Sphingolipids In Regulation Research Articles

Heavy Metal Exposure-Mediated Dysregulation of Sphingolipid Metabolism.

Exposure to heavy metals (HMs) is often associated with inflammation and cell death, exacerbating respiratory diseases including asthma. Most inhaled particulate HM exposures result in the deposition of HM-bound fine particulate matter, PM2.5, in pulmonary cell populations. While localized high concentrations of HMs may be a causative factor, existing studies have mostly evaluated the effects of systemic or low-dose chronic HM exposures. This report investigates the impact of local high concentrations of specific HMs (NaAsO2, MnCl2, and CdCl2) on sphingolipid homeostasis and oxidative stress, as both play a role in mediating responses to HM exposure and have been implicated in asthma. Utilizing an in vitro model system and three-dimensional ex vivo human tissue models, we evaluated the expression of enzymatic regulators of the salvage, recycling, and de novo synthesis pathways of sphingolipid metabolism, and observed differential modulation in these enzymes between HM exposures. Sphingolipidomic analyses of specific HM-exposed cells showed increased levels of anti-apoptotic sphingolipids and reduced pro-apoptotic sphingolipids, suggesting activation of the salvage and de novo synthesis pathways. Differential sphingolipid regulation was observed within HM-exposed lung tissues, with CdCl2 exposure and NaAsO2 exposure activating the salvage and de novo synthesis pathway, respectively. Additionally, using spatial transcriptomics and quantitative real-time PCR, we identified HM exposure-induced transcriptomic signatures of oxidative stress in epithelial cells and human lung tissues.

Regulation of cellular and systemic sphingolipid homeostasis.

One hundred and fifty years ago, Johann Thudichum described sphingolipids as unusual "Sphinx-like" lipids from the brain. Today, we know that thousands of sphingolipid molecules mediate many essential functions in embryonic development and normal physiology. In addition, sphingolipid metabolism and signalling pathways are dysregulated in a wide range of pathologies, and therapeutic agents that target sphingolipids are now used to treat several human diseases. However, our understanding of sphingolipid regulation at cellular and organismal levels and their functions in developmental, physiological and pathological settings is rudimentary. In this Review, we discuss recent advances in sphingolipid pathways in different organelles, how secreted sphingolipid mediators modulate physiology and disease, progress in sphingolipid-targeted therapeutic and diagnostic research, and the trans-cellular sphingolipid metabolic networks between microbiota and mammals. Advances in sphingolipid biology have led to a deeper understanding of mammalian physiology and may lead to progress in the management of many diseases.

Critical Roles of the Sphingolipid Metabolic Pathway in Liver Regeneration, Hepatocellular Carcinoma Progression and Therapy.

The sphingolipid metabolic pathway, an important signaling pathway, plays a crucial role in various physiological processes including cell proliferation, survival, apoptosis, and immune regulation. The liver has the unique ability to regenerate using bioactive lipid mediators involving multiple sphingolipids, including ceramide and sphingosine 1-phosphate (S1P). Dysregulation of the balance between sphingomyelin, ceramide, and S1P has been implicated in the regulation of liver regeneration and diseases, including liver fibrosis and hepatocellular carcinoma (HCC). Understanding and modulating this balance may have therapeutic implications for tumor proliferation, progression, and metastasis in HCC. For cancer therapy, several inhibitors and activators of sphingolipid signaling, including ABC294640, SKI-II, and FTY720, have been discussed. Here, we elucidate the critical roles of the sphingolipid pathway in the regulation of liver regeneration, fibrosis, and HCC. Regulation of sphingolipids and their corresponding enzymes may considerably influence new insights into therapies for various liver disorders and diseases.

Abnormal Regulation of Mitochondrial Sphingolipids during Aging and Alzheimer's Disease.

During pathogenesis of Alzheimer's disease (AD), mitochondria suffer alterations that lead to low energy production and reactive oxygen species formation. However, the mechanism of impaired mitochondria homeostasis in AD is not fully understood. We hypothesized that abnormal sphingolipid metabolism in mitochondria could be one of the contributing factors to mitochondrial dysfunction. Synaptic and non-synaptic mitochondria were isolated from 5xFAD and wild type (WT) mice at 3 and 7 months using Ficoll gradient ultracentrifugation, and their function was analyzed using Seahorse assay. Additionally, mitochondria were analyzed using mass spectrometry for proteomics and sphingolipidomics analyses. Sphingolipid levels were also determined in synaptic and non-synaptic mitochondria isolated from AD patients and healthy controls. We found that synaptic mitochondria isolated from 3-months old 5xFAD mice manifest diminished oxygen consumption as compared to WT. Consistently, proteomics analysis showed that proteins related to respiratory electron transport and oxidative phosphorylation were altered in 5xFAD mice. When quantifying the main sphingolipids in mitochondria, we found that Cer 18:0, Cer 22:0, and Cer 24:1 were increased already at 3 months in 5xFAD mice. No increase in ceramides was detected in mitochondria isolated from AD patients. However, increased levels of sphingosine were found in both 5xFAD mice and AD patients when compared to respective controls. We report that the regulation of sphingolipids in mitochondria is abnormal at 3 months of age in 5xFAD mice, as indicated by the accumulation of long-chain ceramides, which increases with age. Sphingosine levels are increased in both the mitochondria of 5xFAD mice and AD patients. Our data suggest that the sphingolipid composition is dysregulated in mitochondria early during AD pathogenesis.

Transcriptional suppression of sphingolipid catabolism controls pathogen resistance in C. elegans.

Sphingolipids are required for diverse biological functions and are degraded by specific catabolic enzymes. However, the mechanisms that regulate sphingolipid catabolism are not known. Here we characterize a transcriptional axis that regulates sphingolipid breakdown to control resistance against bacterial infection. From an RNAi screen for transcriptional regulators of pathogen resistance in the nematode C. elegans, we identified the nuclear hormone receptor nhr-66, a ligand-gated transcription factor homologous to human hepatocyte nuclear factor 4. Tandem chromatin immunoprecipitation-sequencing and RNA sequencing experiments revealed that NHR-66 is a transcriptional repressor, which directly targets sphingolipid catabolism genes. Transcriptional de-repression of two sphingolipid catabolic enzymes in nhr-66 loss-of-function mutants drives the breakdown of sphingolipids, which enhances host susceptibility to infection with the bacterial pathogen Pseudomonas aeruginosa. These data define transcriptional control of sphingolipid catabolism in the regulation of cellular sphingolipids, a process that is necessary for pathogen resistance.

Sphingolipids Are Depleted in Alcohol-Related Liver Fibrosis

Alcohol disturbs hepatic lipid synthesis and transport, but the role of lipid dysfunction in alcohol-related liver disease (ALD) is unclear. In this biopsy-controlled, prospective, observational study, we characterized the liver and plasma lipidomes in patients with early ALD. We performed mass spectrometry-based lipidomics of paired liver and plasma samples from 315 patients with ALD and of plasma from 51 matched healthy controls. We associated lipid levels with histologic fibrosis, inflammation, and steatosis with correction for multiple testing and adjustment for confounders. We further investigated sphingolipid regulation by means of quantitative real-time polymerase chain reaction sequencing of microRNA, prediction of liver-related events, and tested causality with Mendelian randomization. We detected 198 lipids in the liver and 236 lipids in the circulation from 18 lipid classes. Most sphingolipids (sphingomyelins and ceramides) and phosphocholines were co-down-regulated in both liver and plasma, where lower abundance correlated with higher fibrosis stage. Sphingomyelins showed the most pronounced negative correlation to fibrosis, mirrored by negative correlations in both liver and plasma with hepatic inflammation. Reduced sphingomyelins predicted future liver-related events. This seemed to be characteristic of "pure ALD," as sphingomyelin levels were higher in patients with concomitant metabolic syndrome and ALD/nonalcoholic fatty liver disease overlap. Mendelian randomization in FinnGen and UK Biobanks indicated ALD as the cause of low sphingomyelins, and alcohol use disorder did not correlate with genetic susceptibility to low sphingomyelin levels. Alcohol-related liver fibrosis is characterized by selective and progressive lipid depletion in liver and blood, particularly sphingomyelins, which also associates with progression to liver-related events.

Glial control of sphingolipid levels sculpts diurnal remodeling in a circadian circuit

Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. We show that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior.

099 Ichthyosis associated with sphingosine 1-phosphate lyase deficiency is due to aberrant calcium and sphingolipid regulation

Loss of function of Sphingosine-1-phosphate lyase (SGPL1) is associated with a multi-systemic disorder consisting of primary adrenal insufficiency and steroid-resistant nephrotic syndrome, with a third of patients manifesting ichthyosis. S1P lyase carries out the final degradative step in the sphingolipid pathway, irreversible cleavage of sphingosine-1-phosphate (S1P). Upstream accumulation of sphingolipid intermediates, such as ceramides and sphingosine, which are integral in the formation of the lamellar lipid bilayer, have been identified in multiple SGPL1 KO cell types.

The Role of Ceramide Metabolism and Signaling in the Regulation of Mitophagy and Cancer Therapy.

Simple SummarySphingolipids are membrane-associated lipids that are involved in signal transduction pathways regulating cell death, growth, and migration. In cancer cells, sphingolipids regulate pathways relevant to cancer therapy, such as invasion, metastasis, apoptosis, and lethal mitophagy. Notable sphingolipids include ceramide, a sphingolipid that induces death and lethal mitophagy, and sphingosine-1 phosphate, a sphingolipid that induces survival and chemotherapeutic resistance. These sphingolipids participate in regulating the process of mitophagy, where cells encapsulate damaged mitochondria in double-membrane vesicles (called autophagosomes) for degradation. Lethal mitophagy is an anti-tumorigenic mechanism mediated by ceramide, where cells degrade many mitochondria until the cancer cell dies in an apoptosis-independent manner.Sphingolipids are bioactive lipids responsible for regulating diverse cellular functions such as proliferation, migration, senescence, and death. These lipids are characterized by a long-chain sphingosine backbone amide-linked to a fatty acyl chain with variable length. The length of the fatty acyl chain is determined by specific ceramide synthases, and this fatty acyl length also determines the sphingolipid’s specialized functions within the cell. One function in particular, the regulation of the selective autophagy of mitochondria, or mitophagy, is closely regulated by ceramide, a key regulatory sphingolipid. Mitophagy alterations have important implications for cancer cell proliferation, response to chemotherapeutics, and mitophagy-mediated cell death. This review will focus on the alterations of ceramide synthases in cancer and sphingolipid regulation of lethal mitophagy, concerning cancer therapy.

Sphingolipids as Regulators of Neuro-Inflammation and NADPH Oxidase 2.

Neuro-inflammation accompanies numerous neurological disorders and conditions where it can be associated with a progressive neurodegenerative pathology. In a similar manner, alterations in sphingolipid metabolism often accompany or are causative features in degenerative neurological conditions. These include dementias, motor disorders, autoimmune conditions, inherited metabolic disorders, viral infection, traumatic brain and spinal cord injury, psychiatric conditions, and more. Sphingolipids are major regulators of cellular fate and function in addition to being important structural components of membranes. Their metabolism and signaling pathways can also be regulated by inflammatory mediators. Therefore, as certain sphingolipids exert distinct and opposing cellular roles, alterations in their metabolism can have major consequences. Recently, regulation of bioactive sphingolipids by neuro-inflammatory mediators has been shown to activate a neuronal NADPH oxidase 2 (NOX2) that can provoke damaging oxidation. Therefore, the sphingolipid-regulated neuronal NOX2 serves as a mechanistic link between neuro-inflammation and neurodegeneration. Moreover, therapeutics directed at sphingolipid metabolism or the sphingolipid-regulated NOX2 have the potential to alleviate neurodegeneration arising out of neuro-inflammation.

CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism

The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.

ORMDL3 and Asthma: Linking Sphingolipid Regulation to Altered T Cell Function.

Orosomucoid like 3 (ORMDL3) encodes an ER-resident transmembrane protein that regulates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme for sphingolipid biosynthesis in cells. A decade ago, several genome wide association studies revealed single nucleotide polymorphisms associated with increased ORMDL3 protein expression and susceptibility to allergic asthma. Since that time, numerous studies have investigated how altered ORMDL3 expression might predispose to asthma and other autoimmune/inflammatory diseases. In this brief review, we focus on growing evidence suggesting that heightened ORMDL3 expression specifically in CD4+ T lymphocytes, the central orchestrators of adaptive immunity, constitutes a major underlying mechanism of asthma pathogenesis by skewing their differentiation and function. Furthermore, we explore how sphingolipid modulation in T cells might be responsible for these effects, and how further studies may interrogate this intriguing hypothesis.

Regulation of Serum Sphingolipids in Andean Children Born and Living at High Altitude (3775 m).

Recent studies on Andean children indicate a prevalence of dyslipidemia and hypertension compared to dwellers at lower altitudes, suggesting that despite similar food intake and daily activities, they undergo different metabolic adaptations. In the present study, the sphingolipid pattern was investigated in serum of 7 underweight (UW), 30 normal weight (NW), 13 overweight (OW), and 9 obese (O) Andean children by liquid chromatography-mass spectrometry (LC-MS). Results indicate that levels of Ceramides (Cers) and sphingomyelins (SMs) correlate positively with biochemical parameters (except for Cers and Vitamin D, which correlate negatively), whereas sphingosine-1-phosphate (S1P) correlates negatively. Correlation results and LC-MS data identify the axis high density lipoprotein-cholesterol (HDL-C), Cers, and S1P as related to hypoxia adaptation. Specifically UW children are characterized by increased levels of S1P compared to O and lower levels of Cers compared to NW children. Furthermore, O children show lower levels of S1P and similar levels of Cers and SMs as NW. In conclusion, our results indicate that S1P is the primary target of hypoxia adaptation in Andean children, and its levels are associated with hypoxia tolerance. Furthermore, S1P can act as marker of increased risk of metabolic syndrome and cardiac dysfunction in young Andeans living at altitude.

Multiple actions of doxorubicin on the sphingolipid network revealed by flux analysis

Sphingolipids (SLs) have been implicated in numerous important cellular biologies; however, their study has been hindered by the complexities of SL metabolism. Furthermore, enzymes of SL metabolism represent a dynamic and interconnected network in which one metabolite can be transformed into other bioactive SLs through further metabolism, resulting in diverse cellular responses. Here we explore the effects of both lethal and sublethal doses of doxorubicin (Dox) in MCF-7 cells. The two concentrations of Dox resulted in the regulation of SLs, including accumulations in sphingosine, sphingosine-1-phosphate, dihydroceramide, and ceramide, as well as reduced levels of hexosylceramide. To further define the effects of Dox on SLs, metabolic flux experiments utilizing a d17 dihydrosphingosine probe were conducted. Results indicated the regulation of ceramidases and sphingomyelin synthase components specifically in response to the cytostatic dose. The results also unexpectedly demonstrated dose-dependent inhibition of dihydroceramide desaturase and glucosylceramide synthase in response to Dox. Taken together, this study uncovers novel targets in the SL network for the action of Dox, and the results reveal the significant complexity of SL response to even a single agent. This approach helps to define the role of specific SL enzymes, their metabolic products, and the resulting biologies in response to chemotherapeutics and other stimuli.

The ORMDL/Orm–serine palmitoyltransferase (SPT) complex is directly regulated by ceramide: Reconstitution of SPT regulation in isolated membranes

Sphingolipids compose a lipid family critical for membrane structure as well as intra- and intercellular signaling. De novo sphingolipid biosynthesis is initiated by the enzyme serine palmitoyltransferase (SPT), which resides in the endoplasmic reticulum (ER) membrane. In both yeast and mammalian species, SPT activity is homeostatically regulated through small ER membrane proteins, the Orms in yeast and the ORMDLs in mammalian cells. These proteins form stable complexes with SPT. In yeast, the homeostatic regulation of SPT relies, at least in part, on phosphorylation of the Orms. However, this does not appear to be the case for the mammalian ORMDLs. Here, we accomplished a cell-free reconstitution of the sphingolipid regulation of the ORMDL-SPT complex to probe the underlying regulatory mechanism. Sphingolipid and ORMDL-dependent regulation of SPT was demonstrated in isolated membranes, essentially free of cytosol. This suggests that this regulation does not require soluble cytosolic proteins or small molecules such as ATP. We found that this system is particularly responsive to the pro-apoptotic sphingolipid ceramide and that this response is strictly stereospecific, indicating that ceramide regulates the ORMDL-SPT complex via a specific binding interaction. Yeast membranes harboring the Orm-SPT system also directly responded to sphingolipid, suggesting that yeast cells have, in addition to Orm phosphorylation, an additional Orm-dependent SPT regulatory mechanism. Our results indicate that ORMDL/Orm-mediated regulation of SPT involves a direct interaction of sphingolipid with the membrane-bound components of the SPT-regulatory apparatus.

Diurnal regulation of sphingolipids in blood

Key homeostatic functions are regulated in a diurnal manner and a miss-alignment of such rhythms is believed to contribute to the pathophysiology of several diseases. Signaling sphingolipids (SLs) in plasma such as sphingosine 1-phosphate control lymphocytic trafficking, vascular reactivity and platelet activity, physiological functions all of which display a diurnal rhythm themselves. However, the rhythmicity of SL metabolism in plasma and its potential causes have not been sufficiently investigated so far. Therefore, we analyzed blood of mice and healthy adult human subjects by targeted tandem mass-spectrometry at different time points. In order to investigate the influence of the synchronizing hormone melatonin, we compared melatonin proficient C3H/HeN wildtype mice (C3H) with melatonin receptor-1/2 double knockout mice (MT1/2−/−) and melatonin deficient C57BL/6J mice. We found a strong upregulation of plasma S1P with the beginning of the light period in C3H but not in MT1/2−/− or C57BL/6J mice. Accordingly, our study revealed an upregulation of sphingosine 1-phosphate (S1P d18:1) and sphinganine 1-phosphate (S1P d18:0) with the beginning of the light period in humans. Furthermore, plasma S1P d18:1 and S1P d18:0 were inversely correlated with the respective concentrations in platelets, pointing to a possible involvement of platelet SL metabolism. In humans, the diurnal rhythm of SLs was not associated with changes of SL-binding proteins or counts of cellular SL sources. Overall, this study indicates a physiological rhythmicity of plasma and platelet SL metabolism, likely mediated by melatonin, with potentially important implications for physiological diurnal rhythms and the regulation of SL metabolism and its functions.

Sphingolipids regulate neuromuscular synapse structure and function in Drosophila.

Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function, and degeneration. Their precise role in these processes, however, remains obscure. Serine Palmitoyl‐transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction (NMJ) revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to ∼50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig‐domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. In support of this, we found Bsg to be enriched in lipid rafts. Our data points to a role for sphingolipids in the regulation and fine‐tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg.

Sphingolipidomics analysis of large clinical cohorts. Part 2: Potential impact and applications

It has been known for decades that the regulation of sphingolipids (SLs) is essential for the proper function of many cellular processes. However, a complete understanding of these processes has been complicated by the structural diversity of these lipids. A well-characterized metabolic pathway is responsible for homeostatic maintenance of hundreds of distinct SL species. This pathway is perturbed in a number of pathological processes, resulting in derangement of the “sphingolipidome.” Recently, advances in mass spectrometry (MS) techniques have made it possible to characterize the sphingolipidome in large-scale clinical studies, allowing for the identification of specific SL molecules that mediate pathological processes and/or may serve as biomarkers. This manuscript provides an overview of the functions of SLs, and reviews previous studies that have used MS techniques to identify changes to the sphingolipidome in non-metabolic diseases.

An integrated method for direct interrogation of sphingolipid homeostasis in the heart and brain tissues of mice through postnatal development up to reproductive senescence

Development of rapid metabolomic methods poised for pathway discovery is expected to facilitate the identification of therapeutic candidates in the metabolomic approach to translational medicine. Using sphingolipid homeostasis as a prototype, we present herein an integrated method to facilitate a fast interrogation of altered sphingolipid (and phospholipid) metabolism associated with perturbed endolysosomal functions in mammalian systems. Constructed upon high performance liquid chromatography coupled to mass spectrometry, this method allows semi-quantitative measurements of more than 300 individual species within 20 min. The method was applied to investigate cardiac- and neural-specific developmental changes in sphingolipid regulation from the postnatal stage to reproductive senescence in mice, revealing that endogenous lysobisphosphatidic acids and specific complex glycosphingolipids are tightly co-regulated to foster concerted reductions in sphingolipid levels at distinct stages of postnatal development. Our lipidomic data suggest that such changing regulatory patterns in sphingolipid homeostasis is attributed to differential endolysosomal degradation of complex sphingolipids, which may be critical in ensuring efficient sphingolipid catabolism and organismal health at each stage of postnatal development.

Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

The mechanisms by which lung structural cells survive toxic exposures to cigarette smoke (CS) are not well defined but may involve proper disposal of damaged mitochondria by macro-autophagy (mitophagy), processes that may be influenced by pro-apoptotic ceramide (Cer) or its precursor dihydroceramide (DHC). Human lung epithelial and endothelial cells exposed to CS exhibited mitochondrial damage, signaled by phosphatase and tensin homolog-induced putative kinase 1 (PINK1) phosphorylation, autophagy, and necroptosis. Although cells responded to CS by rapid inhibition of DHC desaturase, which elevated DHC levels, palmitoyl (C16)-Cer also increased in CS-exposed cells. Whereas DHC augmentation triggered autophagy without cell death, the exogenous administration of C16-Cer was sufficient to trigger necroptosis. Inhibition of Cer-generating acid sphingomyelinase reduced both CS-induced PINK1 phosphorylation and necroptosis. When exposed to CS, Pink1-deficient ( Pink1-/-) mice, which are protected from airspace enlargement compared with wild-type littermates, had blunted C16-Cer elevations and less lung necroptosis. CS-exposed Pink1-/- mice also exhibited significantly increased levels of lignoceroyl (C24)-DHC, along with increased expression of Cer synthase 2 ( CerS2), the enzyme responsible for its production. This suggested that a combination of high C24-DHC and low C16-Cer levels might protect against CS-induced necroptosis. Indeed, CerS2-/- mice, which lack C24-DHC at the expense of increased C16-Cer, were more susceptible to CS, developing airspace enlargement following only 1 month of exposure. These results implicate DHCs, in particular, C24-DHC, as protective against CS toxicity by enhancing autophagy, whereas C16-Cer accumulation contributes to mitochondrial damage and PINK1-mediated necroptosis, which may be amplified by the inhibition of C24-DHC-producing CerS2.-Mizumura, K., Justice, M. J., Schweitzer, K. S., Krishnan, S., Bronova, I., Berdyshev, E. V., Hubbard, W. C., Pewzner-Jung, Y., Futerman, A. H., Choi, A. M. K., Petrache, I. Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.