Acute Effects of Monoacylglycerol Lipase Inhibitor ABX1431 on Neuronal Hyperexcitability,Nociception,Locomotion,and the Endocannabinoid System in HIV-1 Tat Male Mice

Abstract

Background:

Evidence suggests that monoacylglycerol lipase (MAGL) inhibitors can potentially treat HIV symptoms by increasing the concentration of 2-arachidonoylglycerol (2-AG). We examined a selective MAGL inhibitor ABX1431 in the context of neuroHIV.

Methods:

To assess the effects of ABX1431, we conducted in vitro and in vivo studies. In vitro calcium imaging on frontal cortex neuronal cultures was performed to evaluate the role of ABX1431 (10, 30, 100 nM) on transactivator of transcription (Tat)-induced neuronal hyperexcitability. Following in vitro experiments, in vivo experiments were performed using Tat transgenic male mice. Mice were treated with 4 mg/kg ABX1431 and assessed for antinociception using tail-flick and hot plate assays followed by locomotor activity. After the behavioral experiments, their brains were harvested to quantify endocannabinoids (eCB) and related lipids through mass spectrometry, and cannabinoid type-1 and -2 receptors (CB₁R and CB₂R) were quantified through western blot.

Results:

In vitro studies revealed that adding Tat directly to the neuronal cultures significantly increased intracellular calcium concentration, which ABX1431 completely reversed at all concentrations. Preincubating the cultures with CB₁R and CB₂R antagonists showed that ABX1431 exhibited its effects partially through CB₁R. In vivo studies demonstrated that acute ABX1431 increased overall total distance traveled and speed of mice regardless of their genotype. Mass spectrometry and western blot analyses revealed differential effects on the eCB system based on Tat expression. The 2-AG levels were significantly upregulated following ABX1431 treatment in the striatum and spinal cord. Arachidonic acid (AA) was also upregulated in the striatum of vehicle-treated Tat(+) mice. No changes were noted in CB₁R expression levels; however, CB₂R levels were increased in ABX1431-treated Tat(−) mice only.

Conclusion:

Findings indicate that ABX1431 has potential neuroprotective effects in vitro partially mediated through CB₁R. Acute treatment of ABX1431 in vivo shows antinociceptive effects, and seems to alter locomotor activity, with upregulating 2-AG levels in the striatum and spinal cord.

Introduction

Approximately 39 million people were living with human immunodeficiency virus type-1 (HIV-1) globally, with 1.3 million newly infected in 2022.¹ The advancement of combined antiretroviral therapies (cART) suppresses HIV-1 replication to undetectable levels² but does not eradicate the virus entirely due to its low brain penetration.³ Low levels of viral replication and chronic immune activation still linger, leading to HIV-1-associated neurocognitive disorders (HAND).^4–6 About 50% of cART-treated people living with HIV-1 (PLWH) develop some form of HAND, which includes problems with memory consolidation, attention,^7–9 mood,^10,11 reduced physical activity levels,¹² and increased pain sensitivity.¹³ HAND is associated with synaptodendritic injury caused by inflammatory factors and viral proteins, including transactivator of transcription (Tat), released from infected and/or activated microglia/macrophages.¹⁴

The prevalence of HAND in the cART era raises questions about treating HIV-1-related brain disorders. The endocannabinoid (eCB) system presents promising therapeutic targets for neurocognitive disorders, such as HAND, due to its role in neuroprotection and anti-inflammatory processes.^15,16 Endogenous cannabinoid ligands, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), show neuroprotective effects in several preclinical models of neurodegenerative disease, including Parkinson's disease, Alzheimer's disease, and multiple sclerosis.^17–20 In the context of neuroHIV, reviews have discussed the potential neuroprotective effects of eCB ligands.^21,22 AEA and 2-AG mainly target cannabinoid type-1 and -2 receptors (CB₁R and CB₂R) and are rapidly degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. As direct application of AEA and 2-AG have short durations of action,²³ the strategy of inhibiting their hydrolytic enzymes possesses considerable promise.^24,25

MAGL not only serves as the major 2-AG degradative enzyme but also is a major rate-limiting enzyme for producing arachidonic acid (AA).²⁶ Accordingly, the MAGL inhibitor, JZL184, dampens inflammation in the central²⁷ and peripheral²⁸ nervous system by decreasing 2-AG metabolism and concomitantly reducing the production of its primary metabolite AA, as well as the further downstream proinflammatory prostaglandins.^29,30 Moreover, JZL184 prevents gp-120-induced synapse loss through CB₂R activation and downregulates production of inflammatory cytokines.²⁹

The highly selective MAGL inhibitor, MJN110, displays neuroprotective effects in vitro, by reducing neuronal hyperexcitability and restoring dendritic arborization complexity, and in vivo, by mitigating Tat-induced neurocognitive alterations.³¹ These findings indicate that MAGL inhibitors represent a viable target for treating HAND. ABX1431 (renamed AG06466) is another highly selective MAGL inhibitor, which has undergone ∼16 clinical trials that have been terminated or completed^32,33 and is considered safe to use.

ABX1431 significantly increases 2-AG concentration in the brain and produces antinociceptive effects in rats³⁴ and also showed anticonvulsant effects in a mouse model of Dravet Syndrome by increasing hippocampal 2-AG concentration.³⁵

Given that ABX1431 is a highly selective MAGL inhibitor with demonstrated safety in clinical trials, we examined its effects in the context of neuroHIV. Accordingly, the present study aims to determine the potential neuroprotective effects of ABX1431 against HIV Tat-induced neurotoxicity in vitro and its acute effects on various behavioral outcomes and the eCB system using the HIV Tat transgenic mouse model.

Materials and Methods

Experiments and procedures described below were approved by the University of North Carolina at Chapel Hill and conducted following the National Institute of Health Guide and Use of Laboratory Animals.

Primary neuronal culture and microglia conditioned media

Primary neuronal cultures (Fig. 1) were obtained from the frontal cortex (FC) of embryonic day 17 C57BL/6J mice (Charles River, Raleigh, NC) as previously described.^36,37 Cultures were incubated in neurobasal media supplemented with appropriate nutrients at 37°C in a humidified atmosphere containing 5% CO₂. All experiments were conducted at days in vitro (DIV) 21.

FIG. 1.

Overall outline of the study. (A) Steps involved in in vitro Ca²⁺ imaging studies using primary neuronal and microglial cultures. At DIV21 neurons were treated with Fura-2AM and the appropriate treatment condition, then incubated for 30 min and imaged. (B) In vivo study outline; Tat(−) and Tat(+) male mice were given ABX1431 (4 mg/kg) through oral gavage and after 4 h mice underwent the behavioral tests as shown. Ca²⁺, calcium; DIV, days in vitro; FC, frontal cortex; MCM, microglia conditioned media; Tat, transactivator of transcription; tg, transgenic. Figure created in Biorender.

Primary microglial cultures were derived from whole brain of C57BL/6J (Charles River) pups at 0–1 postnatal days as described.³⁸ After the treatments on DIV14, microglia conditioned media (MCM) were collected and frozen at −20°C. The MCM dilution used for the calcium (Ca²⁺) imaging experiments was 1:9.

Live cell calcium imaging

Live fluorescence cell imaging was conducted on the cultured neurons using Zeiss Axio Observer Z.1 inverted microscope at 20×s objective (Carl Zeiss). Intracellular calcium ([Ca²⁺]_i) was measured by treating neurons with a cell-permeant calcium indicator fura-2AM (k_d=145 nM; 2.5 μM; Molecular Probes) as previously described.³⁷ Neurons were incubated with appropriate treatments for 30 min prior imaging. Tat (100 nM) was bath applied to the cultures at the 1-, 3-, and 5-min mark and the excitation pattern was recorded for a total of 15 min. For the MCM experiments, 1 mL of diluted MCM was applied to the FC neuronal cultures at 1 min. The [Ca²⁺]_i production was calculated as previously described.³⁹ At least three independent experiments were performed for each treatment. For quantitative analysis of ([Ca²⁺]_i levels, 12–15 neurons were randomly selected per treatment per experiment.

Treatments

For in vitro experiments, primary FC neuronal and microglial cultures were treated with HIV-1 Tat_1–86 (100 nM; ImmunoDiagnostic; clade 267B). Tat 100 nM concentration was used based on previous data from our laboratory.^31,37,38 The concentrations of ABX1431 were based on previous activity-based protein profile studies.³⁴ At DIV14 microglial cultures were incubated with ABX1431 for 1 h before Tat treatment. Treatment with CB₁R antagonist rimonabant (100 nM; Tocris) and CB₂R antagonist SR144528 (100 nM; Tocris) was done 30 min before the ABX1431 treatment. Tat was added to cultures and was allowed to incubate for 24 h before media collection.

For behavioral experiments ABX1431 (No. 26023; Cayman Chemical, Ann Arbor, MI) was dissolved in 1:1:18 mixture of ethanol:kolliphor:saline to obtain a 4 mg/kg solution. Based on previous studies,^34,40 it has been determined that oral gavage of ABX1431 at 4 mg/kg significantly increased the 2-AG concentration in the mouse brain; therefore, ABX1431 dose of 4 mg/kg was selected for all the in vivo studies. Administration of vehicle or ABX1431 was done through oral gavage acutely 4 h before behavioral testing, and was randomized in all experiments.

Animals

Doxycycline (DOX)-inducible, brain-restricted HIV-1_IIIB Tat_1–86 transgenic male mice (∼7–9 months of age, N=28) were used and developed on a hybrid C57BL/6J background.^41,42 Transgenic Tat(+) and control Tat(−) mice were fed a special DOX chow containing 6 mg/g DOX (Envigo) for 4 months before the experiment and were housed on a reversed 12-h light/12-h dark cycle.

Behavioral procedure

All the behavioral experiments were blinded for treatment and genotype.

Tail-flick and hot plate assay

ABX1431 4 mg/kg or vehicle were administered to the mice and spontaneous nociception was assessed 4 h posttreatment as previously described⁴³ (Fig. 1). For tail-flick, the distal 1/3 of the tail was placed in a water bath maintained at 56°C±0.1°C. The latency to remove the tail from the bath was recorded. Hot plate was conducted immediately following the tail-flick assay. Mice were placed on the hot plate maintained at 55°C±0.1°C and removed immediately after withdrawing or licking a paw and the time was recorded. A maximum cutoff latency of 10 sec and 15 sec was used for tail-flick and hot plate, respectively, to prevent tissue damage.

Locomotor activity

Mice were habituated to the locomotor activity chambers 24 h before the experiment and then placed into the experimental chambers as previously described⁴⁴ (ENC-307W; 22×18 cm floor; MED Associates). Locomotion was recorded for 10 min using night vision cameras. Videos were analyzed using AnyMaze software.

eCB and eicosanoid analysis

Following locomotor activity, animals were sacrificed by isoflurane-induced anesthesia followed by rapid decapitation. Prefrontal cortex (PFC), striatum, and spinal cord were dissected and snap frozen in liquid nitrogen. Endogenous cannabinoid ligands, including AEA, 2-AG, N-oleoylethanolamide (OEA), N-palmitoylethanolamide (PEA), and AA were quantified from samples of the right hemisphere using ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) as previously described.^31,45

Western blot analysis

Western blot analysis was carried out on the same three central nervous system (CNS) regions from the left hemisphere as previously described.^46,47 Briefly, tissue samples were homogenized on ice in Pierce™ RIPA lysis and extraction buffer (Thermo Scientific) with Halt™ protease and phosphatase inhibitor cocktail. BCA assay was performed on the lysates to determine the protein concentration. Proteins were denatured at 85°C for 10 min and equal amount of protein (20 μg/lane) was resolved at 120 volts for 1.5 h and transferred to nitrocellulose membrane at 1–4°C and 100 volts for 1 h. The membranes were incubated with Intercept blocking buffer at room temperature for 1 h followed by primary antibodies overnight at 4°C and secondary antibody for 1 h the next day.

The membranes were imaged and analyzed in Empiria studio software (LI-COR Biosciences). The following antibodies were used, primary antibodies: anti-CB₁R (rabbit polyclonal; Proteintech), anti-CB₂R (rabbit polyclonal; AbClonal), and anti-GAPDH antibody (mouse monoclonal; Abcam) as a housekeeping protein. Dilutions for primary are 1:1000, except GAPDH and secondary antibodies are 1:10,000 dilution.

Data represent the fold-change with respect to a control sample represented on all blots and normalized to the housekeeping gene GAPDH.

Statistical analysis

All data are presented as mean±the standard error of the mean. In vitro data were analyzed using analyses of variance (ANOVAs). Animal data sets were analyzed by two-/three-way ANOVAs with time (1, 3, 8, 10 min) as a within-subjects factor and/or treatment (two levels: Vehicle, ABX1431) and genotype [two levels: Tat(−), Tat(+)] as between-subjects factors. All ANOVAs were followed up with Tukey's post hoc test when appropriate. An alpha level of p≤0.05 was considered significant for all statistical tests (Table 1). SPSS Statistics 28 and Prism GraphPad 8.0 were used for data analysis and data graphing, respectively.

Table 1.

Statistical Data for All the Studies Conducted

Study	Treatment effect, p	Genotype effect, p	Treatment×genotype effect, p	Time effect, p	Time×treatment effect, p	Statistics
In vitro studies
Direct treatment to neurons	<0.0001	NA	NA	NA	NA	One-way ANOVA with Tukey's post hoc test
MCM treatment to neurons	<0.0001	NA	NA	NA	NA	One-way ANOVA with Tukey's post hoc test
In vivo studies
Hot plate	0.350	0.781	0.148	NA	NA	Two-way ANOVA with Tukey's post hoc test
Tail flick	0.005	0.870	0.008	NA	NA	Two-way ANOVA with Tukey's post hoc test
Total distance (m)	0.041	0.746	0.986	<0.001	0.008	Three-way ANOVA with Tukey's post hoc test
Average speed (m/sec)	0.009	0.704	0.942	<0.001	0.047
Total time immobile (sec)	0.279	0.714	0.206	0.001	<0.001
Mass spectrometry analysis
PFC AEA	0.341	0.222	0.399	NA	NA	Two-way ANOVA with Tukey's post hoc test
PFC 2-AG	0.080	0.488	0.944	NA	NA
PFC AA	0.194	0.562	0.818	NA	NA
Str AEA	0.701	0.171	0.684	NA	NA
Str 2-AG	0.011	0.270	0.778	NA	NA
Str AA	0.299	0.005	0.053	NA	NA
SC AEA	0.791	0.304	0.808	NA	NA
SC 2-AG	0.013	0.907	0.984	NA	NA
SC AA	0.987	0.754	0.334	NA	NA
Western blot analysis
PFC CB₁R	0.086	0.339	0.304	NA	NA	Two-way ANOVA with Tukey's post hoc test
PFC CB₂R	0.119	0.112	0.104	NA	NA
Str CB₁R	0.188	0.295	0.117	NA	NA
Str CB₂R	0.001	0.109	0.035	NA	NA
SC CB₁R	0.095	0.444	0.332	NA	NA
SC CB₂R	0.055	0.134	0.115	NA	NA

Bolded values denote significance at p≤0.05.

2-AG, 2-arachidonoylglycerol; AA, arachidonic acid; AEA, N-arachidonoylethanolamine; ANOVA, analysis of variance; CB₁R, cannabinoid type-1 receptor; CB₂R, cannabinoid type-2 receptor; MCM, microglia conditioned media; NA, not applicable; PFC, prefrontal cortex; SC, spinal cord; Str, striatum.

Results

In vitro studies

To study the role of ABX1431 in the context of neuroHIV, we conducted in vitro Ca²⁺ imaging studies using primary FC neuronal cultures at DIV21. Tat (100 nM) application onto neuronal cultures significantly increased [Ca²⁺]_i concentration, which was significantly reduced by ABX1431 pretreatment at all concentrations (10, 30, 100 nM; p<0.0001; Fig. 2A). Similarly, to understand the role of MAGL inhibition and Tat in mediating microglial neurotoxicity, we assessed [Ca²⁺]_i responses of FC neuronal cultures to MCM derived from ABX1431 and/or Tat-treated microglia in the presence and absence of CB₁R (rimonabant) and CB₂R (SR144528) antagonists.

FIG. 2.

Calcium changes in cultured FC neurons at DIV21. (A) Direct Tat and drug treatment onto neuronal cultures. The graph represents changes in [Ca²⁺]_i spikes upon 100 nM Tat administration. The significant increase in [Ca²⁺]_i levels by Tat was significantly decreased by ABX1431 pretreatments (10, 30, 100 nM) in a concentration-dependent manner. (B) Neuronal cultures exposed to MCM derived from Tat and drug-treated microglia. The graph represents changes in [Ca²⁺]_i levels of neuronal cultures upon MCM treatment. MCM Tat 100 nM caused a significant increase in [Ca²⁺]_i levels, while MCM ABX1431+Tat did not, indicating that ABX1431 100 nM blocked the effects of MCM Tat. Additionally, CB₁R or CB₂R antagonists, rimonabant and SR144528, respectively, did not block ABX1431's protective effects as a significant downregulation in [Ca²⁺]_i levels was noted for both conditions compared with MCM Tat. However, CB₁R seems to partially be involved in ABX1431's protective effects as MCM Tat+ABX1431+rimonabant significantly differed from control. All data are expressed as mean ± the SEM. Statistical significance was assessed by ANOVAs followed by Tukey's post hoc tests; *p<0.0001 versus control, ^ϕp<0.0001 versus Tat, ^δp<0.0001 and ^ξp=0.001 versus MCM Tat. ANOVA, analysis of variance; [Ca²⁺]_i, intracellular calcium; CB₁R, cannabinoid type-1 receptor; CB₂R, cannabinoid type-2 receptor; FC, frontal cortex.

Application of MCM Tat onto neurons resulted in a significant increase in [Ca²⁺]_i^, (p<0.001), which was blocked completely when applying MCM cotreated with ABX1431 and Tat (p<0.0001). MCM derived from cotreatment of ABX1431, Tat, and rimonabant only showed partial neuroprotective effects. Whereas MCM derived from cotreatment of ABX1431, Tat, and SR144528 showed protective effects similar to that of MCM derived from Tat and ABX1431 treatment, which suggests the protective effects of ABX1431 are partially CB₁R mediated only and not by CB₂R (Fig. 2B).

In vivo studies

Tat(−) and Tat(+) male mice were randomly assigned to vehicle or ABX1431-treated groups. No differences were observed in body mass between groups (Fig. 3A). The hot plate and tail-flick assays were conducted 4 h after ABX1431 oral gavage (4 mg/kg) to access heat-evoked nociception. Hot plate assesses supraspinal-related nociception, and no drug or genotype-dependent effects were observed (Fig. 3B). For tail-flick, spinal-related nociception, acute ABX1431 significantly increased pain latencies in Tat(+) male mice only (p=0.002; Fig. 3C).

FIG. 3.

Effect of acute ABX1431 at 4 mg/kg on body mass and spontaneous evoked nociception and locomotor activity. (A) Body mass was not affected by acute ABX1431 dose. (B) For the hot plate assay, no effects were noted by acute ABX1431 administration. (C) For the spinal-related tail-flick assay, ABX1431 administration at 4 mg/kg increased latency to pain signals in Tat(+) mice, whereas no significant differences were found in Tat(−) mice. (D) Locomotor activity was significantly altered by acute ABX1431 administration over 10 min and increased overall locomotor activity. (E) ABX1431 significantly changed the average speed by which mice traveled and increased overall speed of mice. (F) ABX1431 caused increase in immobility especially at 3-min time point irrespective of the genotype. All data are expressed as mean±the SEM. Statistical significance was assessed by ANOVAs followed by Tukey's post hoc tests; *p=0.002 vehicle-treated Tat(+) mice versus ABX1431-treated Tat(+) mice for tail-flick assay; ^#p≤0.001 main time effect; ^γp<0.05 main treatment effect; ^ρp<0.05 time×treatment interaction effect.

Furthermore, to evaluate effects of acute ABX1431 (4 mg/kg) on motor function, we assessed locomotor activity (Fig. 3D) following the hot plate assay. Over the period of 10 min, there was a significant decrease in locomotion (p<0.001); however, when treatment effect was considered, ABX1431 significantly altered this decrease in locomotor activity over the 10 min (p=0.008). Interestingly, ABX1431 increased the overall locomotor activity (p=0.041). Similarly, there were significant differences in average speed (Fig. 3E), with a significant decrease in speed over time (p<0.001), which was altered by acute ABX1431 (p=0.047). Furthermore, acute ABX1431 exposure significantly increased overall speed (p=0.009). Finally, there were no changes in the overall immobility due to ABX1431 (Fig. 3F), but increased immobility over time (p<0.001) was significantly altered by ABX1431 (p<0.001), which was specifically noted for the 3-min time point, where ABX1431 increased the immobility in both the genotypes.

CNS levels of eCB and receptors

Following behavioral experiments, CNS regions (PFC, striatum, spinal cord) were taken, quantified, and analyzed by two-way ANOVA for eCB and eicosanoid levels through UPLC-MS/MS, including AEA, 2-AG, PEA, OEA, and AA (Fig. 4 and Supplementary Table S1). Lipid concentrations (nmol/g) significantly varied between CNS regions (Fig. 4). The order of 2-AG levels was PFC < striatum < spinal cord. Importantly, acute ABX1431 significantly upregulated 2-AG levels in the striatum and spinal cord regardless of genotype (p<0.05). For AEA, no significant drug or genotype effect was noted with the order of AEA levels being PFC > striatum > spinal cord. Finally, AA levels were comparable between CNS regions, with only the striatum showing a drug×genotype interaction (p=0.031). AA was significantly upregulated in vehicle-treated Tat(+) males compared with Tat(−) males (p=0.009).

FIG. 4.

Endocannabinoid levels (nmol/g) in prefrontal cortex, striatum, and spinal cord. (A) 2-AG levels were found to be lowest in the prefrontal cortex and ABX1431 caused a significant upregulation in 2-AG levels in both, striatum and spinal cord, for both genotypes. (B) Acute ABX1431 treatment did not alter AEA levels in any regions regardless of genotype and drug treatment. (C) AA levels were comparable between the three brain regions and only striatum showed a significant drug×genotype interaction with vehicle-treated Tat(+) mice showing a significant upregulation in AA levels as compared with vehicle-treated Tat(−) mice and no change was observed with ABX1431 treatment. All data are expressed as mean±the SEM. Statistical significance was assessed by ANOVAs followed by Tukey's post hoc test where appropriate; *p≤0.05 main effect of drug; ^δp=0.031 genotype×drug effect; ^ɛp=0.009 vehicle-treated Tat(−) mice versus vehicle-treated Tat(+) mice. 2-AG, 2-arachidonoylglycerol; AA, arachidonic acid; AEA, N-arachidonoylethanolamine.

Regions from the left hemisphere were used for western blot analysis (Fig. 5). No alterations were found for CB₁R protein expression levels (Fig. 5B). A significant treatment×genotype effect was seen for CB₂R expression levels in the striatum (Fig. 5C).

FIG. 5.

Cannabinoid receptors 1 and 2 expression levels in prefrontal cortex, striatum, and spinal cord. CB₁R and CB₂R expression levels were assessed in PFC, striatum, and spinal cord of vehicle and acute ABX1431-treated Tat transgenic mice through western blot. (A) Representative immunoblots for CB₁R, CB₂R, and housekeeping protein GAPDH. (B) No genotype-specific and treatment-specific alterations were found in CB₁R levels in any CNS regions. (C) Significant changes in CB₂R levels were found in the striatum. ABX1431 treatment significantly increased CB₂R levels in Tat(−) mice and no changes were observed in Tat(+) mice. All data are expressed as mean±the SEM. Statistical significance was assessed by ANOVAs followed by Tukey's post hoc test where appropriate; *p=0.004 main drug effect; ^#p=0.001 main genotype effect; ^δp=0.005 genotype×drug effect. CNS, central nervous system.

CB₂R was upregulated in ABX1431-treated Tat(−) mice (p=0.004) and no changes were seen in Tat(+) mice. Raw unedited western blot images have been added to Supplementary Figure S1.

Discussion

HIV-1 Tat activates the glutamatergic N-methyl-D-aspartate (NMDA) receptors^36,48–50 and interacts with the lipoprotein receptor-related protein,⁵¹ causing an increase in neuronal intracellular calcium, dendritic damage, and synapse loss.^49,52–54 2-AG and AEA have been previously reported to counter these effects of Tat by downregulating intracellular calcium concentrations.⁴⁷ In contrast to AEA, 2-AG is present at higher concentrations in the brain and acts as a full agonist at both CB₁R and CB₂R.^55–58 The 2-AG is primarily hydrolyzed by MAGL into AA and glycerol, the primary source for cyclooxygenase-2 mediated production of proinflammatory prostaglandins.^26,59 Therefore, the neuroprotective effects of MAGL inhibitors may be due to reduced neuroinflammatory responses because of decreased production of AA and its prostaglandin metabolites, while simultaneously elevating 2-AG to enhance eCB receptor signaling.⁵⁹

Most in vivo studies have used shorter Tat exposure times of ≤2 weeks, but here we chose 16-week exposure to model the chronic elevation reflected human disease. While some studies have reported Tat-induced allodynia, hyperalgesia and damaged nerve fibers,^60–62 other studies have found either no effects⁶³ or even decreased pain sensitivity.¹³ The effect of Tat on nociception is complex and not clearly understood.¹³ Pain sensitivity also depends on the length of Tat exposure, with studies showing hyposensitivity following 3 weeks of Tat induction¹³ and hypersensitivity after over a month of Tat exposure.^60,64

The hyposensitivity may be due to neuronal dysfunction with initial Tat exposure, which is later reversed into hypersensitivity after prolonged exposure.⁴³ It is plausible that von Frey filaments (i.e., mechanical allodynia) may be a more sensitive assay to detect Tat-induced hypernociception compared with the hot plate or tail-flick assays. Various MAGL inhibitors have been shown to attenuate pain primarily through CB₁R activation.^65–69 The present study also showed that an acute ABX1431 (4 mg/kg) dose increased pain latency in Tat(+) male mice.

The mechanisms by which MAGL inhibitors exert their antinociceptive effects include the involvement of CB₁R^67,70,71 or CB₂R⁷² while others suggest the involvement of both CB₁R and CB₂R.^71,73,74 Moreover, another study claims that the therapeutic effects of MAGL inhibitor JZL184 is anti-inflammatory and does not depend on cannabinoid receptors.⁷⁵ We did not see any alterations in CB₁R levels, although 2-AG was upregulated in the striatum and spinal cord.

There is a possibility that the CB₁R functional activity may be altered, which cannot be detected by western blot. On the contrary, it is also possible that acute drug treatment was insufficient for substantial 2-AG-induced CB₁R receptor desensitization and subsequent internalization or loss of surface receptors as loss of CB₁R is demonstrated after prolonged use or chronic exposure to MAGL inhibitors.⁷⁶ These differences in the mechanism of action of MAGL inhibitors could be explained by the transient role of CB₁Rs resulting from 2-AG overload leading to desensitization of cannabinoid receptors following excessive MAGL inhibition.^76,77

Motor deficits, including impaired gait, motor strength, and motor coordination, are comorbidities PLWH face^78,79 leading to physical ailments and inadequate daily activity levels, even lower than most other chronic diseases.¹² Preclinical studies have revealed that the presence of Tat significantly decreases locomotion,^80–83 which is believed to be due to significant changes in the synaptic organization after Tat exposure.⁸⁰ However, we did not find any genotype differences in locomotor activity and speed in our vehicle-treated mice, which is in line with some past studies.^84,85

Discrepancies between studies may arise due to shorter open-field experiment duration, which was 10 min in our study and 30 min in a previously reported study,⁸⁰ or due to longer Tat exposure (6 months).⁸¹ Perhaps studies such as grip strength may have detected genotype differences. For ABX1431, we found that acute ABX1431 significantly increased overall locomotion and speed of mice regardless of their genotype, which supports previous studies where treatment with MAGL inhibitors, including JZL184 and MJN110, increased locomotor activity.^86–88

Differential effects of JZL184 and MJN110 have also been reported, in which JZL184 decreased locomotion, whereas MJN110 increased speed and total distance traveled.⁷¹ These differences in locomotor activity in the presence of various MAGL inhibitors and doses are yet to be understood and require further investigation.

Changes in the eCB system in PLWH have been previously reported.^89,90 There is little information about the levels of endogenous ligands such as AEA and 2-AG in neuroHIV. Although we did not see any changes in AEA levels in the CNS regions, ABX1431 significantly increased 2-AG in striatum and spinal cord of both genotypes. The increase in 2-AG levels in the spinal cord may account for the increased latency in the tail-flick assay in ABX1431-treated mice.

Additionally, AA, known to be a key player in inflammatory responses,^91–93 was significantly upregulated in vehicle-treated Tat(+) mice, suggesting an increase in proinflammatory mediators in the presence of Tat.⁹⁴ This is supported by a recent study where AA cascade and eicosanoid production were upregulated in the brains of HIV-1 gp120 mice.⁹⁵ As ABX1431 is a MAGL inhibitor, we showed 2-AG levels to be upregulated, but AA levels did not change. The modest increase of 2-AG and lack of changes in AA may be due to the route of administration of ABX1431, as previous studies have shown that intraperitoneal injection of JZL184 caused a large increase in 2-AG and decrease in AA concentration.⁹⁶

Also, the dose of JZL184 used in the previous study⁹⁶ (40 mg/kg) was much higher than the present study, which may be responsible for a decrease in AA concentration. It is also important to note that although MAGL is the primary enzyme responsible for the metabolism of 2-AG, serine hydrolases, α/β hydrolase domain 6 and 12 (ABHD6 and 12) are two other enzymes that play a minor but significant role in 2-AG hydrolysis,^97,98 which may be responsible for the increase in AA levels. It is also important to consider that lack of changes in AA may be due to the Tat-induced proinflammatory effect, which releases AA from cell membrane phospholipids through mechanism such as tumor necrosis factor and toll-like receptors, which are independent from MAGL.⁹⁹ Finally, we did not see any genotype-based changes in the CB₁R and CB₂R levels, except for CB₂R expression in the striatum, which was downregulated in vehicle-treated Tat(+) mice when compared with Tat(−) mice.

The lack of CB₁R alterations in neuroHIV has been reported previously^90,100,101; however, most studies^89,100,102 have shown an upregulation in CB₂R in the context of neuroHIV due to its inducible nature upon microglial cell activation leading to its anti-inflammatory function.^103–105 Interestingly, we found that acute ABX1431 administration led to upregulated CB₂R expression in the striatum of Tat(−) mice without affecting Tat(+) mice, which is the opposite of previous research findings where desensitization of the GPCRs was caused by 2-AG overload.^43,76,77 The exact relationship between the effects of MAGL inhibitors on the eCB system and its signaling pathway is yet to be understood, and warrants further investigation.

This study has several limitations; first, we only used male mice in the study. Literature has shown that women are more vulnerable to HAND symptoms.^106–111 Therefore, it is critical to understand the role of MAGL inhibitors using female mice along with the consideration of estrous cycle in future investigations. Second, the HIV Tat transgenic model used in this study is a well-established neuroHIV model; however, it only expresses one of many viral proteins in HIV. Therefore, this is an important consideration when generalizing findings to PLWH, as viral proteins may interact, target various signaling pathways, and modify the CNS compared with a single protein.⁹⁴

Lastly, the use of DOX to induce Tat expression in the Tat transgenic mouse is another limiting factor as DOX on its own has shown to exert neuroprotective effects and might mask some of the effects seen in this study.^112,113 To minimize bias and confound, all animals, including the Tat(−) mice were fed the same DOX chow throughout the study. Furthermore, it would be important to investigate the chronic effects of ABX1431 exerted on nociception, locomotor activity, and eCB levels, along with other comorbidities of HAND, such as anxiety and memory.

Conclusion

In conclusion, the present study demonstrates in vitro neuroprotective effects of a potent MAGL inhibitor on selected behaviors in a neuroHIV mouse model. Specifically, ABX1431 downregulated Tat- and MCM Tat-induced intracellular calcium release through a CB₁R-mediated mechanism. Moreover, acute ABX1431 displayed moderate antinociceptive effects and increased locomotor activity in our HIV Tat transgenic mouse model. While ABX1431 caused an increase in 2-AG levels in the striatum and spinal cord, only CB₂R was found to be upregulated in the striatum. Mechanistic studies are required to investigate how these alterations in the eCB system by MAGL inhibitors correlate with the behavioral outcomes in the context of neuroHIV. Based on previous clinical studies, ABX1431 is safe to use and therefore repurposing it for the treatment of HAND would facilitate future clinical trials.

Footnotes

Acknowledgment

The authors would also like to acknowledge the work of animal care technician Patrick G. Stutts for maintaining the welfare of our animals through the studies.

Authors' Contributions

Conceptualization: B.J.Y.-S., B.M.I.-J., A.H.L., and S.F.; methodology: B.J.Y.-S., H.P.R., K.M.B., H.D., J.L.P., K.J.R., and S.F.; validation: B.J.Y.-S., H.P.R., and S.F.; formal analysis: B.J.Y.-S. and S.F.; investigation: B.J.Y.-S., H.P.R., and S.F.; resources: S.F.; data curation: B.J.Y.-S., H.P.R., J.L.P., and S.F.; writing—original draft preparation: B.J.Y-S., and S.F.; writing—review and editing: B.J.Y.-S., H.P.R., B.M.I.J., A.H.L., J.L.P., K.J.R., and S.F.; visualization: B.J.Y.-S., H.P.R., and S.F.; supervision: B.J.Y.-S. and S.F.; project administration: B.J.Y.-S., and S.F.; funding acquisition: S.F., A.H.L., and B.M.I.-J.

Author Disclosure Statement

The authors declare no conflicts of interest.

Funding Information

This research was funded by the National Institute on Drug Abuse (NIDA), R01 DA055523 (S.F.), R21 DA041903 (S.F.), T32 DA007244 (H.P.R.), and P30 DA033934 (A.H.L.). B.M.I.-J. was supported by Japan Society for Promotion of Science (JSPS) Fellowship for Overseas Researchers (P17388), Kakenhi Grant-in-Aid for JSPS Fellows (17F17388), and Kakenhi Grant for Scientific Research (21K06399).

Supplementary Material

Abbreviations Used

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