Modulation of cannabinoid receptor 2 alters neuroinflammation and reduces formation of alpha-synuclein aggregates in a rat model of nigral synucleinopathy

Abstract

Research into the disequilibrium of microglial phenotypes has become an area of intense focus in neurodegenerative disease as a potential mechanism that contributes to chronic neuroinflammation and neuronal loss in Parkinson’s disease (PD). There is growing evidence that neuroinflammation accompanies and may promote progression of alpha-synuclein (Asyn)-induced nigral dopaminergic (DA) degeneration. From a therapeutic perspective, development of immunomodulatory strategies that dampen overproduction of pro-inflammatory cytokines from chronically activated immune cells and induce a pro-phagocytic phenotype is expected to promote Asyn removal and protect vulnerable neurons. Cannabinoid receptor-2 (CB2) is highly expressed on activated microglia and peripheral immune cells, is upregulated in the substantia nigra of individuals with PD and in mouse models of nigral degeneration. Furthermore, modulation of CB2 protects against rotenone-induced nigral degeneration; however, CB2 has not been pharmacologically and selectively targeted in an Asyn model of PD. Here, we report that 7 weeks of peripheral administration of CB2 inverse agonist SMM-189 reduced phosphorylated (pSer129) Asyn in the substantia nigra compared to vehicle treatment. Additionally, SMM-189 delayed Asyn-induced immune cell infiltration into the brain as determined by flow cytometry, increased CD68 protein expression, and elevated wound-healing-immune-mediator gene expression. Additionally, peripheral immune cells increased wound-healing non-classical monocytes and decreased pro-inflammatory classical monocytes. In vitro analysis of RAW264.7 macrophages treated with lipopolysaccharide (LPS) and SMM-189 revealed increased phagocytosis as measured by the uptake of fluorescence of pHrodo E. coli bioparticles. Together, results suggest that targeting CB2 with SMM-189 skews immune cell function toward a phagocytic phenotype and reduces toxic aggregated species of Asyn. Our novel findings demonstrate that CB2 may be a target to modulate inflammatory and immune responses in proteinopathies.

Background

Research into the disequilibrium of microglial phenotypes has become an area of intense focus in neurodegenerative disease as a potential mechanism that contributes to disease pathophysiology, including chronic neuroinflammation and neuronal loss [1, 2]. More specifically, microglia dynamically transition from homeostatic to disease-associated phenotypes with varying transcriptional and functional profiles. In Parkinson’s disease (PD), activated microglia have been reported in postmortem brain histopathological analyses and single-cell transcriptomic studies have identified disease-specific increases in midbrain microglia clusters involved in the inflammatory response [3,4,5,6]. Findings from animal models demonstrate that overexpression or delivery of fibrillar alpha-synuclein (Asyn) in the brain influences microglial phenotypes and infiltration of peripheral immune cells [5, 7,8,9,10]. From a therapeutic perspective, the development of immunomodulatory strategies that dampen overproduction of pro-inflammatory cytokines from chronically activated immune cells and promote a pro-phagocytic phenotype is predicted to protect vulnerable neurons and promote toxic Asyn removal.

The cannabinoid system has been recently implicated in PD where homozygous loss-of-function diacylglycerol-lipase beta (DAGLB) mutations were linked to early-onset PD in different families of Chinese descent [11]. DAGLB synthesizes 2-Arachidonoylglycerol (2-AG), which is a potent brain endocannabinoid that has high potency for cannabinoid receptors 1 and 2. Cannabinoid receptor-2 (CB2) is highly expressed on activated microglia and peripheral immune cells including circulating monocytes [12]. Due to its abundant presence on immune cells, CB2 has emerged as a immunomodulatory target as it regulates various inflammatory processes including cytokine production, immune cell migration and proliferation. When evaluating the CB2 receptor expression in PD, it has been reported to be upregulated in the substantia nigra of PD patients by immunohistochemical [13, 14] and transcriptional outcome measures [15]. Similarly, it is increased in mouse models of nigral degeneration [16, 17]. Evidence from several labs show that overexpressing CB2 or pharmacologically targeting the receptor in neurotoxin animal models of PD results in dampened inflammation and in some cases neuroprotection [18, 19]. Non-selective cannabinoid receptor agonists also ameliorate neurotoxin-induced neuroinflammation and neurodegeneration in PD animal models [16, 20]. Yet, only one report, using genetic deletion approaches, has evaluated the role of CB2 on the clearance of Asyn aggregates in brain [21], and there has not yet been a study evaluating CB2-selective ligands on Asyn burden in vivo.

To further understand the role of CB2 in Asyn clearance, we sought to determine the effects of peripheral treatment with the novel CB2 inverse agonist, SMM-189, on Asyn-induced pathologies in a rat model of viral vector-mediated Asyn overexpression. Previous studies using SMM-189 demonstrated immunomodulatory effects that improve acute neuronal injury and behavioral outcomes in TBI models potentially explained by a mechanism where SMM-189 suppresses pro-inflammatory markers and increases anti-inflammatory effects [22,23,24]. Inverse agonists bind constitutively-active CB2 receptors and lock them into an inactive conformation state preventing coupling to G_ai/o with a concomitant increase in cyclic AMP (cAMP) production [25]. SMM-189 acts on this canonical pathway in microglia as evidenced by increased levels of nuclear phosphorylated cAMP response-element binding protein (pCREB), triggering downstream transcription of wound-healing cytokines [26]. Therefore, we hypothesize that targeting CB2 will promote clearance of Asyn by altering the central inflammatory environment. In this study, we provide evidence that pharmacological inverse agonism of CB2 alters the inflammatory state of peripheral and central immune cells and reduces levels of phosphorylated human Asyn in the brain.

Methods

Animals and experimental design

Sprague Dawley male rats (16–18 weeks of age, 400–525 g) were purchased from Charles River Laboratories and acclimated to Emory University animal facilities. Rats were pair housed in standard plexiglass cages under normal 12-hr light cycles and handled in accordance with protocols approved by the IACUC of Emory University in Atlanta, GA. Rats were administered unilateral injections of AAV2/5-human wild-type (WT) Asyn at titers of 2.9–4.3 × 10¹¹ vector genomes (vg)/mL (termed “low cohort”), which was previously shown to not induce nigral neurotoxicity [27]. Thereby, generating a model with overexpression of human WT Asyn and Asyn-mediated neuroinflammation. One week after surgery, animals were randomly selected to receive daily systemic injections of either SMM-189 (intraperitoneal (i.p.), 6 mg/kg, n = 12) or vehicle (i.p., n = 12) and followed for 8 weeks (Figs. 1; n = 2 rats per group were removed due to poor nigral targeting). The dose of SMM-189 was based on a pharmacokinetic study in mice determining therapeutic brain-blood ratios as described below. At sacrifice, animals were injected with euthasol (i.p., 150 mg/kg) and cardiac blood collected to isolate peripheral blood mononuclear cells (PBMCs) for inflammatory gene expression using qPCR analysis. After blood collection, animals were transcardially perfused with ice-cold PBS and the frontal cortex frozen for gene analysis, the striatum frozen for western blotting analysis, and the midbrain post-fixated in 4% PFA to be processed for immunohistochemistry. A second low cohort of rats (SMM-189 n = 6 or vehicle n = 3 with n = 2 removed due to poor nigral targeting) completed the study and provided extra tissue to perform additional immunohistochemical and western blotting analyses. Plasma was also collected at baseline, 4 and 8 weeks post-AAV-hAsyn injections and CSF collected prior to perfusion to measure levels of SMM-189.

Fig. 1

Experimental design and outcome measures. Rats were injected unilaterally with AAV2/5-human WT alpha-synuclein (low cohort: 2.9–4.3E11 vg/mL) using stereotaxic coordinates to target the substantia nigra and one week later were dosed daily with peripheral injections of SMM-189, a novel CB2 inverse agonist (6 mg/kg, ip) or vehicle for 7 remaining weeks until perfusion. Blood was collected at baseline and every 4 weeks to measure plasma drug levels. Endpoint brain and PBMCs were evaluated for changes in inflammatory pathway gene expression using qPCR and the midbrain was subjected to immunohistochemistry to evaluate synuclein pathology and immune cells. An additional cohort of rats followed similar experimental design (high cohort: 4.6E12 vg/mL) to evaluate immune cells in blood (PBMCs) and brain by flow cytometry. Blood was collected at baseline, 4 weeks and 8 weeks to measure plasma drug levels and immunophenotype by flow cytometry and brains collected at either 4 or 8 weeks to immunophenotype by flow cytometry

Fig. 2

Modulation of CB2 with SMM-189 decreases accumulation of pSer129 alpha-synuclein in the lesioned nigra. A) Representative nigral images of pSer129 Asyn (scale bar = 250 μm) and B) quantification in nigra of AAV injected rats treated with SMM-189 normalized to vehicle showed reduced pSer129 Asyn compared to vehicle. C) Representative nigral images of proteinase K-resistant Asyn and D) optical density quantification showed reduced intensity of proteinase K-insoluble Asyn. E) Representative nigral images of total human Asyn. F) Total human Asyn did not differ between groups when evaluating the optical density of Asyn-ir within the lesioned nigra normalized by vehicle. G) Representative western immunoblots of pSer129 and total human Asyn from striatal lysates and their corresponding total protein used for normalization. H) Quantification of western bands of hemispheres ipsilateral to the AAV2/5-hAsyn injections demonstrate that pSer129 was significantly reduced following SMM-189 treatment, but human Asyn was not different between treatments. *p < 0.05, **p < 0.01

An additional cohort of rats (termed “high cohort”) was evaluated to determine effects of SMM-189 on immune cell populations in the brain, resident or infiltrating, and the periphery as measured by flow cytometry (Fig. 1). To ensure infiltrating peripheral immune cells, rats were stereotaxically injected with a higher titer of AAV2/5-human WT Asyn (4.6 × 10¹² vg/mL) and underwent an identical drug treatment timeline with animals sacrificed at 4 (SMM-189: n = 5; vehicle: n = 6) and 8 weeks (SMM-189: n = 7; vehicle: n = 7). At sacrifice, animals were deeply anesthetized with euthasol (i.p., 150 mg/kg) and rapid decapitation performed using a sharp guillotine. Brains were collected at endpoints, and brain immune cells isolated and evaluated using multicolor flow cytometry. Additionally, blood was collected at baseline, 4 and 8 weeks (SMM-189: n = 13; vehicle: n = 13) after stereotaxic surgery for PBMCs isolation to immunophenotype using flow cytometry and for plasma evaluation of SMM-189.

Blood brain barrier penetration of SMM-189 in mice

Analysis of the brain-to-blood ratios of SMM-189 were performed by Sai Life Sciences Limited (Telangana, India). All procedures of the study were in accordance with the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) as published in The Gazette of India, December 15, 1998. Prior approval of the Institutional Animal Ethics Committee (IAEC) was obtained before initiation of the study. In brief, 54 mice were weighed and divided as Group 1 (6 mg/kg, i.p.) and Group 2 (6 mg/kg, by oral (p.o.)). Animals in Group 1 and Group 2 were administered intraperitoneally and orally with SMM-189 solution formulation at 6 mg/kg in 5% Ethanol, 5% Cremophor and 90% Normal Saline, respectively. The blood samples were collected under light isoflurane anesthesia at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h (i.p.) and 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h (p.o.) in labeled micro centrifuge tube containing K₂EDTA as anticoagulant. After blood collection, plasma was harvested by centrifugation and stored at -70ºC until analysis. Immediately after collection of blood, brain samples were collected from set of three mice at each time point at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h (Group 1; i.p.) and 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h (Group 2; p.o.). Brain samples were homogenized using ice-cold phosphate buffer saline (pH-7.4) in a ratio of 2 (buffer):1(brain); and homogenates were stored below − 70 ± 10 ºC until analysis. Total homogenate volume was three times the brain weight. All samples were processed for analysis by protein precipitation using acetonitrile and analyzed with fit-for-purpose LC-MS/MS method (LLOQ = 1.02 ng/mL for plasma and 6.09 ng/g for brain).

Non-compartmental-analysis module in Phoenix WinNonlin^® (Version 6.3) was used to assess the pharmacokinetic parameters. Maximum concentration (Cmax) and time to reach the maximum concentration (Tmax) were the observed values. The areas under the concentration time curve (AUClast and AUCinf) were calculated by linear trapezoidal rule. The terminal elimination rate constant, k_e was determined by regression analysis of the linear terminal portion of the log plasma concentration-time curve. The terminal half-life (T1/2) was estimated as 0.693/k_e.

Production of recombinant adeno-associated viral vectors and nigral stereotaxic injections

Recombinant AAV2/5-human WT Asyn was provided by Dr. Manfredsson and previously reported in [27]. Briefly, the transgene was driven by the chicken beta actin/cytomegalovirus enhancer promoter hybrid and AAV construct purified with an iodixanol density gradient and titers determined by viral genome dot-blot measurements as detailed elsewhere [28]. Surgical procedures were performed under isoflurane anesthesia (induced with 5% and maintained at 1–2%). Rats were placed in a stereotaxic frame (Kopf Instruments) and administered two 2 µl unilateral injections of AAV2/5-human WT Asyn [28, 29] into the left nigra at coordinates AP -5.3 mm, ML + 2.0 mm, DV -7.2 mm and AP -6.0 mm, ML + 2.0 mm and DV -7.2 mm using a glass capillary needle fitted to a Hamilton syringe [30]. Injections were delivered at a rate of 0.5 µl/minute and the glass needle remained at the injection site for 5 min before retraction to avoid backflow. After surgery, rodents were placed in a cage on a heating pad and monitored ambulatory and then administered buprenorphine analgesia (i.p., 0.05 mg/kg) every 8–12 h for up to 2 days.

SMM-189 formulation

SMM-189 was provided by Dr. Bob Moore II and formulated with 15% ethanol (v/v), 15% cremophor (w/v) and 70% saline (v/v). In brief, SMM-189 was dissolved in 15% ethanol, cremophor was weighed in a separate vial and dissolved SMM-189 slowly added and mixed gently until combined. Saline was slowly added and mixed until achieving a homogenous solution. The final solution was sterile filtered with a syringe 0.22 μm filter and stored at 4°C in amber sterile-glass vials for up to 2 weeks. Vehicle treatments consisted of the same concentrations of ethanol, cremophor and saline. Daily injections were administered by research staff blinded to cohort treatment history.

Blood and CSF collection

Blood was collected from the tail vein at baseline and every 4 weeks post-AAV (~ 2 h post-treatment injection) for plasma to measure SMM-189 levels in low and high cohorts and for PBMCs to immunophenotype circulating immune cells in the high cohort animals. Animals were placed in an induction box and anesthetized with 3% isofluorane and maintained on 1–2% isofluorane for the remainder of the procedure. Tails were cleaned with ethanol and approximately 400μL of blood drawn from the lateral tail vein using a low dead space 25ga needle and syringe and blood immediately placed into an EDTA tube. Body temperature was maintained using a heating pad during blood draw and while recovering from anesthesia.

CSF was collected at endpoint from low cohort animals. In brief, after euthasol injections, rats were placed in a stereotaxic frame and the head fixed downwards. An incision was made to expose the muscle layer over the cisterna magna. A sterile pulled micropipettes attached to tubing and a syringe punctured into the cisterna magna and clean CSF was aspirated and collected in a 0.2mL Eppendorf and stored at -80°C. CSF was not collected from the high cohort rats because brains were extracted quickly and CSF collection may have compromised brain immune cell composition.

Mass spectrometry and assessment of SMM-189 in plasma and CSF

SMM-189 and d⁵-SMM-189 were synthesized as previously described [31]. A total of 50 µL aliquots of plasma or CSF were prepared by protein precipitation with 100 µL acetonitrile (spiked with the internal standard d⁵-SMM-189) followed by centrifugation at 10,000g for 10 min at 4°C. Chromatographic separation of the supernatant was performed using a Zorbax SB-C18 3.5 μm 4.6 × 150 mm and a Shimadzu HPLC Nexera XR with Solvent Delivery Module LC-20ADXR, CTO-20AC prominence column oven (temperature 40 °C), a DGU-20A5R Degassing Unit, and a SIL-20ACXR autosampler. The aqueous mobile phase (A) was 95% water and 5% acetonitrile (B) was 100% acetonitrile. The flow rate was set to 500 µL/min and the gradient elution initial 50% B, 0.5 to 3 min 50–100%B, 6 to 6.1 min 100–50% B, 6.5 min stop with a column wash using methanol: water (1:1). Sample analysis was performed using SCIEX Triple Quad 5500 tandem mass spectrometer (Framingham, MA) operated in the negative ion mode at ion spray voltage − 4500 V and temperature 600°C. Multiple reaction monitoring mode using the compound-specific mass transfers of m/z 357 → 185 for SMM-189, and m/z 362 → 185 for d⁵-SMM-189 was used for analysis. A calibration curve ranging from 1 to 100 ng/mL was constructed and validated with spiked samples of rat plasma. The peak area ratios of analyte to the internal standard were linear over the tested concentration range.

Immune cell isolation

PBMCs were isolated from blood of low cohort rats at euthanasia (8 weeks) for qPCR analysis of immune genes and from blood of high cohort rats at baseline, 4 and 8 weeks after AAV2/5-human WT Asyn and evaluated with flow cytometry for a longitudinal immunophenotype assessment. Red blood cells were lysed using 1x RBC lysis buffer (Biolegend Cat#420301) for 5 min in the dark. Lysis buffer was neutralized by adding 1x HBSS (Gibco Cat#14175095). Cells were pelleted by centrifugation at 400g for 5 min, supernatant removed, and cells washed with HBSS. For the high cohort, cells were resuspended in 200μL of PBS and transferred to a 96-well v-bottom plate for flow cytometry staining. For low cohort, 10,000 cells were lysed in 600μL RLT lysis buffer (Qiagen Cat#79216) + 1% BME, further homogenized by centrifugation through a QiaShredder column (2 min at 21,130g). Lysate was then stored at -80°C and later processed for RNA extraction.

Brains were collected from animals in the high cohort at either 4 or 8 weeks after AAV2/5-human WT Asyn and brain immune cells were isolated from individual hemispheres as previously described [32]. Hemispheres were finely cut in RPMI 1640 media (Gibco Cat#11875-093) with a scalpel blade and enzymatically dissociated with collagenase VIII (1.4U/mL; Sigma Cat#C2139) and DNAse1 (1 mg/mL; Sigma Cat#DN25) in 37°C for 15 min. Enzymes were neutralized with 10%FBS (heat inactivated, Atlanta Biologicals Cat#S11150) in RPMI 1640 complete media and centrifuged 400g at room temperature. Tissue was resuspended in 1x HBSS and mechanically dissociated using a fire-polished Pasteur pipette until a cell suspension was generated. The suspension was passed through a 70µM cell strainer and strainers rinsed twice with 1x HBSS. To remove myelin, the strained suspension was centrifuged at 300g at 4 °C for 5 min and resuspended in 37% percoll and layered between 70% and 30% percoll gradients. The samples were centrifuged at 400g for 30 min at room temperature with the brake disabled. The top myelin layer was aspirated and demyelinated immune cells were collected from the 37% and 70% percoll interface with a pipette and washed with 1x HBSS twice, resuspended in PBS and placed in a 96-well v-bottom plate for flow cytometry staining.

RNA extraction and qPCR

Total RNA was extracted from the frontal cortex and from PBMCs of low cohort animals and standardized across all samples and groups [33]. In short, tissue was suspended in Trizol and homogenized with a TissueLyser (Retsch). Homogenate was incubated with chloroform and RNA separated by centrifugation of 113,523g for 15 min at 4°C. RNA was further isolated with a RNAeasy mini kit (Qiagen). The RNA (1 µg) was then reverse transcribed to cDNA and analyzed with primers (Integrated DNA Technologies) for pro-inflammatory and anti-inflammatory immune markers and CB2 (Table 1) by qRT-PCR using protocols previously published [33]. Resulting threshold cycle (Ct) values were analyzed by the 2-ΔΔCt method using the AAV2/5-hαsyn + vehicle as the control group and normalized to GAPDH for PBMCs and HPRT1 for frontal cortex as the housekeeping genes.

Table 1 List of rat primers

Multicolor flow cytometry

Peripheral and central immune cell staining was performed similarly as previously published protocols [32]. In brief, immune cells were washed and resuspended in PBS, then incubated for 30 min at room temperature in LIVE/DEAD Fixable Red Dead cell stain (ThermoFisher Scientific Cat#L23102; 1:2000). Following two washes in PBS, Fc receptors were blocked with anti-rat CD32 Fc block diluted 1:100 in FACS buffer (1mM EDTA and 0.1% sodium azide in PBS) for 15 min at 4°C. PBMCs were stained for separate myeloid and lymphoid panels and brain immune cells stained with an antibody panel to identify microglia, monocytes and lymphocytes (Table S1-S3). Fluorophore conjugated antibodies diluted with FACS buffer were incubated at 4 °C for 20 min. Cells stained for surface myeloid markers were fixed in 1% PFA for 25 min and washed in FACS.

PBMCs stained with lymphoid panel were further probed intracellularly for transcriptional marker Foxp3 to label helper T cells. Here, cells were incubated for 30 min in fixation/permeabilization working solution (eBioscience Cat#00-5523-00), washed in 1x permeabilization buffer, additionally blocked with 2% mouse serum for 15 min and anti-rat foxp3-AF647 added to block for an additional 30-minute incubation. Then washed and resuspended in FACs buffer. All samples had 10uL of AccuCheck Counting beads (ThermoFisher Cat#PCB100) added to final suspension and cells were run on a LSRII Instrument (BD Biosciences, Franklin Lakes, NJ) with FACSDiva software (BD Biosciences). Results were analyzed using FlowJo 10.8.1 software and brain immune populations of monocytes (classical and non-classical), lymphocytes (CD4 + and CD8+) or microglia [32, 33] and PBMC populations defined by specific strategies (Figure S1-S3) [34,35,36].

For PBMC analysis, raw frequencies were normalized across serial runs against vehicle-treated values using the following equation: Raw frequency / (average vehicle frequencies of specific run / average baseline frequencies). For brain immune cell analysis of 8 weeks, samples could not be collected and ran the same day so frequencies were normalized across treatment group at each timepoint using the following equation: raw frequency / (average treatment frequency of specific run 1 / average treatment frequency of specific run 2). Brain immune cells measurement from 4-week collection were not normalized as flow cytometry was performed on all samples at the same time.

Immunohistochemistry

Brains were post-fixed in 4%PFA for 48 h and transferred to 30% sucrose and cut at 40 μm on a freezing microtome. Immunohistochemical stains were performed as previously published with antibodies listed in Table S4 [37]. In brief, endogenous peroxidase was quenched with 3% hydrogen peroxide, blocked with 8% normal serum, avidin (Vector Cat#SP-2001) and 0.1% Triton-X and incubated overnight at 4°C with biotin (Vectorlabs Cat#SP-2001), normal serum and primary antibodies specific for Asyn (Clone 4B12; BioLegend Cat# 807801, RRID: AB_2564730), pSer129 Asyn (Clone EP1536Y; Abcam Cat# ab51253, RRID: AB_869973), CD68 (Bio-Rad Cat# MCA341R, RRID: AB_2291300), MHCII (Bio-Rad Cat# MCA46GA, RRID: AB_567369), and CD163 (Bio-Rad Cat# MCA342A, RRID: AB_2074557). Tissue was incubated in appropriate biotinylated secondary antibodies (1:200), amplified with Vectastain ABC kit (Vectorlabs Cat#PK-6100) and developed with diaminobenzidine tablets or diaminobenzidine drop kit with nickel (Vector Cat#SK-4100) for CD68 immunostaining.

To identify aggregated Asyn, proteinase-K resistant Asyn staining was completed with one piece of matched nigral tissue mounted and dried onto positive charged slides. Once rehydrated, a hydrophobic barrier was drawn around the tissue and proteinase K (1:1000 in PBS) incubated for 30 min at room temperature. Proteinase k was cleared with PBS and the remaining staining methods were identical to those listed above with antibodies including Asyn Clone 4B12 1:5000 and horse anti-mouse biotinylated secondary 1:500.

Immunofluorescence

Tissue was blocked with 5% normal serum in TBS + 0.25% Triton-X and further incubated overnight at 4°C with primary antibodies specific for IBA1 (FUJIFILM Wako Pure Chemical Corporation Cat# 019-19741, RRID: AB_839504) and tyrosine hydroxylase (ImmunoStar Cat# 22941, RRID: AB_572268) to identify microglia in the substantia nigra or tyrosine hydroxylase (Millipore Cat# AB152, RRID: AB_390204) and Asyn (Clone 4B12; BioLegend Cat# 807801, RRID: AB_2564730) to evaluate accuracy of stereotaxic injection targets. Tissue was incubated in secondary antibodies conjugated to appropriate fluorophores for 2 h at room temperature and mounted tissue with Vectashield, an aqueous mounting media with DAPI (Vectorlabs Cat#H-2000).

Histological analysis and quantification

Low cohort animals were assessed by an experimenter blinded to the treatment history for rigorous and proper assessment of unilateral targeting of AAV2/5-human WT Asyn into the substantia nigra using immunofluorescent analysis of human Asyn and tyrosine hydroxylase (Figure S4). Low cohort animals were excluded from analysis when poor stereotaxic targeting occurred, including missing human Asyn expression throughout the rostral-caudal axis of the nigra or if expression was seen only in lateral nigra. A total of 6 animals in the low cohorts were removed and the final numbers are reflective in the animals section of the Methods.

IBA1-positive total immune cell counts and area of immunoreactivity (ir) were quantified using Nikon NIS elements software. Four fluorescent z-stacks of IBA1 stained nigra were collected from images of lesioned and non-lesioned nigra from targeted low cohort animals using a Nikon Eclipse 90i microscope and maximum project images were used for quantification. CD68-ir and CD163-ir was quantified from two to three microphotographs from each animal throughout the substantia nigra using ImageJ particle count analysis [38]. Microphotographs of the nigra immunochemically stained for either Asyn, pSer129 Asyn and proteinase K-resistant Asyn were quantified using Image J (version 1.54f). Regions of Interest (ROIs) were drawn on two to three images throughout the nigra and optical density measurements collected and averaged for each animal. Measurements were normalized to the vehicle group so that data from all cohorts could be combined.

SDS-PAGE and immunoblotting

Striatal tissue from successfully targeted low cohort rats was homogenized in ice-cold TriZOL lysed using the TissueLyser system. Homogenates were incubated with 20% chloroform and centrifuged at 13,523g for 15 min at 4°C. Protein pellets were washed in methanol twice and resuspended in 1% SDS and heated to 50°C. Total protein concentrations were measured using Pierce BCA Protein Assay Kit and equal amount of protein (10–20 µg) mixed with Laemmli sample buffer were loaded in 4–20% TGX precast gels (Biorad) for gel electrophoresis. Protein was transferred to a PVDF membrane (Biorad) using a Trans Blot Turbo Transfer System, the immunoblots were immediately fixed with 0.4% paraformaldehyde (PFA) for 30 min and stained for total protein using Revert 700 (Licor) for reliable normalization. Revert staining was captured and measured on the Licor Odyssey at 700 nm for 30 s.

Non-specific protein binding was blocked with 5% milk and 0.1% Tween-20 in TBS for 1 h at room temperature, and membranes incubated with primary antibodies overnight at 4°C. The blots were washed with 0.1%TBS-Tween-20, incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000) at room temperature for 1 h and developed with SuperSignal West Femto Enhancer or Pico Peroxide solutions under a chemiluminescent detection imaging system (Licor Odyssey). The density of the band was quantified using StudioLite software. Relative expression levels were normalized to its own total protein and to vehicle average expression level.

In vitro cell culture

The RAW264.7 macrophage cells (ATCC Cat# TIB-71, RRID: CVCL_0493) were grown in sterile media which consisted of DMEM high glucose, 10% FBS, 2mM L-glutamine, 1x Pen-strep and 1x sodium pyruvate in a humidified 5% CO2 incubator at 37°C. RAW264.7 were maintained in 75cm³ tissue culture flasks and plated at a density of 1 × 10⁴ cells in triplicate in 96-well plates for assays. Cells were stimulated with either lipopolysaccharide (LPS O111:B4; Sigma Cat#L2630) at 100ng/mL or 1000ng/mL (750EU/mL or 7500 EU/mL, respectively). One hour prior to this stimulation the cells were pre-treated with the CB2 inverse agonist SMM-189 at 3μM or 7 μm or its vehicle. Specifically for cell culture experiments, SMM-189 was formulated with 40% ethanol (v/v), 20% tween-20 (v/v) and 40% saline (v/v). A phagocytosis assay was conducted 24 h after LPS.

Phagocytosis assay

The phagocytosis assay was performed using pHrodo green E. coli bioparticles (Invitrogen Cat# P35366) according to the manufacturer’s instructions. Five images per well were then collected every 30 min for 24 h on an EVOS M7000 microscope (Invitrogen, ThermoFisher Scientific). The cells were maintained for this duration using an on-stage incubator kept at 37°C and 5% CO₂. Analysis of the collected images was performed using EVOS Celleste software (version 5.0), which counted the cells and then measured the integrated optical density of fluorescence within each well over the 24-hour time course. Analysis was conducted on the average integrated optical density across triplicate wells. Results were consistent across duplicate experimental replicates.

Statistics

Statistical analysis was conducted using GraphPad Prism software (version9.5.1). In vivo histological, western and transcriptional analyses were analyzed using parametric unpaired t-tests to determine the statistical difference between SMM-189 and vehicle-treated groups. Flow cytometry data of brain immune cells was analyzed using a two-way ANOVA (factors of hemisphere and treatment) and multiple comparisons performed using uncorrected Fishers LSD, while PBMC serial flow data was analyzed using a fixed-effect model (factors of time and treatment) and multiple comparisons performed using uncorrected Fishers LSD. Cell experiments were analyzed using a one-way ANOVA to determine differences between treatment groups across the entire timed experiment using a Dunnet’s multiple comparison test.

Results

Phosphorylated asyn in substantia nigra is decreased by modulation of CB2 with SMM-189

To assess the effect of SMM-189 on Asyn aggregation at levels that do not induce nigral neurodegeneration, midbrains from the low cohorts (previously resulting in intact nigral neurons) were immunohistochemically stained for human phosphorylated (pS129) Asyn (clone EP1536Y). This phosphorylation site is pathologically relevant as it has been found in Lewy bodies in postmortem samples [39, 40] and has been shown to accelerate aggregation of Asyn in vitro and in vivo [41, 42], suggesting that pS129 is linked to aggregation. Optical density analysis of the substantia nigra revealed reduced pS129-ir in SMM-189-treated animals when normalized against vehicle-treated animals (t(23) = 3.353, p = 0.0023; Fig. 2A, B). Total and proteinase-K resistant Asyn was also evaluated in the substantia nigra and found that while levels of total human Asyn (Biolegend, clone 4B12) were not different between treatments, SMM-189 decreased PK resistant Asyn compared to vehicle (t(10) = 2.303, p = 0.044; Fig. 2C-F) when normalized to vehicle nigra measurements. Consistent with histological analyses of pS129 and total Asyn in the nigra, western analysis of the striatum ipsilateral to the overexpression of human Asyn demonstrated reduced pS129 levels in SMM-189-treated rats (t(15) = 3.710, p = 0.0021), and no differences in total human Asyn levels were found between treatments (p = 0.2133; Fig. 2G, H). Additionally, striatal lysates revealed no significant changes in tyrosine hydroxylase, phosphorylated tyrosine hydroxylase or dopamine transporter (trend to increase DAT from SMM-189 treatment p = 0.051; Figure S5).

SMM-189 measurements in CSF confirm penetration into rat brain

Next, to determine that animals administered different concentrations of AAV titer maintained healthy weights and drug levels, we evaluated the weights and levels of SMM-189 from plasma of rats from low and high cohorts. Rats weight increased overtime (p < 0.0001) as expected in both low and high cohorts with no significant differences between treatment groups (Figure S6A, D). Significant increases were detected in the level of plasma SMM-189 in animals receiving SMM-189 treatment in low cohort (treatment F(1,7) = 16.46, p = 0.0048; time F(2,14) = 8.647, p = 0.0036) at 4 (p = 0.0222) and 8 weeks (p = 0.0001) compared to vehicle-treated rats (Figure S6B). Levels of SMM-189 remained low in the CSF with an average of 0.44±0.22SEM ng/mL (Figure S6C) and a brain to plasma ratio average of 0.032±0.02SEM. This is in contrast to the brain to plasma ratios measured for SMM-189 in murine PK studies wherein the brain to plasma ratio of SMM-189 was 0.74 following i.p. injection and 1.63 after p.o. administration (Table 2). Low levels of drug in the CSF are presumed to reflect the extensive portioning of SMM-189 (clogP value of ~ 5.26) into the brain tissue based on high penetration measured in murine PK studies. Similar to low cohort, SMM-189 plasma levels in the high cohort increased with time (treatment: F(1,68), p < 0.0001; time F(2,68) = 22.24, p < 0.0001) in SMM-189-treated rats compared to vehicle at 4 (p < 0.0001) and 8 weeks (p < 0.0001; Figure S6E). CSF was not collected from high cohort rats.

Table 2 Pharmacokinetic results of SMM-189 peripheral administration in mice

SMM-189 promotes an anti-inflammatory environment and shifts immune cell gene expression toward a wound healing phenotype in the CNS

To evaluate the effects of CB2 modulation on neuroinflammation, nigral sections were immunohistochemically examined for immune cell markers from rats that received low titer virus that previously demonstrated Asyn overexpression without nigral degeneration. IBA1 analysis demonstrated lesion effects with significantly increased counts (F(1,22) = 18.72, p = 0.003) and area (F(1,22) = 22.94, p < 0.0001) in the Asyn-lesioned nigra compared to the unlesioned side (Fig. 3A, B). Although there was not a significant effect of treatment, the magnitude of lesion effect on IBA1 was lessened, yet still not fully rescued, in SMM-189-treated rats (counts p = 0.0115; area p = 0.0129) compared to vehicle-treated rats (counts p = 0.0028; area p = 0.0005), potentially highlighting the ability for SMM-189 to dampen Asyn-activated IBA1 + brain myeloid cells. In the striatum, western blot analysis revealed no differences across treatments in myeloid marker IBA1, but a trend to decrease astrocyte marker GFAP expression levels from SMM-189 treatment (p = 0.0746; Figure S7A, B). SMM-189 promoted a phagocytic environment with increased nigral CD68-ir particle counts (t(15) = 3.413, p = 0.0039) in the substantia nigra injected with AAV-hWT-Asyn compared to vehicle-treated conditions as measured using ImageJ (Fig. 3C, D). CD68 is a lysosomal marker and is upregulated in phagocytic innate immune cells when responding to an inflammatory stimuli. CD163, an innate-immune marker identifying cells from the monocyte/macrophage lineage, was also histologically evaluated in the nigra and found to be decreased in SMM-189-treated rats compared to vehicle-treated animals (t(7) = 5.172, p = 0.0013; Fig. 3E, F). No differences were found in the intensity or area coverage of major histocompatibility complex II (MHCII, an antigen presentation marker) between treatment groups in the lesioned nigra (Figure S7C, D).

Fig. 3

Modulation of CB2 with SMM-189 modifies central neuroinflammation. A) Representative microphotographs of midline midbrain sections immunostained for IBA1. B) Quantification of IBA1 + immunofluorescent staining reveals significant lesion effect in both vehicle- and SMM-189-treated rats based on count and area for each treatment group. C) Representative images of midbrain sections immunostained for CD68. Arrows highlight the CD68 + cells included in the quantification. D) CD68 quantification using ImageJ particle counting and normalized to vehicle found significant increases in SMM-189 compared to vehicle-treated rats. E) Representative microphotographs of nigral sections immunostained for CD163 with a magnified insert for each treatment. Arrows identify the CD163 + staining that was included in quantification. F) CD163 quantification within the lesioned nigra was decreased by SMM-189 treatment. G) The mRNA expression of inflammatory cytokines CD206, YM1, TGFβ, CD80, TNF, CB2 are displayed for both frontal cortex or brain H) and PBMCs. Relative PBMC mRNA abundance was normalized to GAPDH, frontal cortex to HPRT1. Scale bars = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001

To further assess inflammation, cytokines transcript levels were evaluated in the frontal cortex brain tissue and PBMCs of low cohort rats by qRTPCR. SMM-189-treated rats displayed increased M2 alternative activation markers YM1 and CD206 gene expression in the frontal cortex and TGFβ in PBMCs relative to vehicle-treated animals (Fig. 3G, H). Endpoint PBMCs also demonstrated decreased pro-inflammatory cytokine TNF transcript after SMM-189 treatment. CB2 gene expression itself was not different between groups confirming that the target is present in immune cells and brain. The tissue cytokine differences between treatments did not extend to the plasma or CSF biofluids as SMM-189- and vehicle-treated rats displayed similar levels of circulating TNF, IL-1b, IL-10, IL-6 and IL-4 (Figure S8).

SMM-189 induced significant time-dependent alterations in profiles of brain-infiltrating immune cells and in peripheral monocyte populations

Next, to investigate the effects of SMM-189 on the infiltration of immune cells from the periphery, brain immune cells were isolated from the high cohort by enzymatic digestion and Percoll gradient separation at both 4- and 8-weeks post-nigral injections. The high cohort of rats (4.6 × 10¹² vg/mL) were used for this experiment to ensure low levels of Asyn-mediated neurotoxicity yet infiltrating peripheral immune cells [27, 43]. Flow cytometry analysis at 4 weeks revealed that SMM-189 treatment globally reduced the frequency of CD163 + macrophages (F(1,17) = 4.45, p = 0.0499; Fig. 4A, B) and CD45hiCD11b myeloid/monocytes (F(1,17) = 5.64, p = 0.0295; Fig. 4C, D). Multiple comparisons analysis revealed significantly less monocytes in the unlesioned hemisphere of SMM-189-treated rats compared to vehicle-treated (p = 0.0187) and compared to the AAV-hWTAsyn lesioned hemisphere of SMM-189-treated rats (p = 0.0494). Furthermore, no treatment differences were detected in more defined classical or non-classical monocyte subset frequencies (p = 0.745, p = 0.169, respectively; data not shown). A two-way ANOVA analysis found no difference in the frequency of microglia across groups (treatment: p = 0.3073; lesion: p = 0.215; Fig. 4E). However, the MFI of CD11b on microglia was reduced in unlesioned hemispheres of SMM-189-treated rats compared to control (p = 0.0478) and compared to its own lesioned hemisphere (p = 0.0379; Fig. 4F) with no changes in MHCII MFI on microglia (Fig. 4G). No CD3 + lymphocytes parent population differences were identified at 4 weeks (treatment: p = 0.691; lesion: p = 0.361; data not shown), yet evaluation of T cell subsets at 4 weeks revealed an overall reduction in the frequencies of CD4 + T cells in brains of SMM-189-treated rats (treatment: F(1,17) = 7.88, p = 0.0121; Fig. 4H, J). CD8 T cell frequencies were not significantly different across the hemispheres administered AAV2/5-human WT Asyn injections (treatment: p = 0.371; lesion: p = 0.29; data not shown). Interestingly, 8 weeks after AAV injections, SMM-189 did not affect the parent brain immune cell population frequencies of monocytes (CD45hiCD11b+; p = 0.45), microglia (CD45loCD11b+; p = 0.59), lymphocytes (CD45 + CD11b-; p = 0.072; data not shown) and macrophages (CD11b + CD163+; p = 0.78; data not shown) compared to control (Fig. 5). Although evaluation of more defined monocyte populations revealed an elevated frequency of MHCII + classical (CD43-) monocytes in lesioned hemispheres from SMM-189 animals compared to vehicle (Fig. 5C). Consistent with the histological analysis in the nigra, the intensity of MHCII on microglia was no different across treatment groups (F(1, 24) = 0.114. p = 0.739; Fig. 5E), however, SMM-189 globally elevated microglial MFI of CD172a (treatment: F(1,24) = 5.659, p = 0.0257; Fig. 5F) and CD11b (treatment: F(1,24) = 7.8, p = 0.01; Fig. 5G) with significant elevation in the unlesioned hemisphere from multiple comparisons (p = 0.0162). Furthermore, independent of the presence of Asyn, CD4 + lymphocytes globally increased (treatment: F(1,24) = 17.52, p = 0.0003), while CD8 + lymphocytes decreased (treatment: F(1,24) = 23.79, p < 0.0001) in SMM-189-treated animal brains compared to vehicle (Fig. 5H-J). Brain immune cell counts (cells/μL) largely align with the frequency results for both the 4- and 8-week analysis, yet greater variability was found in CD4 and CD8 lymphocyte counts that eliminated significance to trends between treatments (Figure S1).

Fig. 4

Modulation of CB2 with SMM-189 globally reduces macrophages and infiltrating monocytes and CD4 T lymphocytes at 4 weeks. Brain immune cells from high cohort rats were isolated from lesioned (+ AAV-human Asyn) and unlesioned (-AAV-human Asyn) rat hemispheres at 4 weeks and flow cytometry analysis conducted to identify monocyte, lymphocyte and microglia populations from CD45 and CD11b expression and further gated into more distinct populations. Macrophage populations (CD163 + CD11b+) were identified from live cells (see Figure S1). A) Representative dot blots of CD163 + macrophages in the unlesioned hemisphere of a vehicle and SMM-189-treated rat at 4 weeks. B) Quantification of CD163 + reveals a global reduction of macrophages from SMM-189 treatment. C) Representative dot blots of CD45 and CD11b in the unlesioned hemisphere of a vehicle and SMM-189-treated rat. D) Quantification of the monocyte population as a frequency of all live cells (CD45hiCD11b+). E) Quantification of microglia (CD45loCD11b+), and microglia MFI of F) CD11b and G) MHCII. H) Representative dot blots of CD3 + lymphocytes stratified by CD4 and CD8 populations in the lesion hemisphere of a vehicle and SMM-189-treated rat. I) Quantification of CD4-expressing T lymphocytes. *p < 0.05

Fig. 5

Modulation of CB2 with SMM-189 promotes innate immune cell activation and altered infiltration of T lymphocytes in the brain at 8 weeks. Brain immune cells from high cohort rats were isolated from lesioned- (+ AAV-human Asyn) and unlesioned (-AAV-human Asyn) hemispheres at 8 weeks and flow cytometry analysis conducted to identify monocyte, lymphocyte and microglia populations using CD45 and CD11b expression and further gated into more distinct populations (see Figure S1). A) Representative dot blots of CD163 + macrophages in the unlesioned hemisphere of a vehicle and SMM-189-treated rat at 8 weeks. B) Quantification of monocyte (CD45hiCD11b+) and C) MHCII-expressing classical monocyte (MHCII + CD43-) frequencies. D) Analysis of microglia (CD45loCD11b+) and microglia MFI of E) MHCII F) CD172a and G) CD11b. H) Dot blots of CD3 + lymphocytes stratified by CD4 and CD8 populations in the lesion hemisphere of a vehicle and SMM-189-treated rat at 8 weeks. I) Quantification of CD4-expressing and J) CD8-expressing T cells. *p < 0.05, **p < 0.01

To determine the effect of CB2 pharmacologic modulation on peripheral immune cell frequencies across time, PBMCs were evaluated by flow cytometry from high titer rats at baseline and every 4 weeks. In the periphery, SMM-189 reduced circulating classically-activated monocytes (CD43-His48+; treatment: F(1,24) = 5.093, p = 0.0334) with multiple comparisons demonstrating a significant decrease at weeks 4 (p = 0.0465) and 8 (p = 0.0384) in PBMCs of SMM-189-treated rats compared to vehicle-treated animals (Fig. 6). Yet the amount of MHCII as determined by MFI was increased on CD43-His48 + classical monocytes at 4 weeks after SMM-189 treatment compared to control (p = 0.0328). Furthermore, at 8 weeks increases in the wound-healing non-classical monocytes (CD43+; p = 0.0466), which circulate and patrol in search of injury, and decreases in granulocytes (p = 0.0355) was found in SMM-189 compared to vehicle-treated using multiple comparisons analysis. Myeloid cell counts were comparable to frequencies (Figure S2). Lymphocyte frequencies of PBMCs did not reveal significant differences in CD4 or CD8 populations (or the CD4 to CD8 ratio) between treatments, however, counts of both CD4 and CD8 + T cells were decreased from SMM-189 (Figure S3). Animals treated with SMM-189 had elevated frequencies of Tregs (CD4 + Foxp3+, p = 0.004; and further defined as CD4 + Foxp3 + CD25+, p = 0.0145) at 8 weeks compared to their own baseline which was mirrored in the cell count analysis (Figure S3). Immune cell populations were also defined in deep cervical lymph nodes (DCLN) and splenocytes of high cohort rats at the 8-week endpoint to evaluate the influence of peripheral SMM-189 treatments on peripheral lymphoid tissues (Figure S9). A trend was found in the CD4 to CD8 lymphocyte ratio to be decreased in DCLN (p = 0.0503) and spleen (p = 0.0962) in SMM-189-treated rats, representing an inverse immune cell composition compared to the brain at 8 weeks. SMM-189 also prompted a trend to increase CD11b + myeloid splenocytes (p = 0.0541) with significant elevation of their MHCII intensity (p = 0.0185).

Fig. 6

Modulation of CB2 with SMM-189 skews PBMC phenotype and increases frequency of circulating wound-healing non-classical monocytes. A) PBMCs were isolated from whole blood at baseline, 4 and 8 weeks after AAV2/5-hAsyn injections from rats in the high cohort. SMM-189 treatment resulted in no change in frequency of His48 + CD43 + non-classical monocytes, but B) decreased circulating His48 + classical monocytes (CD43 - CD11b + CD172a+) at 4 and 8 weeks compared to vehicle treatment. C) Mean fluorescent intensity (MFI) of MHCII on His48 + classical monocytes was elevated on PBMCs from SMM-189-treated rats compared to vehicle at 4 weeks. D) Parent gate CD43 + non-classical monocytes demonstrated increased frequency of CD43 + non-classical monocytes, but E) no changes in parent gate CD43- classical monocytes. F) Frequency of granulocytes as defined by CD3- and granularity (SSC) were decreased at 8 weeks from SMM-189 treatment compared to vehicle (F). *p < 0.05

SMM-189 pretreatment enhances LPS-induced phagocytosis in RAW264.7 macrophages

To better understand the effects of SMM-189 on functional uptake and degradation by innate immune cells, a phagocytosis assay was performed using E. coli phHrodo bioparticles in RAW264.7 macrophage cells. SMM-189 potentiated the LPS-stimulated response as measured by the increased optical density of GFP expressed in the RAW cells (Fig. 7). Specifically, when stimulated with 100ng/mL LPS, there was a dose dependent response to SMM-189 where pre-treatment with drug at either 3–7 μm significantly increased the integrated density of GFP when compared to pre-treatment with vehicle (p = 0.0024, p < 0.0001, respectively; Fig. 7A). However, when stimulated with a higher concentration of LPS (1 μg/mL), both 3 μm and 7 μm doses of SMM-189 intensified phagocytosis of E. coli bioparticles compared to vehicle (p < 0.0001 and p < 0.0001, respectively; Fig. 7B), suggesting a ceiling effect when cells are highly activated. Regardless of LPS dose stimulation, as a control, GFP expression was significantly restricted with the additional incubation of cytochalasin D, an actin polymerization inhibitor.

Fig. 7

Modulation of CB2 with SMM-189 increases RAW264.7 macrophage phagocytosis after an inflammagen challenge. A) Quantification of GFP fluorescent intensity in RAW264.7 macrophage cultures challenged with either 100ng/mL or B) 1000 ng/mL lipopolysaccharide and pre-treated with SMM-189 or vehicle and 24 h later incubated with pHrodo green E. coli. Treatments are analyzed across time using a one-way ANOVA with Dunnett’s multiple comparisons test. Data are representative of 2 separate experiments. C) Representative GFP images of fluorescent pHrodo green E. coli at 12 h for 100ng/mL LPS + vehicle, D) 100ng/mL LPS + 3 μm SMM-189, E) 100ng/mL LPS + 7 μm SMM-189 conditions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Discussion

Evidence of chronic neuroinflammation and dysfunction of the innate immune system have been described in PD, which has given rise to a growing landscape of therapeutic interventions that target microglia and the peripheral immune system to delay or prevent disease progression [44]. The cannabinoid system has become a recent therapeutic target of interest due to its influence on the immune system and the increased legalization across continents of cannabis that directly acts on the cannabinoid receptors. Given the untoward side effects of cannabis intake on psychomotor performance, focus on the immunomodulatory CB2 signaling arm of the pathway represents a significantly novel and more desirable therapeutic direction for the field. This study is the first to evaluate the effects of a CB2-selective ligand on synucleiopathies. The results of the present study demonstrate that CB2 inverse agonism reduces Asyn phosphorylation and aggregation and shifts central and peripheral immune cells toward a wound-healing, phagocytic phenotype in an Asyn rat model of nigral neuroinflammation. These novel results point to a critical role of CB2 in mediating the function of brain immune cells that could be harnessed to prevent or treat synucleinopathies.

Our data demonstrate that SMM-189 treatment reduced phosphorylated Asyn and proteinase-k resistant Asyn. Aggregated Asyn is resistant to digestion from proteinase K as seen in human synucleinopathies and similar to misfolded proteins relevant to prion disease [45, 46]. These results are in line with other published studies demonstrating that CB2-deficient mouse models resulted in reduced protein aggregation. Specifically, transgenic CB2 knockout mice had significantly decreased Amyloid-β-enriched plaque density in the hippocampus, as measured with Methoxy-XO4 staining, and improved cognitive and learning deficits in mouse models of Alzheimer’s disease-like pathology [47, 48]. An additional study found reduced phosphorylated tau levels in insoluble fractions from the hippocampus of CB2-/- mice overexpressing human P301L mutant tau compared to WT mice [49]. These results would suggest that CB2 plays a role in the removal of aggregated proteins. Although the accumulation of Amyloid-β versus Asyn or tau are differentially localized, extracellular versus intracellular, respectively, evidence from others has supported the extracellular propagation of Asyn from cell-to-cell [50, 51]. Furthermore, microglia are major scavengers that clear extracellular Asyn by phagocytosis and degradation through a lysosome-dependent autophagy process, which in turn can limit Asyn spread [52, 53]. In this study, the reduced Asyn phosphorylation and proteinase-k resistant aggregates coupled to the increased phagocytic marker CD68 in the nigra and influence of SMM-189 in the RAW264.7 cell phagocytosis assay may suggest that modulation of CB2 on immune cells promoted clearance of extracellular misfolded Asyn. In the context of aggregated Asyn, only one other study has evaluated the role of CB2 in Asyn clearance and found opposing effects on Asyn accumulation. Feng and colleagues evaluated transgenic CB2-deficient and CB2 WT mice treated with human Asyn pre-formed fibrils (PFF) in the nucleus accumbens and found increased area coverage of Asyn 2 h post-PFF injections in the absence of CB2 [21]. Although the study suggests impaired Asyn clearance, authors also report increased intensity of CD68 immunostaining and morphologically more ameboid (less branches) shaped IBA1 + microglia in CB2-/- compared to WT mice at the same timepoint, both of which would suggest phagocytic microglial phenotypes. Feng and colleagues did not evaluate insoluble or phosphorylated species of Asyn. Multiple studies have identified phosphorylated Asyn to be critical for Asyn inclusion formation [42, 54], suggesting that the reduced pSer129 in our study may have overall therapeutic value. It is difficult to directly compare our Asyn results to this study because of the many differences in experimental design. Important differences of note include pharmacological inverse agonism in our study compared to transgenic CB2 deficiency in the Feng study, the mode of Asyn overexpression (virus-mediated versus PFF) and the inoculated brain region (substantia nigra versus nucleus accumbens). Microglia are heterogenous across brain regions such that midbrain compared to striatal and pre-frontal cortex microglia have distinct transcriptional profiles enriched for immune-related pathways, which possibly account for opposing results on Asyn (PFF) clearance [55].

Our results support that CB2 plays a role in microglial response and phenotype. A previous study reported that CB2-deficient microglia display shorter projections that are less ramified within 24 h after Asyn PFF injections relative to CB2 WT microglia as measured by a rigorous Sholl analysis [21]. Although, we sampled adequately throughout the nigra, our data did not show statistically significant treatment effect in the count or size of IBA1 + cell bodies when evaluated by IHC at 8-weeks. Yet, the magnitude of lesion effect on IBA1 was lessened with SMM-189 suggesting that CB2 inverse agonism may have small effects on IBA1 + morphological phenotypes in the presence of human Asyn; however our method of analysis may not be sensitive enough to detect subtle morphological differences [56]. While other single-cell morphological techniques such as Sholl analysis provide a more detailed quantification, microglia activation can’t be reduced to a structural transition from ramified to ameboid shapes and functional analyses should instead be considered [57]. We therefore performed flow cytometry on a high titer cohort as a more sensitive yet complementary measure to evaluate brain immune cells frequency. Our flow data did not show an effect of lesion in any of the microglial measurements, which was surprising. This discrepancy between the results from the two types of measurements may be due to the different cohorts of animals provided low versus high titers of AAV-hWTAsyn or because of the varied regions evaluated (IHC evaluation of the nigra; flow evaluation of entire hemisphere). Interestingly, at 8 weeks, our flow data did reveal SMM-189 treatment effects including elevated frequency of MHCII + classical monocytes and the MFI of CD11b and CD172a on microglia compared to vehicle, yet no differences in the frequency of microglia or MHCII + microglia between treatment groups. CD172a, also known as the signal regulatory protein A (SIRPa), is an inhibitory receptor that binds to “do not eat me” ligand CD47 on healthy neurons and loss of CD172a from microglia have led to synaptic pruning in mouse models of Alzheimer’s disease [58]. Whereas CD11b, is an integrin and used to identify cells of myeloid lineage, can be induced in states of microglial activation, and the pharmacological activation has been linked to pro-inflammatory macrophage polarization [59, 60]. Our flow data suggests that SMM-189 induces a weak microglia inflammatory profile after 8 weeks of human Asyn but may suggest that microglia are undergoing compensatory mechanisms because of the observed shifts in brain cytokine gene expression and significant shifts in T cell populations. Importantly, consistent with our findings using CB2 inverse agonist treatment, Feng and colleagues similarly identified upregulated CD68 + microglia from the genetic ablation of CB2. It has previously been shown that PD patient monocytes have impaired phagocytic capabilities and reduced ability to clear Asyn [61, 62], therefore modulation of CB2 may lead to a therapeutic gain-of-function involving immune cell uptake and clearance. Phagocytosis analysis of LPS stimulated RAW264.7 macrophages demonstrated that SMM-189 increased phagocytic capacity, suggesting that CB2 inverse agonism can increase immune cell uptake mechanisms. Future studies should be confirmed and extended to microglia cultures where effects may be more subtle due to lower CB2 expression, yet important. Given that SMM-189 had little effect on microglia measures at earlier timepoints (4 weeks) and then increased CD68 and activation in myeloid compartments at later timepoints (8 weeks), we conclude that CB2 modulation can lead to dynamic changes in the extent of microglial activation overtime.

Although our study suggests that the beneficial effects of SMM-189 were due to modulation of the neuroinflammatory response, the mechanism(s) underlying this critical therapeutic effect have not been fully elucidated. Previous work has identified CB2-mediated cAMP/PKA signaling pathways including the phosphorylation of CREB (cAMP response element-binding) to impact the microglial/macrophage phenotype [21, 26, 63, 64]. CREB has been proposed to directly inhibit NF-κB activation which limits the production of pro-inflammatory responses, while cAMP promoted wound-healing M2 phenotypes (CD206, Arg1, YM1) in macrophages [65]. This study found significant increases in CD206 and YM1 in the brain and reduction of TNF gene expression in PBMCs from SMM-189-treated rats, suggesting that the anti-inflammatory effects may be from cAMP downstream signaling. Furthermore, studies outside the field of synucleinopathies, have observed that CB2 signaling can activate the PI3K/Akt and mammalian target of rapamycin (mTOR) pathways which is implicated in the autophagy-lysosome system and associated with PD progression [66, 67]. Recent reports highlight ligand-specific protective effects of CB2 on TFEB regulation, a master regulator of lysosome biogenesis, in an amyloid-β model [68]. Although this study, did not evaluate the mTOR pathway, an increase in CD68 expression was identified in the nigra of SMM-189-treated rats, suggesting a lysosomal phenotype from CB2 inverse agonism. Additionally, recent reports show that microglial transcription of CB2 is dependent on nuclear factor erythroid 2-related factor 2 (NRF2), a well-known regulator of redox homeostasis and inflammation [69]. An antioxidant response element (ARE) sequence was recently found in the promoter region of the microglial CB2 [69]. NRF2 binds ARE in the nucleus to initiate transcription, suggesting that CB2 is linked to microglia-specific NFR2 inflammatory signaling. We were not able to test the specific signaling of SMM-189 responsible for the microglial phenotype transformations. However, in future studies it will be necessary to evaluate the mechanisms of microglial CB2 modulation in the presence of a synucleinopathy as they may be time-dependent in a progressive model such as the one used herein.

Our flow cytometry analyses revealed SMM-189 decreased the extent of macrophages and infiltrating monocytes and CD4 + T cells at 4 weeks following human Asyn overexpression. Other groups have shown that viral-mediated overexpression of Asyn in the brain induces an influx of peripheral chemokine receptor 2 or CCR2 + immune cells into the degenerating area and that knocking out CCR2 or blocking monocyte infiltration is sufficient to protect against neuronal degeneration [43]. In line with reduced peripheral immune cell infiltration, microglia isolated from SMM-189 treated mice had reduced CD11b + MFI at 4 weeks. Together, these data suggest that SMM-189 can modify early peripheral and central crosstalk and responses to insults such as Asyn overexpression. Interesting, at 8 weeks CD163-ir was reduced in the lesioned hemisphere by IHC, yet no differences were found in the frequency of CD163 + macrophages in the brain by flow cytometry at a complementary timepoint (CD163 + was reduced at 4 weeks by flow analysis). The inconsistencies in CD163 analysis at 8 weeks are most likely due to performing the analyses on different cohorts of animals that had either low or high AAV injections or due to the varied regional analysis (nigral compared to entire hemisphere). Expression of CD163 in the brain has been associated with border associated macrophages, which have been identified as the driving cells for immune cell recruitment and infiltration [70]. Therefore, the reduced levels of CD163 may have contributed to the reduced infiltration of monocytes and lymphocytes at 4 weeks. The heightened activation of brain immune cells at 8 weeks, including more CD11b + and CD172a + expression on microglia and frequency of MHCII + monocytes, is likely to have triggered the extravasation and increased presence of CD4 + helper cells and decreased CD8 + cytotoxic cells. The increased CD4 to CD8 ratio found in the brains of SMM-189-treated rats support an activated immune response that is likely to have contributed to the reduced synuclein pathology. Previous reports have found that T cell adoptive transfer to immune deficient mice reduced phosphorylated Asyn after pre-formed fibril injections, suggesting that T cells can have direct modulatory effects on Asyn toxicity [71]. It is possible that CB2 modulation had direct effects on T cells as they also express the receptor and CB2-mediated activation of T cells alter functions including proliferation and migration [72,73,74]. Although our results did show changes in parent CD3 + T cell frequencies in the brain, the peripheral CD4 + and CD8 + T cell subsets frequency were not altered from systemic SMM-189 treatment. Therefore, our data supports a model in which CB2-dependent modulation of brain myeloid cell function serves to attract and activate brain-infiltrating lymphocytes with little effect on peripheral T cells. We were not able to measure subtypes of T cells in the brain or periphery, with the exception of Tregs, a cell essential to regulating and suppressing immune cells. We found increased peripheral Tregs after 8 weeks of peripheral SMM-189 treatment compared to baseline (Figure S3). Positive effects of Tregs in models of synucleiopathy have been reported by several groups [75, 76]. Future studies should be conducted to better characterize the effect of CB2 modulation on circulating T cell subsets and their associated cytokines to better understand CB2-induced adaptive immune cell functions.

Peripheral treatment with CB2 inverse agonist SMM-189 produced body-wide effects that were independent of human Asyn overexpression. SMM-189 was administered peripherally and therefore is expected to elicit widespread effects in the brain as demonstrated by immune gene expression changes in the frontal cortex, an area not exposed directly human Asyn (Fig. 3G). Furthermore, through multiple comparison analysis of the flow cytometry data, some of the immune effects were found in the unlesioned hemisphere and not in the hemisphere overexpressing human Asyn. This is not surprising as intracerebral injections break the dura and could disturb the entire brain immune homeostasis, which in turn can affect immune cell infiltration throughout the brain, not just in the injected hemisphere. Our peripheral flow data at 8 weeks revealed that SMM-189 skewed peripheral monocytes with decreased pro-inflammatory CD43-His48 + classical monocytes and increased wound-healing CD43 + non-classical monocytes. Although neither specific subset of monocytes was significantly altered in the brain, it is possible that monocytes infiltrating the specific region of Asyn overexpression may be skewed toward the non-classical population. Future studies should include more detailed regional analysis by flow cytometry. Additionally, there was a significant shift in peripheral immune phenotype based on PBMC gene expression, with decreased TNF and increased TGFβ which promotes alternative macrophage activation. These findings suggest that SMM-189 altered downstream CB2 signaling and promoted a widespread anti-inflammatory environment in the peripheral circulating immune cells and in the nigra that would not have been captured by evaluating biofluids at a systems level.

Conclusion

The cannabinoid system has become a new and exciting target for immunomodulation in PD and other neurological conditions [20]. Besides the recent genetic link to the cannabinoid system, others have identified elevated protein expression of CB2 in brains of individuals with PD, underscoring the neuroanatomical basis for targeting CB2. Our novel findings demonstrate that a CB2-selective inverse agonist, SMM-189, can alter immune responses both in the periphery and the brain to mitigate toxic species of Asyn in the brain. To our knowledge, this is the first study to highlight CB2 as a promising therapeutic target via which modulation of inflammatory and immune responses could delay, limit or prevent proteinopathies. Future studies should evaluate the effects of other CB2 modulating compounds in Asyn overexpression models to better understand ligand-specific benefits in synucleinopathies.