Family Report for: ABHD12-PHARC

In humans, lysophosphatidylserines (lyso-PSs) are potent lipid regulators of important immunological processes. Given their structural diversity and commercial paucity, here we report the synthesis of methyl esters of lyso-PS (Me-lyso-PSs) containing medium- to very-long-chain (VLC) lipid tails. We show that Me-lyso-PSs are excellent substrates for the lyso-PS lipase ABHD12, and that these synthetic lipids are acted upon by cellular carboxylesterases to produce lyso-PSs. Next, in macrophages we demonstrate that VLC lyso-PSs orchestrate pro-inflammatory responses and in turn neuroinflammation via a Toll-like receptor 2 (TLR2)-dependent pathway. We also show that long-chain (LC) lyso-PSs robustly induce intracellular cyclic AMP production, cytosolic calcium influx, and phosphorylation of the nodal extracellular signal-regulated kinase to regulate macrophage activation via a TLR2-independent pathway. Finally, we report that LC lyso-PSs potently elicit histamine release during the mast cell degranulation process, and that ABHD12 is the major lyso-PS lipase in these immune cells.

The endocannabinoid (eCB) system is an important part of both the human central nervous system (CNS) and peripheral tissues. It is involved in the regulation of various physiological and neuronal processes and has been associated with various diseases. The eCB system is a complex network composed of receptor molecules, their cannabinoid ligands, and enzymes regulating the synthesis, release, uptake, and degradation of the signalling molecules. Although the eCB system and the molecular processes of eCB signalling have been studied extensively over the past decades, the involved molecules and underlying signalling mechanisms have not been described in full detail. An example pose the two poorly characterised eCB-degrading enzymes alpha/beta-hydrolase domain protein six (ABHD6) and ABHD12, which have been shown to hydrolyse 2-arachidonoyl glycerol-the main eCB in the CNS. We review the current knowledge about the eCB system and the role of ABHD6 and ABHD12 within this important signalling system and associated diseases. Homology modelling and multiple sequence alignments highlight the structural features of the studied enzymes and their similarities, as well as the structural basis of disease-related ABHD12 mutations. However, homologies within the ABHD family are very low, and even the closest homologues have widely varying substrate preferences. Detailed experimental analyses at the molecular level will be necessary to understand these important enzymes in full detail.

Endogenous ligands for cannabinoid receptors ("endocannabinoids") include the lipid transmitters anandamide and 2-arachidonoylglycerol (2-AG). Endocannabinoids modulate a diverse set of physiological processes and are tightly regulated by enzymatic biosynthesis and degradation. Termination of anandamide signaling by fatty acid amide hydrolase (FAAH) is well characterized, but less is known about the inactivation of 2-AG, which can be hydrolyzed by multiple enzymes in vitro, including FAAH and monoacylglycerol lipase (MAGL). Here, we have taken a functional proteomic approach to comprehensively map 2-AG hydrolases in the mouse brain. Our data reveal that approximately 85% of brain 2-AG hydrolase activity can be ascribed to MAGL, and that the remaining 15% is mostly catalyzed by two uncharacterized enzymes, ABHD6 and ABHD12. Interestingly, MAGL, ABHD6, and ABHD12 display distinct subcellular distributions, suggesting that they may control different pools of 2-AG in the nervous system.

In humans, lysophosphatidylserines (lyso-PSs) are potent lipid regulators of important immunological processes. Given their structural diversity and commercial paucity, here we report the synthesis of methyl esters of lyso-PS (Me-lyso-PSs) containing medium- to very-long-chain (VLC) lipid tails. We show that Me-lyso-PSs are excellent substrates for the lyso-PS lipase ABHD12, and that these synthetic lipids are acted upon by cellular carboxylesterases to produce lyso-PSs. Next, in macrophages we demonstrate that VLC lyso-PSs orchestrate pro-inflammatory responses and in turn neuroinflammation via a Toll-like receptor 2 (TLR2)-dependent pathway. We also show that long-chain (LC) lyso-PSs robustly induce intracellular cyclic AMP production, cytosolic calcium influx, and phosphorylation of the nodal extracellular signal-regulated kinase to regulate macrophage activation via a TLR2-independent pathway. Finally, we report that LC lyso-PSs potently elicit histamine release during the mast cell degranulation process, and that ABHD12 is the major lyso-PS lipase in these immune cells.

Lysophosphatidylserine (lyso-PS), a lysophospholipid derived from phosphatidylserine (PS), has emerged as a potent signaling lipid in mammalian physiology. In vivo, the metabolic serine hydrolases ABHD16A and ABHD12 are major lipases that biosynthesize and degrade lyso-PS respectively. Of biomedical relevance, deleterious mutations to ABHD12 causes accumulation of lyso-PS in the brain, and this deregulated lyso-PS metabolism leads to the human genetic neurological disorder PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract). While the roles of ABHD16A and ABHD12 in lyso-PS metabolism in the mammalian brain are well established, the anatomical and (sub)cellular localizations of both lipases, and the functional cross-talk between them towards regulating lyso-PS lipids remain under investigated. Here, using subcellular organelle fractionation, biochemical assays and immunofluorescence based high resolution microscopy, we show that the PS lipase ABHD16A is an endoplasmic reticulum (ER) localized enzyme, an organelle intricately regulating cellular PS levels. Further, leveraging immunohistochemical analysis using genetic ABHD16A and ABHD12 knockout mice as important controls, we map the anatomical distribution of both these lipases in tandem in the murine brain, and show for the first time, the distinct localization of these lipases to different regions and cells of the cerebellum. We complement the aforementioned immunohistochemical studies by quantitatively measuring lyso-PS concentrations in various brain regions using mass spectrometry, and find that the cerebellar lyso-PS levels are most affected by ABHD16A (decreased) or ABHD12 (increased) deletion. Taken together, our studies provide new insights into lyso-PS signaling in the cerebellum, the most atrophic brain region in human PHARC subjects.

Reactive oxygen species (ROS) are transient, highly reactive intermediates or byproducts produced during oxygen metabolism. However, when innate mechanisms are unable to cope with sequestration of surplus ROS, oxidative stress results, in which excess ROS damage biomolecules. Oxidized phosphatidylserine (PS), a proapoptotic 'eat me' signal, is produced in response to elevated ROS, yet little is known regarding its chemical composition and metabolism. Here, we report a small molecule that generates ROS in different mammalian cells. We used this molecule to detect, characterize and study oxidized PS in mammalian cells. We developed a chemical-genetic screen to identify enzymes that regulate oxidized PS in mammalian cells and found that the lipase ABHD12 hydrolyzes oxidized PS. We validated these findings in different physiological settings including primary peritoneal macrophages and brains from Abhd12(-/-) mice under inflammatory stress, and in the process, we functionally annotated an enzyme regulating oxidized PS in vivo.

The endocannabinoid (eCB) system is an important part of both the human central nervous system (CNS) and peripheral tissues. It is involved in the regulation of various physiological and neuronal processes and has been associated with various diseases. The eCB system is a complex network composed of receptor molecules, their cannabinoid ligands, and enzymes regulating the synthesis, release, uptake, and degradation of the signalling molecules. Although the eCB system and the molecular processes of eCB signalling have been studied extensively over the past decades, the involved molecules and underlying signalling mechanisms have not been described in full detail. An example pose the two poorly characterised eCB-degrading enzymes alpha/beta-hydrolase domain protein six (ABHD6) and ABHD12, which have been shown to hydrolyse 2-arachidonoyl glycerol-the main eCB in the CNS. We review the current knowledge about the eCB system and the role of ABHD6 and ABHD12 within this important signalling system and associated diseases. Homology modelling and multiple sequence alignments highlight the structural features of the studied enzymes and their similarities, as well as the structural basis of disease-related ABHD12 mutations. However, homologies within the ABHD family are very low, and even the closest homologues have widely varying substrate preferences. Detailed experimental analyses at the molecular level will be necessary to understand these important enzymes in full detail.

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a rare genetic human neurological disorder caused by null mutations to the Abhd12 gene, which encodes the integral membrane serine hydrolase enzyme ABHD12. Although the role that ABHD12 plays in PHARC is understood, the thorough biochemical characterization of ABHD12 is lacking. Here, we report the facile synthesis of mono-1-(fatty)acyl-glycerol lipids of varying chain lengths and unsaturation and use this lipid substrate library to biochemically characterize recombinant mammalian ABHD12. The substrate profiling study for ABHD12 suggested that this enzyme requires glycosylation for optimal activity and that it has a strong preference for very-long-chain lipid substrates. We further validated this substrate profile against brain membrane lysates generated from WT and ABHD12 knockout mice. Finally, using cellular organelle fractionation and immunofluorescence assays, we show that mammalian ABHD12 is enriched on the endoplasmic reticulum membrane, where most of the very-long-chain fatty acids are biosynthesized in cells. Taken together, our findings provide a biochemical explanation for why very-long-chain lipids (such as lysophosphatidylserine lipids) accumulate in the brains of ABHD12 knockout mice, which is a murine model of PHARC.

Lysophosphatidylserines (lyso-PSs) are a class of signaling lipids that regulate immunological and neurological processes. The metabolism of lyso-PSs remains poorly understood in vivo. Recently, we determined that ABHD12 is a major brain lyso-PS lipase, implicating lyso-PSs in the neurological disease polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC), which is caused by null mutations in the ABHD12 gene. Here, we couple activity-based profiling with pharmacological and genetic methods to annotate the poorly characterized enzyme ABHD16A as a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. We describe a small-molecule inhibitor of ABHD16A that depletes lyso-PSs from cells, including lymphoblasts derived from subjects with PHARC. In mouse macrophages, disruption of ABHD12 and ABHD16A respectively increases and decreases both lyso-PSs and lipopolysaccharide-induced cytokine production. Finally, Abhd16a(-/-) mice have decreased brain lyso-PSs, which runs counter to the elevation in lyso-PS in Abhd12(-/-) mice. Our findings illuminate an ABHD16A-ABHD12 axis that dynamically regulates lyso-PS metabolism in vivo, designating these enzymes as potential targets for treating neuroimmunological disorders.

Advances in human genetics are leading to the discovery of new disease-causing mutations at a remarkable rate. Many such mutations, however, occur in genes that encode for proteins of unknown function, which limits our molecular understanding of, and ability to devise treatments for, human disease. Here, we use untargeted metabolomics combined with a genetic mouse model to determine that the poorly characterized serine hydrolase alpha/beta-hydrolase domain-containing (ABHD)12, mutations in which cause the human neurodegenerative disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract), is a principal lysophosphatidylserine (LPS) lipase in the mammalian brain. ABHD12(-/-) mice display massive increases in a rare set of very long chain LPS lipids that have been previously reported as Toll-like receptor 2 activators. We confirm that recombinant ABHD12 protein exhibits robust LPS lipase activity, which is also substantially reduced in ABHD12(-/-) brain tissue. Notably, elevations in brain LPS lipids in ABHD12(-/-) mice occur early in life (2-6 mo) and are followed by age-dependent increases in microglial activation and auditory and motor defects that resemble the behavioral phenotypes of human PHARC patients. Taken together, our data provide a molecular model for PHARC, where disruption of ABHD12 causes deregulated LPS metabolism and the accumulation of proinflammatory lipids that promote microglial and neurobehavioral abnormalities.

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a neurodegenerative disease marked by early-onset cataract and hearing loss, retinitis pigmentosa, and involvement of both the central and peripheral nervous systems, including demyelinating sensorimotor polyneuropathy and cerebellar ataxia. Previously, we mapped this Refsum-like disorder to a 16 Mb region on chromosome 20. Here we report that mutations in the ABHD12 gene cause PHARC disease and we describe the clinical manifestations in a total of 19 patients from four different countries. The ABHD12 enzyme was recently shown to hydrolyze 2-arachidonoyl glycerol (2-AG), the main endocannabinoid lipid transmitter that acts on cannabinoid receptors CB1 and CB2. Our data therefore represent an example of an inherited disorder related to endocannabinoid metabolism. The endocannabinoid system is involved in a wide range of physiological processes including neurotransmission, mood, appetite, pain appreciation, addiction behavior, and inflammation, and several potential drugs targeting these pathways are in development for clinical applications. Our findings show that ABHD12 performs essential functions in both the central and peripheral nervous systems and the eye. Any future drug-mediated interference with this enzyme should consider the potential risk of long-term adverse effects.

Endogenous ligands for cannabinoid receptors ("endocannabinoids") include the lipid transmitters anandamide and 2-arachidonoylglycerol (2-AG). Endocannabinoids modulate a diverse set of physiological processes and are tightly regulated by enzymatic biosynthesis and degradation. Termination of anandamide signaling by fatty acid amide hydrolase (FAAH) is well characterized, but less is known about the inactivation of 2-AG, which can be hydrolyzed by multiple enzymes in vitro, including FAAH and monoacylglycerol lipase (MAGL). Here, we have taken a functional proteomic approach to comprehensively map 2-AG hydrolases in the mouse brain. Our data reveal that approximately 85% of brain 2-AG hydrolase activity can be ascribed to MAGL, and that the remaining 15% is mostly catalyzed by two uncharacterized enzymes, ABHD6 and ABHD12. Interestingly, MAGL, ABHD6, and ABHD12 display distinct subcellular distributions, suggesting that they may control different pools of 2-AG in the nervous system.