ABHD12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease PHARC

Abstract

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 α/β-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.

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 5′ and 3′ 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) Lack of 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. Fluorescent gel shown in grayscale.

Fig. 2.

ABHD12^−/− mice develop age-dependent PHARC-related behaviors. (A–C) Eleven- to 12-month-old (B) and 17- to 18-mo-old (C), but not 5- to 6-mo-old (A), ABHD12^−/− mice (black triangles) showed significantly reduced auditory startle reflex compared with ABHD12^+/− (gray squares) and ABHD12^+/+ (white diamonds) littermate controls (n = 8–19 per group). (D) The auditory brainstem response (ABR) was impaired in 9- to 10-mo-old ABHD12^−/− mice (black bars), which showed delayed interpeak latency of waves I–IV. ABHD12^+/− mice (gray bars) also showed a modest defect in ABR compared with ABHD12^+/+ mice (white bars) that was only observed at the lowest auditory stimulus (n = 5 per genotype). (E and F) Seventeen- to 18-mo-old ABHD12^−/− mice displayed mild ataxia (E) and a reduced latency to fall (F) in the rotarod and hanging wire test, respectively (n = 16–19 per genotype). Data represent the averages ± SEM. Variance among genotypes was assessed by two-way ANOVA and Fisher’s protected least-significant difference (PLSD) test. The Student’s t test was used to determine significance for each individual dB pulse tested in A–C. *P < 0.05; **P < 0.01; ***P < 0.001 vs. ABHD12^+/+ mice.

Fig. 3.

ABHD12 disruption causes age-dependent increases in microglial activation in the mouse brain. (A–C) Representative images of Iba1 immunostaining for microglia in the cerebellum of 18-mo-old (A), 12-mo-old (B), and 6-mo-old (C) ABHD12^+/+ and ABHD12^−/− mice. (D–F) Quantifying the number of enlarged cells (>200 μm²) revealed significantly more activated microglia in cerebellar sections from 18-mo-old (D) and 12-mo-old (E) ABHD12^−/− mice compared with ABHD12^+/+ mice. No genotype differences were observed at 6 mo of age (F). Data represent the averages ± SEM. ***P < 0.001 (n = 3–4 per genotype per age group). (Scale bar, 100 μm.)

Fig. 4.

Deregulated (lyso)phosphatidylserine [(L)PS] metabolites in brain tissue from ABHD12^−/− mice. (A) Untargeted metabolomics identified several metabolites with altered brain levels in 2- to 6-mo-old ABHD12^−/− mice compared with ABHD12^+/+ mice. The m/z values with the largest fold-change differences between genotypes are marked. (B) Relative peak intensity of a metabolite with an m/z value of 524.3, corresponding to 18:0 lysophosphatidylserine (LPS), in ABHD12^−/− vs. ABHD12^+/+ brain tissue. (C and D) The m/z 524.3 metabolite (black trace) exhibited the same LC elution time (C) and MS/MS fragmentation pattern (D) as a synthetic 18:0 LPS standard (red trace). Key daughter ions in the MS/MS analysis include 153.0 (dehydro-glycerophosphate) and 437.3 [18:0 lysophosphatidic acid (LPA)]. (E and F) Targeted MRM measurements of LPS (E) and PS (F) species from brain tissue of ABHD12^+/+ (white bars), ABHD12^+/− (gray bars), or ABHD12^−/− (black bars) mice. Data are presented as average values ± SEM (n = 3–5 per group). *P < 0.05; **P < 0.01; ***P < 0.001 vs. ABHD12^+/+ (Student’s t test).

Fig. 5.

ABHD12 is a major brain LPS lipase. (A) Membrane homogenates from HEK293T cells transfected with a murine ABHD12 cDNA (gray bars) displayed increased hydrolytic activity compared with mock-transfected cell membranes (black bars) toward several lysophospholipids and other singly acylated glycerolipids (e.g., MAG, BMP). In contrast, doubly acylated glycerolipids (e.g., DAG, PA, PC, PE, PI, PG, PS) were not hydrolyzed by ABHD12. See Table S7 for more information on lipid species tested as ABHD12 substrates. (Inset) Active ABHD12 expression in transfected cells was confirmed by ABPP with the FP-rhodamine probe. (B) ABHD12^−/− brain membrane homogenates (black bars) displayed significantly reduced hydrolytic activity toward 18:1 LPS, LPG, LPI, and BMP compared with ABHD12^+/+ brain homogenates (white bars). Data are presented as average values ± SEM (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001 vs. mock or ABHD12^+/+ homogenates (Student’s t test).

Fig. 6.

Working model to explain the molecular and cellular basis for ABHD12-dependent neuropathology. Our results point to a model where loss of ABHD12 leads to accumulation of the enzyme’s endogenous LPS substrates, as well as corresponding (indirect) alterations in PS lipids. These metabolic changes occur in young mice (<6 mo), before the appearance of cellular or behavioral signs of neuropathology. By 12 mo of age, neuroinflammatory markers, such as cerebellar microglial activation, are observed in ABHD12^−/− mice, which are then followed by PHARC-related behavioral deficits that intensify with age. FFA, free fatty acid; GPS, glycerophosphate.