7) and presence (n=6) of 10 µM AH6809 and neurons treated with 3 ng mL−1 IL-1β for 4 h in the absence (n=11) and presence (n=8) of AH6809. ***p<0.001 relative to untreated control, †p<0.05 relative to AH6809 treated control (one-way ANOVA with Tukey’s post-test).
Figure 7
Figure 7
Summary scheme shows the hypothesized mechanism of the synapse protection induced by the inhibition of MGL. (A) Diagram illustrates the JZL184-induced increase in 2-AG that activates CB2R-dependent synapse protection and the reduced AA production that decreases PGE2-mediated synapse loss. (B) Scheme shows 2-AG and AA dependent pathways affected by JZL184 to reduce synapse loss. Solid arrows indicate flow of signaling pathways. Green lines highlight prostaglandin signaling and red lines highlight eCB signaling.

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