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Inhibition of de novo ceramide biosynthesis affects aging phenotype in an in vitro model of neuronal senescence.

ABSTRACT: Although aging is considered to be an unavoidable event, recent experimental evidence suggests that the process can be counteracted. Intracellular calcium (Ca2+i) dyshomeostasis, mitochondrial dysfunction, oxidative stress, and lipid dysregulation are critical factors that contribute to senescence-related processes. Ceramides, a pleiotropic class of sphingolipids, are important mediators of cellular senescence, but their role in neuronal aging is still largely unexplored. In this study, we investigated the effects of L-cycloserine (L-CS), an inhibitor of thede novoceramide biosynthesis, on the aging phenotype of cortical neurons cultured for 22 days, a setting employed as anin vitromodel of senescence. Our findings indicate that, compared to control cultures, 'aged' neurons display dysregulation of [Ca2+]ilevels, mitochondrial dysfunction, increased generation of reactive oxygen species (ROS), altered synaptic activity as well as the activation of neuronal death-related molecules. Treatment with L-CS positively affected the senescent phenotype, a result associated with recovery of neuronal [Ca2+]isignaling and reduction of mitochondrial dysfunction and ROS generation. The results suggest that thede novoceramide biosynthesis represents a critical intermediate in the molecular and functional cascade leading to neuronal senescence and identify ceramide biosynthesis inhibitors as promising pharmacological tools to decrease age-related neuronal dysfunctions.

SUBMITTER: Granzotto A

PROVIDER: S-EPMC6738398 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Inhibition of de novo ceramide biosynthesis affects aging phenotype in an in vitro model of neuronal senescence.

Granzotto Alberto A Bomba Manuela M Castelli Vanessa V Navarra Riccardo R Massetti Noemi N d'Aurora Marco M Onofrj Marco M Cicalini Ilaria I Del Boccio Piero P Gatta Valentina V Cimini Annamaria A Piomelli Daniele D Sensi Stefano L SL

Aging 20190829 16

Although aging is considered to be an unavoidable event, recent experimental evidence suggests that the process can be counteracted. Intracellular calcium (Ca2+i) dyshomeostasis, mitochondrial dysfunction, oxidative stress, and lipid dysregulation are critical factors that contribute to senescence-related processes. Ceramides, a pleiotropic class of sphingolipids, are important mediators of cellular senescence, but their role in neuronal aging is still largely unexplored. I ...[more]

PMID: 31467258

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Project description:BackgroundWe previously showed that cardiomyocyte Krϋppel-like factor (KLF) 5 regulates cardiac fatty acid oxidation. As heart failure has been associated with altered fatty acid oxidation, we investigated the role of cardiomyocyte KLF5 in lipid metabolism and pathophysiology of ischemic heart failure.MethodsUsing real-time polymerase chain reaction and Western blot, we investigated the KLF5 expression changes in a myocardial infarction (MI) mouse model and heart tissue from patients with ischemic heart failure. Using 2D echocardiography, we evaluated the effect of KLF5 inhibition after MI using pharmacological KLF5 inhibitor ML264 and mice with cardiomyocyte-specific KLF5 deletion (αMHC [α-myosin heavy chain]-KLF5-/-). We identified the involvement of KLF5 in regulating lipid metabolism and ceramide accumulation after MI using liquid chromatography-tandem mass spectrometry, and Western blot and real-time polymerase chain reaction analysis of ceramide metabolism-related genes. We lastly evaluated the effect of cardiomyocyte-specific KLF5 overexpression (αMHC-rtTA [reverse tetracycline-controlled transactivator]-KLF5) on cardiac function and ceramide metabolism, and rescued the phenotype using myriocin to inhibit ceramide biosynthesis.ResultsKLF5 mRNA and protein levels were higher in human ischemic heart failure samples and in rodent models at 24 hours, 2 weeks, and 4 weeks post-permanent left coronary artery ligation. αMHC-KLF5-/- mice and mice treated with ML264 had higher ejection fraction and lower ventricular volume and heart weight after MI. Lipidomic analysis showed that αMHC-KLF5-/- mice with MI had lower myocardial ceramide levels compared with littermate control mice with MI, although basal ceramide content of αMHC-KLF5-/- mice was not different in control mice. KLF5 ablation suppressed the expression of SPTLC1 and SPTLC2 (serine palmitoyltransferase [SPT] long-chain base subunit ()1 2, respectively), which regulate de novo ceramide biosynthesis. We confirmed our previous findings that myocardial SPTLC1 and SPTLC2 levels are increased in heart failure patients. Consistently, αMHC-rtTA-KLF5 mice showed increased SPTLC1 and SPTLC2 expression, higher myocardial ceramide levels, and systolic dysfunction beginning 2 weeks after KLF5 induction. Treatment of αMHC-rtTA-KLF5 mice with myriocin that inhibits SPT, suppressed myocardial ceramide levels and alleviated systolic dysfunction.ConclusionsKLF5 is induced during the development of ischemic heart failure in humans and mice and stimulates ceramide biosynthesis. Genetic or pharmacological inhibition of KLF5 in mice with MI prevents ceramide accumulation, alleviates eccentric remodeling, and increases ejection fraction. Thus, KLF5 emerges as a novel therapeutic target for the treatment of ischemic heart failure.

Project description:BackgroundReproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo. Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes, imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns.ResultsAn in vitro culture system was developed from immature mouse oocytes that were grown to germinal vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% O2) and hypoxia (5% oxygen, 5% O2). The cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNA-seq libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes, global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% O2 condition, transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcription. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bodies of grown oocytes presented a lower methylation profile.ConclusionsOur observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% O2 had a higher correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentration is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mechanisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.

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Inhibition of de novo ceramide biosynthesis affects aging phenotype in an in vitro model of neuronal senescence.

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Inhibition of <i>de novo</i> ceramide biosynthesis affects aging phenotype in an <i>in vitro</i> model of neuronal senescence.

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