Abstract
Cardiolipin (CL) is the signature phospholipid of the inner mitochondrial membrane, where it stabilizes electron transport chain protein complexes1. The final step in CL biosynthesis relates to its remodelling: the exchange of nascent acyl chains with longer, unsaturated chains1. However, the enzyme responsible for cleaving nascent CL (nCL) has remained elusive. Here, we describe ABHD18 as a candidate deacylase in the CL biosynthesis pathway. Accordingly, ABHD18 converts CL into monolysocardiolipin (MLCL) in vitro, and its inactivation in cells and mice results in a shift to nCL in serum and tissues. Notably, ABHD18 deactivation rescues the mitochondrial defects in cells and the morbidity and mortality in mice associated with Barth syndrome. This rare genetic disease is characterized by the build-up of MLCL resulting from inactivating mutations in TAFAZZIN (TAZ), which encodes the final enzyme in the CL-remodelling cascade1. We also identified a selective, covalent, small-molecule inhibitor of ABHD18 that rescues TAZ mutant phenotypes in fibroblasts from human patients and in fish embryos. This study highlights a striking example of genetic suppression of a monogenic disease revealing a canonical enzyme in the CL biosynthesis pathway.
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Data availability
All the data used to generate the figures can be found in the Supplementary Tables. Raw data for genetic screens and RNA sequencing are available at NCBI GEO (Accession: GSE297454 and PRJNA1301228). The Bio-ID data are available through MassIVE (Accession: PXD064381 (MSV000098022)). Links to the publicly available datasets analysed in this manuscript are as follows: DepMap, https://depmap.org. Tissue expression data (GTEX), https://gtexportal.org; and tissue expression and subcellular localization data, https://www.proteinatlas.org.
Code availability
No new code was generated for this study.
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Acknowledgements
We thank J. C. Bozelli Jr; the late R. Epand for characterizing HAP1 TAZ clonal cell lines for altered mCL levels using NMR spectroscopy; the proteomics team at SPARC BioCentre at the Hospital for Sick Children for isolating biotinylated preys with KingFisher Flex, mass spectrometry data generation and analysis, and use of Agilent Seahorse equipment; Cayman Chemicals for lipidomics sample preparation and mass spectrometry data generation and analysis; the Imaging Facility and Nanoscale Biomedical Imaging Facility at the Hospital for Sick Children for confocal microscope use; A. Darbandi for TEM sample preparation and instrument use; J. Burgess, A. Zalazar, S. Prokop, M. Carpenter and T. Cunningham for animal care and zebrafish-facility maintenance at the Hospital for Sick Children; the SickKids Heart Centre Biobank Registry for access to patient samples; the Temerty Faculty of the Medicine Flow Cytometry Facility at the University of Toronto for instrument use; and the core staff in the BSU, Histology and Molecular Technology facilities at the CRUK Scotland Institute. S.N.M. was the recipient of Mito2i graduate fellowships. This work was partly supported by the Azrieli PCHP Catalyst Program to I.C.S., the German Research Foundation (DFG; TRR259_397484323) to M.B., a BTHS IDEA grant to J.M., Cancer Research UK to the CRUK Scotland Institute (A31287), the Canadian Institutes for Health Research (PJT-GMX-463531 to J.M.), the Ontario Research Fund (RE11 to C.B., J.M. and B.J.A.). J.M. is the GlaxoSmithKline chair in Genetics and Genome Biology at the Hospital for Sick Children.
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Contributions
Conceptualization: S.N.M., A. Srivastava, P.M., S.M.B.N., V.A.B. and J.M. Methodology: S.N.M., A. Srivastava, V.S.E., P.M., I.C.S., M.J.N. and D.S. Data acquisition: S.N.M., A. Srivastava, V.S.E., P.M., E.A., J.W., D.T.T., A.G.F., O.S.C., L.C., J.v.A., F.N., L.M., C.S., L.W.-G., N.N., R.M.S., F.M.V., B.E., R.L., S.M., H.V., D.S., M.J.N., B.M.M. and L.M.J.N. Analysis and interpretation: S.N.M., A. Srivastava, V.S.E., P.M., L.V.B., N.M., A. Shaw, S.v.H., B.M.M., S.P., L.M.J.N., K.R.B., R.L., M.B., M.J.N. and D.S. Visualization: S.N.M., P.M., A. Srivastava, L.V.B., K.R.B., S.M.B.N., V.A.B. and J.M. Drafting of the manuscript: S.N.M., P.M., A. Srivastava, S.M.B.N., V.A.B. and J.M., with input from all authors. Technical support: K.C., A.H.Y.T., T.P., O.S., A.H., L.N., M.C. and M.L. Funding and supervision: B.J.A., C.L.M., T.R.B., C.B., I.C.S., S.M.B.N., V.A.B. and J.M.
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S.M. serves on the Advisory Committee of Bristol Myers Squibb, Tenaya Therapeutics and Rocket Pharmaceuticals. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Quality control and validation of CRISPR and gene-trap screens.
(a) Precision-recall curves for five biological replicate CRISPR screens in HAP1 TAZ KO cells using the reference core essential gene set (CEG2) defined in Hart et al. 2017. (b) Scatterplots representing TAZ qGIs derived from all possible pairwise combinations of five replicate screens. Pearson’s correlation based on comparison of qGI scores calculated on all grey points (|qGI | >0.5, FDR < 0.5) is reported in all cases, with R and p-value (TAZ_051 vs. TAZ_186 P = 3.6667×10−91, TAZ_203 vs. TAZ_208 P = 9.31965×10−210, TAZ_203 vs. TAZ_264 P = 1.68248×10−163, TAZ_208 vs. TAZ_264 P = 3.79×10−83) determined using the stat_cor() method in the ggpubr R package. Significant points in both screens highlighted (red). (c) Fitness effect (LFC) distributions for reference core essential (CEG2) and non-essential gene sets across five TAZ KO query screens, the ABHD18-c1 screen and the TAZ;ABHD18-c2 screen respectively. (d) Enrichment for Gene Ontology (GO), molecular function, GO bioprocesses and Reactome terms among genes that exhibited a significant negative GI with TAZ (significant in at least 2 TAZ replicates, |qGI | >0.5, FDR < 0.5) (top), and positive GI with TAZ (bottom). Number of genes overlapping a particular term and term size are indicated. P-values determined by Fisher’s one-tailed test using gProfileR. (e) Mutagenesis-based viability screens in HAP1 WT, and two independent TAZ cell lines. X-axis: log10-transformed mean number of insertions (± strand). Y-axis: ratio, defined as # sense reads/ (# sense + # antisense reads). Significant genes (P adj<1e-3, as compared to four WT controls) are dark grey. Selected genes are marked in red. (f) Overview of unique insertions in the ABHD18 gene in both TAZ KO (s1 and s2 clones) gene-trap screens. Gene transcript (top), sense integrations (middle), and antisense insertions (bottom). (g) Competitive growth assays. Co-cultures of TAZ;ABHD18 double KO cells with either TAZ KO cells or WT HAP1 cells followed over time by flow cytometry and normalised to day 0. (h) Relative proliferation of WT, TAZ KO, ABHD18 KO and ABHD18;TAZ double-KO RPE1 pools, presented as means of n = 2 biological replicates). (i) Relative proliferation of WT, TAZ KO, ABHD18 KO and ABHD18;TAZ double-KO H1-iCas9 pools (left; WT n = 5, TAZ-KO1 n = 4, ABHD18-KO1 n = 3, ABHD18-KO1: TAZ KO-1 n = 4 biological replicate assays) and proportion of pluripotent cells in selected pools (right; WT n = 4, TAZ-KO1 n = 4, ABHD18-KO1 n = 3, ABHD18-KO1: TAZ-KO1 n = 3 biological replicate assays). Data presented as means ± s.d. with significance calculated using one-way ANOVA followed by Dunnett’s post-hoc test. (j) Representative flow cytometric analysis of OCT4 & SOX2 expression in WT, TAZ, ABHD18 and ABHD18;TAZ H1 hPSC pools. (k) Bar plot showing relative fitness effects when overexpressing either TAZ (TAZ OE) or ABHD18 (ABHD18 OE) across double mutant backgrounds. OE of either TAZ [clone 2 P = 0, clone 8 P = 0.0007] or ABHD18 [clone 2 P = 0, clone 8 P = 0.0001] reduces fitness in TAZ;ABHD18 double-KO cells. Data presented are means ± s.d. (n = 3 biological replicates) with significance determined by one-way ANOVA followed by Tukey’s multiple comparisons test. (l) Volcano plot showing qGI scores and associated significance (-log10(P value)) for TAZ;ABHD18-c2 CRISPR screen. (m) Scatterplot of the overlap between TAZ;ABHD18-c2 and ABHD18-c1 qGIs showing GI overlap. Yellow: Shared positive GIs, blue: shared negative GIs (qGI < −0.5, >0.5; FDR < 0.5) in both screens. (n) Heat map displaying genes (y axis) with significant interactions with TAZ across five replicate screens and at least one additional screened query (x-axis) (|qGI | >0.5, FDR < 0.5). Positive and negative qGI scores are indicated by yellow and blue, respectively. (o) Bar plot illustrating relative cell proliferation of partial LOF mutant TAZ;ABHD18-c8 (partial LOF of ABHD18) compared with WT HAP1. Data presented as means ± s.d. (n = 3 biological replicates) with significance determined by one-way ANOVA followed by Dunnett’s post-hoc test. (p) Scatterplot displaying profile similarity of ABHD18 across genome-wide DepMap CRISPR/Cas9 screens. Similarity was quantified by taking all pairwise gene-gene Pearson correlation coefficients of CERES score profiles across 563 screens (19Q2 DepMap data release). Distribution of 17,633 CERES profile similarity is plotted as a quantile-quantile plot (right). Pathway analysis of ABHD18 profile similarity for all 17,634 genes represented in DepMap were mean summarized by pathway as defined in the HumanCyc standard (left). Pathway similarity and dissimilarity with ABHD18 were tested using a two-sided Wilcoxon rank-sum test with multiple-hypothesis correction using the Benjamini and Hochberg procedure.
Extended Data Fig. 2 BioID enrichments support mitochondrial localization of ABHD18.
(a) Confocal images of ABHD18-FLAG HEK293 Flp-In T-Rex cells stained for FLAG (green) and HSP60 (red). Intensity measurements across the indicated white line are indicated in the bottom right panel. At least 30 cells from 2 biological replicate experiments were analyzed and a representative image shown. (b) GO biological process (BP), molecular function (MF), and cellular compartment (CC) enrichments for BioID preys captured by both N- and C-term miniTurbo-tagged ABHD18 in either glucose or galactose growth conditions. Enrichment terms for significantly enriched gene sets (p < 0.05, maximum term size 105) are indicated and bars depict -log10 (FDR) determined by p-values calculated using gProfileR. (c) Scatterplots of interaction specificity of N- and C-term tagged ABHD18 detected preys captured with BioID in either glucose or galactose growth conditions. Average spectral counts of preys captured in proximity to either N- or C-term miniTurbo BirA-tagged ABHD18 are plotted against their specificity across hundreds of baits from the Human Cell Map BioID dataset. Avg Spec was calculated using SAINTexpress v3.6.1. Data are representative of three biologically independent experiments per condition. (d) Representative TEM images of HAP1 parental, TAZ KO, ABHD18 KO, TAZ;ABHD18 double-KO cells, green: mitochondria. Scale bars, 1 μm. Representative of n = 4 biological replicate cell pellets per genotype.
Extended Data Fig. 3 ABHD18 regulates mitochondrial respiratory chain assembly and generates MLCL from nCL.
(a) BN-PAGE immunoblots (total OxPHOS, CII, CIII, CIV, CV) and in-gel activity assays (CI, CIV) from isolated mitochondria of HAP1 mutant cell lines. Blots probed for individual complexes and supercomplexes. Total OXPHOS (n = 5 biological replicates) obtained using antibody cocktail targeting CI, CII, CIII, CIV, and CV. CIII and CIV blots (n = 3 biological replicates), and CIV activity are representative (n = 2 biological replicates). (b) Bar plots of Seahorse Cell Mito Stress Test for respiratory parameters: basal respiration, maximal respiration, mitochondrial ATP generation, and glycolytic ATP generation across mutant HAP1 cell lines. Data presented are means ± s.d. of n = 4 technical replicates. Scatterplots of OCR and ECAR measurements taken during seahorse assays (right). (c) Dot plots of lipidomics analysis of CL and MLCL abundance in HAP1 WT and mutant cell lines (left and middle panel respectively). Relative abundance shown as bar plot above dot plot for each genotype. Bar plot quantifying MLCL/CL ratio changes across HAP1 cell lines using total CL and MLCL species abundance (right). (d) Dot plots showing lipidomics analysis of CL and MLCL abundance in an additional set of HAP1 mutant cell lines (n = 3 clonal mutant cell lines, n = 4 WT cell lines). (e) Thin Layer Chromatography (TLC) showing wild-type ABHD18[WT], but not catalytic dead ABHD18[S199A] recombinant protein deacylates different cardiolipin species to yield MLCL and DLCL, and deacylates MLCL to yield DLCL. Negative control does not contain any ABHD18 protein present, only buffer.
Extended Data Fig. 4 Taz−/YAbhd18−/− mice are fertile and demonstrate normal heart function.
(a) Representative CL spectrum of control wildtype mice (n = 3 animals, left), and Abhd18 knockout mice (n = 3 animals, right) obtained by blood spot assay. Blue: summation of carbon atoms in the fatty acid side chains. Red: CL peaks for each carbon group along with number of double bonds. (b) qRT-PCR analysis of Taz and Abhd18 expression levels in mice heart tissue. Expression was normalized to Gapdh. Data are presented as means ± s.e.m. from three animals. (c) Schematic showing sterility test cross between Taz−/Y:Abhd18−/− double KO male mice and Taz+/−Abhd18−/− female mice. (d) Representative ultrasound images of mouse hearts recorded in M mode (WT n = 8, Taz−/Y n = 3, Abhd18−/− n = 7, Abhd18−/−Taz−/Y n = 6 mice). Six measurements were taken for each image using ImageJ: 1. Anterior Wall Thicknesss (AWT) – diastole (d), 2. Left Ventricle End Diastole Diameter (LVEDD), 3. Posterior Wall Thickness (PWT) – diastole (d), 4. AWT – systole (s), 5. Left Ventricle End Systole Diameter (LVESD) and 6. PWT – systole (s).
Extended Data Fig. 5 ABD646 treatment mimics ABHD18 LOF phenotypes.
(a) Dot plots showing lipidomic analysis of CL and MLCL abundance in HAP1 and TAZ-c1 KO cells treated with either DMSO or 1μM of ABD646 for 5 days (left and middle panel respectively). Bar plot quantifying MLCL/CL ratio changes across HAP1 cell lines using total CL and MLCL species abundance (right). (b) Bar plots illustrating the proliferative effect of 10 μm ABD646 on HAP1 and TAZ KO cells. All values normalized to HAP1 DMSO (n = 3 biological replicates), with significance determined by one-way ANOVA followed by Dunnett’s post-hoc test. (c) TEM images of HAP1 TAZ KO cells treated with DMSO or ABD646 for five days. Green: mitochondria. Scale bars, 1 μm (top) and 200 nm (bottom). (d) Schematic illustrating mode of action of three substrate oxidation pathway inhibitors for glucose/pyruvate (UK5099), long chain fatty acids (LCFA, Etomoxir), and glutamine oxidation (BPTES). (e) Bar plots of seahorse substrate oxidation tests using Etomoxir, BPTES, and UK5099 on HAP1 WT and mutant cells, with ABD646 or DMSO treatment. Data are maximal respiration values (OCR pmol/min, n = 6 technical replicates). (f) Representative TEM images showing partial restoration of mitochondrial morphologies observed in BTHS fibroblasts treated with 1μM of ABD646 for 5 days. Green: mitochondria. Scale bars: 1 μm, and 200 nm. n = 2 biological replicate cell pellets per treatment. (g) Flow cytometry analysis of mitochondrial mass (Mitotracker Green FM) and membrane potential (Mitotracker CMXROS) in fibroblasts from patients with BTHS treated with vehicle or 5μM ABD646 for five days. (h) BN-PAGE immunoblots and in-gel activity assays for mitochondrial complexes in fibroblasts from patients with BTHS treated with vehicle or 5μM ABD646 for five days. (i) Dot plots showing lipidomics analysis of CL and MLCL abundance in BTHS fibroblasts treated with either DMSO or 1μM of ABD646 for 5 days (left and middle panel respectively). Bar plot quantifying MLCL/CL ratio changes across HAP1 cell lines using total CL and MLCL species abundance (right). (j) Representative images at day 5 of zebrafish embryos injected with increasing concentrations of TAZ MOs (2.5ng-10.0 ng) (left; scale bars, 200 μm). Bar plot quantifying observed phenotypes across doses of TAZ MOs (right). (k) 24 h acute treatment of ABD646 has chronic benefit for TAZ MO zebrafish. Representative images of zebrafish embryos following either a 24-h treatment with ABD646 on WT (n = 45), and TAZ MO treated (n = 40) embryos. Embryos were washed once a day for 5 consecutive days with imaging and quantification on day 5 (left; scale bars, 200 μm). Right, quantification of embryos demonstrating “rescue” or near WT phenotypes per wash day. (l) Box plot of ABHD18 gene expression across HAP1 and TAZ mutant clones in nutrient rich (IMDM) or nutrient limiting (DMEM) media conditions. Data are median read counts ± interquartile range (IQR), n = 3 biological replicates per media condition. Significance determined by one-way ANOVA followed by Dunnett’s post hoc test. (m) Bar plot of spearman correlations between TAZ and ABHD18 gene expression across tissue samples. Data from the Genotype-Tissue Expression (GTEx) Project.
Supplementary information
Supplementary Information
This file contains the methods for the chemical synthesis of ABD646, Supplementary Figs. 1 and 2, Supplementary Methods Tables 1–4 and full descriptions for Supplementary Videos 1–4 and Supplementary Tables 1–4 (supplied separately).
Supplementary Table 1
Genetic screens and related data analyses.
Supplementary Table 2
ABHD18 Bio-ID interaction data.
Supplementary Table 3
Lipidomics data.
Supplementary Table 4
RNA-seq data from mouse hearts.
Supplementary Video 1
Video of a wild-type GFP-labelled fish heart showing cardiac contractions following treatment with drug vehicle (see Methods for details).
Supplementary Video 2
Video of a wild-type GFP-labelled fish heart treated for 5 days with ABD646 showing cardiac contractions (see Methods for details).
Supplementary Video 3
Video of a GFP-labelled fish heart showing cardiac contractions following treatment with Taz morpholinos and subsequently with drug vehicle (see Methods for details).
Supplementary Video 4
Video of a GFP-labelled fish heart showing cardiac contractions following treatment with Taz morpholinos and subsequently with ABD646 for 5 days (see Methods for details).
Supplementary Information
Supplementary Video Summary Slide.
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Masud, S.N., Srivastava, A., Mero, P. et al. Genetic suppression features ABHD18 as a Barth syndrome therapeutic target. Nature 645, 1029–1038 (2025). https://doi.org/10.1038/s41586-025-09373-5
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DOI: https://doi.org/10.1038/s41586-025-09373-5
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