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16 pages, 1600 KiB

Open AccessArticle

Peripheral Upregulation of Parkinson’s Disease-Associated Genes Encoding α-Synuclein, β-Glucocerebrosidase, and Ceramide Glucosyltransferase in Major Depression

by Razvan-Marius Brazdis, Claudia von Zimmermann, Bernd Lenz, Johannes Kornhuber and Christiane Mühle

Int. J. Mol. Sci. 2024, 25(6), 3219; https://doi.org/10.3390/ijms25063219 - 12 Mar 2024

Viewed by 1158

Abstract

Due to the high comorbidity of Parkinson’s disease (PD) with major depressive disorder (MDD) and the involvement of sphingolipids in both conditions, we investigated the peripheral expression levels of three primarily PD-associated genes: α-synuclein (SNCA), lysosomal enzyme β-glucocerebrosidase (GBA1), [...] Read more.

Due to the high comorbidity of Parkinson’s disease (PD) with major depressive disorder (MDD) and the involvement of sphingolipids in both conditions, we investigated the peripheral expression levels of three primarily PD-associated genes: α-synuclein (SNCA), lysosomal enzyme β-glucocerebrosidase (GBA1), and UDP-glucose ceramide glucosyltransferase (UGCG) in a sex-balanced MDD cohort. Normalized gene expression was determined by quantitative PCR in patients suffering from MDD (unmedicated n = 63, medicated n = 66) and controls (remitted MDD n = 39, healthy subjects n = 61). We observed that expression levels of SNCA (p = 0.036), GBA1 (p = 0.014), and UGCG (p = 0.0002) were higher in currently depressed patients compared to controls and remitted patients, and expression of GBA1 and UGCG decreased in medicated patients during three weeks of therapy. Additionally, in subgroups, expression was positively correlated with the severity of depression and anxiety. Furthermore, we identified correlations between the gene expression levels and PD-related laboratory parameters. Our findings suggest that SNCA, GBA1, and UGCG analysis could be instrumental in the search for biomarkers of MDD and in understanding the overlapping pathological mechanisms underlying neuro-psychiatric diseases. Full article

(This article belongs to the Special Issue Molecular Research on Depression)

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Peripheral gene expressions of SNCA (a), GBA (b), and UGCG (c) were significantly higher in patients with current MDE (combined unmedicated patients (PU) and medicated patients (PM)) at inclusion compared to unaffected individuals (combined remitted patients (PR) and healthy subjects (HC)). These levels remained for SNCA (d) but decreased between inclusion (T1) and follow-up (T2) after on average three weeks of treatment as usual for GBA1 (e) and UGCG (f) in the group of initially medicated patients. Normalized gene expression relative to reference genes is shown on a logarithmic y-axis. The numbers of individuals are provided below the x-axis. p-values from Mann–Whitney U test (a–c) and Wilcoxon test for paired values (d–f). Box plots with median and interquartile range. Full article ">Figure 2
Positive correlations between depression severity assessed by HAM-D (a–c), MADRS (d–f), and STAI trait (g–i) with peripheral gene expressions of SNCA, GBA, and UGCG in patients with remitted major depressive disorder (PR) separated in female (red dots) and male (blue dots) subgroups at inclusion. Linear regression line for the combined group with 95% confidence interval and statistics (Spearman correlation, in bold for p < 0.05). Sex-stratified statistical data are in <a href="#ijms-25-03219-t003" class="html-table">Table 3</a>. Full article ">

14 pages, 1977 KiB

Open AccessArticle

Interaction of Fabry Disease and Diabetes Mellitus: Suboptimal Recruitment of Kidney Protective Factors

by Maria D. Sanchez-Niño, Maria I. Ceballos, Sol Carriazo, Aranzazu Pintor-Chocano, Ana B. Sanz, Moin A. Saleem and Alberto Ortiz

Int. J. Mol. Sci. 2023, 24(21), 15853; https://doi.org/10.3390/ijms242115853 - 1 Nov 2023

Viewed by 1661

Abstract

Fabry disease is a lysosomal disease characterized by globotriaosylceramide (Gb3) accumulation. It may coexist with diabetes mellitus and both cause potentially lethal kidney end-organ damage. However, there is little information on their interaction with kidney disease. We have addressed the interaction between Fabry [...] Read more.

Fabry disease is a lysosomal disease characterized by globotriaosylceramide (Gb3) accumulation. It may coexist with diabetes mellitus and both cause potentially lethal kidney end-organ damage. However, there is little information on their interaction with kidney disease. We have addressed the interaction between Fabry disease and diabetes in data mining of human kidney transcriptomics databases and in Fabry (Gla-/-) and wild type mice with or without streptozotocin-induced diabetes. Data mining was consistent with differential expression of genes encoding enzymes from the Gb3 metabolic pathway in human diabetic kidney disease, including upregulation of UGCG, the gene encoding the upstream and rate-limiting enzyme glucosyl ceramide synthase. Diabetic Fabry mice displayed the most severe kidney infiltration by F4/80+ macrophages, and a lower kidney expression of kidney protective genes (Pgc1α and Tfeb) than diabetic wild type mice, without a further increase in kidney fibrosis. Moreover, only diabetic Fabry mice developed kidney insufficiency and these mice with kidney insufficiency had a high expression of Ugcg. In conclusion, we found evidence of interaction between diabetes and Fabry disease that may increase the severity of the kidney phenotype through modulation of the Gb3 synthesis pathway and downregulation of kidney protective genes. Full article

(This article belongs to the Section Molecular Endocrinology and Metabolism)

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Impact of diabetes on genes encoding enzymes in the Gb3 pathway and on kidney function in wild type and Fabry mice. (a) Scheme of Gb3 synthesis and metabolism: glucosylceramide synthase encoded by Ugcg, Gb3 synthase encoded by A4galt, acid ceramidase encoded by Asah1, and alpha-galactosidase A encoded by Gla. Arrows present a summary of data shown in panels (b–d). (b–d) Kidney mRNA was measured by real time RT-PCR in diabetic wild-type or Fabry mice or vehicle controls. (b) Ugcg, (c) A4galt, (d) Asah1. (e) Plasma creatinine and (f) plasma urea. Data expressed as mean ± SEM of 5–15 animals per group. * p < 0.05 vs. respective vehicle group. Significance (p < 0.05) was assessed by Student’s t-test for two groups of data and ANOVA for three or more groups with Bonferroni post-hoc correction. Full article ">Figure 2
Impact of diabetes on kidney inflammation in Fabry mice. (a) Mcp1, (b) Rantes and (c) Fn14 mRNA expression in kidneys was measured by real time RT-PCR. (d) Correlation between serum urea and kidney Fn14 mRNA expression as assessed by linear regression. (e) Immunohistochemistry assessment of F4/80+ macrophages: representative images and quantitation. * p < 0.05 vs. vehicle WT. ** p < 0.05 vs. vehicle Fabry.Original magnification ×20. Data expressed as mean ± SEM of 5–15 animals per group. Significance (p < 0.05) was assessed by Student’s t-test for two groups of data and ANOVA for three or more groups with Bonferroni post-hoc correction. Full article ">Figure 3
Impact of diabetes on kidney fibrosis in Fabry mice. (a) Kidney Col1a2 and (b) Fn1 mRNA expression was measured by real time RT-PCR. (c) Sirius red staining for collagen deposition: representative images and quantitation. (d) Immunohistochemistry disclosed milder fibronectin deposition in diabetic Fabry mice than in diabetic WT mice. Original magnification ×20. * p < 0.05 vs. vehicle WT. ** p < 0.01 vs. WT diabetic, *** p < 0.0005 vs. WT diabetic. Data expressed as mean ± SEM of 5–15 animals per group. Significance (p < 0.05) was assessed by Student’s t-test for two groups of data and ANOVA for three or more groups with Bonferroni post-hoc correction. Full article ">Figure 4
Impact of diabetes on the expression of kidney nephroprotective genes in Fabry mice. (a) Kidney Klotho mRNA, (b) Pgc1α mRNA, (c) Tfeb mRNA. * p < 0.05 vs. WT vehicle, ** p < 0.05 vs. WT diabetic. Data expressed as mean ± SEM of 5–15 animals per group. Significance (p < 0.05) was assessed by Student’s t-test for two groups of data and ANOVA for three or more groups with Bonferroni post-hoc correction. Full article ">

13 pages, 865 KiB

Open AccessReview

Update on Glycosphingolipids Abundance in Hepatocellular Carcinoma

by Frances L. Byrne, Ellen M. Olzomer, Nina Lolies, Kyle L. Hoehn and Marthe-Susanna Wegner

Int. J. Mol. Sci. 2022, 23(9), 4477; https://doi.org/10.3390/ijms23094477 - 19 Apr 2022

Cited by 5 | Viewed by 2638

Abstract

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer. Low numbers of HCC patients being suitable for liver resection or transplantation and multidrug resistance development during pharmacotherapy leads to high death rates for HCC patients. Understanding the molecular mechanisms of [...] Read more.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer. Low numbers of HCC patients being suitable for liver resection or transplantation and multidrug resistance development during pharmacotherapy leads to high death rates for HCC patients. Understanding the molecular mechanisms of HCC etiology may contribute to the development of novel therapeutic strategies for prevention and treatment of HCC. UDP-glucose ceramide glycosyltransferase (UGCG), a key enzyme in glycosphingolipid metabolism, generates glucosylceramide (GlcCer), which is the precursor for all glycosphingolipids (GSLs). Since UGCG gene expression is altered in 0.8% of HCC tumors, GSLs may play a role in cellular processes in liver cancer cells. Here, we discuss the current literature about GSLs and their abundance in normal liver cells, Gaucher disease and HCC. Furthermore, we review the involvement of UGCG/GlcCer in multidrug resistance development, globosides as a potential prognostic marker for HCC, gangliosides as a potential liver cancer stem cell marker, and the role of sulfatides in tumor metastasis. Only a limited number of molecular mechanisms executed by GSLs in HCC are known, which we summarize here briefly. Overall, the role GSLs play in HCC progression and their ability to serve as biomarkers or prognostic indicators for HCC, requires further investigation. Full article

(This article belongs to the Section Molecular Oncology)

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GSLs and HCC related publications and ceramide concentrations in glycosphingolipid-enriched microdomains (GEMs) in NMuLi/UGCG OE and control cells. (A) Publications identified by the key word combinations HCC glucosylceramide, HCC glycosphingolipids, Gaucher HCC, Gaucher liver cancer, liver cancer glycosphingolipids, hepatocellular carcinoma gangliosides, hepatocellular carcinoma globosides, hepatocellular carcinoma hexosylceramide, and hepatocellular carcinoma lactosylceramide between 1967 and 2021 [<a href="#B17-ijms-23-04477" class="html-bibr">17</a>]. Notably, the majority of these studies were identified by the keywords liver, cancer, and glycosphingolipids. Blue bars represent years 2010 to 2021 (increase in published studies). (B) GEMs were isolated by sucrose density centrifugation and GEMs verified by cholesterol and GlcCer concentration determination. NMuLi/EV-2 = empty vector control; NMuLi/UGCG OE = UGCG overexpressing cells. The GEM verification for these samples is published in [<a href="#B18-ijms-23-04477" class="html-bibr">18</a>]. Data are represented as a mean of n = 3 ± SEM. Unpaired t test with Welch’s correction. ** p < 0.01. Full article ">Figure 2
Schematic overview of ganglioside-mediated effects in HCC. (A) Ganglioside-mediated effects on cell migration. (B) Ganglioside-mediated effects on cancer stem cells and immune response. Full article ">

16 pages, 3066 KiB

Open AccessArticle

Inhibition of Ceramide Glycosylation Enhances Cisplatin Sensitivity in Cholangiocarcinoma by Limiting the Activation of the ERK Signaling Pathway

by Piyasiri Chueakwon, Peeranat Jatooratthawichot, Krajang Talabnin, James R. Ketudat Cairns and Chutima Talabnin

Life 2022, 12(3), 351; https://doi.org/10.3390/life12030351 - 28 Feb 2022

Cited by 5 | Viewed by 2825

Abstract

Cholangiocarcinoma (CCA) is an aggressive tumor of the biliary epithelium with poor survival that shows limited response to conventional chemotherapy. Increased expression of glucosylceramide synthase (GCS) contributes to drug resistance and the progression of various cancers; the expression profiles of GCS (UGCG) and [...] Read more.

Cholangiocarcinoma (CCA) is an aggressive tumor of the biliary epithelium with poor survival that shows limited response to conventional chemotherapy. Increased expression of glucosylceramide synthase (GCS) contributes to drug resistance and the progression of various cancers; the expression profiles of GCS (UGCG) and the genes for glucocerebrosidases 1, 2, and 3 (GBA1, GBA2, and GBA3) were therefore studied in CCA. The biological functions of GCS for cell proliferation and cisplatin sensitivity in CCA were explored. GCS expression was higher in CCA tumor tissues than that of GBA1, GBA2, and GBA3. Verification of GCS expression in 29 paired frozen CCA tissues showed that 8 of 29 cases (27.6%) had high GCS expression. The expression of GCS and GBA2 was induced in CCA cell lines following low-dose cisplatin treatment. Suppression of GCS by either palmitoylamino-3-morpholino-1-propanol (PPMP), GCS knockdown or a combination of the two resulted in reduced cell proliferation. These treatments enhanced the effect of cisplatin-induced CCA cell death, increased the expression of apoptotic proteins and reduced phosphorylation of ERK upon cisplatin treatment. Taken together, inhibition of the GCS increased cisplatin-induced CCA apoptosis via the inhibition of the ERK signaling pathway. Thus, targeting GCS might be a strategy for CCA treatment. Full article

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Expression of ceramide-metabolizing enzymes in paired CCA tissues. (A) Diagram of the sphingolipids biosynthesis pathways. Expression data were obtained from the GEO database (GSE76297 dataset) for a comparison of: (B) GCS mRNA expression, (C) GBA1 mRNA expression, (D) GBA2 mRNA expression and (E) GBA3 mRNA expression. The Mann-Whitney was used to compare the expression between tumor (n = 92) and non-tumor tissues (n = 91) as well as paired tissues. ***, p < 0.001 versus non-tumor tissues. Full article ">Figure 2
GCS expression in 29 paired frozen CCA tissues. (A) GCS mRNA expression was determined by qPCR. (B) Cut-off at 1.5-fold change (tumor/adjacent normal) was used for dichotomized GCS mRNA expression in 2 groups. Cut-off value ≤ 1.5-fold change and > 1.5-fold change denoted as low and high GCS expression, respectively. (C) Kaplan-Meier analysis of overall survival in low and high GCS cases. Log Rank test was used for survival analysis. *, p < 0.05 versus adjacent normal tissues. Full article ">Figure 3
Changes of ceramide-metabolizing enzymes upon cisplatin treatment. (A) Respective basal mRNA expression of GCS, GBA1, and GBA2 were determined in two CCA cell lines (KKU-100 and KKU-213A). (B) KKU-100 and KKU-213A were treated with cisplatin at 0, 10, 20, 40, and 60 µM for 24 and 48 h. (C) Respective mRNA expression of GCS, GBA2, and GCS/GBA2 ratio was determined in KKU-100 and KKU-213A after 24 h of cisplatin treatment. Values are expressed as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01 versus cisplatin at 0 µM. ND: not detected. Full article ">Figure 4
Suppression of GCS reduces CCA cell growth. (A) mRNA and protein levels of GCS after suppression by siRNAs for 24 and 48 h. (B) Cell proliferation after GCS suppression by siRNAs for 0–48 h, (C) PPMP (GCS inhibitor), and (D) co-treatment by siRNAs and PPMP. (E,F) The relative mRNA expression of growth-related genes in siGCS-treated KKU-213A or PPMP-treated KKU-213A were determined using qPCR. Values are expressed as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 versus siCTRL. The original unedited blot was presented in <a href="#app1-life-12-00351" class="html-app">Figure S1</a>. Full article ">Figure 5
Suppression of GCS enhances cisplatin sensitivity. (A) Inducible mRNA expression of GCS following low-dose cisplatin (0, 10, 20, and 40 μM). Cell viability of KKU-213A was determined after (B) GCS inhibition by siRNAs for 24 h and treated with cisplatin for 48 h, (C) GCS inhibition by PPMP for 24 h and in combination with cisplatin for 48 h, and (D) co-treatment of GCS with siRNAs and PPMP for 24 h plus cisplatin for 48 h. Values are expressed as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 versus cisplatin at 0 µM. Full article ">Figure 6
Suppression of GCS induces CCA cell apoptosis via the attenuation of ERK activation. KKU-213A was exposed by PPMP alone (10 µM), cisplatin alone (10 or 20 µM), or the combination of both for 24 h. Protein expression potentially involved (A) apoptosis and (B) survival signaling pathway observed by Western blot assay. Relative protein expression was measured by Image J software (version 1.53a). Values are expressed as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 versus cisplatin at 0 µM. # p < 0.05; ## p < 0.01; ### p < 0.001 versus cisplatin at 10 or 20 µM. The original unedited blot was presented in <a href="#app1-life-12-00351" class="html-app">Figure S2</a>. Full article ">

19 pages, 2832 KiB

Open AccessFeature PaperArticle

The Sphingolipid Asset Is Altered in the Nigrostriatal System of Mice Models of Parkinson’s Disease

by Victor Blokhin, Maria Shupik, Ulyana Gutner, Ekaterina Pavlova, Albert T. Lebedev, Olga Maloshitskaya, Vsevolod Bogdanov, Sergey Sokolov, Alice Alessenko and Michael Ugrumov

Biomolecules 2022, 12(1), 93; https://doi.org/10.3390/biom12010093 - 6 Jan 2022

Cited by 4 | Viewed by 2371

Abstract

Parkinson’s disease (PD) is a neurodegenerative disease incurable due to late diagnosis and treatment. Therefore, one of the priorities of neurology is to study the mechanisms of PD pathogenesis at the preclinical and early clinical stages. Given the important role of sphingolipids in [...] Read more.

Parkinson’s disease (PD) is a neurodegenerative disease incurable due to late diagnosis and treatment. Therefore, one of the priorities of neurology is to study the mechanisms of PD pathogenesis at the preclinical and early clinical stages. Given the important role of sphingolipids in the pathogenesis of neurodegenerative diseases, we aimed to analyze the gene expression of key sphingolipid metabolism enzymes (ASAH1, ASAH2, CERS1, CERS3, CERS5, GBA1, SMPD1, SMPD2, UGCG) and the content of 32 sphingolipids (subspecies of ceramides, sphingomyelins, monohexosylceramides and sphinganine, sphingosine, and sphingosine-1-phosphate) in the nigrostriatal system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models of the preclinical and clinical stages of PD. It has been shown that in PD models, the expression of five of the nine studied genes (CERS1, CERS5, ASAH1, ASAH2, and GBA1) increases but only in the substantia nigra (SN) containing dopaminergic cell bodies. Changes in the expression of enzyme genes were accompanied by an increase in the content of 7 of the 32 studied sphingolipids. Such findings suggest these genes as attractive candidates for diagnostic purposes for preclinical and clinical stages of PD. Full article

(This article belongs to the Section Biomacromolecules: Lipids)

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22 pages, 10787 KiB

Open AccessArticle

Blockade of Glycosphingolipid Synthesis Inhibits Cell Cycle and Spheroid Growth of Colon Cancer Cells In Vitro and Experimental Colon Cancer Incidence In Vivo

by Richard Jennemann, Martina Volz, Felix Bestvater, Claudia Schmidt, Karsten Richter, Sylvia Kaden, Johannes Müthing, Hermann-Josef Gröne and Roger Sandhoff

Int. J. Mol. Sci. 2021, 22(19), 10539; https://doi.org/10.3390/ijms221910539 - 29 Sep 2021

Cited by 11 | Viewed by 3075

Abstract

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is [...] Read more.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC. Full article

(This article belongs to the Special Issue Activity, Function and Druggability of Cancer-Related Enzymes)

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17 pages, 1155 KiB

Open AccessReview

Roles of Gangliosides in Hypothalamic Control of Energy Balance: New Insights

by Kei-ichiro Inamori and Jin-ichi Inokuchi

Int. J. Mol. Sci. 2020, 21(15), 5349; https://doi.org/10.3390/ijms21155349 - 28 Jul 2020

Cited by 10 | Viewed by 3134

Abstract

Gangliosides are essential components of cell membranes and are involved in a variety of physiological processes, including cell growth, differentiation, and receptor-mediated signal transduction. They regulate functions of proteins in membrane microdomains, notably receptor tyrosine kinases such as insulin receptor (InsR) and epidermal [...] Read more.

Gangliosides are essential components of cell membranes and are involved in a variety of physiological processes, including cell growth, differentiation, and receptor-mediated signal transduction. They regulate functions of proteins in membrane microdomains, notably receptor tyrosine kinases such as insulin receptor (InsR) and epidermal growth factor receptor (EGFR), through lateral association. Studies during the past two decades using knockout (KO) or pharmacologically inhibited cells, or KO mouse models for glucosylceramide synthase (GCS; Ugcg), GM3 synthase (GM3S; St3gal5), and GD3 synthase (GD3S; St8sia1) have revealed essential roles of gangliosides in hypothalamic control of energy balance. The a-series gangliosides GM1 and GD1a interact with leptin receptor (LepR) and promote LepR signaling through activation of the JAK2/STAT3 pathway. Studies of GM3S KO cells have shown that the extracellular signal-regulated kinase (ERK) pathway, downstream of the LepR signaling pathway, is also modulated by gangliosides. Recent studies have revealed crosstalk between the LepR signaling pathway and other receptor signaling pathways (e.g., InsR and EGFR pathways). Gangliosides thus have the ability to modulate the effects of leptin by regulating functions of such receptors, and by direct interaction with LepR to control signaling. Full article

(This article belongs to the Section Molecular Neurobiology)

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3071 KiB

Open AccessArticle

Role of Intracellular Lipid Logistics in the Preferential Usage of Very Long Chain-Ceramides in Glucosylceramide

by Toshiyuki Yamaji, Aya Horie, Yuriko Tachida, Chisato Sakuma, Yusuke Suzuki, Yasunori Kushi and Kentaro Hanada

Int. J. Mol. Sci. 2016, 17(10), 1761; https://doi.org/10.3390/ijms17101761 - 21 Oct 2016

Cited by 19 | Viewed by 5313

Abstract

Ceramide is a common precursor of sphingomyelin (SM) and glycosphingolipids (GSLs) in mammalian cells. Ceramide synthase 2 (CERS2), one of the six ceramide synthase isoforms, is responsible for the synthesis of very long chain fatty acid (C20–26 fatty acids) (VLC)-containing ceramides (VLC-Cer). It [...] Read more.

Ceramide is a common precursor of sphingomyelin (SM) and glycosphingolipids (GSLs) in mammalian cells. Ceramide synthase 2 (CERS2), one of the six ceramide synthase isoforms, is responsible for the synthesis of very long chain fatty acid (C20–26 fatty acids) (VLC)-containing ceramides (VLC-Cer). It is known that the proportion of VLC species in GSLs is higher than that in SM. To address the mechanism of the VLC-preference of GSLs, we used genome editing to establish three HeLa cell mutants that expressed different amounts of CERS2 and compared the acyl chain lengths of SM and GSLs by metabolic labeling experiments. VLC-sphingolipid expression was increased along with that of CERS2, and the proportion of VLC species in glucosylceramide (GlcCer) was higher than that in SM for all expression levels of CERS2. This higher proportion was still maintained even when the proportion of C16-Cer to the total ceramides was increased by disrupting the ceramide transport protein (CERT)-dependent C16-Cer delivery pathway for SM synthesis. On the other hand, merging the Golgi apparatus and the endoplasmic reticulum (ER) by Brefeldin A decreased the proportion of VLC species in GlcCer probably due to higher accessibility of UDP-glucose ceramide glucosyltransferase (UGCG) to C16-rich ceramides. These results suggest the existence of a yet-to-be-identified mechanism rendering VLC-Cer more accessible than C16-Cer to UGCG, which is independent of CERT. Full article

(This article belongs to the Section Biochemistry)

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Full article ">Figure 1
Isolation of CERS2-deficient clones. (A) The target site of the TALEN-CERS2 pair in the human CERS2 gene and indel analysis in TALEN-CERS2–treated (TAL-CERS2) clones. The target sequences (CERS2 Fw and Rv) are located in intron 1 and exon 2, respectively. The sequence between the TALEN target sequences contains the start codon (yellow box) and the splicing acceptor (SA) at the end of intron 1 (blue lined box). Green boxes highlight the portions corresponding to the start codon and the SA. Deletions are shown in red and their lengths are specified on the right of the sequences; (B) Western blot analysis of CERS2 in the TAL-CERS2 clones. Cell lysates from the parent cells and CERS2-deficient mutants were immunoblotted with anti-CERS2 antibody (top) and anti-α-tubulin antibody (middle). Part of the enlarged view of the top image is shown at the bottom. The arrow and arrowhead indicate CERS2; P, parent cell line; 16, TAL-CERS2#16 clone; 18, TAL-CERS2#18 clone; 13, TAL-CERS2#13 clone; (-), no transfection; M, mock; and C2, CERS2 cDNA restoration. Note that overexpression of CERS2 resulted in formation of high molecular weight aggregates. Full article ">Figure 2
Metabolic labeling of lipids in TAL-CERS2 clones. (A) Metabolic labeling of sphingolipids with radioactive serine in the parent cells and TAL-CERS2 clones shown in <a href="#ijms-17-01761-f001" class="html-fig">Figure 1</a>B. Cells were cultured with [14C]serine for 16 h, and lipids extracted from the cells were separated by normal-phase thin-layer chromatography (TLC) (Method 1). Radioactive image of an analyzed TLC plate is shown. Cer, ceramide; GalCer, galactosylceramide; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine. P, parent cell line; 16, TAL-CERS2#16 clone; 18, TAL-CERS2#18 clone; 13, TAL-CERS2#13 clone; (-), no transfection; M, mock; C2, CERS2 cDNA restoration. The darker image is shown in <a href="#app1-ijms-17-01761" class="html-app">Figure S3A</a>; (B) comparison of the proportion of the upper band intensities (indicating the proportion of very long chain (VLC)) of GlcCer and sphingomyelin (SM). The enlarged and darker images of [14C]serine-labeled GlcCer and SM shown in (A) are aligned. The proportions of VLC-containing lipids are expressed as the upper (VLC-containing) band intensity as a percentage of the sum of the upper (VLC) and lower (C16 long chain fatty acid (FA) (LC)-containing) band intensities of GlcCer and SM for each cell line shown in <a href="#ijms-17-01761-f001" class="html-fig">Figure 1</a>B: mean percentage ± standard deviation (SD) obtained from five independent experiments. The Student’s t-test was used. * p < 0.05. Full article ">Figure 3
Different fatty acid preferences of GlcCer and SM. (A,B) Metabolic labeling of sphingolipids with radioactive serine for a short time. Cells were cultured with [14C]serine for 2 h, and lipids extracted from the cells were separated by TLC. (A) Separation between upper (VLC-containing) and lower (LC-containing) bands of ceramide by normal-phase TLC (Method 3 described in the “Experimental Section”). Non-radiolabeled ceramide standards (C16:0, C24:1, and C24:0) were developed simultaneously and visualized by iodine vapor. The positions of the spot origin (ori) and the solvent front (top) in two images were aligned; (B) separation of upper and lower bands of GlcCer and SM by normal-phase TLC according to Method 1. Radioactive images of analyzed TLC plates are shown. The darker image is shown in <a href="#app1-ijms-17-01761" class="html-app">Figure S6B</a>; (C) comparison of the proportion of upper band intensities (indicating the proportion of VLC) of ceramide, GlcCer, and SM shown in (A,B). The enlarged and darker images of [14C]serine-labeled ceramide, GlcCer, and SM shown in (A,B) are aligned. The proportions of VLC-containing lipids are expressed as described in <a href="#ijms-17-01761-f002" class="html-fig">Figure 2</a>B: mean percentage ± SD obtained from three independent experiments. The Bonferroni corrected Student’s t-test was used for multiple comparisons. * p < 0.017. Full article ">Figure 4
CERT gene disruption by the TALEN–CERT pair in the TAL-CERS2#18 clone. (A) The target site of the TALEN–CERT pair in the human CERT gene and indel analysis of the TAL-CERS2#18-CERT#10 (#18-10) clones. The target sequences (CERT Fw and Rv) are located in exon 2, which codes for part of the pleckstrin homology (PH) domain. Deletions and replacements are shown in red and their lengths are specified on the right of the sequences. WT, wild type; (B) Western blot analysis of CERT in the TAL-CERS2#18 clones. Cell lysates from the parent cells and the indicated mutants were immunoblotted with anti-CERT antibody (top) and anti-α-tubulin antibody (bottom). The arrow indicates CERT at 68 kD. P, parent cell line; 18, TAL-CERS2#18 clone; 18-10, TAL-CERS2#18-CERT#10 clone. Full article ">Figure 5
Effects of CERT disruption and Brefeldin A (BFA) treatment on the proportion of VLC species in sphingolipids. (A,B) Metabolic labeling of sphingolipids with radioactive serine in the parent cells (P), TAL-CERS2#18 clone (#18), and TAL-CERS2#18-CERT#10 clone (#18-10). Cells were cultured with [14C]serine in the presence (+) or absence (-) of BFA for 2 h, and lipids extracted from the cells were separated by Method 1 for GlcCer and SM in (A) and Method 3 for ceramide analysis in (B). The darker images are shown in <a href="#app1-ijms-17-01761" class="html-app">Figures S7A and S7C</a>; (C) comparison of the proportion of VLC of ceramide, GlcCer, and SM shown in (A,B). The method used is as described in <a href="#ijms-17-01761-f002" class="html-fig">Figure 2</a>B. The enlarged and darker images of [14C]serine-labeled ceramide, GlcCer, and SM shown in (A,B) are aligned. The proportions of VLC-containing lipids are expressed as described in <a href="#ijms-17-01761-f002" class="html-fig">Figure 2</a>B: mean percentage ± SD obtained from three independent experiments. The mean percentage in the TAL-CERS2#18 clone was compared with that in the TAL-CERS2#18-CERT#10 clone (#18-10) and that in the BFA-treated TAL-CERS2#18 clone, and the Bonferroni corrected Student’s t-test was used for the multiple comparisons. * p < 0.025. Full article ">Figure 6
Effect of UGCG overexpression on the proportion of VLC in GlcCer. (A) Western blot analysis of UGCG in UGCG-overexpressed TAL-CERS2#18 cells. Cell lysates from TAL-CERS2#18 cells and UGCG-overexpressed TAL-CERS2#18 cells were immunoblotted with anti-HA antibody (HA), anti-UGCG antibody (top), and anti-α-tubulin antibody (bottom). 18, TAL-CERS2#18 clone; UGCG, UGCG overexpression. The whole images are shown in <a href="#app1-ijms-17-01761" class="html-app">Figure S8</a>. (-), no transfection; UGCG, UGCG cDNA overexpression; (B) metabolic labeling of sphingolipids with radioactive serine in TAL-CERS2#18 and UGCG-overexpressed TAL-CERS2#18 cells. Cells were cultured with [14C]serine for 2 h, and lipids extracted from the cells were separated by Method 1 for GlcCer; (C) comparison of the proportions of VLC-GlcCer between endogenous GlcCer in TAL-CERS2#18 cells and overexpressed GlcCer in UGCG-overexpressed TAL-CERS2#18 cells. The proportions of VLC-containing GlcCer are expressed as described in <a href="#ijms-17-01761-f002" class="html-fig">Figure 2</a>B: mean percentage ± SD obtained from three independent experiments. The Student’s t-test was used. * p < 0.05. Full article ">

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