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) alpha-synuclein 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.

Keywords: Parkinson’s disease; alpha-synuclein; cannabinoid receptor-2; microglia phenotype.

Figure 1.. Experimental design and outcome measures.

Rats were injected unilaterally with AAV2/5-human wild-type 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 (6mg/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.

Figure 2.. Modulation of CB2 with SMM-189 decreases accumulation of pSer129 alpha-synuclein in the lesioned nigra.

Representative images of pSer129 Asyn (A, scale bar=250μm), proteinase K-insoluble Asyn (C), and total human Asyn (E) in rats that received AAV2/5-hAsyn and treated with either vehicle or SMM-189. Quantification of low cohort rats treated with SMM-189 normalized to vehicle showed reduced pSer129 Asyn (abcam ab512253, B) compared to vehicle and reduced intensity of proteinase K-insoluble Asyn (D). Total human Asyn did not differ between groups when evaluating the optical density of Asyn-ir within the lesioned nigra normalized by vehicle (F). Representative western immunoblots of pSer129 and total human synuclein from striatal lysates and their corresponding total protein used for normalization (G). 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 (H). *p<0.05, **p<0.01

Figure 3.. Modulation of CB2 with SMM-189 modifies central neuroinflammation.

Representative microphotographs of midline midbrain sections immunostained for IBA1 (A). Quantification of IBA1+ immunofluorescent staining reveals significant lesion effect based on count and area for each treatment group. Scale bar = 100μm (B). Representative images of midbrain sections immunostained for CD68, scale bar = 500μm (C). Arrows highlight the CD68+ cells included in the quantification. CD68 quantification using ImageJ particle counting and normalized to vehicle found significant increases in SMM-189 compared to vehicle-treated rats, while CD163 quantification within the lesioned nigra was decreased by SMM-189 treatment (D). The mRNA expression of inflammatory cytokines CD206, YM1, TGFβ, CD80, TNF, CB2 are displayed for both frontal cortex or brain (E) and PBMCs (F). Relative PBMC mRNA abundance was normalized to GAPDH, frontal cortex to HPRT1. *p<0.05, **p<0.01, ***p<0.001

Figure 4.. Modulation of CB2 with SMM-189 globally reduces infiltrating monocytes and brain myeloid cell activation at 4 weeks.

Brain immune cells from high cohort rats were isolated from lesioned (+human Asyn) and unlesioned (−human Asyn) rat hemispheres at 4 weeks and monocyte, lymphocyte and microglia populations initially identified from CD45 and CD11b expression and further gated into more distinct populations. Quantification of immune cell population frequencies are shown here for monocytes (CD11b+CD172a+), microglia (CD45loCD11b+) and lymphocytes (CD3+) of their parent gate (A-C), with significant reductions in monocyte frequencies in SMM-189 rats (A) and no differences in microglia (B) or CD3+ lymphocytes (C). Quantification of non-classical (CD43+CD172a+), classical (CD43−CD172+) monocytes and MCHII+ classical monocytes show no differences across groups (D-F). Mean fluorescent intensity (MFI) was evaluated for monocyte and microglia populations and found alterd CD172a on classical monocytes (G) with no effect of CD172a on microglia (H). MFI of CD11b was reduced in unlesioned hemispheres of those with SMM-189 treatment compared to vehicle and also found significantly lower than SMM-189 lesioned hemispheres (I). T cell subsets were quantified and found that CD4+ T cells were reduced in lesioned hemispheres of SMM-189 rats compared to vehicle (J). Although no differences were seen in CD8+ T cells (K), a reduced CD4:CD8 ratio was identified in the presence of Asyn (L). *p<0.05, **p<0.01

Figure 5.. Modulation of CB2 with SMM-189 promotes monocyte infiltration and increased innate and adaptive immune cell activation in the brain.

Brain immune cells from high cohort rats were isolated from lesioned- (+human Asyn) and unlesioned (−human Asyn) hemispheres at 8 weeks and monocyte, lymphocyte and microglia populations initially identified using CD45 and CD11b expression and further gated into more distinct populations. Quantification of immune cell population frequencies are shown here for microglia (CD45loCD11b+), monocytes (CD11b+CD172a+) and lymphocytes (CD3+) of their parent gate (A-C). Monocyte frequencies were increased in the brains of SMM-189-treated animals compared to vehicle-treated animals in both hemispheres (A), but no differences in microglia (B) or CD3+ lymphocytes (C). Quantification of non-classical (CD43+CD172a+), classical (CD43−CD172+) monocytes show no differences across groups (D-E), yet treatment with SMM-189 increased the frequency of MCHII+ classical monocytes in the presence of Asyn compared to treatment with vehicle (F). Mean fluorescent intensity (MFI) of CD172a on classical monocytes was not different between conditions (G), but was globally increased on CD172a+microglia from SMM-189-treated rats (H). MFI of CD11b on microglia was increased in unlesioned hemispheres of those with SMM-189 treatment compared to vehicle (I). CD4 and CD8 T cell subsets were quantified and found that CD4+ T cells were increased (J), while CD8+ T cells were reduced (K) in both hemispheres of SMM-189-treated rats compared to vehicle, resulting in increased CD4:CD8 ratios across treatment groups (L). *p<0.05, **p<0.01, ***p<0.001

Figure 6.. Modulation of CB2 with SMM-189 skews PBMC phenotype and increases frequency of circulating wound-healing non-classical monocytes.

Peripheral blood mononuclear cells 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+ non-classical monocytes (A), but decreased circulating His48+ classical monocytes (CD43+CD11b+CD172a+) at 4 and 8 weeks compared to vehicle treatment (B). 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 (C). Parent gate CD43+ non-classical monocytes demonstrated increased frequency of CD43+ non-classical monocytes (D), but no changes in parent gate CD43− classical monocytes (E). 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

Figure 7.. Modulation of CB2 with SMM-189 increases RAW264.7 macrophage phagocytosis after an inflammagen challenge.

Quantification of GFP fluorescent intensity in RAW264.7 macrophage cultures challenged with either 100ng/mL (A) or 1000 ng/mL (B) lipopolysaccharide and pre-treated with SMM-189 or vehicle and 24 hours 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. Representative GFP images of fluorescent pHrodo green E. coli at 12 hours for 100ng/mL LPS + vehicle (C), 100ng/mL LPS + 3uM SMM-189 (D), 100ng/mL LPS + 7uM SMM-189 (E) conditions. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001