ABHD12 Controls Brain Lysophosphatidylserine Pathways That Are Deregulated in a Murine Model of the Neurodegenerative Disease PHARC

ABHD12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease PHARC Jacqueline L. Blankmana,b, Jonathan Z. Longa,b, Sunia A. Traugerc, Gary Siuzdakc, and Benjamin F. Cravatta,b,1 aThe Skaggs Institute for Chemical Biology and Departments of bChemical Physiology and cMolecular Biology, The Scripps Research Institute, La Jolla, CA 92037 Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved November 30, 2012 (received for review October 1, 2012) Advances in human genetics are leading to the discovery of new could contribute to the metabolism of the endogenous canna- disease-causing mutations at a remarkable rate. Many such muta- binoid 2-arachidonoylglycerol (2-AG) in the nervous system. tions, however, occur in genes that encode for proteins of unknown Nonetheless, the physiological metabolites regulated by ABHD12 function, which limits our molecular understanding of, and ability to in vivo, and the molecular and cellular mechanisms by which this enzyme contributes to PHARC, are unknown. Here, we have devise treatments for, human disease. Here, we use untargeted −/− metabolomics combined with a genetic mouse model to determine addressed these important questions by generating ABHD12 α β mice and analyzing these animals for metabolomic and behavioral that the poorly characterized serine hydrolase / -hydrolase do- −/− < main-containing (ABHD)12, mutations in which cause the human phenotypes. Young ABHD12 mice ( 6 mo old) were mostly normal in their behavior; however, as these animals age, they neurodegenerative disorder PHARC (polyneuropathy, hearing loss, develop an array of PHARC-related phenotypes, including de- ataxia, retinosis pigmentosa, and cataract), is a principal lysophos- −/− fective auditory and motor behavior, with concomitant cellular phatidylserine (LPS) lipase in the mammalian brain. ABHD12 mice pathology indicative of a neuroinflammatory response. Metab- − − display massive increases in a rare set of very long chain LPS lipids olomic characterization of brain tissue from ABHD12 / mice that have been previously reported as Toll-like receptor 2 activators. revealed striking elevations in a series of lysophosphatidylserine We confirm that recombinant ABHD12 protein exhibits robust LPS fl NEUROSCIENCE − − (LPS) lipids that occur before the onset of neuroin ammatory lipase activity, which is also substantially reduced in ABHD12 / fi − − and behavioral defects. We con rmed that LPS lipids are direct brain tissue. Notably, elevations in brain LPS lipids in ABHD12 / substrates for ABHD12 and that this enzyme is a major contri- mice occur early in life (2–6 mo) and are followed by age-dependent butor to bulk brain LPS lipase activity. These data, taken to- − − increases in microglial activation and auditory and motor defects gether, demonstrate that ABHD12 / mice are a suitable animal that resemble the behavioral phenotypes of human PHARC patients. model for investigating the mechanistic basis of PHARC and Taken together, our data provide a molecular model for PHARC, point to a functional role for an ABHD12-regulated LPS pathway where disruption of ABHD12 causes deregulated LPS metabolism in this disease. and the accumulation of proinflammatory lipids that promote microglial and neurobehavioral abnormalities. Results Targeted Disruption of the Abhd12 Gene. To generate mice lacking lipidomics | neuroinflammation ABHD12, a targeting construct was designed to delete exons 8–10 of the Abhd12 gene, including the predicted catalytic serine S246 contained within a canonical GXSXG motif, upon homologous enome mapping and sequencing have facilitated determi- recombination in C57BL/6-derived embryonic stem (ES) cells Gnation of the genetic basis for over 3,500 inherited diseases (Fig. 1A). One targeted ES clone, 96, was identified by Southern in humans (1), with additional pathogenic mutations still being blotting (Fig. 1B) and used to generate chimeric mice. These discovered. For many Mendelian disorders, however, the mo- chimeras gave germ-line transmission of the targeted mutation, as lecular and cellular mechanisms that link genotype to disease determined by Southern blotting (Fig. 1C) and PCR genotyping − − phenotype remain poorly understood. This problem is perhaps (Fig. 1D). Mice homozygous for the targeted allele (ABHD12 / most apparent for diseases where the causative mutations occur mice) were viable and born at the expected Mendelian frequency. in genes that encode for proteins with unannotated or poorly Loss of ABHD12 mRNA (Fig. 1E), protein (Fig. 1F), and activity − − characterized functions. Here, we focus on one such disease - the (Fig. 1G) were confirmed in the brains of ABHD12 / mice by neurodegenerative disorder PHARC (polyneuropathy, hearing RT-PCR, liquid chromatography–mass spectrometry (LC-MS) loss, ataxia, retinosis pigmentosa, and cataract). proteomics, and activity-based protein profiling (ABPP) (6), re- PHARC [Mendelian Inheritance in Man (MIM) no. 612674; spectively. Levels of other protein constituents of the endocanna- Online Mendelian Inheritance in Man database, http://omim.org] binoid system were unaffected by ABHD12 disruption (Fig. 1F). is a rare, autosomal recessive disorder that causes polymodal − − sensory and motor defects associated with demyelination of sen- ABHD12 / Mice Develop an Age-Dependent, PHARC-Related Phenotype. − − somotor neurons, retinal dystrophy, and cerebellar atrophy (2). ABHD12 / mice were fertile and largely indistinguishable from + + + − PHARC symptoms are slowly progressive and begin in the child- their WT (ABHD12 / ) and heterozygous (ABHD12 / ) litter- + + + − hood or teenage years; heterozygous carriers are unaffected (3). In mates at early stages of life. ABHD12 / , ABHD12 / , and 2010, PHARC was reported to be caused by homozygous mutations in ABHD12, which codes for the poorly characterized serine hydrolase enzyme α/β-hydrolase domain-containing (ABHD)12 Author contributions: J.L.B. and B.F.C. designed research; J.L.B. and S.A.T. performed (3). To date, five distinct ABHD12 mutations have been identified research; J.L.B. and J.Z.L. contributed new reagents/analytic tools; J.L.B., G.S., and B.F.C. in patients with PHARC, all of which are expected to lead to analyzed data; and J.L.B. and B.F.C. wrote the paper. complete loss of ABHD12 expression (3, 4). PHARC, therefore, The authors declare no conflict of interest. likely represents a human ABHD12 null model. This article is a PNAS Direct Submission. We demonstrated previously that ABHD12 is a membrane- 1To whom correspondence should be addressed. E-mail: [email protected]. bound enzyme that exhibits monoacylglycerol (MAG) lipase This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activity in vitro (5). These initial data suggested that ABHD12 1073/pnas.1217121110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1217121110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Fig. 1. Generation and initial characterization of − − ABHD12 / mice. (A) ABHD12-targeting vector designed to replace Abhd12 exons 8–10 with a neomycin (Neo) selection cassette upon homologous recombination. (B) Targeted clone no. 96 was identified by Southern analysis of EcoRI-digested ES cell genomic DNA with external 59 and 39 probes. Germ-line transmission was confirmed by Southern (C) and PCR (D) genotyping of genomic tail DNA. (E) RT-PCR confirmed loss of ABHD12 mRNA in ABHD12−/− mice. (F) Untargeted MS-based proteomics confirmed absence of ABHD12 protein in − − ABHD12 / brain tissue. Protein levels of other measured constituents of the endocannabinoid system were equivalent between genotypes. Values represent the mean ± SEM (n = 3). (G)Lackof ABHD12 activity in ABHD12−/− mice was confirmed by ABPP of brain membrane proteomes with the serine hydrolase probe fluorophosphonate (FP)- rhodamine. Other serine hydrolase activities were − − unaltered in ABHD12 / brain proteome. Fluores- cent gel shown in grayscale. − − ABHD12 / mice were assessed in a battery of behavioral tests auditory information, auditory brainstem responses (ABRs) were potentially relevant to the symptoms of PHARC disease. Initial measured in a cohort of 9- to 10-mo-old mice after confirming − − behavioral studies were performed with relatively young mice (5– that ABHD12 / mice exhibit a reduced auditory startle response 6 mo old), but considering that neurodegenerative phenotypes at this age (Fig. S1). The sinusoidal waveform of the brainstem − − often only become apparent in aged mice, even for disease auditory evoked potential (BAEP) was intact in ABHD12 / models with childhood onset in humans (7–10), this panel of tests mice (Fig. S1), and no genotypic differences were detected in was repeated when mice were 11–12 and 17–18 mo old. peak amplitude (Table S1). However, absolute latencies of waves − − Young ABHD12 / mice behaved similarly to control litter- IV, V, and VII (Table S1) and interpeak latencies of waves I–IV mates except for modest hypermotility, which was transitory and were delayed in ABHD12-disrupted mice (Fig. 2D), confirming not observed during the later testing sessions (Fig. S1). A slight impaired auditory signaling in these animals and suggesting that reduction in the auditory startle reflex was also observed in 5- to the hearing loss induced by ABHD12 disruption may be caused − − 6-mo-old ABHD12 / mice, although this effect failed to reach by reduced auditory conductance. − − statistical significance

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