GPR18 drives FAAH inhibition-induced neuroprotection against HIV-1 Tat-induced neurodegeneration

Abstract

Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7-11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845's effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CB₁R & CB₂R). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition.

Keywords: Anandamide; Endocannabinoid; Fatty acid amide hydrolase; G protein coupled receptor; HIV-1 Tat; Matrix metalloproteinase; Microglia; Monocyte chemoattractant protein 1; Neuroinflammation; Neuroprotection.

Fig. 1.

(A) Pseudocolor images of neuronal ratiometric calcium imaging comparing frontal cortex neurons (DIV 7–11) treated with MCM Control or MCM Tat conditions over 30 min. (B-B′) MCM Tat 100 nM caused significant increases in [Ca²⁺]_i levels while MCM PF3845 + Tat caused no significant changes over 30 min (B), which is also supported by the last 10 min of calcium assessment (B′), indicating that PF3845 100 nM blocked MCM Tat effects. Additionally, MCM heat inactivated Tat (100 nM) did not elicit any significant [Ca²⁺]_i response. (C) MCM Tat-induced increases in [Ca²⁺]_i levels are significantly downregulated by PF3845 indicated by the MCM PF3845 + Tat condition. (D) Incubation with the CB₁R antagonist, SR141 (100 nM), failed to block PF3845’s neuroprotective effect as seen in the MCM SR141 + PF3845 + Tat condition for the last 10 min of the time course data. (E) Likewise, the CB₂R antagonist, SR144 (100 nM), failed to block PF3845’s neuroprotective effect as seen in the MCM SR144 + PF3845 + Tat condition for the last 10 min of the time course data. (F) Interestingly, the GPR18 antagonist, CID (100 nM), significantly blocked the neuroprotective effects of PF3845 in the MCM CID + PF3845 + Tat condition for the last 10 min of the time course data where the change in [Ca²⁺]_i was similar to the MCM Tat condition, suggesting that GPR18-related activity was necessary for the observed protective effect. Statistical significance was assessed by ANOVAs followed by Bonferroni’s post hoc tests. Bonferroni’s post hoc test were conducted on the last 10 min of the time course data (bar graphs); *p < 0.001 vs. MCM Control, ^#p < 0.05 vs. MCM Tat, ^$p < 0.05 vs. MCM antagonists + Tat; φp < 0.001 vs. MCM PF3845 + Tat (at least three independent experiments). Scale bars = 50 um. MCM, microglia conditioned media; hiTat, heat inactivated Tat; SR141, SR141716A (CB₁R antagonist); SR144, SR144528 (CB₂R antagonist); CID, CID-85469571 (GPR18 antagonist). Please see Supplementary Fig. 2 for the actual time course data of the CB₁R, CB₂R and GPR18 antagonists experiments over a 30 min time period.

Fig. 2.

Live cell calcium imaging experiments of frontal cortex neurons (DIV 7–11) treated with MCM derived from FAAH WT and FAAH KO microglia over a 30 min time period. The MCM FAAH KO + Tat condition induced significantly less [Ca²⁺]_i levels than the MCM FAAH WT + Tat condition indicating that decreased FAAH activity reduced the neurotoxicity of the MCM. Likewise, the MCM FAAH KO + CID + Tat condition displayed significantly higher levels of neuronal [Ca²⁺]_i compared to the MCM FAAH KO + Tat condition suggesting that this observed neuroprotective effect was dependent on GPR18 activity. Statistical significance was assessed by ANOVAs followed by a Bonferroni’s post hoc test that was conducted on the last 10 min of the time course data (bar graph); *p < 0.05 vs. MCM FAAH WT, ^#p < 0.05 vs. MCM FAAH WT + Tat, ^$p < 0.05 vs. MCM FAAH KO + CID + Tat (at least three independent experiments). MCM, microglia conditioned media; WT, wildtype; KO, knockout.

Fig. 3.

(A & B) Pseudocolor images of phalloidin-stained frontal cortex neuronal cultures (DIV 21) treated with various MCM conditions for 24 h. (A′ & B′) Inset images are depicted on the main image by white boxes and the white arrows denote phalloidin stained punctate structures. (C) MCM Tat treatment significantly decreased average gray pixel value of phalloidin staining while MCM PF3845 + Tat enhanced average gray pixel value of phalloidin staining, relative to MCM Control treatment. MCM CID + PF3845 + Tat did not cause the protective effect seen in MCM PF3845 + Tat treatment, suggesting that PF3845 treatment blocks the loss of actin in dendritic structures induced by Tat in MCM via a GPR18-related mechanism. (D) MCM Tat treatment significantly decreased phalloidin labelled puncta number while MCM PF3845 + Tat enhanced phalloidin labelled puncta number, relative to MCM Control treatment. This suggests that PF3845 protected and increased the number of actin-containing synaptic structures against Tat-related neurotoxicity through microglia. MCM CID + PF3845 + Tat did not cause the protective effect seen in MCM PF3845 + Tat treatment, indicating that GPR18 appears to be a necessary receptor in this PF3845 neuroprotective effect. Statistical significance was assessed by ANOVAs followed by Bonferroni’s post hoc tests; *p < 0.001 vs. MCM Control, ^#p < 0.001 vs. MCM Tat, ^$p ≤ 0.001 vs. MCM CID + PF3845 + Tat. Scale bars: Primary = 100 μm, Inset = 30 μm. MCM, microglial conditioned media; CID, CID-85469571.

Fig. 4.

(A) MCM Tat caused a significant increase in MCP-1/CCL2 levels compared to MCM Control treatment while incubation with PF3845 blocked this increase and further decreased MCP-1/CCL2 levels compared to control. Antagonizing GPR18 with CID blocked these PF3845-mediated decreases in MCP-1/CCL2 as seen in the MCM CID + PF3845 + Tat condition. (B) MCM Tat caused a significant increase in proMMP-9 levels compared to control and incubating with PF3845 blocked this effect. Antagonizing GPR18 with CID blocked this PF3845-mediated decrease in proMMP-9 as seen in the MCM CID + PF3845 + Tat condition. (C) MCM Tat caused a significant increase in MMP-9 levels compared to control and incubation with PF3845 blocked this effect. Antagonizing GPR18 with CID blocked the PF3845-mediated decrease in MMP-9 as seen in the MCM CID + PF3845 + Tat condition. Collectively, these data suggest that Tat enhanced the excretion of neurotoxins, while PF3845 blunted this effect at least partially through GPR18-related activity. Statistical significance was assessed by ANOVAs followed by Bonferroni’s post hoc tests; *p < 0.05 vs. MCM Control, ^#p < 0.05 vs. MCM Tat, ^$p < 0.05 vs. MCM CID + PF3845 + Tat. MCM, microglial conditioned media; CID, CID-85469571.

Fig. 5.

(A) An overlay of a phase image of cultured microglia with outlines of randomly selected microglia processed for ImageJ FracLac analysis. (A′) Phase image of cultured microglia showing a range of amoeboid and branched morphologies. (A″) Outlines of randomly selected microglia processed for ImageJ FracLac analysis with an inset image that is represented on the primary image by a black box showing microglial ruffles. (C) Microglia treated with PF3845 and PF3845 + Tat treatment conditions demonstrated a significant increase in fractal dimension (D_F) compared to other treatment conditions, indicating microglia morphology following PF3845 treatment is more associated with an intermediate activation state and less with the activated amoeboid state. (D) Microglia treated with PF3845 and PF3845 + Tat treatment conditions demonstrated a significant decrease in lacunarity (Λ) compared to other treatment conditions, suggesting that the PF3845 treated microglia have more overall morphological homogeneity. No effects were noted for Tat treatment potentially as microglia grown in culture in vitro under control conditions reside in a more amoeboid phenotype/shape. Statistical significance was assessed by ANOVAs followed by Bonferroni’s post hoc tests; *p < 0.05 vs. Control. Scale Bars: Primary = 100 μm, Inset = 30 μm. CID, CID-85469571.

Fig. 6.

Concentrations of AEA in MCM were assessed using LC/MS/MS analysis following 24 h of PF3845, Tat and/or CID exposure to cultured microglia. All MCM conditions treated with PF3845 had significantly enhanced levels of AEA compared to MCM Control and MCM Tat. This suggests that PF3845 was effective in inhibiting microglial FAAH’s ability to degrade AEA. No effects on AEA levels were noted when CID was added to MCM PF3845 + Tat, indicating that blocking AEA’s activity on GPR18 receptors had no effect on AEA levels itself. Statistical significance was assessed by ANOVAs followed by a Bonferroni’s post hoc test; *p < 0.05 vs. MCM Control, ^#p < 0.05 vs. MCM Tat. MCM, microglial conditioned media; CID, CID-85469571.

Fig. 7.

(A) Shows the effects of treatment conditions on microglia morphology. Tat does not show any effects on fractal dimention (D_F) and lacunarity (Λ) whereas PF3845 via GPR18 increases D_F and decreases Λ, which is associated with a less activated microglia state and more overall morphological homogeneity, respectively. (B) Shows the soluble factors released from treated microglia into the microglial conditioned media (MCM). MCM conditions derived from Tat treated microglia (MCM Tat) show upregulated proinflammatory responses with increased levels in MCP-1/CCL2, MMP-9, and proMMP-9. In contrast, MCMs derived from microglia pretreated with PF3845 (MCM PF3845) show increased AEA levels by inhibiting the catabolic FAAH enzyme via GPR18-related mechanisms, which results in anti-inflammatory responses by decreasing MCP-1/CCL2, MMP-9, and proMMP-9 levels. (C) Shows neuronal changes that occur when frontal cortex neurons are treated with MCM conditions derived from treated microglia. MCM Tat increases neuronal Ca²⁺ dysregulation and synaptodendritic damage whereas MCMs derived from microglia pretreated with PF3845 alongside Tat (MCM PF3845 + Tat) results in reduction of Ca²⁺ dysregulation and synaptodendritic damage via GPR18-related mechanisms. Λ, lacunarity; Ca²⁺, calcium; CID-85469571, GPR18 antagonist; D_F, fractal dimension.