Activation of CB2 receptor is required for the therapeutic effect of ABHD6 inhibition in experimental autoimmune encephalomyelitis

Abstract

Alpha/beta-hydrolase domain 6 (ABHD6) is a novel 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, that can fine-tune the endocannabinoid signaling in the central nervous system. Recently we and others have demonstrated the protective effect of ABHD6 inhibition in the animal models of traumatic brain injury and epileptic seizures. In this study, we investigated the role of targeting ABHD6 in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Post-symptom treatment with an ABHD6 inhibitor WWL70 increased the brain levels of 2-AG and ameliorated the clinical signs of EAE, T cells infiltration, microglia activation and the expression of activated leukocyte cell adhesion molecules. The production of iNOS, COX-2, TNF-α and IL-1β and the phosphorylation of NF-κB were also significantly reduced by WWL70 treatment. The neuroprotective effect of WWL70 was demonstrated by increased survival of mature oligodendrocytes, reduced demyelination and axonal loss in WWL70 treated EAE mouse spinal cord. The therapeutic effect of WWL70 on EAE was absent by co-administration of CB2 receptor antagonist, but not CB1 receptor antagonist. Consistently, WWL70 did not afford any protection in CB2 receptor knockout mice after EAE induction. Given the increased expression of ABHD6 in microglia/macrophages, but not in T cells, we speculated that inhibition of ABHD6 might enhance 2-AG signaling particularly in microglia/macrophages to exert anti-inflammatory effects via activation of CB2 receptors. These results suggest that inhibition of ABHD6 might be used as an ideal strategy for the treatment of MS and other neurodegenerative diseases.

WWL70 attenuates PGE2 production derived from 2-arachidonoylglycerol in microglia by ABHD6-independent mechanism

“…Before addition to the enzyme-linked immunoassay (EIA), the medium was centrifuged at 5000 rpm for 2 min with a table top centrifuge to exclude residual cells. To determine the role of WWL70 on PGE 2 production in vivo, EAE was induced by subcutaneous injection of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG 35–55 ) in 8-week-old female C57BL/6 mice, and the clinical score was assessed as we reported recently [23]. WWL70 (10 mg/kg, i.p.)…”

“…The resultant mixture was applied to a prostaglandin EIA to measure the total amount of prostaglandins including PGE 2 and PGF 2α . The PGE 2 biosynthesis assay was performed using microsomes derived from BV2 cells treated with LPS for 8 h or brain tissue from EAE mouse at day 21 post-immunization [23]. BV2 cells were suspended with 0.1 M potassium phosphate (pH 7.4), 0.25 M sucrose, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 1 μM MAFP, and protease inhibitor cocktail (Roche) and disrupted by sonication for 15 s, while the brain tissue was homogenized with a Potter homogenizer with the potassium phosphate buffer at 4 °C.…”

“…Although the results from our laboratory and others have shown that only ~4% of 2-AG hydrolysis in the mouse brain and spinal cord is attributable to ABHD6 under physiological conditions [21], ABHD6 contributes significantly to the 2-AG hydrolysis in microglia/macrophages, in which there is no or little expression of MAGL [18, 19]. Recently, we have found that WWL70 exerts therapeutic effects in mouse models of traumatic brain injury [22] and experimental autoimmune encephalomyelitis (EAE) [23] through attenuating microglial activation, COX-2 expression, and the impaired function of the blood-brain barrier. In addition, WWL123, a derivative of WWL70 with high permeability to the blood-brain barrier, was also protective in a mouse model of epilepsy [24].…”

Biochemical characterization of human alpha/beta hydrolase domain 6 as a therapeutic target

“…ABHD6 expression negatively regulates AMPA subunit surface expression 46 , and in a mouse model of multiple sclerosis, it was found that therapeutic effects seen by ABHD6 inhibition are mediated through the activation of the CB2 receptor. 45 Overall ABHD6 is a very attractive drug target, but only a few specific inhibitor chemotypes have been identified so far, namely triazoleureas 19 , glycine sulfonamides 20 , and thiadiazole carbamates 21 . In the absence of the ABHD6 atomic structure, biochemical characterization through mutational and functional analysis is key in the continued progression and improvement in developing potent, selective ABHD6-inhibitors.…”

“…The native fatty acids oleic acid and palmitic acid were paired with 6-methoxy-4-methylcoumarin with an ester bond and analyzed. Both palmitic and oleic acid paired with coumarin were able to be hydrolyzed by hMGL with high affinity, but 45 with a large reduction in maximal velocity; neither fatty acid was compatible with hABHD6 ( Figure 15D and 15E, Table 5). …”