Pharmacological blockade of 2-AG degradation ameliorates clinical, neuroinflammatory and synaptic alterations in experimental autoimmune encephalomyelitis

doi:10.21203/rs.3.rs-3334131/v1

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Pharmacological blockade of 2-AG degradation ameliorates clinical, neuroinflammatory and synaptic alterations in experimental autoimmune encephalomyelitis

https://doi.org/10.21203/rs.3.rs-3334131/v1

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published 31 Mar, 2024

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The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, the neuroprotective and anti-inflammatory effects of the ECS have been studied, and inhibiting the degradation of the endocannabinoid 2-arachydonoylglycerol (2-AG) is emerging as a promising strategy to counteract brain damage in MS. In this study, a systemic and preventive in vivo treatment with MAGLi 432, the reversible inhibitor of monoacylglycerol lipase (MAGLi), was performed in experimental autoimmune encephalomyelitis (EAE) mice. Clinical, biochemical, electrophysiological and immunofluorescence analyses were carried out to evaluate the impact of the drug on motor disability, neuroinflammation and synaptic damage. MAGLi 432 induced a less severe EAE disease, accompanied by an increase of 2-AG and a reduction of acid arachidonic (AA) and prostaglandins (PGs) brain levels. MAGLi 432 reversed the striatal synaptic hyperexcitability promoted by EAE neuroinflammatory insult. Furthermore, MAGLi 432 reduced both astrogliosis and microgliosis in EAE, as microglia/macrophages density was reduced, in parallel with a less reactive morphology. By flow cytometry analysis, we also observed less infiltrating CD45 + and CD3 + cells in the brain of MAGLi 432-treated EAE mice.

In conclusion, in vivo MAGL inhibition promotes a significant amelioration of EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects, including an enhanced 2-AG tone and reduced PGs levels, gliosis, and lymphocyte infiltration. Overall, these data provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and reveal the therapeutic potential of MAGLi-based drugs in MS inflammatory and neurodegenerative brain damage.

Multiple Sclerosis (MS)

Microglia

Neuroinflammation

Synaptic transmission

Excitotoxicity

Endocannabinoid

Monoacylglycerol lipase (MAGL)

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system (CNS) and presenting both demyelinating and neurodegenerative features (1). Clinical and preclinical data revealed that lymphocyte infiltration, together with microglial and astroglial activation are key events in the onset and progression of the disease. The early stages of the disease are characterized by brain and spinal cord invasion by peripheral immune cells, including macrophages, CD8 + T cells, CD4 + T cells and B cells, while microglia and astrocyte activation are the main responsible of brain damage in the chronic phases (2, 3). Recent advances in MS and in its mouse model experimental autoimmune encephalomyelitis (EAE), clearly showed that neuroinflammation unbalances synaptic transmission towards more pronounced hyperexcitability, ultimately resulting in excitotoxic neurodegeneration. Remarkably, inflammatory synaptopathy is an attractive therapeutic target in MS, since early synaptic alterations are potentially reversible.

Soluble mediators of inflammation released by infiltrating lymphocytes and by microglia/macrophages are master players of inflammatory synaptopathy. Accordingly, in the EAE striatum we observed that the two major proinflammatory cytokines, IL-1β and TNF, acting at pre- or postsynaptic sites respectively, enhance glutamatergic transmission and induce dendritic pathology (4–7). Endocannabinoids (eCBs), including 2-Arachidonoylglycerol (2-AG), are endogenous lipid-derived messengers able to bind to two G protein-coupled receptors cannabinoid receptor 1 (CB1) and 2 (CB2) and to non-CB1/ non-CB2 receptors such as TRPV channels (8, 9). In MS, dysregulation of the endocannabinoid system (ECS) is involved in the progression of the disease, and animal research supports the utility of cannabinoid compounds for MS treatment (10, 11). Different therapeutic strategies can increase the eCB tone. Targeting the eCBs metabolism to indirectly manipulate this pro-homeostatic system is emerging as an important pharmacological strategy, which avoids the unwanted side effects of direct CB receptor agonists (12). Enhancing anandamide (AEA) tone by inhibiting its degradation by fatty acid amide hydrolase (FAAH) provided significant clinical and synaptic protection in EAE (13), while the effects of 2-AG in EAE pathophysiology is less explored.

Numerous findings support monoacylglycerol lipase (MAGL) as an interesting target for the treatment of many pathological conditions, including MS (14). MAGL is the major enzyme that controls the levels of 2-AG, and is expressed in neurons, astrocytes, oligodendrocytes, microglia and pericytes (14, 15). Several highly potent and selective MAGL inhibitors (e.g., JZL184, KLM29 and MJN110) are available and considered as useful tools to explore a wide range of positive effects induced by 2-AG in animal models. JZL184, which acts as an irreversible inhibitor, was found to reduce demyelination in association with less microglia/macrophage activation in the spinal cord of EAE mice (16), to control spasticity (17), and to decrease neurological deficits (16, 18–21). Unfortunately, chronic administration of JZL184 led to a generalized desensitization of its central action, recapitulating the effects observed in mice lacking the MAGL gene(21). For this reason, reversible MAGL inhibitors have been recently developed (14, 22–24), in an attempt to take more advantage of 2-AG neuroprotective and anti-inflammatory effects. Since the molecular and cellular mechanisms underlying the potential beneficial effects of MAGL inhibition in EAE are still unclear, here we investigated the effect of a novel reversible MAGL inhibitor (MAGLi 432) (15) in the context of EAE.

Animals

Female C57BL/6 mice (6–8 weeks of age) were purchased from Charles-River (Italy) and stabilized, four-caged, in Type 3H cages for at least seven days after delivery. Mice were housed under constant conditions with a light/dark cycle of 12 h, in a temperature-controlled environment and free access to food and water. Animal experiments were carried out according to the Internal Institutional Review Committee, the European Directive 2010/63/EU and the European Recommendations 526/2007 and the Italian D.Lgs 26/2014. The experimental protocol was approved by the Italian Ministry of Health (Aut.n. 337/2021-PR). All efforts were made to minimize the number of animals used and their suffering. The choice of female sex is of relevance for MS, due to the high prevalence of the disease in female versus male subjects. All experiments were performed blind. A total of two independent immunizations of a minimum size of 9 animals per group were performed.

EAE induction and treatment protocols

Chronic-progressive EAE was induced in eight- to ten-week-old mice by active immunization with an emulsion of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35-55) in Complete Freund’s Adjuvant (CFA), followed by intravenous administration of pertussis toxin (500 ng) twice (at days 0 and 2) as previously described (4). Control mice, hereafter referred to as CFA, received the same treatment as EAE mice without the MOG peptide, including complete CFA and Pertussis toxin. EAE clinical score was recorded daily according to a 0–5 scale. For each animal, the onset day was recorded as the day post-immunization (dpi) when it showed the first clinical manifestations (score > 0). EAE mice were further randomly divided in two groups and were daily treated with the drug (MAGLi 432; i.p., 2 mg/Kg/day) or vehicle (VHL, 80% NaCl 0.9%+10% DMSO + 10% Tween 80) via intraperitoneal injection at a volume of 200 microliter/mouse, starting from 0-day post immunization (0 dpi). The clinical course of the disease was followed until the acute phase around 20–25 dpi (acute phase refers to the post peak symptomatic phase) and early chronic phase of the disease (30–35 dpi). Mice were killed after 1 hour from MAGLi 432/VHL administration.

Behavioral assessment

Animals were tested during the light period (9:00–12:00 am) in a dedicated room with a constant temperature (26 ± 1 ◦C). Each session was preceded by at least 1 h habituation in the behavioral room.

Grip strength test

Immunized mice were tested for grip strength performance using the Grip Strength Meter (Ugo Basile, Italy) at the onset of disease (8 and 14 dpi) and at the acute phase (21 dpi) (25). The apparatus for this test consisted of a steel wire grid (8 × 8 cm) connected to an isometric force transducer Mice were lifted by their tail so that they grasped the grid with their paws. Then gently pulled backward until they released the grid and the maximal force in newtons (N) exerted by the mouse before losing the grip was measured. The mean of three consecutive measurements for each animal was calculated.

Immunofluorescence and confocal microscopy

Mice were deeply anesthetized and intracardially perfused with ice-cold 4% paraformaldehyde (PFA) at 24 dpi and at 35 dpi (N = 3–4 per group). Collected brains were post-fixed in 4% PFA for 2 h and equilibrated with 30% sucrose for at least one night. Thirty-micrometer-thick coronal sections were serially cut on a frozen microtome to include the whole striatum to perform immunofluorescence experiments as previously described (25). Brain regions were identified using a mouse brain atlas, and for each animal, at least 6 serial sections (one section every six in the bregma interval from 1.34 to -0,10) containing the striatum, were processed for immunofluorescence. Slices were permeabilized in PBS with Triton X-100 0.25% (Tx-PBS). All following incubations were performed in Tx-PBS. To block non-specific sites, sections were pre-incubated with 10% normal donkey serum solution for 1 h RT and then incubated with the primary antibody overnight at + 4°C. Then, after washing, they were incubated with secondary antibodies for 2 h RT and rinsed. Primary antibodies were used as follows: rabbit or Goat anti-IBA1 (1:750, Wako); Rabbit anti-TMEM119 (0,25 µg/ml, Abcam); Rabbit Anti-GFAP (1:500, Dako); Rat Anti-CD3 (1:300, R&D systems); Rabbit anti-Collagen IV (1:1000, Biorad). These were used in combination with the following secondary antibodies: Cy3-conjugated donkey anti-rabbit or anti-Goat (1:200; Jackson); Alexa-488-conjugated donkey anti-rabbit or anti-rat (1:200; Invitrogen), Alexa-647-conjugated donkey anti-rabbit (1:200; Invitrogen). Nuclei were stained with [4′, 6-diamidino-2-phenylindole (DAPI)] (1 µl/ml; Sigma Aldrich).

Images were acquired using a Nikon Eclipse TI2 confocal laser-scanner microscope with 20x and 40x, 60x objectives and were processed using ImageJ software. All images had a pixel resolution of 1024×1024. The confocal pinhole was kept at 1.0, the gain and the offset were lowered to prevent saturation in the brightest signals, and sequential scanning for each channel was performed. Z-stack acquisitions (20x and 40x objective, zoom 1x with 2 µm intervals for a total of 17 steps; 60x, zoom 2 with 1 µm intervals for a total of 34 steps) were made applying the same intensity and exposure time. A large image function generates a single high-magnification image (capturing 2 images, according to Bregma level). A z-projection image derived from all captured images was produced. All images were adjusted for reducing noise by applying smooth and background subtraction as required by the NIH ImageJ. In the z-projections, the IBA + density analysis to evaluate microglia density was performed and a bilateral Roi (region of interest) including dorsal striatum was drawn. To obtain cellular density, the number of IBA1 + cells was automatically determined, and divided by the area covered by the Roi. Data were expressed as the number of cells per mm². ImageJ software was used to define microglia morphology as in (26). A gray scale image of IBA-1 was generated and then cells were cropped and thresholded to generate a binary (black and white) image (See supplementary Fig. 1). For the Sholl analysis the ImageJ plugin Sholl Analysis was used (starting radius: 2 µm, ending radius: 60 µm, radius step size: 2 µm). For skeleton analysis, the same binary image created for Sholl Analysis was used. The image was skeletonized using the ImageJ Plugin Skeletonize and was subsequently analyzed using the plugin Analyze Skeleton 2D (27). ImageJ software was used to quantify the astroglia total area (GFAP + surface %) in the Roi, generating intensity threshold images (binary images), and the measure of astroglial surface was calculated by the software.

Electrophysiology

Mice (11–13 wks) were killed by cervical dislocation and the brains were removed. Then, corticostriatal coronal slices (200 µm) were cut by means of a vibratome (Leica VT1200 - Leica biosystems, Wetzlar, Germany) and transferred to a recording chamber with continuously flowing artificial CSF (ACSF) (34°C, 2–3 mL/min) gassed with 95% O₂-5% CO₂. The composition of ACSF was (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 Glucose, 25 NaHCO3. Whole-cell patch-clamp recordings were made with borosilicate glass pipettes (1.8 mm outer diameter; 2–4 MΩ), in voltage-clamp mode, at the holding potential of -80 mV. Only data from putative medium spiny projection neurons (MSNs). Spontaneous excitatory and inhibitory post-synaptic currents (EPSCs, IPSCs) recording, data storing, and analysis were performed as previously described (28). Corticostriatal slices derived from EAE mice in vivo treated with MAGLi 432 or VHL (sEPSC, 20–24 dpi; sIPSC: 30–34 dpi) were used to record sEPSCs and sIPSCs. In some experiments, AM281 (2 µM; Sigma), a potent and selective CB1 antagonist, was acutely applied during the electrophysiological recordings.

For ex vivo experiments, sEPSCs were recorded from EAE corticostriatal slices (20–24 dpi) incubated with MAGLi 432 (1µM, 1 hour), or pre-incubated with AM281 and subsequently with AM281 plus MAGLi 432 (1 hour). Synaptic events were stored using PCLAMP (Axon Instruments - Molecular Devices, San Jose, CA, USA) and analysed offline on a personal computer with Mini Analysis 6.0.7 (Synaptosoft, Leonia, NJ, USA) software.

Infiltrating mononuclear cell isolation

Mice were deeply anesthetized and intracardially perfused with ice-cold 1X PBS at 21–23 dpi (EAE VHL N = 12; EAE MAGLi N = 8). Collected brains and spinal cords of 2 mice for each experimental group were pooled and mechanically disaggregated. Cell suspension was passed through a 70-µm cell strainer to remove cell debris, centrifuged and subjected to a gradient separation. Cells were resuspended in 4 ml of 37% Percoll in room-temperature PBS and overlay onto 3 ml of 70% Percoll in room-temperature PBS in a 15ml tube. Cells were centrifuged at 500 g for 30 min at room temperature without brake. The fat on top of the tube was removed and the cells from the interface were collected and the sample washed twice in PBS. Two pairs of samples were finally combined to obtain a single pool derived from 4 mice for each experimental group.

Flow cytometry

Immediately after isolation, CNS infiltrating immune cells were prepared for flow cytometric analysis. Cells were incubated with Zombie UV™ Fixable Viability kit (BioLegend, San Diego CA) according to manufacturer’s instruction. Briefly, cells were washed in PBS and then resuspended in 100 µl of PBS diluted Zombie UV™ dye (diluted 1:100 from stock solution). Cells were incubated 15 minutes at room temperature in the dark, then washed with PBS 0.5% BSA. After staining with the vital dye, cells were labeled with fluorochrome-conjugated antibodies against membrane markers. The anti-mouse antibodies used were: anti-CD45 APC, anti-CD3 PE-Cy7, anti-CD4 APC-Cy7, anti-CD8a PE, anti-CD19 PE-Cy7, anti-F4/80 PE-CF594(all antibodies from BD Biosciences, Milan, Italy). Cells were incubated at 4°C for 20 minutes in the dark with the appropriate mix of antibodies, then were washed and resuspended in PBS 0.5% BSA. Samples were acquired immediately on a BD LSR Fortessa X-20 and analyzed using FACS Diva software (v8.0.2). At least 10000 events in the region of living (zombie uv negative) cells were acquired.

Liquid chromatography/mass spectrometry (LC/MS) and Activity-based protein profiling (ABPP) analysis

For the in vivo LC/MS and ABPP analysis derived from MAGLi or VHL treated mice (N = 6 mice per experimental group), cerebellum and left cerebral hemisphere were dissected at 24 dpi and placed in separated 1,5 mL vials and snap frozen in dry ice and stored at -80°. For ex vivo experiments about LC/MS and ABPP analysis, mice (N = 2 mice) were euthanized (at 20–25 dpi) like previously described and corticostriatal slices (190 µm thickness) were placed in 1,5 mL vials (two slices for vials) and snap frozen in dry ice. The vials were stored at -80°C.

ABPP. Cerebellum samples and corticostriatal slices were homogenized in ABPP lysis buffer (20 mM HEPES, 2 mM DTT, 1 mM MgCl2, 0.1% Benzonase® Nuclease, ≥ 250 units/µL), incubated for 30 minutes on ice and centrifuged at 13,000 rmp, for 10 minutes at 4°C. The supernatants were collected and the total protein concentration was determined using Pierce 660 nm Protein Assay (ThermoFisher, cat.nr. 22662) following the manufacturer’s instruction. The total protein concentration of each sample was adjusted to 2 or 1 mg/mL with ABPP storage buffer (20 mM HEPES, 2 mM DTT). In a 96 well plate, 19.5 µL of samples were incubated with 0.5 µL 80 nM MAGL-specific fluorescent probe (15) for 25 minutes at RT. The reaction was stopped by the direct addition of 12.5 µL dH2O, 12.5 µL NuPAGETM LDS Sample Buffer (4x, ThermoFisher, cat.nr. NP0007) and 5 µL NuPAGETM Sample Reducing Agent (10x, ThermoFisher, cat.nr. NP0004) followed by incubation of the plate on ice for 30 minutes. The samples were then separated by gel electrophoresis. 25 µL of samples were loaded in a 12% polyacrylamide gel (NuPAGETM 12% Bis-Tris, 1.0mm, Mini Protein Gel, 10-well, cat.nr, NP341BOX) and run for 55 minutes at 200V in NuPAGETM MES SDS Runnung Buffer (diluted to 1X in dH2O, cat.nr. NP0002). The picture acquisition was performed using the ChemiDoc system (BioRad) with Cy5 filter settings and automated acquisition time. An actin Western Blot was performed as loading control. The gels were transferred on nitrocellulose membranes using iBlotTM 2 Transfer Stacks (ThermoFisher, cat.nr. IB23001) and the iBlotTM 2 Gel Transfer Device (ThermoFisher, cat.nr. IB21001) following the manufacturer’s instructions. The membranes were blocked with 5% nonfat milk in PBS-Tween 0.1% for one hour at RT and stained with an HRP-labeled anti-β-actin antibody (Abcam, cat.nr ab49900) diluted 1:25000 in 5% nonfat milk,PBS-Tween 0.1%, overnight, at 4°C while shaking. The membranes were washed three times for 5 minutes with PBS-Tween 0.1% and developed with SuperSignalTM West Dura Extended Duration Substrate (ThermoFisher, cat.nr. 37071) following the manufacturer’s instructions.The membrane picture were taken using ChemiDoc system (BioRad), using a minimum exposure time of 1 second.

Liquid chromatography/mass spectrometry. Frozen brains or brain slices were placed into 7 mL hard tissue homogenizing vials prefilled with ceramic beads (Bertin Instruments, Cat.No. P000935-LYSK0-A.0) and homogenized in methanol using a Precellys homogenizer (Bertin Instruments, Cat.No. P000062-PEVO0-A) operating at 3x 10s, 6000rpm giving a final concentration of 100 mg tissue/mL methanol. Brain homogenates were purified by protein precipitation followed by centrifugation (18000g for 10min). Aliquots of the cleaned samples were further diluted 1:2 with methanol containing internal standards prior to analysis. Undiluted precipitates were used to measure AEA, PGE2 and PGD2 levels, whereas precipitates were diluted 1:100 to measure 2-AG and arachidonic acid (2 µL of each sample was injected). Calibration samples were prepared accordingly using cleaned aliquots of pooled brain homogenates spiked with 10-fold concentrated calibration standards serially diluted in methanol. The internal standards for arachidonic acid, 2-AG, AEA, PGE2 and PGD2 were AA-d8, 2-AG-d5, AEA-d8, PGE2-d4 or PGD2-d9, respectively (all from Cayman Chemicals, Ann Arbor, MI, USA). All calibration curves were fitted using a linear regression function with 1/y weighting, excluding zero and absolute concentrations were calculated by dividing the peak area ratio (= peak area analyte/peak area internal standard) by the slope of the calibration curve.

Statistical Analysis

Statistical analysis was performed with Prism GraphPad version 9.0. Data distribution was tested for normality by using Kolmogorov–Smirnov test. Non-normally distributed data were analyzed through non-parametric tests. Data were presented as the mean ± S.E.M and the significance level was established at p < 0. 05. Differences between two groups were analysed using two-tailed Paired or Unpaired Student’s t test, as appropriate. Multiple comparisons were performed by One-Way ANOVA followed by Tukey’s HSD or Two-Way ANOVA followed by Bonferroni Post Hoc. Differences between groups in clinical score analysis were tested by Mann–Whitney test and by log-rank test. The exact statistical test used for each experiment and its details can be found in the figure legends. Throughout the text “N” refers to the number of animals. For electrophysiological experiments and quantitative immunofluorescence analysis “n” refers to the number of cells recorded or slices analyzed, respectively.

1. In vivo treatment with MAGLi 432 ameliorates clinical score and motor performance in EAE

We assessed the effects of a systemic and preventive MAGL inhibition on MOG_{35 − 55} EAE animal model of EAE. To this aim, two groups of mice were daily treated with MAGLi 432 (2 mg/kg/day) or VHL via intraperitoneal injections, starting from 0 day post immunization (0 dpi) and the clinical course of the disease was followed until the acute phase of the disease (25 dpi).

We observed that MAGLi 432 induced a less severe EAE disease compared to mice treated with VHL, with a significant difference emerging around 16–25 dpi (Fig. 1A).

To evaluate whether these beneficial effects were accompanied by an improvement in neuromuscular function, we assessed the maximal force of the mice using the grip strength test before the appearance of clinical deficits and during the acute phase of the disease (Fig. 1B). During the early phase of the disease (14 dpi), MAGLi 432-treated mice showed a better performance in terms of strength of both fore and hind-limb relative to the vehicle group, an effect that disappeared at 21 dpi.

ABPP biochemical analyses performed on mouse brain samples at 24 dpi demonstrated the MAGL target occupancy of MAGLi 432 (Fig. 1C). LC/MS analysis of 24 dpi brain hemisphere showed a significant increase of 2-AG levels combined with a concomitant and significant decrease of AA, PGD2 and PGE2 levels (Fig. 1D) thus confirming MAGL target engagement in the brain of the MAGLi 432 treated mice. Notably, anandamide (AEA) was not modulated by the treatment, supporting the specificity of MAGLi 432 for MAGL enzyme.

The beneficial effect of MAGLi on EAE clinical disability was confirmed in a second experimental set where a significant difference was also observed in terms of disease incidence (Supplementary Fig. 2)

2. MAGLi 432 in vivo treatment reduces striatal microgliosis and astrogliosis in EAE mice

The effect of MAGLi 432 treatment on neuroinflammation was evaluated by immunofluorescence and confocal analysis. The immunostaining of microglia/macrophages (IBA1/TMEM positive cells) and astroglia (GFAP positive cells) were performed in the striatum, a brain region highly sensitive to the neuroinflammatory and neurodegenerative processes of MS/EAE (4, 5, 29)and strongly modulated by the ECS (30–32).

Quantification of microglia/macrophages density showed a significant reduction of IBA1 + cells in EAE-MAGLi compared to EAE-VHL at 24 dpi (Fig. 2A-A’). Such an effect was maintained during the chronic phase of the disease (35 dpi), as revealed in the second experimental set (Supplementary Fig. 3).

A main feature of microglia is its rapid morphological change upon activation, characterized by gradual acquisition of an amoeboid shape resulting in a complex multistage and multifaced activation process. Thus, by means of Sholl and Skeleton analyses, we carried out a detailed analysis of microglia branching ramification in the dorsal striatum, considering defined brain regions distant or close to the inflammatory lesion site. Lesion sites were defined by an increased cellularity denoted by the nuclear stain DAPI at the levels of the corpus callosum (CC) and dorsal striatum (DS) (33). Double immunostaining for TMEM119 /IBA1 was performed to distinguish infiltrating macrophages from resident microglia close to the lesion site (34) (Supplementary Fig. 4). Interestingly, we showed that the ramification rate of EAE microglia was significantly decreased next to the lesion compared to cells distant from this site, revealing for the first time a region-dependent microglial phenotype within the EAE striatum (Fig. 2B-B’ EAE-VHL; 2C-C’ EAE-MAGLi and relative tables). Only the maximum branch length parameter was unchanged.

To better define the microglia activation state in the selected region distant from the lesion site, we compared the Sholl analysis between EAE and CFA microglia. The number of intersections, the ramification index, and the critical value (radius of highest count of intersection), were increased in EAE striatum compared to CFA condition, indicating a hyper-ramified intermediate phenotype of EAE microglia (35) (Fig. 3A and see table in Fig. 3A’; Supplementary Fig. 5). Interestingly, the hyper-ramified activated microglia phenotype was significantly attenuated in EAE MAGLi treated animals (Fig. 3A and see table in Fig. 3A’; Supplementary Fig. 5), corroborating the anti-inflammatory effect of the drug previously described.

The skeleton analysis also showed that the number of branches, junctions and end-points of microglia were increased in EAE compared to CFA mice, revealing an elevated branching density and complexity of EAE microglia. Notably, in accordance with previous results, MAGLi 432 attenuated EAE microgliosis (see table in Fig. 3B). Moreover, the maximum branch length or average branch length of microglia were reduced in EAE-VHL compared to CFA mice, whereas both parameters were unaffected by MAGLi treatment (see table in Fig. 3B).

Furthermore, we detected a hypertrophic change of microglia cell bodies following EAE induction. As shown in Fig. 3, the mean cell body area was increased in the EAE group compared to CFA mice, and significantly reduced in EAE-MAGLi group (Fig. 3C).

Regarding the analysis of microglia morphology in the region next to the lesion site, we did not detect any difference between EAE-VHL and EAE-MAGLi mice, in terms of number of intersections (Fig. 4A), ramification index and critical value (see table in Fig. 4A’). Skeleton analysis parameters were also unaffected by the treatment (Fig. 4B).

Finally, by performing GFAP immunostaining, we investigated for the first time the effect of MAGLi treatment on striatal astrogliosis, an event markedly associated with EAE induction (36) (Fig. 5A and Supplementary Fig. 6). Since the morphology of astroglia cells and their spread on EAE striatum can misrepresent the analysis of GFAP + cell density, we evaluated astrogliosis by measuring the percentage of astroglia area. High magnification and binary images showed a significant attenuation of astrogliosis in the striatum and corpus callosum of EAE MAGLi mice compared to EAE-VHL mice (Fig. 5B).

3. MAGLi 432 in vivo treatment reduces immune cell recruitment into CNS

MS disease is characterized by inflammatory infiltration of the CNS and, among infiltrating immune cells, autoreactive T cells initiate CNS demyelinating lesions and, likely, microglial activation and inflammatory synaptopathy (2, 37). To evaluate whether MAGLi treatment could reduce effectively infiltration and/or favor resolution of inflammation, we isolated lymphocytes infiltrating the brain and spinal cord of EAE VHL and MAGLi treated mice. By means of flow cytometry, we quantified and defined the populations of peripheral infiltrates in the acute phase of the disease (Fig. 6A). We observed a reduced number of all nucleated hematopoietic cells (CD45 + cells, unpaired t test p < 0.05) as well as T cells (CD3 + cells) in the CNS of EAE MAGLi animals compared to VHL mice. Within CD3₊ cells, we noticed a decreasing trend, although not significant, of both CD4 and CD8 sub-populations. On the other hand, no effects were observed on B cells and macrophages. Flow-cytometry experiment was corroborated by qualitative immunofluorescence analyses of CD3 + infiltrating lymphocytes in lesion sites of the corpus callosum/dorsal striatum of EAE MAGLi mice (Fig. 6B-B’). The inflammatory lesion area was defined by nuclear DAPI staining (33) and by anti-Collagen IV staining to visualize capillary walls. Co-staining of CD3 and Collagen IV suggests that the infiltration of CD3 + T cells, typically enhanced in EAE brain/spinal cord lesion sites, was reduced in EAE MAGLi mice compared to EAE VHL mice, especially nearby and within capillaries (Fig. 6B-B’).

4. In vivo MAGLi 432 treatment ameliorates glutamatergic alterations in the EAE striatum

Based on the complex interaction between neuroinflammation and ECS in modulating EAE synaptopathy, we investigated a potential beneficial effect of MAGLi 432 treatment on EAE synaptic dysfunction. To this aim, we performed whole-cell voltage-clamp recordings from medium spiny neurons (MSNs) to evaluate both spontaneous glutamatergic (sEPSCs) (20–24 dpi) and GABAergic (sIPSCs) transmission (30–34 dpi) of EAE mice in vivo treated with MAGLi or VHL.

Remarkably, daily systemic treatment with MAGLi ameliorated glutamatergic alterations that characterizes the EAE striatum. Indeed, EAE treated mice showed values of sEPSC frequency similar to healthy mice and significantly different compared to EAE VHL mice (Fig. 7A) whereas the sEPSC kinetics were not affected (Supplementary Fig. 7). The frequency and amplitude of the EAE GABAergic transmission were not affected by the treatment (Supplementary Fig. 7). Finally, we investigated the involvement of CB1 receptors, whose activation is known to mediate a tonic suppression of glutamatergic transmission. However, as shown in Fig. 7B, incubation of AM281, a potent and selective CB1 antagonist, did not affect sEPSC frequency of EAE-MAGLi slices suggesting a chronic, rather than tonic, effect of the treatment.

5. Activation of CB1 receptor promoted by MAGLi 432 rescues EAE striatal glutamatergic alterations

To explore a possible direct effect of MAGLi on EAE striatal synaptic transmission, we performed ex vivo experiments incubating corticostriatal slices derived from EAE mice (20–24 dpi; N = 8) for 1-hour with MAGLi 432 (1µM) or VHL directly. Acute MAGL inhibition led to a complete MAGL target occupancy, as demonstrated by ABPP (Fig. 8A), and a strong modulation of 2-AG levels (Fig. 8B), as previously observed after in vivo treatment with MAGLi in EAE mice. Conversely, the treatment did not significantly decrease AA, PGD2 and PGE2 levels (Fig. 8B). In parallel, ex vivo treatment with MAGLi 432 in EAE slices completely rescued the exacerbation of EAE glutamatergic frequency (Fig. 8C). Such an effect was abolished by concomitant incubation with AM281 (2 µM) the CB1 antagonist, suggesting a direct involvement of CB1 receptor in this acute MAGLi-mediated synaptic modulation.

In the present study, we investigated the neuroprotective effects of a selective and reversible MAGL inhibitor on EAE disease, uncovering for the first time a relevant impact of 2-AG metabolism on inflammatory-mediated synaptopathy. We demonstrated that daily peripheral administration of MAGLi 432 ameliorates EAE clinical disability, attenuating synaptic damage and neuroinflammation. In particular, 24 days of pharmacological treatment increased brain levels of 2-AG and reduced prostaglandin synthesis in the brain. Moreover, MAGLi attenuated T lymphocytes infiltration in the CNS and microglia/astroglia activation. The reduction of the inflammatory status together with a CB1-dependent suppression of glutamatergic transmission could account for the amelioration of striatal synaptopathy and clinical disability.

The enhancement of 2-AG promoted by the inhibition of MAGL enzyme could induce different interconnected mechanisms that underlie the beneficial effects here observed: the endogenous stimulation of both CB1 and CB2, and the reduction of eicosanoid production. Indeed, 2-AG is a full agonist of both CB receptors, and acid arachidonic (AA), its main metabolite, is the precursor of pro-inflammatory prostaglandins (PGs) (38, 39).

Within the CNS, 2-AG could exert both neuroprotective and anti-inflammatory effects. CB1 is expressed by glial cells (oligodendrocytes, astrocytes, macrophages/microglia), pericytes and endothelial cells, although the most important location for this receptor is neuronal (40). Indeed, eCBs primarily regulate synaptic function by suppressing neurotransmitter release acting on the presynaptic terminal at excitatory and inhibitory synapses (41). CB2 is expressed only by a few neuronal populations (42, 43) while is expressed by cells of the immune system (e.g. B and T lymphocytes) and endothelial cells. Noteworthy, CB2 is abundantly expressed by activated astrocytes and microglia (42, 43). In the last years, the regulation of microglia M2 polarization mediated by eCBs has been described (44), including the attenuation of microglial proinflammatory cytokine release (45, 46) and the modulation of microglial phagocytic function (45). Moreover, a role of cannabinoids in the modulation of astrocytic response was also pointed out, particularly through mechanisms involving AEA and palmitoylethanolamide (PEA). Importantly, astrocytes have a well-recognized role on eCB degradation and astrocytic-specific deletion of MAGL was shown to attenuate neuroinflammation (47).

MS is characterized by massive CNS inflammatory infiltrates comprising an intricate coordination of the innate and adaptive immune systems. Notably, histopathology of post-mortem tissue derived from MS patients showed CB receptor expression in infiltrated T-lymphocytes and revealed CB2-immunoreactive microglia/macrophages in MS lesions (48).

Our data demonstrate that MAGLi 432 modulates inflammatory response in EAE brain. The increased 2-AG tone and the decreased AA levels could attenuate neuroinflammation by different mechanisms that include: i) an immunoregulatory action on T cells and monocytes ii) a modulation of the blood-brain barrier (BBB) permeability targeting endothelial cells (49) iii) a direct interplay with microglia and astroglia cells.

Our data from flow cytometry data suggest that monocyte activation and infiltration were not affected by MAGLi 432 treatment in EAE mice. Moreover, in a different neuroinflammatory condition and under a different pharmacological regimen, it was recently shown that MAGLi 432 did not ameliorate lipopolysaccharide-induced blood–brain barrier permeability (15). Conversely, Piro and colleagues observed that a different MAGL inhibitor, CPD-4645, reduced BBB permeability in both LPS-challenged and ischemic inflammatory injured mice, partially through CB1 and CB2 signaling (49). Based on these observations, we can speculate that, in our condition, MAGLi 432 attenuates neuroinflammation by modulating peripheral T lymphocytes infiltration and/or by directly mitigating astrogliosis and microgliosis. Evidence suggests cannabinoids as critical players in promoting a reparative state of microglia in EAE, however the precise mechanisms underlying this effect need to be further defined (44, 50). Cannabinoid agonists have been shown to reduce the clinical severity of murine models of EAE and were associated with decreased microglia/macrophage activation in the CNS (51–53). Alongside their effect on reducing microglia/macrophage activation and clinical severity, synthetic CB2-selective agonists have also been shown to mitigate T cell infiltration, oxidative damage and axonal loss in the spinal cord of EAE mice, as well as to mitigate microgliosis and astrogliosis in the corpus callosum of cuprizone mice, thus accelerating remyelination process (54, 55). Interestingly, de Lago et al. (56) have shown that the beneficial effects of cannabinoid-based therapies on EAE severity and microglial/macrophage activation in the CNS appear to be mediated through CB1 receptors (56). In support of this finding, treatment of EAE mice with the synthetic cannabinoid CB52 alleviates clinical disease and reduces spinal cord microglia activation, T cell infiltration and damage. These effects were mediated by the activation of CB1 receptors (57).

In the present study, the detailed analysis of microglia morphology describes for the first time the complex changes of microglia phenotype in EAE striatum and highlights the impact of MAGLi on these processes. Microglia and infiltrating macrophages are known to participate in both beneficial and detrimental processes during MS development, and this dichotomic behavior could be explained by different activation states of these cells (58). This complex and rapid cellular process includes changes in cell numbers and cellular morphology resulting in a context- and time-dependent signature of microglia cells. For these reasons, microglia morphology modifications cannot be simply described as a transition from ramified to ameboid shape. Our results showed that in the EAE striatum, the region distant from the inflammatory lesion site, is characterized by hyper-ramified microglia with an intermediate activation state characterized by a “bushy-like” appearance (35, 59, 60). On the other hand, next to the lesion site, EAE microglia reduced their ramification rate displaying transition into ameboid morphology. Of note, MAGLi 432 treatment mitigated microglia activation state by reducing striatal cell density and modulating cellular morphological phenotype. In particular, the drug effect on morphological activation was mainly appreciable in cells distant from the lesion site where, probably, brain damage is not exacerbated and where, importantly, electrophysiological recordings were performed.

Together with microglia, astrocytes play a key role in EAE neuroinflammation and respond to brain damage by upregulating GFAP expression which is used as a marker for astrogliosis. This activation process is associated with functional, morphological and biochemical changes (61). While some data described the effect of MAGLi on microglia, very few data exist on MAGLi and reactive astrocytes. Here, we observed for the first timethat MAGLi treatment promotes a strong attenuation of astroglia activation in EAE striatum, thus contributing to the resolution of neuroinflammation.

In parallel, the pharmacological treatment with MAGLi 432 mitigated the aberrant glutamatergic transmission that characterizes EAE striatum by recovering glutamate-mediated sEPSC frequency. Notably, glutamatergic and GABAergic synapses are privileged sites of action for eCBs that act in concert with proinflammatory cytokines (11, 62). Activation of CB1 reduces the frequency of spontaneous glutamate-mediated synaptic currents in striatal neurons, thus contrasting the effects of IL-1β on glutamate release from presynaptic nerve terminals (32). eCBs are generally synthesized “on demand” and can suppress transmitter release in a transient (short-) or long-lasting manner (41). Importantly, neuroinflammation promotes synaptic dysfunction in EAE striatum and the anti-inflammatory effect mediated by MAGLi 432 could support the modulation of glutamate transmission. Thus, the anti-inflammatory effect combined with the direct CB1-dependent suppression of neurotransmitter release could account for the anti-synaptotoxic effect observed during MAGLi 432 treatment. Of note, acute application of CB1 antagonist (AM281) after a long- lasting- treatment with MAGLi 432 did not change glutamate frequency, indicating a consolidated process of reduced presynaptic glutamate release. Conversely, concomitant bath application of MAGLi 432 and CB1 antagonist, abolished the acute effect of MAGLi on sEPSCs, confirming that inhibition of the degradative enzyme of 2-AG promotes the stimulation of CB1 presynaptic receptors.

Noteworthy the significance of the pharmacological approach used in this study relies on the fact that the reversible inhibition of MAGL has the capacity to increase the 2-AG tone avoiding strong side effects associated with direct CB agonist (24).

Disease course and progression of MS are heterogeneous and the impact of ECS on CNS homeostasis is equally disparate. This study provides valuable insights into the role of MAGL inhibition in Multiple Sclerosis (Fig. 9). Notably, several clinical trials are ongoing with MAGL inhibitors, such as ABX-1431, which is entering clinical phase 2 studies for neurological disorders including MS. Our study sheds light on broad beneficial anti-inflammatory effects of MAGL inhibition on EAE disease thus supporting the potential efficacy of MAGL inhibitor-based therapy in MS disease.

Arachydonoylglycerol (2-AG); acid arachidonic (AA); activity-based protein profiling (ABPP); anandamide (AEA); artificial CSF (ACSF); blood-brain barrier (BBB); cannabinoid receptor 1 (CB1) and 2 (CB2); central 2nervous system (CNS); complete Freund’s Adjuvant (CFA); day post-immunization (dpi); endocannabinoid system (ECS); endocannabinoids (eCBs); experimental autoimmune encephalomyelitis (EAE); fatty acid amide hydrolase (FAAH); inhibitor of monoacylglycerol lipase (MAGLi); liquid chromatography/mass spectrometry (LC/MS); medium spiny projection neurons (MSNs); monoacylglycerol lipase (MAGL); multiple sclerosis (MS)

myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55); paraformaldehyde (PFA); prostaglandins (PGs); spontaneous postsynaptic glutamatergic current (sEPSC); spontaneous postsynaptic inhibitory current (sIPSC); vehicle (VHL).

Ethics approval and consent to participate

Animals experiments followed the guidelines of Animal Research Reporting In Vivo Experiments (ARRIVE, (Kilkenny 2010)) and were carried out according to the Internal Institutional Review Committee, the European Directive 2010/63/EU, the European Recommendations 526/2007 and the Italian D.Lgs 26/2014 (Prot. n°337/2021-PR).

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request

Competing interests

DC is the recipient of an Institutional grant from Roche. No personal compensation was received. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. SS, AP, IR, LC are paid employment by the company F. Hoffmann-La Roche. This does not alter the authors’ adherence to all the BMC policies on sharing data and materials. The other authors declare that they have no competing interests.

Funding

The study was supported by: a F. Hoffmann-La Roche grant to Diego Centonze; Ministero della Salute (Ministry of Health, Italy): Progetto Ricerca Corrente to IRCCS Neuromed; Ministero della Salute (Ministry of Health, Italy): Progetto Ricerca Corrente to IRCCS San Raffaele; Ministero della Salute (Ministry of Health, Italy): Francesca de Vito GR-2021-12373337; Fondazione Italiana Sclerosi Multipla (FISM): Diego Centonze cod. 2019/S/1 and financed or co-financed with the ‘5 per mille’ public funding; Funded by Ministry of University and Research and the European Union - Next Generation EU - NRRP M6C2 - Investment 2.1 Enhancement and strengthening of biomedical research in the NHS"- PNRR MNESYS: Centonze, Mandolesi and Musella; Project ‘Nuovi Biomarker Diagnostici e Terapeutici delle Malattie Neurodegenerative’— co-funded by FOE 2021—funding from CNR to Georgia Mandolesi.

Authors' contributions

LG, GM, DC, and AM conceived and designed the study. LG, FDV, SB, SC, VV, DF, AT, KS, LV, MN, VR, SS, AP, IR and LC, acquired and analyzed the data LG, GM, DC, and AM drafted the manuscript. All the authors critically revised the article for important intellectual content and approved the version to be published. LG and GM contributed equally to this work, as first authors. DC and AM contributed equally to this work, as senior authors.

Acknowledgments

The authors thank Massimo Tolu for helpful technical assistance. The graphical abstract was created with BioRender.com.

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Competing interest reported. DC is the recipient of an Institutional grant from Roche. No personal compensation was received. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. SS, AP, IR, LC are paid employment by the company F. Hoffmann-La Roche. This does not alter the authors’ adherence to all the BMC policies on sharing data and materials. The other authors declare that they have no competing interests.

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Pharmacological blockade of 2-AG degradation ameliorates clinical, neuroinflammatory and synaptic alterations in experimental autoimmune encephalomyelitis

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Abstract

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BACKGROUND

MATERIALS AND METHODS

Animals

EAE induction and treatment protocols

Behavioral assessment

Grip strength test

Immunofluorescence and confocal microscopy

Electrophysiology

Infiltrating mononuclear cell isolation

Flow cytometry

Liquid chromatography/mass spectrometry (LC/MS) and Activity-based protein profiling (ABPP) analysis

Statistical Analysis

RESULTS

5. Activation of CB1 receptor promoted by MAGLi 432 rescues EAE striatal glutamatergic alterations

DISCUSSION

CONCLUSIONS

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Declarations

References

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