Browsing by Subject "Sphingolipids"
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Item Brain and blood metabolite signatures of pathology and progression in Alzheimer disease: A targeted metabolomics study(Public Library of Science, 2018-01-25) Varma, Vijay R.; Oommen, Anup M.; Varma, Sudhir; Casanova, Ramon; An, Yang; Andrews, Ryan M.; O’Brien, Richard; Pletnikova, Olga; Troncoso, Juan C.; Toledo, Jon; Baillie, Rebecca; Arnold, Matthias; Kastenmueller, Gabi; Nho, Kwangsik; Doraiswamy, P. Murali; Saykin, Andrew J.; Kaddurah-Daouk, Rima; Legido-Quigley, Cristina; Thambisetty, Madhav; Radiology and Imaging Sciences, School of MedicineBACKGROUND: The metabolic basis of Alzheimer disease (AD) is poorly understood, and the relationships between systemic abnormalities in metabolism and AD pathogenesis are unclear. Understanding how global perturbations in metabolism are related to severity of AD neuropathology and the eventual expression of AD symptoms in at-risk individuals is critical to developing effective disease-modifying treatments. In this study, we undertook parallel metabolomics analyses in both the brain and blood to identify systemic correlates of neuropathology and their associations with prodromal and preclinical measures of AD progression. METHODS AND FINDINGS: Quantitative and targeted metabolomics (Biocrates AbsoluteIDQ [identification and quantification] p180) assays were performed on brain tissue samples from the autopsy cohort of the Baltimore Longitudinal Study of Aging (BLSA) (N = 44, mean age = 81.33, % female = 36.36) from AD (N = 15), control (CN; N = 14), and "asymptomatic Alzheimer's disease" (ASYMAD, i.e., individuals with significant AD pathology but no cognitive impairment during life; N = 15) participants. Using machine-learning methods, we identified a panel of 26 metabolites from two main classes-sphingolipids and glycerophospholipids-that discriminated AD and CN samples with accuracy, sensitivity, and specificity of 83.33%, 86.67%, and 80%, respectively. We then assayed these 26 metabolites in serum samples from two well-characterized longitudinal cohorts representing prodromal (Alzheimer's Disease Neuroimaging Initiative [ADNI], N = 767, mean age = 75.19, % female = 42.63) and preclinical (BLSA) (N = 207, mean age = 78.68, % female = 42.63) AD, in which we tested their associations with magnetic resonance imaging (MRI) measures of AD-related brain atrophy, cerebrospinal fluid (CSF) biomarkers of AD pathology, risk of conversion to incident AD, and trajectories of cognitive performance. We developed an integrated blood and brain endophenotype score that summarized the relative importance of each metabolite to severity of AD pathology and disease progression (Endophenotype Association Score in Early Alzheimer's Disease [EASE-AD]). Finally, we mapped the main metabolite classes emerging from our analyses to key biological pathways implicated in AD pathogenesis. We found that distinct sphingolipid species including sphingomyelin (SM) with acyl residue sums C16:0, C18:1, and C16:1 (SM C16:0, SM C18:1, SM C16:1) and hydroxysphingomyelin with acyl residue sum C14:1 (SM (OH) C14:1) were consistently associated with severity of AD pathology at autopsy and AD progression across prodromal and preclinical stages. Higher log-transformed blood concentrations of all four sphingolipids in cognitively normal individuals were significantly associated with increased risk of future conversion to incident AD: SM C16:0 (hazard ratio [HR] = 4.430, 95% confidence interval [CI] = 1.703-11.520, p = 0.002), SM C16:1 (HR = 3.455, 95% CI = 1.516-7.873, p = 0.003), SM (OH) C14:1 (HR = 3.539, 95% CI = 1.373-9.122, p = 0.009), and SM C18:1 (HR = 2.255, 95% CI = 1.047-4.855, p = 0.038). The sphingolipid species identified map to several biologically relevant pathways implicated in AD, including tau phosphorylation, amyloid-β (Aβ) metabolism, calcium homeostasis, acetylcholine biosynthesis, and apoptosis. Our study has limitations: the relatively small number of brain tissue samples may have limited our power to detect significant associations, control for heterogeneity between groups, and replicate our findings in independent, autopsy-derived brain samples. CONCLUSIONS: We present a novel framework to identify biologically relevant brain and blood metabolites associated with disease pathology and progression during the prodromal and preclinical stages of AD. Our results show that perturbations in sphingolipid metabolism are consistently associated with endophenotypes across preclinical and prodromal AD, as well as with AD pathology at autopsy. Sphingolipids may be biologically relevant biomarkers for the early detection of AD, and correcting perturbations in sphingolipid metabolism may be a plausible and novel therapeutic strategy in AD.Item Crystal structure of RahU, an aegerolysin protein from the human pathogen Pseudomonas aeruginosa, and its interaction with membrane ceramide phosphorylethanolamine(Springer Nature, 2021-03-22) Kočar, Eva; Lenarčič, Tea; Hodnik, Vesna; Panevska, Anastasija; Huang, Yunjie; Bajc, Gregor; Kostanjšek, Rok; Naren, Anjaparavanda P.; Maček, Peter; Anderluh, Gregor; Sepčić, Kristina; Podobnik, Marjetka; Butala, Matej; Pediatrics, School of MedicineAegerolysins are proteins produced by bacteria, fungi, plants and protozoa. The most studied fungal aegerolysins share a common property of interacting with membranes enriched with cholesterol in combination with either sphingomyelin or ceramide phosphorylethanolamine (CPE), major sphingolipids in the cell membranes of vertebrates and invertebrates, respectively. However, genome analyses show a particularly high frequency of aegerolysin genes in bacteria, including the pathogenic genera Pseudomonas and Vibrio; these are human pathogens of high clinical relevance and can thrive in a variety of other species. The knowledge on bacterial aegerolysin-lipid interactions is scarce. We show that Pseudomonas aeruginosa aegerolysin RahU interacts with CPE, but not with sphingomyelin-enriched artificial membranes, and that RahU interacts with the insect cell line producing CPE. We report crystal structures of RahU alone and in complex with tris(hydroxymethyl)aminomethane (Tris), which, like the phosphorylethanolamine head group of CPE, contains a primary amine. The RahU structures reveal that the two loops proximal to the amino terminus form a cavity that accommodates Tris, and that the flexibility of these two loops is important for this interaction. We show that Tris interferes with CPE-enriched membranes for binding to RahU, implying on the importance of the ligand cavity between the loops and its proximity in RahU membrane interaction. We further support this by studying the interaction of single amino acid substitution mutants of RahU with the CPE-enriched membranes. Our results thus represent a starting point for a better understanding of the role of P. aeruginosa RahU, and possibly other bacterial aegerolysins, in bacterial interactions with other organisms.Item Dual Anti-Inflammatory and Anti-Angiogenic Action of miR-15a in Diabetic Retinopathy.(Elsevier, 2016-09) Wang, Qi; Navitskaya, Svetlana; Chakravarthy, Harshini; Huang, Chao; Kady, Nermin; Lydic, Todd A.; Chen, Y. Eugene; Yin, Ke-Jie; Powell, Folami Lamoke; Martin, Pamela M.; Grant, Maria B.; Busik, Julia V.; Department of Ophthalmology, IU School of MedicineActivation of pro-inflammatory and pro-angiogenic pathways in the retina and the bone marrow contributes to pathogenesis of diabetic retinopathy. We identified miR-15a as key regulator of both pro-inflammatory and pro-angiogenic pathways through direct binding and inhibition of the central enzyme in the sphingolipid metabolism, ASM, and the pro-angiogenic growth factor, VEGF-A. miR-15a was downregulated in diabetic retina and bone marrow cells. Over-expression of miR-15a downregulated, and inhibition of miR-15a upregulated ASM and VEGF-A expression in retinal cells. In addition to retinal effects, migration and retinal vascular repair function was impaired in miR-15a inhibitor-treated circulating angiogenic cells (CAC). Diabetic mice overexpressing miR-15a under Tie-2 promoter had normalized retinal permeability compared to wild type littermates. Importantly, miR-15a overexpression led to modulation toward nondiabetic levels, rather than complete inhibition of ASM and VEGF-A providing therapeutic effect without detrimental consequences of ASM and VEGF-A deficiencies.Item Human Brain Lipidomics: Utilities of Chloride Adducts in Flow Injection Analysis(MDPI, 2021-04-28) Wood, Paul L.; Hauther, Kathleen A.; Scarborough, Jon H.; Craney, Dustin J.; Dudzik, Beatrix; Cebak, John E.; Woltjer, Randall L.; Psychiatry, School of MedicineCeramides have been implicated in a number of disease processes. However, current means of evaluation with flow infusion analysis (FIA) have been limited primarily due to poor sensitivity within our high-resolution mass spectrometry lipidomics analytical platform. To circumvent this deficiency, we investigated the potential of chloride adducts as an alternative method to improve sensitivity with electrospray ionization. Chloride adducts of ceramides and ceramide subfamilies provided 2- to 50-fold increases in sensitivity both with analytical standards and biological samples. Chloride adducts of a number of other lipids with reactive hydroxy groups were also enhanced. For example, monogalactosyl diacylglycerols (MGDGs), extracted from frontal lobe cortical gray and subcortical white matter of cognitively intact subjects, were not detected as ammonium adducts but were readily detected as chloride adducts. Hydroxy lipids demonstrate a high level of specificity in that phosphoglycerols and phosphoinositols do not form chloride adducts. In the case of choline glycerophospholipids, the fatty acid substituents of these lipids could be monitored by MS2 of the chloride adducts. Monitoring the chloride adducts of a number of key lipids offers enhanced sensitivity and specificity with FIA. In the case of glycerophosphocholines, the chloride adducts also allow determination of fatty acid substituents. The chloride adducts of lipids possessing electrophilic hydrogens of hydroxyl groups provide significant increases in sensitivity. In the case of glycerophosphocholines, chloride attachment to the quaternary ammonium group generates a dominant anion, which provides the identities of the fatty acid substituents under MS2 conditions.Item Inhibition of acid sphingomyelinase disrupts LYNUS signaling and triggers autophagy(American Society for Biochemistry and Molecular Biology, 2018-04) Justice, Matthew J.; Bronova, Irina; Schweitzer, Kelly S.; Poirier, Christophe; Blum, Janice S.; Berdyshev, Evgeny V.; Petrache, Irina; Biochemistry and Molecular Biology, School of MedicineActivation of the lysosomal ceramide-producing enzyme, acid sphingomyelinase (ASM), by various stresses is centrally involved in cell death and has been implicated in autophagy. We set out to investigate the role of the baseline ASM activity in maintaining physiological functions of lysosomes, focusing on the lysosomal nutrient-sensing complex (LYNUS), a lysosomal membrane-anchored multiprotein complex that includes mammalian target of rapamycin (mTOR) and transcription factor EB (TFEB). ASM inhibition with imipramine or sphingomyelin phosphodiesterase 1 (SMPD1) siRNA in human lung cells, or by transgenic Smpd1+/- haploinsufficiency of mouse lungs, markedly reduced mTOR- and P70-S6 kinase (Thr 389)-phosphorylation and modified TFEB in a pattern consistent with its activation. Inhibition of baseline ASM activity significantly increased autophagy with preserved degradative potential. Pulse labeling of sphingolipid metabolites revealed that ASM inhibition markedly decreased sphingosine (Sph) and Sph-1-phosphate (S1P) levels at the level of ceramide hydrolysis. These findings suggest that ASM functions to maintain physiological mTOR signaling and inhibit autophagy and implicate Sph and/or S1P in the control of lysosomal function.Item LIPIDOMIC PROFILING OF DICTYOSTELIUM DISCOIDEUM(2012-08-27) Birch, Garrison L.; Minto, Robert; Blacklock, Brenda J.; McLeish, Michael J.The lipid profile of Dictyostelium discoideum, a cellular slime mold found evolutionarily between plants and animals, has never been clearly defined. To address this, the fatty acid content of vegetative cells was analyzed by gas chromatography-mass spectrometry of fatty acid methyl esters and their identities verified with synthesized authentic standards. The synthetic scheme developed to produce the unusual fatty acids found in D. discoideum was engineered to afford the labeling of compounds (2H) for use in feeding studies to elucidate the fatty acid elongation and desaturation pathways present in D. discoideum. After establishing the fatty acid profile and acyl metabolic pathway, an initial understanding the complex lipids present in D. discoideum, chiefly sphingolipids, was sought. Triple quadrupole and quadrupole time-of flight mass spectrometers equipped with electrospray ionization sources were used to identify these complex lipids.Item Multi-Omic analyses characterize the ceramide/sphingomyelin pathway as a therapeutic target in Alzheimer's disease(Springer Nature, 2022-10-08) Baloni, Priyanka; Arnold, Matthias; Buitrago, Luna; Nho, Kwangsik; Moreno, Herman; Huynh, Kevin; Brauner, Barbara; Louie, Gregory; Kueider-Paisley, Alexandra; Suhre, Karsten; Saykin, Andrew J.; Ekroos, Kim; Meikle, Peter J.; Hood, Leroy; Price, Nathan D.; The Alzheimer’s Disease Metabolomics Consortium; Doraiswamy, P. Murali; Funk, Cory C.; Hernández, A. Iván; Kastenmüller, Gabi; Baillie, Rebecca; Han, Xianlin; Kaddurah-Daouk, Rima; Radiology and Imaging Sciences, School of MedicineDysregulation of sphingomyelin and ceramide metabolism have been implicated in Alzheimer's disease. Genome-wide and transcriptome-wide association studies have identified various genes and genetic variants in lipid metabolism that are associated with Alzheimer's disease. However, the molecular mechanisms of sphingomyelin and ceramide disruption remain to be determined. We focus on the sphingolipid pathway and carry out multi-omics analyses to identify central and peripheral metabolic changes in Alzheimer's patients, correlating them to imaging features. Our multi-omics approach is based on (a) 2114 human post-mortem brain transcriptomics to identify differentially expressed genes; (b) in silico metabolic flux analysis on context-specific metabolic networks identified differential reaction fluxes; (c) multimodal neuroimaging analysis on 1576 participants to associate genetic variants in sphingomyelin pathway with Alzheimer's disease pathogenesis; (d) plasma metabolomic and lipidomic analysis to identify associations of lipid species with dysregulation in Alzheimer's; and (e) metabolite genome-wide association studies to define receptors within the pathway as a potential drug target. We validate our hypothesis in amyloidogenic APP/PS1 mice and show prolonged exposure to fingolimod alleviated synaptic plasticity and cognitive impairment in mice. Our integrative multi-omics approach identifies potential targets in the sphingomyelin pathway and suggests modulators of S1P metabolism as possible candidates for Alzheimer's disease treatment.Item Pharmacological sphingosine-1 phosphate receptor 1 targeting in cigarette smoke-induced emphysema in mice(American Physiological Society, 2022) Goel, Khushboo; Schweitzer, Kelly S.; Serban, Karina A.; Bittman, Robert; Petrache, Irina; Medicine, School of MedicinePrimarily caused by chronic cigarette smoking (CS), emphysema is characterized by loss of alveolar cells comprising lung units involved in gas exchange and inflammation that culminate in airspace enlargement. Dysregulation of sphingolipid metabolism with increases of ceramide relative to sphingosine-1 phosphate (S1P) signaling has been shown to cause lung cell apoptosis and is emerging as a potential therapeutic target in emphysema. We sought to determine the impact of augmenting S1P signaling via S1P receptor 1 (S1P1) in a mouse model of CS-induced emphysema. DBA2 mice were exposed to CS for 4 or 6 mo and treated with pharmacological agonists of S1P1: phosphonated FTY720 (FTY720-1S and 2S analogs; 0.01–1.0 mg/kg) or GSK183303A (10 mg/kg). Pharmacological S1P1 agonists ameliorated CS-induced lung parenchymal apoptosis and airspace enlargement as well as loss of body weight. S1P1 agonists had modest inhibitory effects on CS-induced airspace inflammation and lung functional changes measured by Flexivent, improving lung tissue resistance. S1P1 abundance was reduced in chronic CS-conditions and remained decreased after CS-cessation or treatment with FTY720-1S. These results support an important role for S1P-S1P1 axis in maintaining the structural integrity of alveoli during chronic CS exposure and suggest that increasing both S1P1 signaling and abundance may be beneficial to counteract the effects of chronic CS exposure.Item Protein phosphatase 2A (PP2A) holoenzymes regulate death associated protein kinase (DAPK) in ceramide-induced anoikis(2010-05-03T19:42:36Z) Widau, Ryan Cole; Gallagher, Patricia J.; Herring, B. Paul; Rhodes, Simon J.; Skalnik, David GordonModulation of sphingolipid-induced apoptosis is a potential mechanism to enhance the effectiveness of chemotherapeutic drugs. Ceramide is a pleiotropic, sphingolipid produced by cells in response to inflammatory cytokines, chemotherapeutic drugs and ionizing radiation. Ceramide is a potent activator of protein phosphatases, including protein phosphatase 2A (PP2A) leading to dephosphorylation of substrates important in regulating mitochondrial dysfunction and apoptosis. Previous studies demonstrated that death associated protein kinase (DAPK) plays a role in ceramide-induced apoptosis via an unknown mechanism. The tumor suppressor DAPK is a calcium/calmodulin regulated serine/threonine kinase with an important role in regulating cytoskeletal dynamics. Auto-phosphorylation within the calmodulin-binding domain at serine308 inhibits DAPK catalytic activity. Dephosphorylation of serine308 by a hitherto unknown phosphatase enhances kinase activity and proteasomal mediated degradation of DAPK. In these studies, using a tandem affinity purification procedure coupled to LC-MS/MS, we have identified two holoenzyme forms of PP2A as DAPK interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Serine308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK protein levels by enhancing proteasomal-mediated degradation of the kinase, as a means to attenuate prolonged kinase activation. These studies also demonstrate that ceramide causes a caspase-independent cell detachment in HeLa cells, a human cervical carcinoma cell line. Subsequent to detachment, these cells underwent caspase-dependent apoptosis due to lack of adhesion, termed anoikis. Overexpression of wild type DAPK induced cell rounding and detachment similar to cells treated with ceramide; however, this effect was not observed following expression of a phosphorylation mutant, S308E DAPK. Finally, the endogenous interaction of DAPK and PP2A was determined to be required for ceramide-induced cell detachment and anoikis. Together these studies have provided exciting and essential new data regarding the mechanisms of cell adhesion and anoikis. These results define a novel cellular pathway initiated by ceramide-mediated activation of PP2A and DAPK to regulate inside-out signaling and promote anoikis.Item Role of Acid Sphingomyelinase in Shifting the Balance Between Proinflammatory and Reparative Bone Marrow Cells in Diabetic Retinopathy(Wiley, 2016-04) Chakravarthy, Harshini; Navitskaya, Svetlana; O'reilly, Sandra; Gallimore, Jacob; Mize, Hannah; Beli, Eleni; Wang, Qi; Kady, Nermin; Huang, Chao; Blanchard, Gary J.; Grant, Maria B.; Busika, Julia V.; Ophthalmology, School of MedicineThe metabolic insults associated with diabetes lead to low-grade chronic inflammation, retinal endothelial cell damage, and inadequate vascular repair. This is partly due to the increased activation of bone marrow (BM)-derived proinflammatory monocytes infiltrating the retina, and the compromised function of BM-derived reparative circulating angiogenic cells (CACs), which home to sites of endothelial injury and foster vascular repair. We now propose that a metabolic link leading to activated monocytes and dysfunctional CACs in diabetes involves upregulation of a central enzyme of sphingolipid signaling, acid sphingomyelinase (ASM). Selective inhibition of ASM in the BM prevented diabetes-induced activation of BM-derived microglia-like cells and normalized proinflammatory cytokine levels in the retina. ASM upregulation in diabetic CACs caused accumulation of ceramide on their cell membrane, thereby reducing membrane fluidity and impairing CAC migration. Replacing sphingomyelin with ceramide in synthetic membrane vesicles caused a similar decrease in membrane fluidity. Inhibition of ASM in diabetic CACs improved membrane fluidity and homing of these cells to damaged retinal vessels. Collectively, these findings indicate that selective modulation of sphingolipid metabolism in BM-derived cell populations in diabetes normalizes the reparative/proinflammatory cell balance and can be explored as a novel therapeutic strategy for treating diabetic retinopathy.