SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc- SMPDL3B

Sphingolipids are important structural components of cell membranes and prominent signaling molecules controlling cell growth, differentiation, and apoptosis. Sphingolipids are particularly abundant in the brain, and defects in sphingolipid degradation are associated with several human neurodegenerative diseases. However, molecular mechanisms governing sphingolipid metabolism remain unclear. Here we report that sphingolipid degradation is under transcriptional control of SIRT1, a highly conserved mammalian NAD+-dependent protein deacetylase, in mouse embryonic stem cells (mESCs). Deletion of SIRT1 results in accumulation of sphingomyelin in mESCs, primarily due to reduction of SMPDL3B, a GPI-anchored plasma membrane bound sphingomyelin phosphodiesterase. Mechanistically, SIRT1 regulates transcription of Smpdl3b through c-Myc. Functionally, SIRT1 deficiency-induced accumulation of sphingomyelin increases membrane fluidity and impairs neural differentiation in vitro and in vivo. Our findings discover a key regulatory mechanism for sphingolipid homeostasis and neural differentiation, further imply that pharmacological manipulation of SIRT1-mediated sphingomyelin degradation might be beneficial for treatment of human neurological diseases.


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To validate that defective neural differentiation of SIRT1 KO mESCs is related to 304 its accumulation of sphingomyelin, we analyzed whether adding back SMPDL3B in these to intrauterine growth retardation when dams were fed on a regular chow diet (containing 337 4% fat) ( Figure 9A, chow). The mRNA levels of Smpdl3b were significantly reduced in the 338 brain of SIRT1 KO E18.5 embryos ( Figure 9B). However, these embryos did not display 339 any detectable defects in brain sphingomyelin levels, nor in expression of a number of 340 neural progenitor and neuron markers ( Figure 9C and 9D, chow) despite reported 341 developmental defects in other systems(19-21). Since high-fat diet (HFD) feeding has 342 been shown to induce sphingolipid biosynthesis and turnover of sphingolipids in multiple 343 tissues (9), we tested whether maternal HFD feeding could induce sphingomyelin 344 accumulation and disrupt neural development in SIRT1 KO embryos. Intriguingly, 345 maternal feeding of an HFD diet containing 36% fat for 4-8 weeks before breeding 346 significantly reduced intrauterine growth of embryos, particularly on SIRT1 KO embryos, 347 at E18.5 ( Figure 9A, HFD). Maternal HFD feeding also elevated sphingomyelin contents 348 in all tested regions of brain in SIRT1 KO but not WT E18.5 embryos ( Figure 9C, HFD).

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These maternal HFD feeding-induced gross and metabolic alterations were associated

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It will be of great interest to test this possibility in future studies.

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As the most conserved mammalian NAD + -dependent protein deacetylase, SIRT1      Table S1), additional studies are needed to assess how the SIRT1-Myc 415 regulatory axis coordinates diverse metabolic processes to shape stem cell fates in 416 response to different environmental signals. It is also worth noting that SIRT1 KO mESCs 417 have additional lipid metabolic defects, including depletion of monoacyglycerols, 418 accumulation of plasmalogens, acetylcholine, and monohydroxy fatty acids, and altered 419 phospholipids, regardless of medium methionine concentrations ( Figure S1B and Table   420 S1). It will be of importance to evaluate the contribution of these lipid metabolic defects to 421 the observed hypersensitivity of SIRT1 KO embryos to maternal HFD feeding-induced 422 intrauterine growth retardation ( Figure 7A) in future studies.

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Our study has a number of important implications. Firstly, the previously   (Table S4)

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Mice were randomly assigned to experimental groups after they were allowed to 505 acclimate for at least one week prior to experiments.

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The primers used in RT-PCR are listed in (Table S4).

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They were then incubated with 1% glycine/PBS for 10 minutes, and cell membrane was 563 permeabilized with 0.3% Triton X-100 in 1% glycine/PBS for 10 minutes. Cells were further 564 blocked with 1% BSA and 0.05% Tween 20 in PBS for 30 minutes, incubated with primary 565 antibodies (Table S5)   legend. Significant differences between the means were analyzed by the two-tailed,

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Additional information on DEGs is available from Table S3.

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Metabolomics data (lipid alterations) between WT and SIRT1 KO mESCs is available in 735 Table S1.

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Sphingolipid profiles between WT and SIRT1 KO mESCs and hESCs are available in 737 Table S2.

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All oligos used in the study are available in Table S4.

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All antibodies used in the study are available in Table S5.

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List of Supplemental Tables   742   Table S1. Lipid alterations in WT and SIRT1 KO mESCs analyzed by metabolomics.

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This study was also supported in part by grants from National Nature Science Foundation

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A mono-allelic bivalent chromatin domain controls tissue-specific imprinting at Grb10.            Figure 9. Maternal high-fat diet feeding impairs neural development in SIRT1 deficient embryos.

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(A) Maternal high-fat diet feeding reduces body weight of embryos. Maternal high-fat diet (HFD) feeding was performed 3-4 weeks before pregnancy (pre-feeding) as described in Methods. Body weight of E14.5 and E18.5 embryos were measured (*p<0.05, **p<0.01, ***p<0.001). (B) SIRT1 KO embryos have reduced expression of Smpdl3b in brains. The mRNA levels of Smpdl3b in brain of E18.5 embryos from chow fed dams or HFD fed dams were analyzed by qPCR (n=6 embryos, *p<0.05, **p<0.01). (C) Maternal high-fat diet feeding induces sphingomyelin accumulation in brains of SIRT1 KO embryos. Maternal high-fat diet (HFD) feeding was performed 3-4 weeks before pregnancy (pre-feeding) as described in Methods. Brains from E18.5 embryos were dissected into three parts and the endogenous sphingomyelins were extracted and measured (n=6 embryos, *p<0.05). (D) Maternal high-fat diet feeding induces defective expression of neural markers in brains of SIRT1 KO embryos. The mRNA levels of indicated neural markers in brain of E18.5 embryos from chow fed dams or HFD fed dams were analyzed by qPCR (n=6 embryos, *p<0.05, **p<0.01, ***p<0.001).       Figure S5. SIRT1 deficiency in mESCs significantly reduces the expression of genes involved in membrane signaling.

WT V KO V KO c-Myc-WT KO c-Myc-KR KO c-Myc-KQ
(A) Genes involved in membrane function are significantly enriched in genes downregulated in SIRT1 KO mESCs. (B) Two major impaired membrane pathways in SIRT1 KO mESCs. GSEA analysis of transcriptomes from WT and SIRT1 KO mESCs was performed as described in Methods. Two major downregulated membrane pathways were shown (n=3 biological replicates).