Activation of central cannabinoid type 2 receptors, but not on peripheral immune cells, is required for endocannabinoid-mediated neuroprotection in Parkinson´s disease

doi:10.21203/rs.3.rs-4752464/v1

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Activation of central cannabinoid type 2 receptors, but not on peripheral immune cells, is required for endocannabinoid-mediated neuroprotection in Parkinson´s disease

https://doi.org/10.21203/rs.3.rs-4752464/v1

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Background

Neuroinflammation is a key feature of Parkinson´s disease (PD), a neurodegenerative disease for which there is no cure. The cannabinoid receptor type 2 (CB2R) is expressed by cells of the innate and adaptive immune systems. Inhibition of monoacylglycerol lipase increases the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG), which is neuroprotective in an experimental model of PD. We hypothesize that the beneficial effect of MAGL inhibition with JZL184 is mediated by the activation of CB2R on specific immune cell populations.

Methods

Experimental parkinsonism was induced by chronic administration of MPTP and probenecid for 5 weeks in wild-type (WT), CB2R transgenic and knockout (KO) mice. Motor behavior and histological techniques were used to determine the status of the nigrostriatal pathway. Myeloid and lymphoid subpopulations and their TNFα⁺ production were analyzed by low cytometry in the striatum and ventral midbrain. To distinguish whether a central or peripheral CB2R activation is required for neuroprotection, mice were treated with the CB2R agonists JWH133 and RO304. In addition, WT mice were irradiated and transplanted with CB2R KO hematopoietic stem cells to generate chimeric animals lacking CB2R in the peripheral immune system. Finally, we analyzed the transcriptomic profile of the endocannabinoid system in midbrain microglia and astrocytes from PD patients.

Results

Parkinsonian mice experimented a specific increase in CD4⁺ T cell infiltration in the midbrain. The neuroprotective effect of JZL184 was accompanied by a reduction in CD4⁺ T cell infiltration, these effects were abolished in CB2R KO mice. CB2R expression was restricted to myeloid cells and lymphocytes, and increased in microglia under parkinsonian conditions. Administration of JWH133, but not RO304, exerted a neuroprotective and immunomodulatory effect similar to that of JZL184. Using chimeric mice, we demonstrated that central CB2R activation is required for neuroprotection. Transcripts related to 2-AG biosynthesis are downregulated in the midbrain microglia from PD patients.

Conclusions

Activation of CB2R in the brain prevents nigrostriatal degeneration, CD4⁺ T cell infiltration and TNFα production in the midbrain of parkinsonian mice. The reduced 2-AG signaling in microglia from PD patients suggests that activation of microglial CB2R may be an interesting strategy for the treatment of PD.

Parkinson’s disease

neuroprotection

endocannabinoid system

cannabinoid receptor type 2

microglia and CD4+ T cells.

The versatility of the endocannabinoid system (ECS) places it in a key position to control the effects of neurodegeneration. Endocannabinoids are produced by both immune and neural cells (1, 2). The central location of the various elements, receptors and endocannabinoid synthesizing/degrading enzymes in the brain, allows the modulation of crucial processes such as neuroinflammation for the correct balance between neuron survival and degeneration (3). The immunomodulatory properties of the non-psychoactive cannabinoid type 2 receptor (CB2R) have long been known. CB2R was first described as a receptor that was not expressed in the brain, but was particularly abundant in immune cells and tissues (4, 5). Subsequent studies showed a low expression of CB2R in microglia at steady-state conditions and in some neurons (6, 7), which was increased in microglia under pathological conditions (8–10). Interestingly, CB2R is expressed on cells that constitute the innate and adaptive arms of the immune system and are involved in the pathophysiology of neurodegenerative diseases.

Parkinson´s disease (PD) is an age-related neurodegenerative disorder characterized by the loss of the dopaminergic nigrostriatal pathway. Neuroinflammation, a key feature of PD, has been placed at the intersection of the major risk factors, age, genetic background and environment (11). The identification of several single-nucleotide polymorphisms in PD associated with immune system functions (12, 13) and common genetic variants between PD and autoimmune diseases (14, 15) highlight the relevance of the immune system in this neurodegenerative disease. The interaction between innate and T cells may be a critical immunological driver of disease pathogenesis in PD (16). Proinflammatory microglia with increased HLA-DR⁺ levels (17, 18) and expressing tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β) and IL6 have been described in postmortem tissue from PD patients (18, 19). Experimental models of PD show a dynamic activation of microglia and astrocytes in early stages of neurodegeneration that correlates with the immune cell infiltration into the midbrain (20, 21). Evidence also points to the involvement of adaptive immune system in PD pathogenesis, but there is no consensus on the exact role played by these cells or the T cell subset involved in the disease (22, 23).

Circulating monocytes are altered in PD, with an increased ratio in proinflammatory classical monocytes accompanied by an activation of the CCR2-CCL2 axis (24, 25). Genes involved in immune activation have been detected in monocytes isolated at early stages of PD (26). A recent study concluded that perturbations of peripheral innate cell compartment in PD correlate with time and symptom severity, while changes are smaller in prodromal stages (27). Infiltrating lymphocytes have been described in the midbrain of PD patients (17, 22, 23). CD4⁺ T cells with an altered migratory potential are observed in the peripheral blood of PD patients (28). Data from a longitudinal study suggest that changes in the peripheral adaptive immune system in PD are associated with early or acute disease events in PD (27).

Modulation of specific elements of the ECS exerts different effects in experimental models of PD, symptomatic (29), neuroprotective (30) or accelerating (31) the time course of the disease. In this study we have focused on the neuroprotective effect of monoacylglycerol lipase (MAGL) inhibition with JZL184 in vivo (30). MAGL is the major hydrolyzing enzyme of the endocannabinoid 2-arachydonoyl glycerol (2-AG) in the brain (32). One consequence of MAGL inhibition is an increase in 2-AG signaling, a potent modulator of peripheral and central immunity (33), which could modulate the inflammatory response via CB2R (34) or reduce the availability of arachidonic acid required for prostaglandin synthesis (35). In vitro studies from our group suggest that CB2Rs are involved in the neuroprotective effect of JZL184 (36), but it is unknown whether they are also involved in vivo. Here, we hypothesize that CB2R activation at specific locations, central vs periphery or innate vs adaptive immune cells, would be responsible for the neuroprotective effect of MAGL inhibition. Our results show that the neuroprotective effect of the MAGL inhibitor JZL184 is mediated by CB2R in vivo and we demonstrate that activation of CB2R in the brain, but not in peripheral immune cells is required for neuroprotection.

2.1. Animals

Adult male C57BL6J (WT) mice, aged 2 months, were obtained from Envigo (Barcelona, Spain). CB2R KO and EGFP-CB2R mice were generated and donated by Julián Romero (37). Mice were housed in cages with a maximum of 6 mice and maintained in a temperature (21°C) and humidity-controlled room. Standard rodent pellet chow (Envigo) and water were provided ad libitum. Light was maintained on a 12 h light/dark cycle. All procedures involving animals were carried out in accordance with the Spanish National Research Council’s Guide for the Care and Use of Laboratory Animals. The experimental designs were approved by the Animal Experimentation Ethics Committee of the University of Navarra (Ref: 109 − 18, 109-18E3, 109-18E4).

2.2. MPTP intoxication and drug administration

To generate the chronic PD mouse model, 4-month-old mice received 10 i.p. injections of MPTP (20 mg/kg in saline; MedChemExpress, Shanghai, China) plus probenecid (250 mg/kg in saline; Life Technologies, Oregon, USA) (MPTPp) to delay renal MPTP clearance. The compounds were co-administered in two consecutive injections twice a week for 5 weeks. Control and MPTPp mice were randomly divided into two groups and treated with JZL184 (8 mg/kg; Cayman Chemical, Ann Arbor, MI, USA), or JWH133 (0.2 mg/kg; Tocris Bioscience, Minneapolis, MN, USA), or RO6871304 (RO304, 10 mg/kg; Roche Pharma, Switzerland) or with vehicle. JZL184 and JWH133 were dissolved in saline (Braun, Barcelona, Spain) containing 15% dimethyl sulfoxide (DMSO, Sigma-Aldrich), 5% poly(ethylene glycol) (PEG, Sigma-Aldrich) and 5% Tween-80 (Guinama, Valencia, Spain). RO304 was dissolved in saline containing 29.1% DMSO and 5.3% Cremophor® EL (Sigma-Aldrich). JZL184 was administered 5 days per week, while mice were treated with JWH133 and RO304 daily for 5 weeks. Control mice received only probenecid (250 mg/kg) in the same dosing regimen as MPTPp.

2.3. Hematopoietic stem cell extraction

WT and CB2R KO donor mice were euthanized by CO₂ asphyxiation. The femur and tibia were removed and the epiphyseal ends were clipped to rinse the bone marrow from the bone cavity with cold cytometry buffer (CB): 5mM EDTA (Thermo Fisher Scientific), 0.5% fetal bovine serum (FBS, Gibco, Paisley), 100 U/mL penicillin G (Gibco), 100 µg/mL streptomycin (Gibco) in phosphate-buffered saline (PBS, Lonza, Basel, Switzerland), using a 23-gauge needle. Cells were filtered through a 70 µm nylon cell strainer and centrifuged at 300 g for 6 min. Pooled hematopoietic stem cells (HSCs) were lysed with red blood cell lysis buffer (RBC) for 5 min at room temperature (RT). Lysis was stopped by adding CB and after centrifugation, HSCs were resuspended in PBS at 50 x 10⁶ cells/mL. HSCs were prepared less than 2 h before and kept on ice prior to injection.

2.4. Generation of bone marrow chimeras

Recipient WT mice (2 months) were irradiated with two fractions of 600 rads with a difference of 6 h. For bone marrow reconstitution, 5 x 10⁶ HSCs from WT or CB2R KO animals were transplanted by intravenous administration 24 h after irradiation. Mice were treated with antibiotic (Kariflox, 5 mg/mL) in the drinking water for 4 weeks. Bone marrow was considered fully reconstituted 7 weeks after transplantation. MPTPp and JZL184 treatments started after this time.

2.5. Motor behavior

Pole, bar and rotarod tests were performed to analyze the motor behavior of the mice. The pole and bar tests were performed 4 h after the last MPTP injection and the rotarod 16 h later. Behavioral tests were performed under low light conditions. In the pole test, animals were placed upright on the top of a vertical wooden pole, 50 cm high and 1 cm in diameter, covered with a bandage. Animals were pre-trained until they were able to turn their head down and to descend from the pole in less than 5 s. The mean time to turn their head down and completely descend from the pole was measured in two trials with a rest period of 15 min between each trial. In the bar test, animals were placed with their forepaws on a bar parallel 4 cm above the ground. The mean time to move the forepaws to the ground was measured in three consecutive trials. In the rotarod test (LE8200, Panlab, Barcelona, Spain), animals were placed over a moving rod, which was programmed to rotate at increasing speeds from 4 rpm to 40 rpm in 5 min. Animals were pre-trained on two consecutive days until they were able to remain over the rod for at least 1 min. The average time that each mouse remained on the rod was recorded in two trials with a 30 min rest period in between.

2.6. Blood extraction

Submandibular blood samples (50 µL) were collected in EDTA microvette-coated tubes (BD Biosciences, Franklin Lakes, NJ, USA), mixed with 750 µL of RBC and incubated for 15 minutes at RT on a rotating shaker. Samples were centrifuged at 1200 g for 15 min at RT, cells were washed and the pellet was resuspended in 100 µL of CB for flow cytometry.

2.7. Preparation of brain cell suspensions

Adult mice were anesthetized with i.p. ketamine/xylazine and perfused transcardially with ice-cold PBS. The brain was removed and the striatum and ventral midbrain were dissected on ice. The tissue was digested with a collagenase D mixture (400 units/mL, Roche, Mannheim, Germany) or a papain mixture (2 mg/mL, Worthington, Lake-wood, NJ, USA), containing the enzyme DNase-I (50 µg/mL, Sigma-Aldrich) in Dulbecco’s PBS (DPBS, Lonza, Basel, Switzerland). Digestion was performed at 37°C with rotation for 15 min with collagenase D or for 30 min with papain. The samples were cooled down and 10 µL of EDTA 500 mM (Invitrogen, Carlsbad, CA, EEUU) were added to stop the reaction. Brain tissue was mechanically dissociated with a glass Pasteur pipette, filtered through a 70 µm nylon cell and centrifuged at 300 g for 15 min at 4°C. Samples were resuspended in 25% Percoll (GE Healthcare, Chicago, IL, EEUU) and centrifuged at 1000 g for 10 min at RT to remove cell debris and myelin. The white layer was carefully removed and the cell pellet was resuspended in 100 µL of CB or RPMI medium (Invitrogen) for flow cytometry.

2.8. Flow cytometry

Cell pellets obtained from the brain were resuspended in 100 µL of CB and incubated with Zombie NIR dye (BioLegend, San Diego, CA, USA) for 5 min at RT to assess the percentage of cell viability (50 µL/sample; 1:2000 dilution in PBS). Zombie NIR dye was quenched with 150 µL of CB and cells were centrifuged at 2000 rpm for 1 min. Cells were labeled with a panel of fluorescent antibodies diluted in CB and the FcR blocking reagent (1:50; Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4°C: BV510 anti-CD11b (1:500, M1/70, BioLegend) or VioBlue anti-CD11b (1:100, M1/70.15.11.5, Miltenyi Biotec), BV421 anti-CD45 (1:1000, 30F11, BioLegend) or BV510 anti-CD45 (1:1000, 30F11, BioLegend), APC anti-ACSA2 (1:50, IH3-18A3, Miltenyi Biotec), BUV395 anti-CD8a (1:200, 53 − 6.7, BD Bioscience), BV711 anti-CD4 (1:200, GK1.5, BioLegend) and PerCP-Vio700 anti-CD3 (1:50, 145-2C11, Miltenyi Biotec). In EGFP-CB2R transgenic mice, EGFP was detected directly using a 488 nm laser. Cell pellets obtained from blood samples were incubated with Zombie NIR and labeled with the following panel of fluorescent antibodies: BV510 anti-CD11b (1:500, M1/70, BioLegend), BV421 anti-CD45 (1:1000, 30F11, BioLegend) and PerCP-Cy5.5 anti-CD3 (1:200, 145-2C11, BioLegend). After centrifugation, they were labeled with a primary antibody rabbit anti-CB2R (1:25, Cayman Chemical) in CB during 15 min at RT, washed and incubated with a secondary antibody Alexa Fluor 647 goat anti-rabbit (1:100, Invitrogen) under the same conditions. After labeling, samples were centrifuged, resuspended in CB, acquired on a CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA) and analyzed using the CytExpert 2.3 (Beckman Coulter) and FlowJo 10.0.7r2 (BD Biosciences, Franklin Lakes, NJ) software.

2.9. In vitro T cell stimulation for intracellular cytokine profiling

To assess TNFα production by lymphocytes, midbrain cell pellets were resuspended in 100 µL of RPMI supplemented with 10% FBS, 100 U/mL penicillin G, 100 µg/mL streptomycin, 10 µg/mL gentamicin solution, 50 µM β-mercaptoethanol and 12.5 mM HEPES buffer instead of CB. A combined solution of phorbol 12-myristate 13-acetate [PMA 0.05 µg/mL (Sigma-Aldrich)/ionomycin 0.5 µg/mL (Sigma-Aldrich)] was then added to stimulate T cell cytokine production. Brefeldin-A 5 µg/mL (BioLegend) was used to block cytokine transport. After incubation at 37°C for 4 h, samples were centrifuged at 2000 rpm for 1 min and washed with PBS. Cells were first labeled with cell surface markers for flow cytometry as described previously, cells were incubated with a fixation/permeabilization solution (Invitrogen) for 7 min at 4°C in the dark. The cells were then centrifuged under the same conditions and washed with PermWASH solution (Invitrogen). After further centrifugation, cells were labeled with an intracellular panel of fluorescent antibodies diluted in PermWASH solution during 15 min at 4°C in the dark: PE-Cy7 anti-TNFα (1:400, MP6-XT22, BioLegend). After labeling, samples were centrifuged, resuspended in CB, collected on a CytoFLEX LX flow cytometer and analyzed using the CytExpert 2.3 and FlowJo 10.0.7r2 software.

2.10. Histological techniques

Animals were anesthetized with i.p. ketamine/xylazine and perfused transcardially with Ringer’s solution (145.4 mM NaCl, 3.4 mM KCl, 2.4 mM NaHCO₃, pH 7.4) for 5 min at a rate of 9.5 mL/min and with 4% paraformaldehyde (PFA; Panreac, Barcelona, Spain) in 0.125 M PBS (pH 7.4) for 10 min. Brains were removed, post-fixed for 24 h in 4% PFA and stored in 30% sucrose/PBS until decanting. Coronal 40 µm thick sections were cut on a Leica SM2000R sliding microtome (Leica, Wetzlar, Germany). Free-floating sections were washed with PBS and endogenous peroxidase activity was inactivated for 30 min with 0.03% H₂O₂ (Sigma-Aldrich)/methanol (Panreac). The sections were washed with PBS and incubated with blocking solution [4% normal goat serum, 0.05% Triton X-100 (Sigma Aldrich) and 4% BSA (Merck, Darmstadt, Germany) in PBS] for 40 min. Rabbit anti-tyrosine hydroxylase (TH; 1:1000, Merck Millipore) was diluted in blocking solution and incubated overnight at RT. Sections were then incubated with a biotinylated secondary antibody goat anti-rabbit (1:500, Jackson ImmunoResearch, Ely) in blocking solution for 2 h at RT, with peroxidase-conjugated avidin (1:5000, Sigma-Aldrich) for 90 min at RT and, after washing with PBS, with 0.05% diaminobenzidine (Sigma-Aldrich), 0.03% H₂O₂ and Trizma-HCl buffer (pH 7.6). Tissues were mounted on glass slides in a 0.2% solution of gelatin in 0.05 M Tris-HCl buffer (pH 7.6) (Sigma-Aldrich), dried overnight and dehydrated in toluene (Panreac) for 12 min. Finally, the slides were coverslipped with DPX (BDH Chemicals, Poole).

2.11. Image analysis

Images were captured on an Aperio C52 digital pathology slide scanner (Leica) at a 20× magnification. Optical density values of TH immunoreactivity were obtained from 6 striatal sections per animal taken at equal intervals (360 µm) by using ImageJ (National Institutes of Health, MD). For TH density analysis, a random region of the cortex was used as a blank and its value was subtracted from the intensity of both striatal hemispheres. TH⁺ neurons in the SNpc were counted in 6–7 coronal sections per animal taken at equal intervals (160 µm) covering the entire nucleus. Unbiased design-based stereology was performed using a Bx61 microscope (Olympus, Hicksville, NY) equipped with a DP71 camera (Olympus), a stage connected to a xyz stepper (H101BX, PRIOR) and Stereo Investigator software (version 2021.1.1; MBF Bioscience, Williston, VT). The reference volume (Vr) was calculated from images obtained with the 2× objective using a point count array according to Cavalieri principles (38). Measurement of the cross-sectional area of the nucleus and estimation of the Vr were determined using the following equation:

$$\:Vr=T\frac{a}{p}ƩPi$$

where T is the section thickness, a/p is the area of each point and Pi corresponds to the number of points falling within the SNpc. SNpc masks were outlined with the 10× objective to estimate the area. TH⁺ neurons were counted at 100× magnification under oil immersion, using randomized meander sampling and the optical dissector methods. To count a minimum of 100–150 cells/animal a sampling frame of 4900 µm² and sampling steps of 181 µm× 181 µm (dx, dy) were used for control mice and 140 µm× 140 µm (dx, dy) were used for MPTPp mice. The total number of TH⁺ neurons (N) was calculated using the following formula:

$$\:N=\:Ʃ{Q}^{-}\frac{t}{h}\:\frac{1}{asf}\frac{1}{ssf}$$

where ƩQ⁻ is the number TH⁺ cells counted, t is the mean of the section thickness, h is the height of the optical dissector, asf is the area sampling fraction, and ssf is the section sampling fraction. The density of TH⁺ neurons (D) was determined using the following formula: D = N/Vr. Gunderson's coefficients of error were < 0.1 for all stereological quantifications.

2.12. Analysis of processed snRNA-seq data from Parkinson´s disease patients

Single nuclei RNA sequencing (snRNAseq) data from the midbrain of idiopathic PD (IPD) patients (39) were analyzed to determine the transcriptome profile of the ECS in glial cells. Differentially expressed genes in the IPD-midbrain microglia (CD74) and astrocytes (AQP4) with q < 0.05 were downloaded. Transcripts related to synthesis, degradation and intracellular signaling of the endocannabinoids were investigated in the gene set lists and upregulated (estimate > 0) and downregulated (estimate < 0) genes were graphed.

2.13. Statistics

GraphPad Prism version 8.0 was used to generate the graphs for statistical analysis. All data are presented as means with 95% confidence intervals (CI). The normal distribution of the data was analyzed with a Shapiro-Wilk test. To compare two experimental groups, student’s t-test (two-tailed) for equal variances was used for data following a normal distribution, and the Welch’s correction was applied if variances were significantly different. Mann-Whitney U test was used to evaluate data that do not follow a normal distribution. For the analysis of one variable, we used one-way ANOVA followed by Bonferroni’s multiple comparison test for data following a normal distribution and Kruskal-Wallis followed by Dunn’s multiple comparison test for data not following a normal distribution. Two-way ANOVA followed by Bonferroni’s post hoc test was used to analyze two variables. p-values < 0.05 were considered as statistically significant.

3.1. CD4 ⁺ T cell infiltration in the ventral midbrain correlates with the extent of neuronal degeneration in the MPTPp mouse model

MAGL inhibition with JZL184 increases brain 2-AG levels and protects the nigrostriatal pathway from MPTP-induced dopaminergic degeneration (Suppl. Figure 1) (30). Administration of the radioligand [¹²³I]-Ioflupane to visualize the presynaptic dopamine transporter (DAT) showed that JZL184-treated animals had similar radioligand binding in the basal ganglia as control mice (Suppl. Figure 2). These results demonstrate that JZL184 preserves dopaminergic terminals in the MPTPp mouse model and this may account for the improvement in motor behavior. In this context, we asked whether modulation of the immune system might be involved in the neuroprotective effect of JZL184. We prepared cell suspensions from the striatum and the ventral midbrain to analyze immune cells by flow cytometry. The gating strategy to select immune cell subpopulations is shown in Fig. 1A. Myeloid cells were identified by the expression of CD11b and CD45, the CD11b⁺CD45^low gate was assigned to resident microglia and the CD11b⁺CD45^high gate to myeloid infiltrating cells and resident activated microglia. CD4⁺ and CD8⁺ T cells were selected from the CD11b⁻CD45^high population (Fig. 1A). A specific increase in CD4⁺ T cell infiltration was observed in the ventral midbrain of MPTPp mice (F_1,20 = 6.3, p_int = 0.02), but not in JZL184-treated animals (Fig. 1B). No differences were detected in striatal CD4⁺ T cell infiltration (Fig. 1B), CD8⁺ T cells (Fig. 1C), CD11b⁺CD45^low (Fig. 1D) or CD11b⁺CD45^high (Fig. 1E). These results suggest a link between CD4⁺ T cell infiltration in the ventral midbrain and dopaminergic neuronal cell death.

3.2. CB2R in immune cells is required for the neuroprotective effect of JZL184

We next asked whether immune cells are directly involved in the neuroprotective effect of JZL184 or are indirectly affected by the improvement in neuronal survival. First, we examined the cellular selectivity of CB2R expression by flow cytometry using transgenic EGFP-CB2R mice (Fig. 2A). The CB2R expression pattern was similar in the striatum and in the ventral midbrain, with CD11b⁺CD45^high myeloid cells exhibiting the highest levels of CB2R followed by CD11b⁺CD45^low and CD3⁺ cells. No EGFP signal was observed in astrocytes (ACSA2⁺) and in the negative fraction (Fig. 2A). Intoxication with MPTPp differentially modulated CB2R levels in the striatum and in the ventral midbrain (Fig. 2B). In the midbrain, CB2R expression was increased in CD11b⁺CD45^low cells and decreased in CD11b⁺CD45^high cells, whereas a trend was observed in striatal CD3⁺ cells (Fig. 2B). These results suggest that CB2R expressed in myeloid and T cells may be involved in the neuroprotective effect of JZL184.

To determine whether the beneficial effect of JZL184 was mediated by CB2R, constitutive CB2R KO mice were intoxicated with MPTPp and treated with the drug for 5 weeks. Analysis of motor behavior in the pole, rotarod and bar tests showed that JZL184 did not improve motor behavior in treated parkinsonian mice (Fig. 3A). Under these conditions CD4⁺ T cells infiltrating the ventral midbrain remained significantly elevated (Fig. 3B). These results suggest that CB2R is required for the action of JZL184.

To validate the neuroprotective effect of CB2R activation, MPTPp mice were treated with the CB2R agonist JWH133 (0.2 mg/kg) (40, 41). Motor behavior (Fig. 4A) was significantly improved in the pole (F_1,20 = 11, p_int = 0.003), rotarod (F_1,20 = 29.5, p_int < 0.0001) and bar tests (F_1,20 = 10.8, p_int = 0.004). Immunohistochemical analysis showed a specific increase in TH immunostaining in the striatum (Fig. 4B, F_1,20 = 22, p_int = 0.0001) and a higher number of TH⁺ neurons in the SNpc compared to untreated animals (Fig. 4C, F_1,20 = 8.6, p_int = 0.008). These results demonstrate that CB2R activation is neuroprotective in the MPTPp mouse model of PD.

3.4. Activation of CB2R in microglia, but not in lymphocytes, protects the nigrostriatal pathway

We next asked whether activation of CB2R present in the brain or in peripheral immune cells was required for neuroprotection. To this end, MPTPp mice were treated daily for 5 weeks with RO304 (10 mg/kg), a CB2R agonist that does not cross the blood brain barrier (BBB) (42). RO304 did not improve motor behavior in the pole, rotarod and bar tests (Fig. 5A). Histological assessment of the nigrostriatal pathway showed a lack of protection of striatal TH⁺ terminals (Fig. 5B) and dopaminergic neurons in the SNpc (Fig. 5C). The infiltration of CD4⁺ T cells in the striatum and in the midbrain was maintained in MPTPp mice treated with RO304 and vehicle, and was significantly reduced by JWH133 treatment (Fig. 6A), while no effect was observed for CD8⁺ T cells (Fig. 6B). Myeloid CD11b⁺CD45^low cells increased significantly with both treatments (Fig. 6C) and CD11b⁺CD45^high cells increased with RO304 (Fig. 6D) in the ventral midbrain. These results demonstrate that administration of RO304 is unable to reproduce the neuroprotective effect of JWH133, suggesting that activation of CB2R in the brain parenchyma is necessary for neuroprotection. JZL184 and JWH133 had a similar effect on immune cells in the midbrain, but differed in the striatum, where JWH133 reduced CD4⁺ T cell infiltration in this region but JZL184 did not. Independently of their differential effect on striatal T cell infiltration, both neuroprotective compounds reduced the MPTP-induced TNFα production in CD4⁺ and in CD8⁺ (Fig. 6E) T cells in the ventral midbrain. No significant differences in TNFα positive lymphocytes in this region were observed between the MPTPp mice treated with vehicle or RO304.

To determine which CB2R-expressing cells in the brain parenchyma are necessary for neuroprotection, we generated bone marrow chimeric mice. Irradiated WT mice were transplanted with HSCs from WT (Chi-WT) or CB2R KO (Chi-CB2R KO) donor mice (Fig. 7A). A significant decrease in CB2R expression was observed in CD3⁺ and CD11b⁺CD45^high cells in the periphery 7 weeks after the transplant (Fig. 7B). MPTPp intoxicated chimeric mice treated with JZL184 performed similarly to control mice in the pole, rotarod and bar tests and significantly better than untreated MPTPp mice (Fig. 7C). TH immunostaining in the striatum (Fig. 7D) and stereological quantification of TH⁺ neurons in the SNpc (Fig. 7E) showed that the neuroprotective effect was maintained in the absence of peripheral CB2R. Taken together, these results indicate that CB2R activation in the brain, probably microglial cells, but not in infiltrating peripheral immune cells is necessary for neuroprotection. CB2R agonists do not appear to have a direct effect on T cells, but the indirect reduction in TNFα production may contribute to their neuroprotective effect.

3.4. The 2-AG biosynthesis is downregulated in the midbrain microglia of PD patients

To determine the transcriptomic profile of the ECS in PD, we took advantage of single nuclei RNA sequencing (snRNA-seq) data obtained from the midbrain of 6 idiopathic PD patients with severe neuronal loss and 5 age-matched controls (39). Differentially displayed transcripts associated with the ECS (Fig. 8A) were searched for in glial cell populations. Differentially expressed genes in glia were more related to 2-AG metabolism than to anandamide (AEA) (Fig. 8B). In microglia (CD74), a decrease in 2-AG biosynthesis related transcripts (DAGLB, PLCB2, PDIA3) and an increase in the degradation enzyme ABHD3 suggest a decrease in 2-AG production. In astrocytes (AQP4), the increase in both, biosynthesis (PLCH1, PLCG2, PLCB4, PDIA3) and degradation related transcripts, such as MGLL, indicate an accelerated turnover of this molecule. Transcripts related to the cannabinoid transporter type 1 or type 2 expression were not differentially expressed in human PD midbrain cells. This may be related to the advanced stage of the neuronal loss in the subjects analyzed (Lewy body Braak stage 5/6). Considering the decrease in 2-AG production and the resulting insufficient CB2R activation in microglia from PD patients to resolve the insult and promote an anti-inflammatory environment in the brain parenchyma, our results suggest that pharmacological CB2R activation could be an interesting therapy for PD.

In the present study, we have investigated the mechanisms underlying the neuroprotective effect of MAGL inhibition with JZL184. We identified a specific increase in CD4⁺ T cell infiltration in the ventral midbrain of MPTPp mice, which was not observed in JZL184-treated animals. Experiments with CB2R KO mice suggest that the mechanism of action of JZL184 is mediated by CB2R. Using selective CB2R agonists and chimeric mice we have shown that CB2R activation in the brain, probably in microglia, but not in peripheral immune cells, is required for neuroprotection in the chronic experimental model of PD. The decreased expression of 2-AG synthesizing enzymes in human microglia suggests a reduced production of this endogenous CB2R ligand in PD and supports the potential beneficial effect of CB2R ligands that reach the CNS for the treatment of this neurodegenerative disease.

Inflammation is a central feature of PD, to develop effective immunomodulatory therapies it is necessary to understand the interactions between the immune system and neuronal survival. Among the different experimental models of PD, we have selected the MPTPp mouse model for this study because, as we have recently described, it is the model that best recapitulates the glial activation described in the human midbrain (21). The activation state of glial cells in experimental models of PD based on α-synuclein overexpression or MPTPp intoxication correlates with CD4⁺ T cell infiltration in the midbrain (21, 43, 44). Infiltrated CD4⁺ T cells have been described in the midbrain of PD patients (17, 22). Dynamic changes in the number of CD4⁺ lymphocytes and activation are associated with PD progression, and an increase in CD4⁺ naïve T cells after clinical diagnosis of PD suggests that neurodegeneration may contribute to CD4⁺ T cell recruitment to the CNS (27). Neuroprotection exerted by JZL184 and JWH133 correlates with a decreased CD4⁺ T cell infiltration into the midbrain, an effect that is not observed in CB2R KO mice. This could be due to the inhibition of CXCL12-induced T-cell chemotaxis at the peak of CB2R expression induced by 2-AG and JWH133 (45).

The anti-inflammatory properties of CB2R agonists make them a promising pharmacological alternative to counteract inflammatory response in multiple sclerosis and stroke (46–48). In experimental models of Alzheimer´s disease, mice lacking CB2R show a reduced proinflammatory response without affecting memory and learning (49). In contrast, neuroinflammation induced by graft-vs-host disease is ameliorated by CB2R inverse agonist/antagonists and CB2R deletion (50). In the last inflammatory context, the general functions of CB2Rs are to promote leukocyte recruitment into the brain and to regulate the balance between TNFα and IFNγ (50). In the MPTPp mouse model, we achieve neuroprotection by the same mechanisms proposed by Moe et al. (2024), reduced CD4⁺ T cell recruitment and TNFα production, which are induced by CB2R activation and abolished in CB2R KO mice. We also show that host activation of CB2R in the brain of chimeric mice is required for neuroprotection. Chimeric host mice lacking CB2R were highly sensitive to MPTPp and most animals did not reach the endpoint of the experiment. A similar observation has been reported for acute MPTP administration to mice lacking CB2R (51). These results suggest that CB2R expression in the brain, mainly in microglia, counteracts the effects of neuronal death by MPTP administration. As we have previously reported, the effect of MAGL inhibition on neuronal survival is mediated by CB2R and depends on the presence of glial cells (52). In addition, our group has shown that the basal inflammatory tone of the midbrain is different from other brain regions and, consequently, the inflammatory response elicited by lipopolysaccharide (LPS) is region dependent (53). The different transcriptomic profile between striatal and midbrain myeloid cells (53) may trigger alternative inflammatory pathways upon CB2R activation. In the time course of MPTPp-induced dopaminergic degeneration, the progressive increase in CB2R expression parallels the acquisition of a phagocytic and proinflammatory profile in microglia (21). CB2R is not required for the generation of LPS/IFNγ-induced microglial activation (54), but it is involved in the acquisition of an alternative and phagocytic phenotype (55). In the MPTPp mouse model, CB2R activation by increased 2-AG levels may polarize towards a neuroprotective phenotype.

The enzymatic hydrolysis of 2-AG is primarily mediated by MAGL, which is the major regulator of 2-AG levels in the brain (35, 56). Microglia express the components required for eCB autocrine or paracrine signaling (57). In the midbrain from human PD patients, downregulation of 2-AG synthesizing enzymes (DAGLB, PLCB2 and PLD1) and upregulation of 2-AG degrading enzyme (ABDHD3) suggest a reduced production of this endocannabinoid by microglia. The transcriptomic profile in the same cells (39) predicts an upregulation of the Th1 pathway, IFNγ signaling and T cell infiltration (21). Upregulation of IFNγ signaling in the human midbrain could interfere with 2-AG production and neuroprotection (58). The increased CB2R receptor expression observed in microglial cells in the SNpc of PD patients (10) is reproduced in the experimental MPTPp mouse model. Taken together, these data support the potential benefits of MAGL inhibition to increase 2-AG levels in the context of dopaminergic degeneration. Several studies have described an increased Th1 response in peripheral CD4⁺ T cells in PD patients, suggesting a potential entry of lymphocytes across the BBB. The reduction in TNFα production by CD4⁺ and CD8⁺ T cells may contribute to the neuroprotective effect. These findings are consistent with the reduced incidence of PD in patients treated with anti-TNFα therapy (59).

Our results show that the neuroprotective effect of JZL184 in the MPTPp mouse model of PD is mediated by CB2R. The BBB-permeable CB2R agonist (JWH133) reproduced the effect of JZL184, whereas the peripheral CB2R agonist (RO304) failed to ameliorate the parkinsonian condition. CB2R activation in the brain is required for neuroprotection and prevention of CD4 T cell recruitment to the midbrain and T cell production of TNFα. The expression of the ECS elements in midbrain microglia of PD patients supports the potential therapeutic benefit of MAGL inhibition or central CB2R activation for the treatment of PD.

2-AG: 2-arachidonyl glycerol

AEA: Anandamide

BBB: Blood brain barrier

CB: Cytometry buffer

CB2R: Cannabinoid type 2 receptor

Chi-CB2R KO: Chimeric mice transplanted with CB2R KO HSCs

Chi-WT: Chimeric mice transplanted with WT HSCs

CI: Confidence intervals

Ctrl: Control

DAT: Dopamine transporter

ECS: Endocannabinoid system

EGFP: Enhanced green fluorescent protein

FBS: Fetal bovine serum

HSCs: Hematopoietic stem cells

i.p: Intraperitoneal

IFN: Interferon

IL: Interleukin

IPD: Idiopathic Parkinson’s disease

MAGL: Monoacylglycerol lipase

MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MPTPp: MPTP + probenecid

PBS: Phosphate-buffered saline

PD: Parkinson’s disease

Prob: Probenecid

RBC: Red blood cell

RT: Room temperature

SNpc: Substantia nigra pars compacta

snRNA-seq: Single nuclei RNA sequencing

SPECT: Single photon emission computed tomography

SUV: Standardized uptake value

TH: Tyrosine hydroxylase

TNFα: Tumor necrosis factor alpha

Veh: Vehicle

WT: Wild-type

Ethics approval and consent to participate

All procedures involving animals were carried out in accordance with the Spanish National Research Council’s Guide for the Care and Use of Laboratory Animals. The experimental designs were approved by the Animal Experimentation Ethics Committee of the University of Navarra (Ref: 109-18, 109-18E3, 109-18E4). Data from human midbrain were downloaded from the open access publication by Smajic et al., 2022.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

CJH is a scientific advisor and has equity in Formulate Biosciences, Inc. UG is a full employee of F. Hoffmann-La Roche Ltd.

Funding

This work was supported by the Spanish Government Instituto de Salud Carlos III, ISCIII-FEDER PI20/01063, Ministerio de Ciencia e Innovación MCIN/AEI/FEDER PID2022-138461OB-I00 and Fundación Gangoiti. LA was funded by FPU19/03255, LB was funded by FPU: FPU018/02244; AT was funded by FPU21/01545.

Authors' contributions

LA, MAB and CV administered MPTP to generate the mouse models and assessed the motor behavior. LA, MAB and SH generated the chimeric mice. LA, CV, LB and AT conducted the histological techniques and LA and EL performed the stereological counting under the supervision of EM. LA, MAB and CV performed the flow cytometry experiments under the supervision of SH. MC and IP performed the DaTSCAN. SM, CJH and JR generated and provided the CB2R transgenic and KO mice. UG provided the RO304. PC provided assessment with the selection and analysis of human data. LA and MSA performed the statistical analysis wrote the manuscript. MSA conceived and supervised the whole project. All authors critically reviewed and approved the final manuscript.

Acknowledgements

We acknowledge the support of Diego Alignani from the flow cytometry facility for his guidance with the FACS analysis. We acknowledge the technical support of Beatriz Paternain and Amaya Trigo.

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Activation of central cannabinoid type 2 receptors, but not on peripheral immune cells, is required for endocannabinoid-mediated neuroprotection in Parkinson´s disease

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Abstract

Background

Methods

Results

Conclusions

Figures

1. BACKGROUND

2. MATERIAL AND METHODS

2.1. Animals

2.2. MPTP intoxication and drug administration

2.3. Hematopoietic stem cell extraction

2.4. Generation of bone marrow chimeras

2.5. Motor behavior

2.6. Blood extraction

2.7. Preparation of brain cell suspensions

2.8. Flow cytometry

2.9. In vitro T cell stimulation for intracellular cytokine profiling

2.10. Histological techniques

2.11. Image analysis

2.12. Analysis of processed snRNA-seq data from Parkinson´s disease patients

2.13. Statistics

3. RESULTS

3.2. CB2R in immune cells is required for the neuroprotective effect of JZL184

3.4. Activation of CB2R in microglia, but not in lymphocytes, protects the nigrostriatal pathway

3.4. The 2-AG biosynthesis is downregulated in the midbrain microglia of PD patients

4. DISCUSSION

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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