The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors

Journal name:
Nature Neuroscience
Volume:
13,
Pages:
951–957
Year published:
DOI:
doi:10.1038/nn.2601
Received
Accepted
Published online

Abstract

The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB1 cannabinoid receptors on neurons and CB2 cannabinoid receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase α-β-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB2-mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activity-dependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB1-dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.

At a glance

Figures

  1. ABHD6 hydrolyzes 2-AG in BV-2 cells and controls the efficacy of 2-AG at CB2 receptors.
    Figure 1: ABHD6 hydrolyzes 2-AG in BV-2 cells and controls the efficacy of 2-AG at CB2 receptors.

    (a) Cartoon scheme of the ABPP-MudPIT procedure used to identify serine hydrolases expressed by BV-2 cells. (b) [3H]–2-AG hydrolysis in homogenates prepared from BV-2 cells infected with shRNAs targeting ABHD6, ABHD12, NTE or FAAH, compared to scrambled shRNA. **P < 0.01 compared to scrambled response (ANOVA one-way, Dunnett's post test). (c) [3H]–2-AG hydrolysis in intact BV-2 cells infected with shRNA targeting either ABHD6 or FAAH, compared to scrambled shRNA. ***P < 0.0001 compared to scrambled response (Student's t test). (d) 2-AG-induced cell migration of BV-2 cells infected with shRNA targeting ABHD6 or FAAH, compared to scrambled shRNA. Data are expressed as percentage of basal migration (migration of the shRNA-treated clones in the presence of 0.1% DMSO). The 2-AG response is inhibited by pretreatment with the CB2 receptor antagonist SR144528 (SR2, 200 nM). Data are shown as mean ± s.e.m. of 3–5 independent experiments, each performed in triplicate. The percentage knockdown was systematically verified for each infection and reported in Supplementary Table 1.

  2. Effect of the ABHD6 inhibitor WWL70 and the MAGL inhibitor JZL184 on [3H]-2-AG hydrolysis in cell homogenates.
    Figure 2: Effect of the ABHD6 inhibitor WWL70 and the MAGL inhibitor JZL184 on [3H]–2-AG hydrolysis in cell homogenates.

    [3H]–2-AG hydrolysis in homogenates prepared from (a,b) COS-7 cells transfected with mouse ABHD6 (5 μg of protein per reaction) or with mouse MAGL (0.25 μg of protein per reaction); (c) BV-2 cells (10 μg of protein per reaction); and (d) mouse neurons in primary culture (1 μg of protein per reaction). For the combination of the two inhibitors in d, WWL70 was used at 10 μM and JZL184 at 1 μM. Data are mean ± s.e.m. of 3 to 4 independent experiments. Each experiment was performed in triplicate with homogenates or cells from independent transfections and cell preparations. Transfection of COS-7 cells with ABHD6 increased [3H]–2-AG hydrolysis by about twofold compared to COS-7 cells transfected with empty vector, whereas transfection of COS-7 cells with MAGL increased [3H]–2-AG hydrolysis by 17-fold compared to COS-7 cells transfected with empty vector (data not shown).

  3. Effect of WWL70 and JZL184 on [3H]-2-AG hydrolysis and 2-AG accumulation in intact neurons in primary culture.
    Figure 3: Effect of WWL70 and JZL184 on [3H]–2-AG hydrolysis and 2-AG accumulation in intact neurons in primary culture.

    (a) [3H]–2-AG hydrolysis by intact primary neurons after 30 min pretreatment with WWL70 (10 μM) or JZL184 (1 μM). The data are expressed as % of control hydrolysis (pretreatment with 0.1% DMSO). (b,c) Levels of 2-AG in intact primary neurons pretreated for 30 min with WWL70 (10 μM), JZL184 (1 μM) or vehicle (0.1% DMSO, control; b) and stimulated with glutamate (100 μM) and carbachol (1 mM) for 2.5 min, after which lipids were extracted and 2-AG amounts measured by GC-MS (c). Treatment with glutamate plus carbachol led to a 2.5-fold increase in 2-AG amounts (in fmol per 100,000 cells: 18 to 44). Data are shown as mean ± s.e.m. of 3–6 independent experiments. Experiments were performed in triplicate for the hydrolysis assay and in duplicate for 2-AG quantification by GC-MS, using cells from independent cell culture preparations. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA one-way, Bonferroni's post test).

  4. Visualization of ABHD6 protein in different cell types.
    Figure 4: Visualization of ABHD6 protein in different cell types.

    (ah) Representative images of COS-7 cells transfected with plasmids encoding ABHD6 and GFP (ac; scale bar, 50 μm), BV-2 cells (d; scale bar, 100 μm) and mouse neurons in primary culture (eh; scale bar, 20 μm). Fluorescence detection of primary antibodies recognizing ABHD6 (a, d, e; red in c, h), CB1 receptor (f, green in h), or MAP2 (g, blue in h). Fluorescence detection of GFP (b, green in c). Insets show pre-incubation with 5 μg per ml of the ABHD6 immunizing peptide (ad).

  5. Localization of ABHD6 protein in mouse prefrontal cortex.
    Figure 5: Localization of ABHD6 protein in mouse prefrontal cortex.

    (af) Fluorescent immunohistochemical staining of adult mouse prefrontal cortex (scale bars: ac, 100 μm; df, 20 μm) using antibodies that recognize ABHD6 (a,d; red in c,f) and CB1 receptors (b,e; green in c,f). Inset in a shows pre-incubation with 5 μg per ml of the ABHD6 immunizing peptide. (g) Electron micrograph of antibodies recognizing ABHD6 (arrowheads) in adult mouse prefrontal cortex, labeled with silver-enhanced gold particles (scale bar, 200 nm). Note that ABHD6 is predominantly found on the postsynaptic side of the synapse (arrow) in dendritic spines (ds), and not in presynaptic boutons (b). (h) Quantitative analysis of all electron microscopy images using ImageJ software. The number of gold particles on pre- and postsynaptic sides of identifiable synapses were manually counted and then normalized to the area of the respective structures for comparison. Data are mean ± s.e.m. of 154 identifiable synapses, in two animals. ***P < 0.001 (unpaired two-tailed t-test).

  6. Effect of WWL70 and JZL184 on CB1-dependent LTD in mouse prefrontal cortex.
    Figure 6: Effect of WWL70 and JZL184 on CB1-dependent LTD in mouse prefrontal cortex.

    Field potentials measured in layer 5 after stimulation in layer 2 (arrow, 5 min at 10 Hz). Both the area and amplitude of the field excitatory postsynaptic potential (fEPSP) were measured (graphs depict area). Slices were preincubated for 10 min with WWL70 (10 μM), JZL184 (1 μM) or vehicle (0.01% DMSO, control), and the inhibitors remained present in the superfusion medium throughout the experiment. (a) Time course and (b) summary of the 40-min time point. Data are mean ± s.e.m. of at least ten different brain slices. Statistical significance (above bars) was calculated relative to control (Mann Whitney).

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Author information

  1. These authors contributed equally to this work.

    • Jacqueline L Blankman &
    • Eric A Horne

Affiliations

  1. Neurobiology and Behavior Graduate Program, University of Washington, Seattle, Washington, USA.

    • William R Marrs &
    • Susan Fung
  2. The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.

    • Jacqueline L Blankman,
    • Jessica P Alexander,
    • Jonathan Z Long,
    • Weiwei Li &
    • Benjamin F Cravatt
  3. Department of Pharmacology, University of Washington, Seattle, Washington, USA.

    • Eric A Horne,
    • Yi Hsing Lin,
    • Jonathan Coy,
    • Cong Xu &
    • Nephi Stella
  4. INSERM U862, Equipe Physiopathologie de la plasticité synaptique, Bordeaux, France.

    • Aurore Thomazeau,
    • Mathieu Lafourcade &
    • Olivier J Manzoni
  5. Department of Otolaryngology, University of Washington, Seattle, Washington, USA.

    • Agnes L Bodor
  6. Bioanalysis and Pharmacology of Bioactive Lipids Laboratory, CHAM7230, Louvain Drug Research Institute, Université catholique de Louvain, Bruxelles, Belgium.

    • Giulio G Muccioli
  7. Department of Psychological and Brain Sciences, Gill Center for Biomolecular Science, Indiana University, Bloomington, Indiana, USA.

    • Sherry Shu-Jung Hu &
    • Ken Mackie
  8. Neurobiology Undergraduate Program, University of Washington, Seattle, Washington, USA.

    • Grace Woodruff
  9. Department of Neurology, University of Washington, Seattle, Washington, USA.

    • Thomas Möller
  10. Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington, USA.

    • Nephi Stella

Contributions

W.R.M. prepared the cell cultures, performed the hydrolysis experiments, conducted the data analysis and wrote the manuscript. J.L.B. performed the ABPP experiments and contributed to the data analysis. E.A.H. performed the GC-MS and immunofluoresence experiments and contributed to the electron microscopy experiments and data analysis. A.T. and M.L. performed the electrophysiology experiments. Y.H.L. prepared the cell culture transfections and the shRNA constructs, and performed the qPCR experiments. J.C. contributed to the immunofluorescence experiments. A.L.B. performed the electron microscopy experiments. G.G.M. contributed to the hydrolysis experiments. S.S.-J.H. contributed to antibody production. G.W. and S.F. performed the cell migration experiments. J.P.A. contributed to the ABPP experiments. J.Z.L. and W.L. produced the hydrolase inhibitors. C.X. contributed to the cell culture experiments. T.M. provided the transgenic mice. K.M. provided antibodies. O.J.M. supervised the electrophysiology experiments. B.F.C. supervised the ABPP experiments and the development of hydrolase inhibitors. N.S. supervised the project and wrote the manuscript.

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The authors declare no competing financial interests.

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