1-Deoxydihydroceramide causes anoxic death by impairing chaperonin-mediated protein folding

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Abstract

Ischaemic heart disease and stroke are the most common causes of death worldwide. Anoxia, defined as the lack of oxygen, is commonly seen in both these pathologies and triggers profound metabolic and cellular changes. Sphingolipids have been implicated in anoxia injury, but the pathomechanism is unknown. Here we show that anoxia-associated injury causes accumulation of the non-canonical sphingolipid 1-deoxydihydroceramide (DoxDHCer). Anoxia causes an imbalance between serine and alanine resulting in a switch from normal serine-derived sphinganine biosynthesis to non-canonical alanine-derived 1-deoxysphinganine. 1-Deoxysphinganine is incorporated into DoxDHCer, which impairs actin folding via the cytosolic chaperonin TRiC, leading to growth arrest in yeast, increased cell death upon anoxia–reoxygenation in worms and ischaemia–reperfusion injury in mouse hearts. Prevention of DoxDHCer accumulation in worms and in mouse hearts resulted in decreased anoxia-induced injury. These findings unravel key metabolic changes during oxygen deprivation and point to novel strategies to avoid tissue damage and death.

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Fig. 1: Anoxia-sensitive worm CerS mutants accumulate non-canonical DoxDHCer.
Fig. 2: Anoxic metabolism leads to production of lethal non-canonical 1-deoxysphingolipids.
Fig. 3: DoxDHCer that is produced by specific CerSs is lethal to yeast and worms.
Fig. 4: Non-canonical DoxDHCer is sufficient to cause ischaemia–reperfusion injury in mouse hearts.
Fig. 5: Cytosolic chaperonin TRiC is a target of non-canonical 1-deoxysphingolipids.
Fig. 6: Ischaemia–reperfusion leads to DoxDHCer-dependent impairment of TRiC.

Data availability

The data that support the findings are available in the supporting data files. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE48 partner repository with the dataset identifier PXD014573.

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Acknowledgements

We would like to thank L. Pineau for construction of yeast strains, I. Riezman for help with LC–MS analysis, V. Nesatyy and F. David for help with lipidomics analysis, M. Müller for help with statistical analysis, J. Dessimoz for histological support and I. Castanon for helpful comments on the manuscript. Worms and bacterial strains were received from the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440), from National BioResource Project (Tokyo Women’s Medical University, Tokyo, Japan), from NemaGENETAG. Worm strain GK454 was a kind gift from K. Sato. J.T.H. was supported by an EMBO/Marie-Curie Long-Term Fellowship. This work was supported by grants from the Swiss National Science Foundation (SNSF) (to H.R. and J.-C.M.), the National Centre of Competence in Research (NCCR) Chemical Biology and SystemsX (evaluated by the SNSF) and the Canton of Geneva. A.Z. was supported by an SNSF assistant professorship.

Author information

J.T.H. and H.R. designed the study, J.T.H. performed lipidomic and metabolomic analyses, as well as functional studies in worms and yeast. A.G.H. constructed the sptl-1C121W worm and performed yeast actin staining. S.G. isolated and characterized the ttc-17(gnv3) suppressor mutant under the guidance of J.-C.M. M.P. and L.G. designed, M.P., L.G., B.P. and H.T. performed, and M.P. and L.G. interpreted the mouse ischaemia–reperfusion experiments under the guidance of M.O. A.Z. synthesized iso-branched C17 DoxSa. D.A. performed proteomics analysis under the guidance of A.A. N.G. developed peak identification and quantification for non-targeted lipidomics. J.T.H. and H.R. wrote and revised the manuscript.

Correspondence to Howard Riezman.

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Extended data

Extended Data Fig. 1 Non-canonical DoxDHCer increases during anoxia while normal ceramide is depleted.

a,b, Targeted lipid analysis of DoxDHC (a) and ceramide (b) species from wild-type and hyl-2(tm2031) worms, and the respective animals expressing the sptl-1C121W allele (+C121W) either after 3 d of normoxia (solid columns) or after an additional 20 h of anoxia (shaded columns); log2-transformed data from n = 4 independent biological replicates; bars are means with s.e.m. c,d, Correlations of DoxDHCer (c) and ceramide (d) amounts in pmole per nmoles inorganic phosphate between normoxic and anoxic conditions, for the same dataset as in in a and b; n = 4 independent biological replicates, points are means with s.e.m.; slopes of the linear regressions for each strain are given as m with a P value testing the difference to a line with the slope 1. Source data

Extended Data Fig. 2 Activity and substrate specificity of mammalian CerS is conserved when expressed in yeast, and anoxia-reoxygenation survival of hyl-2(gnv1) mutant worms is extended by knock down of ttc-17, which is widely expressed in L4 larvae and young adults.

a, Yeast dilution growth assay of lag1Δlac1Δ CerS double mutants showing rescue with all six mammalian CerSs CerS1–CerS6 on rich medium. The experiment was repeated twice with similar results. b, Representative structure of an inositol phosphoceramide (IPC), which is the S. cerevisiae higher sphingolipid analogous to mammalian sphingomyelin and contains canonical serine-derived phytosphingosine (blue). c, Heat maps of specific IPC species levels in wild-type and CerS mutants rescued with individual mammalian CerSs showing IPC levels of the major molecular species produced as nM per 25 OD600 or percent of total. d, Yeast dilution growth assay of wild-type yeast expressing control (EV) and the six mammalian CerSs CerS1–CerS6 on rich medium (YPD) containing 0.05% tergitol and vehicle (EtOH), plus either 5 µM DoxSa or 5 µM and 100 µM fumonisin B1 (FB1). The experiment was repeated twice with similar results. e, Survival of hyl-2(gnv1) after 48 h of anoxia and 24 h reoxygenation following 3-d feeding on bacteria expressing empty vector (L4440) or RNAi vectors targeting ttc-17 or the positive control (daf-2) and the negative control (mre-11); n = 3 independent experiments with 2 independent biological replicates each; bars are means with s.e.m., P values are determined by unpaired two-sided Student’s t-test. f, cDNA quantification via PCR shows knock down of ttc-17 mRNA in response to ttc-17 RNAi compared with empty vector (L4440). The experiment was repeated three times with similar results. gj, Confocal microscopy images of adults (g,i) and L4 larva (h) expressing a GFP reporter from a ttc-17 promoter showing widespread expression, especially in the hypodermis (arrow head), head neurons, the intestine and the developing vulva (arrow). j, Confocal microscopy image of a wild-type adult not expressing any fluorescent protein to show background signal mainly from gut granules in the intestine. Similar results for gj were obtained in three independent experiments. Scale bars represent 100 μm. Source data

Extended Data Fig. 3 Both non-canonical as well as canonical sphingolipids are upregulated upon ischaemia–reperfusion in mouse hearts overexpressing wild-type or mutant (C133W) SPTLC1 constructs.

a, Mouse hearts expressing wild-type or mutant SPTLC1 constructs for 1 week after adeno-associated viral (AAV) infection were analysed either following sham treatment or ischaemia–reperfusion. bf, Both non-canonical (b,c) and canonical (dg) sphingolipids are upregulated upon ischaemia–reperfusion, as can be seen for DoxSa (b), DoxDHCer (c), sphinganine (d), sphingosine (e), ceramide (f) and DHCer (g). While wild-type worms and C133W mutants are mostly similar upon ischaemia–reperfusion, C133W shows a weak increase in DoxDHCer in non-ischaemic sham (c) and lower (dihydro-)ceramide levels than WT after ischaemia–reperfusion (f,g), but surprisingly also significantly lower DoxDHCer levels than wild type after ischaemia–reperfusion (c). This shows that C133W mutation causes an increase of DoxDHCer relative to wild type only in sham conditions in which sufficient serine is available as substrate for the wild-type enzyme. Serine is not used as substrate by C133W that uses alanine instead. In ischaemic conditions, wild-type SPT readily uses alanine as a substrate, even more efficiently than C133W; n = 4 for sham and n = 8 for ischaemia–reperfusion are individual animals, bars are means with s.d. All statistical tests in bg are unpaired two-sided Student’s t-tests. h, Plot representation of area of necrosis relative to area at risk for animals with AAV expression of wild-type and mutant (C133W) SPTLC1. Under these conditions, very little difference can be seen compared with the vehicle-treated control animals (grey line), but hearts with higher DoxDHCer levels do show a tendency of more necrosis. Vehicle linear regression from Fig. 4h; for AAV, n = 8 individual animals; coefficient of determination (r2) given for each linear regression and P as determined by ANCOVA. Source data

Extended Data Fig. 4 Non-canonical DoxSa reverts myriocin cardioprotection by increasing levels of non-canonical 1-deoxy(dihydro)ceramide, but not canonical ceramide.

ad, While animals treated with control vehicle or myriocin were comparable, mice involved in the group treated with myriocin plus DoxSa (Myr+DoxSa) by chance happened to show significantly increased body weight (a), left ventricle (LV) weight (b) and area at risk in left ventricle (c). While experiments were performed blinded, the batch of mice used for Myr+DoxSa was slightly larger at time of surgery. Still, relative area at risk (d) was comparable between all three conditions, n = 14 (Vehicle), n = 12 (Myriocin) and n = 10 (Myriocin+DoxSa) are individual animals; bars are means with s.d. e,f, LC–MS analysis of free sphingoid bases from mouse hearts after ischaemia–reperfusion treatment, whose hearts had been preconditioned either with vehicle, myriocin or myriocin and DoxSa (Myr+DoxSa), shows a significant decrease upon myriocin treatment for both sphinganine (e) and sphingosine (f); n = 10–11 animals. g,h, Direct infusion lipid analysis of ceramide levels from the same hearts as in e,f, showing downregulation of ceramide (g) and dihydroceramides (h) upon myriocin treatment, which is not reverted by myriocin plus DoxSa treatment (Myr+DoxSa) for ceramide (g). Apparent reversal of dihydroceramides (h) levels upon Myr+DoxSa treatment can be explained by LC–MS (ik) as 1-deoxyceramides have the same m/z ratio as dihydroceramides and need to be separated by LC15. Indeed, only the reduced levels upon myriocin treatment in DoxDHC (i) and 1-deoxyceramides (j) but not in the dihydroceramides (k) are reverted by myriocin plus DoxSa treatment; n = 12 (Vehicle), n = 12 (Myriocin) and n = 10 (Myriocin+DoxSa) are individual animals, bars are means with s.d . All statistical tests are unpaired two-sided Student’s t-tests. ln, Analyses of area of necrosis in mg relative to the amount of DoxDHCer (l) and ceramide (m) show a low positive correlation, while DHCer (n) shows only negligible correlation; r values are the correlation coefficients from the linear regressions (solid lines), discontinuous lines are 95% confidence intervals; n = 25 (l), n = 24 (m,n) individual animals. Source data

Extended Data Fig. 5 Non-canonical 1-deoxysphingolipids impair protein folding and disrupt the cytoskeleton.

ac, Larger images of mCherry::ACT-5 (mC::ACT-5) intestinal signals of wild-type animals (a) upon treatment with empty vector (L4440) or RNAi knock down feeding constructs targeting all eight subunits of the chaperonin TRiC complex under normoxia or after 24- and 48-h anoxia and 24-h reoxygenation treatment (b), and after treatment with exogenous worm DoxSa (iso-branched C17 DoxSa) (c); scale bars, 25 µm. Experiments were repeated three times with similar results. d, Yeast cells expressing mammalian CerS3 showing F-actin staining with phalloidin upon DoxSa treatment for 3 h; scale bar, 2 µm. Experiments were repeated five times with similar results. e, Yeast dilution growth assay of wild-type yeast and thermo-sensitive TRiC chaperonin mutants expressing control or mammalian CerS3 constructs on rich medium containing 0.05% tergitol as well as vehicle (EtOH) or 2, 4 and 8 µM DoxSa (DoxSa) at a permissive temperature of 24 °C. Experiments were repeated three times with similar results. f, Yeast dilution growth assay of diploid wild-type yeast and haplo-insufficient TRiC chaperonin mutants expressing control or mammalian CerS3 constructs on rich medium containing 0.05% tergitol as well as vehicle (EtOH) or 4, 8 and 10 µM DoxSa at 30 °C. Experiments were repeated twice with similar results. g, Yeast dilution growth assay of wild-type yeast (BY4741) expressing mammalian CerS3 as well as a control construct, or overexpressing all 8 chaperonin subunits from a genomic Tdh3 promoter on rich medium (YPD) and rich medium containing 0.05% tergitol as well as vehicle (EtOH) or 2.5, 5, 7.5, and 10 µM DoxSa at 30 °C. Experiments were repeated five times with similar results.

Extended Data Fig. 6 Ischaemia–reperfusion and non-canonical DoxSa treatments both damage the cytoskeleton by reducing TRiC chaperonin and actin colocalization in cardiomyocytes.

a, Immunohistochemical detection of the TRiC chaperonin complex CCT5 subunit after ischaemia–reperfusion injury showing irregular staining in the area at risk of the left ventricle; staining around the right ventricle (RV) is more regular. Scale bar, 1 mm. Stainings were repeated with similar results on hearts from nine individual animals, using staining without primary antibody as negative control. b, Close-up of H&E staining (upper), anti-CCT5 immunohistochemical staining (middle) and PTAH staining (lower) in area at risk in the left ventricle showing both necrotic (N) and healthy (H) cardiomyocyte fibres, as well as edema (E); scale bars, 100 µm. Stainings were repeated with similar results on hearts from at least two individual animals. c, Immunofluorescence imaging of cardiomyocytes treated with vehicle, myriocin or myriocin plus DoxSa after ischaemia–reperfusion in the left ventricle and in non-ischaemic right ventricle; anti-CCT5 staining is shown in cyan, anti-actin in magenta and DAPI in yellow. Upper panels show overviews with scale bars of 20 µm, while lower panels show close-ups with scale bars at 5 µm. Experiments were repeated four times with similar results.

Extended Data Fig. 7 Non-targeted high-mass-accuracy lipidomics screen identifies significantly altered sphingolipid species in hyl-2(gnv1) mutant animals.

Columns are as follows: measured mass per charge ratios, as detected with the LTQ orbitrap mass spectrometer and identified with in-house software; relative change between WT(N2) and hyl-2(gnv1) (increased species have red borders, and decreased species have blue borders); previously detected sphingolipid changes from Menuz et al.13 (colours as in the previous column); statistical significance as determined by unpaired two-sided Student’s t-test; FDR-corrected q-values; assigned lipid species with mass error below 2 ppm (most significantly increased assigned species are highlighted in red, and most significantly decreased assigned species are in blue); molecular formula of identified lipid species; theoretical mass per charge ratios; mass errors between measured and theoretical mass per charge ratio in parts per million. n = 6 (wild type) and 3 (hyl-2) independent biological replicates. Hex, hexose. Short hand for total back-bone carbons was used: AA:b, where AA is the total ceramide backbone carbons, and b is the number of double bonds. -OH, number of additional hydroxylations. Short hand for detailed species description was used: ihMM:n/X:Y:Z where I is iso-branched, h is sphingoid base hydroxyl groups (m, one; d, two; t, three), MM is the number of sphingoid base carbons, n is the number of sphingoid base double bonds, X is the number of fatty-acid carbons, Y is the number of fatty-acid double bonds and Z is the number of fatty acid hydroxylations.

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Hannich, J.T., Haribowo, A.G., Gentina, S. et al. 1-Deoxydihydroceramide causes anoxic death by impairing chaperonin-mediated protein folding. Nat Metab 1, 996–1008 (2019) doi:10.1038/s42255-019-0123-y

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