Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

Hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine, and this hydrolysis activity drives tissue damage and inflammation and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and result in secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcriptional regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional readthrough of the cerN transcription terminator. Evidence for these conclusions came from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and reverse transcriptase PCR (RT-PCR) showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observed that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to the regulation of plcH transcription as a site for the integration of multiple host-derived signals. IMPORTANCE: PlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of Pseudomonas aeruginosa. Here, we show that sphingosine, which presents itself or as a product of P. aeruginosa sphingomyelinase and ceramidase activity, leads to the induction of plcH transcription. This transcriptional induction occurs from the promoter of the upstream ceramidase gene generating a conditional operon. The transcript on which plcH resides, therefore, is different depending on which host molecule or condition leads to induction, and this may have implications for PlcH post-transcriptional regulation. This work also adds to our understanding of P. aeruginosa with host-derived sphingolipids.

The Importance of Sphingomyelin Phosphodiesterase Acid-Like 3b (SMPDL-3b) Levels in Kidney Biopsy Specimens of Children With Nephrotic Syndrome.

OBJECTIVE: It remains unclear whether the low amount of SMPDL-3b required for rituximab binding is the cause of treatment resistance in patients with treatment-resistant nephrotic syndrome with advanced podocyte injury. Given the limited number of studies on the relationship between rituximab and SMPDL-3b, this study was conducted to assess whether SMPDL-3b levels in pretreatment renal biopsy specimens can be used to predict the clinical effectiveness of immunosuppressive drugs, especially rituximab, in children with nephrotic syndrome. METHODS: Kidney biopsy specimens from 44 patients diagnosed with idiopatic nephrotic syndrome were analyzed using immunohistochemical staining with an anti-SMPDL-3b antibody and real-time polymerase chain reaction (PCR) for SMPDL-3b mRNA expression. RESULTS: We showed that SMPDL-3b mRNA expression and anti-SMPDL-3b antibody staining did not differ significantly between the patient groups with different responses to immunosuppressive therapies. CONCLUSION: Our results suggest that SMPDL-3b may actually be an indicator of disease progression rather than a marker for predicting response to a particular immunosuppressive agent.

Neutral sphingomyelinase blockade enhances hematopoietic stem cell fitness through an integrated stress response.

Hematopoietic stem and progenitor cell (HSPC) transplantation serves as a curative therapy for many benign and malignant hematopoietic disorders and as a platform for gene therapy. However, growing needs for ex vivo manipulation of HSPC-graft products are limited by barriers in maintaining critical self-renewal and quiescence properties. The role of sphingolipid metabolism in safeguarding these essential cellular properties has been recently recognized, but not yet widely explored. Here, we demonstrate that pharmacologic and genetic inhibition of neutral sphingomyelinase 2 (nSMase-2) leads to sustained improvements in long-term competitive transplantation efficiency after ex vivo culture. Mechanistically, nSMase-2 blockade activates a canonical integrated stress response (ISR) and promotes metabolic quiescence in human and murine HSPCs. These adaptations result in part from disruption in sphingolipid metabolism that impairs the release of nSMase-2-dependent extracellular vesicles (EVs). The aggregate findings link EV trafficking and the ISR as a regulatory dyad guarding HSPC homeostasis and long-term fitness. Translationally, transient nSMase-2 inhibition enables ex vivo graft manipulation with enhanced HSPC potency.

m6A modification-tuned sphingolipid metabolism regulates postnatal liver development in male mice.

Different organs undergo distinct transcriptional, epigenetic and physiological alterations that guarantee their functional maturation after birth. However, the roles of epitranscriptomic machineries in these processes have remained elusive. Here we demonstrate that expression of RNA methyltransferase enzymes Mettl3 and Mettl14 gradually declines during postnatal liver development in male mice. Liver-specific Mettl3 deficiency causes hepatocyte hypertrophy, liver injury and growth retardation. Transcriptomic and N6-methyl-adenosine (m6A) profiling identify the neutral sphingomyelinase, Smpd3, as a target of Mettl3. Decreased decay of Smpd3 transcripts due to Mettl3 deficiency results in sphingolipid metabolism rewiring, characterized by toxic ceramide accumulation and leading to mitochondrial damage and elevated endoplasmic reticulum stress. Pharmacological Smpd3 inhibition, Smpd3 knockdown or Sgms1 overexpression that counteracts Smpd3 can ameliorate the abnormality of Mettl3-deficent liver. Our findings demonstrate that Mettl3-N6-methyl-adenosine fine-tunes sphingolipid metabolism, highlighting the pivotal role of an epitranscriptomic machinery in coordination of organ growth and the timing of functional maturation during postnatal liver development.

Acid sphingomyelinase expression is associated with survival in resectable pancreatic ductal adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) is one of the most common cancers worldwide. Unfortunately, the prognosis of PDAC is rather poor, and for instance, in the USA, over 47,000 people die because of pancreatic cancer annually. Here, we demonstrate that high expression of acid sphingomyelinase in PDAC strongly correlates with long-term survival of patients, as revealed by the analysis of two independent data sources. The positive effects of acid sphingomyelinase expression on long-term survival of PDAC patients were independent of patient demographics as well as tumor grade, lymph node involvement, perineural invasion, tumor stage, lymphovascular invasion, and adjuvant therapy. We also demonstrate that genetic deficiency or pharmacological inhibition of the acid sphingomyelinase promotes tumor growth in an orthotopic mouse model of PDAC. This is mirrored by a poorer pathologic response, as defined by the College of American Pathologists (CAP) score for pancreatic cancer, to neoadjuvant therapy of patients co-treated with functional inhibitors of the acid sphingomyelinase, in particular tricyclic antidepressants and selective serotonin reuptake inhibitors, in a retrospective analysis. Our data indicate expression of the acid sphingomyelinase in PDAC as a prognostic marker for tumor progression. They further suggest that the use of functional inhibitors of the acid sphingomyelinase, at least of tricyclic antidepressants and selective serotonin reuptake inhibitors in patients with PDAC, is contra-indicated. Finally, our data also suggest a potential novel treatment of PDAC patients with recombinant acid sphingomyelinase. KEY MESSAGES: Pancreatic ductal adenocarcinoma (PDAC) is a common tumor with poor prognosis. Expression of acid sphingomyelinase (ASM) determines outcome of PDAC. Genetic deficiency or pharmacologic inhibition of ASM promotes tumor growth in a mouse model. Inhibition of ASM during neoadjuvant treatment for PDAC correlates with worse pathology. ASM expression is a prognostic marker and potential target in PDAC.

Ellagic acid and its metabolites urolithins A/B ameliorate most common disease phenotypes in cellular and mouse models for lysosomal storage disorders by enhancing extracellular vesicle secretion.

Niemann Pick diseases types A (NPDA) and C (NPDC) are lysosomal storage disorders (LSDs) leading to cognitive impairment, neurodegeneration, and early death. NPDA and NPDC have different genetic origins, being caused by mutations in the acid sphingomyelinase (ASM) or the cholesterol transport protein NPC1, respectively. However, they share a common pathological hallmark in the accumulation of lipids in the endolysosomal compartment. Here, we tested the hypothesis that polyphenols reduce lipid overload in NPD cells by enhancing the secretion of extracellular vesicles (ECVs). We show that among the polyphenols tested, the ellagic acid metabolites, urolithin A and B, were the safest and most efficient in increasing ECV secretion. They reduced levels of accumulating lipids and lysosomal size and permeabilization in cultured bone marrow-derived macrophages and neurons from ASMko and NPC1 mutant mice, which mimic NPDA and NPDC, respectively. Moreover, oral treatment with ellagic acid reduced lipid levels, ameliorated lysosomal alterations, and diminished microglia activation in the brain of NPD mice. These results support the therapeutic value of ECV secretion and polyphenols for NPDs, which may also help treat other LSDs characterized by intracellular lipid overload.

SMPD1 expression profile and mutation landscape help decipher genotype-phenotype association and precision diagnosis for acid sphingomyelinase deficiency.

BACKGROUND: Acid sphingomyelinase deficiency (ASMD) disorder, also known as Niemann-Pick disease (NPD) is a rare genetic disease caused by mutations in SMPD1 gene, which encodes sphingomyelin phosphodiesterase (ASM). Except for liver and spleen enlargement and lung disease, two subtypes (Type A and B) of NDP have different onset times, survival times, ASM activities, and neurological abnormalities. To comprehensively explore NPD's genotype-phenotype association and pathophysiological characteristics, we collected 144 NPD cases with strict quality control through literature mining. RESULTS: The difference in ASM activity can differentiate NPD type A from other subtypes, with the ratio of ASM activity to the reference values being lower in type A (threshold 0.045 (4.45%)). Severe variations, such as deletion and insertion, can cause complete loss of ASM function, leading to type A, whereas relatively mild missense mutations generally result in type B. Among reported mutations, the p.Arg3AlafsX76 mutation is highly prevalent in the Chinese population, and the p.R608del mutation is common in Mediterranean countries. The expression profiles of SMPD1 from GTEx and single-cell RNA sequencing data of multiple fetal tissues showed that high expressions of SMPD1 can be observed in the liver, spleen, and brain tissues of adults and hepatoblasts, hematopoietic stem cells, STC2_TLX1-positive cells, mesothelial cells of the spleen, vascular endothelial cells of the cerebellum and the cerebrum of fetuses, indicating that SMPD1 dysfunction is highly likely to have a significant effect on the function of those cell types during development and the clinicians need pay attention to these organs or tissues as well during diagnosis. In addition, we also predicted 21 new pathogenic mutations in the SMPD1 gene that potentially cause the NPD, signifying that more rare cases will be detected with those mutations in SMPD1. Finally, we also analysed the function of the NPD type A cells following the extracellular milieu. CONCLUSIONS: Our study is the first to elucidate the effects of SMPD1 mutation on cell types and at the tissue level, which provides new insights into the genotype-phenotype association and can help in the precise diagnosis of NPD.

Immunotherapy targeting plasma ASM is protective in a mouse model of Alzheimer's disease.

Acid sphingomyelinase (ASM) has been implicated in neurodegenerative disease pathology, including Alzheimer's disease (AD). However, the specific role of plasma ASM in promoting these pathologies is poorly understood. Herein, we explore plasma ASM as a circulating factor that accelerates neuropathological features in AD by exposing young APP/PS1 mice to the blood of mice overexpressing ASM, through parabiotic surgery. Elevated plasma ASM was found to enhance several neuropathological features in the young APP/PS1 mice by mediating the differentiation of blood-derived, pathogenic Th17 cells. Antibody-based immunotherapy targeting plasma ASM showed efficient inhibition of ASM activity in the blood of APP/PS1 mice and, interestingly, led to prophylactic effects on neuropathological features by suppressing pathogenic Th17 cells. Our data reveals insights into the potential pathogenic mechanisms underlying AD and highlights ASM-targeting immunotherapy as a potential strategy for further investigation.

Dietary and genetic disruption of hepatic methionine metabolism induce acid sphingomyelinase to promote steatohepatitis.

Alcoholic (ASH) and nonalcoholic. (NASH).steatohepatitis are advanced.stages.of.fatty.liver.disease.Methionine adenosyltransferase 1A (MAT1A) plays a key role in hepatic methionine metabolism and germline Mat1a deletion in mice promotes NASH. Acid sphingomyelinase (ASMase) triggers hepatocellular apoptosis and liver fibrosis and has been shown to downregulate MAT1A expression in the context of fulminant liver failure. Given the role of ASMase in steatohepatitis development, we investigated the status of ASMase in Mat1a-/- mice and the regulation of ASMase by SAM/SAH. Consistent with its role in NASH, Mat1a-/- mice fed a choline-deficient (CD) diet exhibited macrosteatosis, inflammation, fibrosis and liver injury as well as reduced total and mitochondrial GSH levels. Our data uncovered an increased basal expression and activity of ASMase but not neutral SMase in Mat1a-/- mice, which further increased upon CD feeding. Interestingly, adenovirus-mediated shRNA expression targeting ASMase reduced ASMase activity and protected Mat1a-/- mice against CD diet-induced NASH. Similar results were observed in CD fed Mat1a-/- mice by pharmacological inhibition of ASMase with amitriptyline. Moreover, Mat1a/ASMase double knockout mice were resistant to CD-induced NASH. ASMase knockdown protected wild type mice against NASH induced by feeding a diet deficient in methionine and choline. Furthermore, Mat1a-/- mice developed acute-on-chronic ASH and this outcome was ameliorated by amitriptyline treatment. In vitro data in primary mouse hepatocytes revealed that decreased SAM/SAH ratio increased ASMase mRNA level and activity. MAT1A and ASMase mRNA levels exhibited an inverse correlation in liver samples from patients with ASH and NASH. Thus, disruption of methionine metabolism sensitizes to steatohepatitis by ASMase activation via decreased SAM/SAH. These findings imply that MAT1A deletion and ASMase activation engage in a self-sustained loop of relevance for steatohepatitis.

Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis.

BACKGROUND: Our recent studies suggest that sphingomyelin levels in the plasma membrane influence TF (tissue factor) procoagulant activity. The current study was performed to investigate how alterations to sphingomyelin metabolic pathway would affect TF procoagulant activity and thereby affect hemostatic and thrombotic processes. METHODS: Macrophages and endothelial cells were transfected with specific siRNAs or infected with adenoviral vectors to alter sphingomyelin levels in the membrane. TF activity was measured in factor X activation assay. Saphenous vein incision-induced bleeding and the inferior vena cava ligation-induced flow restriction mouse models were used to evaluate hemostasis and thrombosis, respectively. RESULTS: Overexpression of SMS (sphingomyelin synthase) 1 or SMS2 in human monocyte-derived macrophages suppresses ATP-stimulated TF procoagulant activity, whereas silencing SMS1 or SMS2 increases the basal cell surface TF activity to the same level as of ATP-decrypted TF activity. Consistent with the concept that sphingomyelin metabolism influences TF procoagulant activity, silencing of acid sphingomyelinase or neutral sphingomyelinase 2 or 3 attenuates ATP-induced enhanced TF procoagulant activity in macrophages and endothelial cells. Niemann-Pick disease fibroblasts with a higher concentration of sphingomyelin exhibited lower TF activity compared with wild-type fibroblasts. In vivo studies revealed that LPS+ATP-induced TF activity and thrombin generation were attenuated in ASMase-/- mice, while their levels were increased in SMS2-/- mice. Further studies revealed that acid sphingomyelinase deficiency leads to impaired hemostasis, whereas SMS2 deficiency increases thrombotic risk. CONCLUSIONS: Overall, our data indicate that alterations in sphingomyelin metabolism would influence TF procoagulant activity and affect hemostatic and thrombotic processes.

Compound Heterozygote Mutation in the SMPD1 Gene Leading to Nieman-Pick Disease Type A.

BACKGROUND Niemann-Pick disease (NPD) type A is an autosomal recessive lipid storage disorder caused by acid sphingomyelinase deficiency due to a mutation in the SMPD1 gene. Type A is the most severe phenotype of NPD, with early onset in infancy and unfavorable outcome in early childhood. CASE REPORT An 11-month-old boy with hepatosplenomegaly, elevated liver transaminases, and faltering growth was admitted to our hospital for further assessment of potential liver disease. He had severe generalized muscular hypotonia, muscular hypotrophy, reduced muscular strenght, joint laxity, weak deep tendon reflexes, and severe motor developmental delay. Leukodystrophy was seen on the brain MRI, and brainstem auditory evoked potentials were characteristic for auditory neuropathy. A chest X-ray showed signs of interstitial lung disease, which was not further evaluated due to absence of respiratory distress. Liver biopsy histopathologic findings were indicative for lipid storage disease. Genetic analysis showed that the patient is a compound heterozygote in the SMPD1 gene - (NM_000543.5): c.573delT p.(Ser192Alafs*65), which was inherited from the mother and c.1267C>T p.(His423Tyr) was inherited from the father. Both variants were previously individually reported in NPD type A and B. The clinical phenotype in our patient was characteristic of NPD type A, with an early onset and a rapidly progresive neurodegeneration. The patient was included in multidisciplinary follow-up, providing him symptomatic treatment and support. CONCLUSIONS We present a case of NPD type A caused by a rare compound heterozygote mutation in the SMPD1 gene. Most clinical findings and the disease course were typical for NPD type A, except for bilateral auditory neuropathy, which seems to be an uncommon finding in this phenotype and could be underestimated due to infrequent testing for auditory dysfunction.

SMPDL3b modulates radiation-induced DNA damage response in renal podocytes.

The kidneys are radiosensitive and dose-limiting organs for radiotherapy (RT) targeting abdominal and paraspinal tumors. Excessive radiation doses to the kidneys ultimately lead to radiation nephropathy. Our prior work unmasked a novel role for the lipid-modifying enzyme, sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), in regulating the response of renal podocytes to radiation injury. In this study, we investigated the role of SMPDL3b in DNA double-strand breaks (DSBs) repair in vitro and in vivo. We assessed the kinetics of DSBs recognition and repair along with the ATM pathway and nuclear sphingolipid metabolism in wild-type (WT) and SMPDL3b overexpressing (OE) human podocytes. We also assessed the extent of DNA damage repair in SMPDL3b knock-down (KD) human podocytes, and C57BL6 WT and podocyte-specific SMPDL3b-knock out (KO) mice after radiation injury. We found that SMPDL3b overexpression enhanced DSBs recognition and repair through modulating ATM nuclear shuttling. OE podocytes were protected against radiation-induced apoptosis by increasing the phosphorylation of p53 at serine 15 and attenuating subsequent caspase-3 cleavage. SMPDL3b overexpression prevented radiation-induced alterations in nuclear ceramide-1-phosphate (C1P) and ceramide levels. Interestingly, exogenous C1P pretreatment radiosensitized OE podocytes by delaying ATM nuclear foci formation and DSBs repair. On the other hand, SMPDL3b knock-down, in vitro and in vivo, induced a significant delay in DSBs repair. Additionally, increased activation of apoptosis was induced in podocytes of SMPDL3b-KO mice compared to WT mice at 24 h post-irradiation. Together, our results unravel a novel role for SMPDL3b in radiation-induced DNA damage response. The current work suggests that SMPDL3b modulates nuclear sphingolipid metabolism, ATM nuclear shuttling, and DSBs repair.

Mechanisms underlying Nrf2 nuclear translocation by non-lethal levels of hydrogen peroxide: p38 MAPK-dependent neutral sphingomyelinase2 membrane trafficking and ceramide/PKCζ/CK2 signaling.

Hydrogen peroxide is an aerobic metabolite playing a central role in redox signaling and oxidative stress. H2O2 could activate redox sensitive transcription factors, such as Nrf2, AP-1 and NF-κB by different manners. In some cells, treatment with non-lethal levels of H2O2 induces rapid activation of Nrf2, which upregulates expression of a set of genes involved in glutathione (GSH) synthesis and defenses against oxidative damage. It depends on two steps, the rapid translational activation of Nrf2 and facilitation of Nrf2 nuclear translocation. We review the molecular mechanisms by which H2O2 induces nuclear translocation of Nrf2 in cultured cells by highlighting the role of neutral sphingomyelinase 2 (nSMase2), a GSH sensor. H2O2 enters cells through aquaporin channels in the plasma membrane and is rapidly reduced to H2O by GSH peroxidases to consume cellular GSH, resulting in nSMase2 activation to generate ceramide. H2O2 also activates p38 MAP kinase, which enhances transfer of nSMase2 from perinuclear regions to plasma membrane lipid rafts to accelerate ceramide generation. Low levels of ceramide activate PKCζ, which then activates casein kinase 2 (CK2). These protein kinases are able to phosphorylate Nrf2 to stabilize and activate it. Notably, Nrf2 also binds to caveolin-1 (Cav1), which protects Nrf2 from Keap1-mediated degradation and limits Nrf2 nuclear translocation. We propose that Cav1serves as a signaling hub for the control of H2O2-mediated phosphorylation of Nrf2 by kinases, which results in release of Nrf2 from Cav1 to facilitate nuclear translocation. In summary, H2O2 induces GSH depletion which is recovered by Nrf2 activation dependent on p38/nSMase2/ceramide signaling.

Acid Sphingomyelinase-Ceramide Induced Vascular Injury Determines Colorectal Cancer Stem Cell Fate.

BACKGROUND/AIMS: It is unknown whether cancer stem cells respond differentially to treatment compared with progeny, potentially providing therapeutic vulnerabilities. Our program pioneered use of ultra-high single dose radiotherapy, which cures diverse metastatic diseases at a higher rate (90-95%) than conventional fractionation (~65%). Single dose radiotherapy engages a distinct biology involving microvascular acid sphingomyelinase/ceramide signaling, which, via NADPH oxidase-2-dependent perfusion defects, initiates an adaptive tumor SUMO Stress Response that globally-inactivates homologous recombination repair of double stand breaks, conferring cure. Accumulating data show diverse stem cells display heightened-dependence on homologous recombination repair to repair resolve double stand breaks. METHODS: Here we use colorectal cancer patient-derived xenografts containing logarithmically-increased Lgr5+ stem cells to explore whether optimizing engagement of this acid sphingomyelinase dependent biology enhances stem cell dependent tumor cure. RESULTS: We show radioresistant colorectal cancer patient-derived xenograft CLR27-2 contains radioresistant microvasculature and stem cells, whereas radiosensitive colorectal cancer patient-derived xenograft CLR1-1 contains radiosensitive microvasculature and stem cells. Pharmacologic or gene therapy enhancement of single dose radiotherapy-induced acid sphingomyelinase/ceramide-mediated microvascular dysfunction dramatically sensitizes CLR27-2 homologous recombination repair inactivation, converting Lgr5+ cells from the most resistant to most sensitive patient-derived xenograft population, yielding tumor cure. CONCLUSION: We posit homologous recombination repair represents a vulnerability determining colorectal cancer stem cell fate, approachable therapeutically using single dose radiotherapy.

Influence of sphingomyelin metabolism during epithelial-mesenchymal transition associated with aging in the renal papilla.

The renal collecting ducts (CD) are formed by a fully differentiated epithelium, and their tissue organization and function require the presence of mature cell adhesion structures. In certain circumstances, the cells can undergo de-differentiation by a process called epithelial-mesenchymal transition (EMT), in which the cells lose their epithelial phenotype and acquire the characteristics of the mesenchymal cells, which includes loss of cell-cell adhesion. We have previously shown that in renal papillary CD cells, cell adhesion structures are located in sphingomyelin (SM)-enriched plasma membrane microdomains and the inhibition of SM synthase 1 activity induced CD cells to undergo an EMT process. In the present study, we evaluated the influence of SM metabolism during the EMT of the cells that form the CD of the renal papilla during aging. To this end, primary cultures of renal papillary CD cells from young, middle-, and aged-rats were performed. By combining biochemical and immunofluorescence studies, we found experimental evidence that CD cells undergo an increase in spontaneous and reversible EMT during aging and that at least one of the reasons for this phenomenon is the decrease in SM content due to the combination of decreased SM synthase activity and an increase in SM degradation mediated by neutral sphingomyelinase. Age is a risk factor for many diseases, among which renal fibrosis is included. Our findings highlight the importance of sphingolipids and particularly SM as a modulator of the fate of CD cells and probably contribute to the development of treatments to avoid or reverse renal fibrosis during aging.

Sphingomyelinase activity promotes atrophy and attenuates force in human muscle fibres and is elevated in heart failure patients.

BACKGROUND: Activation of sphingomyelinase (SMase) as a result of a general inflammatory response has been implicated as a mechanism underlying disease-related loss of skeletal muscle mass and function in several clinical conditions including heart failure. Here, for the first time, we characterize the effects of SMase activity on human muscle fibre contractile function and assess skeletal muscle SMase activity in heart failure patients. METHODS: The effects of SMase on force production and intracellular Ca2+ handling were investigated in single intact human muscle fibres. Additional mechanistic studies were performed in single mouse toe muscle fibres. RNA sequencing was performed in human muscle bundles exposed to SMase. Intramuscular SMase activity was measured from heart failure patients (n = 61, age 69 ± 0.8 years, NYHA III-IV, ejection fraction 25 ± 1.0%, peak VO2 14.4 ± 0.6 mL × kg × min) and healthy age-matched control subjects (n = 10, age 71 ± 2.2 years, ejection fraction 60 ± 1.2%, peak VO2 25.8 ± 1.1 mL × kg × min). SMase activity was related to circulatory factors known to be associated with progression and disease severity in heart failure. RESULTS: Sphingomyelinase reduced muscle fibre force production (-30%, P < 0.05) by impairing sarcoplasmic reticulum (SR) Ca2+ release (P < 0.05) and reducing myofibrillar Ca2+ sensitivity. In human muscle bundles exposed to SMase, RNA sequencing analysis revealed 180 and 291 genes as up-regulated and down-regulated, respectively, at a FDR of 1%. Gene-set enrichment analysis identified 'proteasome degradation' as an up-regulated pathway (average fold-change 1.1, P = 0.008), while the pathway 'cytoplasmic ribosomal proteins' (average fold-change 0.8, P < 0.0001) and factors involving proliferation of muscle cells (average fold-change 0.8, P = 0.0002) where identified as down-regulated. Intramuscular SMase activity was ~20% higher (P < 0.05) in human heart failure patients than in age-matched healthy controls and was positively correlated with markers of disease severity and progression, and with several circulating inflammatory proteins, including TNF-receptor 1 and 2. In a longitudinal cohort of heart failure patients (n = 6, mean follow-up time 2.5 ± 0.2 years), SMase activity was demonstrated to increase by 30% (P < 0.05) with duration of disease. CONCLUSIONS: The present findings implicate activation of skeletal muscle SMase as a mechanism underlying human heart failure-related loss of muscle mass and function. Moreover, our findings strengthen the idea that SMase activation may underpin disease-related loss of muscle mass and function in other clinical conditions, acting as a common patophysiological mechanism for the myopathy often reported in diseases associated with a systemic inflammatory response.

Neutral Sphingomyelinase 2 Mediates Oxidative Stress Effects on Astrocyte Senescence and Synaptic Plasticity Transcripts.

We have shown that deficiency of neutral sphingomyelinase 2 (nSMase2), an enzyme generating the sphingolipid ceramide, improves memory in adult mice. Here, we performed sphingolipid and RNA-seq analyses on the cortex from 10-month-old nSMase2-deficient (fro/fro) and heterozygous (+ /fro) mice. fro/fro cortex showed reduced levels of ceramide, particularly in astrocytes. Differentially abundant transcripts included several functionally related groups, with decreases in mitochondrial oxidative phosphorylation and astrocyte activation transcripts, while axon guidance and synaptic transmission and plasticity transcripts were increased, indicating a role of nSMase2 in oxidative stress, astrocyte activation, and cognition. Experimentally induced oxidative stress decreased the level of glutathione (GSH), an endogenous inhibitor of nSMase2, and increased immunolabeling for ceramide in primary + /fro astrocytes, but not in fro/fro astrocytes. ß-galactosidase activity was lower in 5-week-old fro/fro astrocytes, indicating delayed senescence due to nSMase2 deficiency. In fro/fro cortex, levels of the senescence markers C3b and p27 and the proinflammatory cytokines interleukin 1ß, interleukin 6, and tumor necrosis factor α were reduced, concurrent with twofold decreased phosphorylation of their downstream target, protein kinase Stat3. RNA and protein levels of the ionotropic glutamate receptor subunit 2B (Grin2b/NR2B) were increased by twofold, which was previously shown to enhance cognition. This was consistent with threefold reduced levels of exosomes carrying miR-223-3p, a micro-RNA downregulating NR2B. In summary, our data show that nSMase2 deficiency prevents oxidative stress-induced elevation of ceramide and secretion of exosomes by astrocytes that suppress neuronal function, indicating a role of nSMase2 in the regulation of neuroinflammation and cognition.

Nuclear Ceramide Is Associated with Ataxia Telangiectasia Mutated Activation in the Neocarzinostatin-Induced Apoptosis of Lymphoblastoid Cells.

Ceramide is a bioactive sphingolipid that mediates ionizing radiation- and chemotherapy-induced apoptosis. Neocarzinostatin (NCS) is a genotoxic anti-cancer drug that induces apoptosis in response to DNA double-strand breaks (DSBs) through ataxia telangiectasia mutated (ATM) activation. However, the involvement of ceramide in NCS-evoked nuclear events such as DSB-activated ATM has not been clarified. Here, we found that nuclear ceramide increased by NCS-mediated apoptosis through the enhanced assembly of ATM and the meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 (MRN) complex proteins in human lymphoblastoid L-39 cells. NCS induced an increase of ceramide production through activation of neutral sphingomyelinase (nSMase) and suppression of sphingomyelin synthase (SMS) upstream of DSB-mediated ATM activation. In ATM-deficient lymphoblastoid AT-59 cells compared with L-39 cells, NCS treatment showed a decrease of apoptosis even though ceramide increase and DSBs were observed. Expression of wild-type ATM, but not the kinase-dead mutant ATM, in AT-59 cells increased NCS-induced apoptosis despite similar ceramide accumulation. Interestingly, NCS increased ceramide content in the nucleus through nSMase activation and SMS suppression and promoted colocalization of ceramide with phosphorylated ATM and foci of MRN complex. Inhibition of ceramide generation by the overexpression of SMS suppressed NCS-induced apoptosis through the inhibition of ATM activation and assembly of the MRN complex. In addition, inhibition of ceramide increased by the nSMase inhibitor GW4869 prevented NCS-mediated activation of the ATM. Therefore, our findings suggest the involvement of the nuclear ceramide with ATM activation in NCS-mediated apoptosis. SIGNIFICANCE STATEMENT: This study demonstrates that regulation of ceramide with neutral sphingomyelinase and sphingomyelin synthase in the nucleus in double-strand break-mimetic agent neocarzinostatin (NCS)-induced apoptosis. This study also showed that ceramide increase in the nucleus plays a role in NCS-induced apoptosis through activation of the ataxia telangiectasia mutated/meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 complex in human lymphoblastoid cells.