Fig 1: Brain ABHD12 levels increase upon an inflammatory stimulus. (a) Representative immunofluorescence images and quantification of ABHD12 levels on cerebellar microglia from wild type (+/+) mice, following intraperitoneal injection of vehicle (1× PBS) or LPS (10 mg/kg body weight, 4 h), showing a ~2-fold increase in ABHD12 levels following lipopolysaccharide (LPS) treatment. Data (bars) are represented as mean ± standard deviation from six independent experiments (biological replicates) per experimental group. Scale bar in the immunofluorescence images is 10 µm. (b) Representative blot and quantification from western blot analysis of membrane lysates of the cerebellum from wild type (+/+) mice, following intraperitoneal injection of vehicle (1× PBS) or LPS (10 mg/kg body weight, 4 h), showing a ~2-fold increase in ABHD12 levels following LPS treatment. The levels of the PS lipase ABHD16A in the cerebellum did not change following LPS treatment. Actin was used as a loading control for this experiment. This experiment was done three times (3 independent biological replicates) with reproducible results each time. For both (a) and (b), ***p < .001 versus (+/+) group by Student's two-tailed unpaired parametric t test
Fig 2: Deletion of ABHD12 in primary microglial cells results in increased phagocytosis in a fluorescent bead uptake assay. (a) Quantification of the total percentage of primary microglial cells obtained from wild type (+/+) and ABHD12 knockout (-/-) mice engulfing fluorescent beads, showing no significant differences between the two genotypes. Data (bars) are represented as mean ± standard deviation from six independent experiments (biological replicates) per genotype. (b) Representative microscopy images of primary microglial cells obtained from wild type (+/+) and ABHD12 knockout (-/-) mice, engulfing fluorescent latex beads, showing increased phagocytosis activity towards engulfing the fluorescent latex beads in the ABHD12-null primary microglial cells. Scale bar in the microscopy images is 10 µm, and (*) represents centroid of the nucleus. (C) The number of fluorescent beads phagocytosed by primary microglial cells obtained from wild type (+/+) and ABHD12 knockout (-/-) mice, showing a significant increase of number of phagocytosed fluorescent latex beads by ABHD12-null primary microglial cells. Data (bars) are represented as mean ± standard deviation from six independent experiments (biological replicates) per genotype. ***p < .001 versus vehicle group by Student's two-tailed unpaired parametric t test; ns = not statistically significant
Fig 3: The biochemical pathways regulated by the lipase ABHD12. Loss of ABHD12 lipase activity causes accumulation of lysophosphatidylserine (lyso-PS) (Blankman et al., 2013) and oxidized PS lipids (Kelkar et al., 2019) in the brain that contribute to neuroinflammation that manifests into the neurobehavioral defects associated with the human neurological disorder PHARC (Fiskerstrand et al., 2010). GPS = glycerophosphoserine and FFA = free fatty acid. This figure is adapted with modifications from a previous study from our lab (Kelkar et al., 2019)
Fig 4: ABHD12-null activated microglia in the cerebellum have increased LAMP1 expression. Representative immunofluorescence images and quantification of LAMP1 levels on activated microglia in the cerebellum of wild type (+/+) and ABHD12 knockout (-/-) mice following intraperitoneal injection of lipopolysaccharide (LPS) (10 mg/kg body weight, 4 hours), showing a ~2-fold increase in the levels of LAMP1 on activated microglia from ABHD12 knockout mice. Data (bars) are represented as mean ± standard deviation from six independent experiments (biological replicates) per experimental group. Scale bar in the immunofluorescence images is 10 µm. ***p < .001 versus (+/+) group by Student's two-tailed unpaired parametric t test
Fig 5: Pharmacological inhibition of ABHD12 results in increased phagocytosis by microglial cells in a fluorescent bead uptake assay. (a) Quantification of the total percentage of microglial cells (BV-2 and N9) engulfing fluorescent beads following vehicle (DMSO) or JJH350 (10 µM, 6 h) treatment, showing no significant differences between the two experimental groups. Data (bars) are represented as mean ± standard deviation from six independent experiments (biological replicates) per experimental group. (b) Representative microscopy images of microglial cells (BV-2 and N9) engulfing fluorescent beads following vehicle (DMSO) or JJH350 (10 µM, 6 h) treatment, showing increased phagocytosis of fluorescent beads in the JJH350-treated microglial cells. Scale bar in the microscopy images is 10 µm, and (*) represents centroid of the nucleus. (c) The number of fluorescent beads phagocytosed by microglial cells (BV-2 and N9) following vehicle (DMSO) or JJH350 (10 µM, 6 h) treatment, showing a significant increase of number of phagocytosed fluorescent beads by the JJH350-treated microglial cells (BV-2 and N9). Data (bars) are represented as mean ± standard deviation from 12 independent experiments (biological replicates) per experimental group. ***p < .001 versus vehicle group by Student's two-tailed unpaired parametric t test; ns = not statistically significant
Supplier Page from Abcam for Anti-ABHD12 antibody [EPR13683-72]