118 research outputs found
Sphingosine kinase 1/S1P receptor signaling axis controls glial proliferation in mice with Sandhoff disease
No full textSphingosine-1-phosphate (S1P) is a lipid-signaling molecule produced by sphingosine kinase in response to a wide number of stimuli. By acting through a family of widely expressed G protein-coupled receptors, S1P regulates diverse physiological processes. Here we examined the role of S1P signaling in neurodegeneration using a mouse model of Sandhoff disease, a prototypical neuronopathic lysosomal storage disorder. When sphingosine kinase 1 (Sphk1) was deleted in Sandhoff disease mice, a milder disease course occurred, with decreased proliferation of glial cells and less-pronounced astrogliosis. A similar result of milder disease course and reduced astroglial proliferation was obtained by deletion of the gene for the S1P3 receptor, a G protein-coupled receptor enriched in astrocytes. Our studies demonstrate a functional role of S1P synthesis and receptor expression in astrocyte proliferation leading to astrogliosis during the terminal stages of neurodegeneration in Sandhoff disease mice. Because astrocyte responses are involved in many types of neurodegeneration, the Sphk1/S1P receptor signaling axis may be generally important during the pathogen-esis of neurodegenerative diseases
Natural history study of glycan accumulation in large animal models of GM2 gangliosidoses
No full textbeta-hexosaminidase is an enzyme responsible for the degradation of gangliosides, glycans, and other glycoconjugates containing beta-linked hexosamines that enter the lysosome. GM2 gangliosidoses, such as Tay-Sachs and Sandhoff, are lysosomal storage disorders characterized by beta-hexosaminidase deficiency and subsequent lysosomal accumulation of its substrate metabolites. These two diseases result in neurodegeneration and early mortality in children. A significant difference between these two disorders is the accumulation in Sandhoff disease of soluble oligosaccharide metabolites that derive from N- and O-linked glycans. In this paper we describe our results from a longitudinal biochemical study of a feline model of Sandhoff disease and an ovine model of Tay-Sachs disease to investigate the accumulation of GM2/GA2 gangliosides, a secondary biomarker for phospholipidosis, bis-(monoacylglycero)-phosphate, and soluble glycan metabolites in both tissue and fluid samples from both animal models. While both Sandhoff cats and Tay-Sachs sheep accumulated significant amounts of GM2 and GA2 gangliosides compared to age-matched unaffected controls, the Sandhoff cats having the more severe disease, accumulated larger amounts of gangliosides compared to Tay-Sachs sheep in their occipital lobes. For monitoring glycan metabolites, we developed a quantitative LC/MS assay for one of these free glycans in order to perform longitudinal analysis. The Sandhoff cats showed significant disease-related increases in this glycan in brain and in other matrices including urine which may provide a useful clinical tool for measuring disease severity and therapeutic efficacy. Finally, we observed age-dependent increasing accumulation for a number of analytes, especially in Sandhoff cats where glycosphingolipid, phospholipid, and glycan levels showed incremental increases at later time points without signs of peaking. This large animal natural history study for Sandhoff and Tay-Sachs is the first of its kind, providing insight into disease progression at the biochemical level. This report may help in the development and testing of new therapies to treat these disorders
Very long chain sphingolipids: Tissue expression, function and synthesis
No full textAbstractForecort membranes of all mammalian cells contain, in addition to phosphoglycerolipids and cholesterol, substantial amounts of sphingolipids. In most cells the acyl moieties of these sphingolipids are of long chain type (C16–24) and often saturated. However, epidermal keratinocytes and male germ cells largely express sphingolipids with very long chain-acyl moieties (C26–C36) during their differentiation and maturation. This expression seems to depend on CerS3, one of six ceramide synthases. However, the complex biosynthetic sequence for both epidermal and testicular sphingolipids has not been elucidated completely. Whereas it is established that omega-hydroxylated very long chain-sphingolipids are essential for proper skin barrier function, the role of polyunsaturated very long chain-sphingolipids (ceramides, sphingomyelins and glycosphingolipids) in differentiating spermatocytes/spermatids is just beginning to be revealed
Natural history study of glycan accumulation in large animal models of GM2 gangliosidoses.
No full textβ-hexosaminidase is an enzyme responsible for the degradation of gangliosides, glycans, and other glycoconjugates containing β-linked hexosamines that enter the lysosome. GM2 gangliosidoses, such as Tay-Sachs and Sandhoff, are lysosomal storage disorders characterized by β-hexosaminidase deficiency and subsequent lysosomal accumulation of its substrate metabolites. These two diseases result in neurodegeneration and early mortality in children. A significant difference between these two disorders is the accumulation in Sandhoff disease of soluble oligosaccharide metabolites that derive from N- and O-linked glycans. In this paper we describe our results from a longitudinal biochemical study of a feline model of Sandhoff disease and an ovine model of Tay-Sachs disease to investigate the accumulation of GM2/GA2 gangliosides, a secondary biomarker for phospholipidosis, bis-(monoacylglycero)-phosphate, and soluble glycan metabolites in both tissue and fluid samples from both animal models. While both Sandhoff cats and Tay-Sachs sheep accumulated significant amounts of GM2 and GA2 gangliosides compared to age-matched unaffected controls, the Sandhoff cats having the more severe disease, accumulated larger amounts of gangliosides compared to Tay-Sachs sheep in their occipital lobes. For monitoring glycan metabolites, we developed a quantitative LC/MS assay for one of these free glycans in order to perform longitudinal analysis. The Sandhoff cats showed significant disease-related increases in this glycan in brain and in other matrices including urine which may provide a useful clinical tool for measuring disease severity and therapeutic efficacy. Finally, we observed age-dependent increasing accumulation for a number of analytes, especially in Sandhoff cats where glycosphingolipid, phospholipid, and glycan levels showed incremental increases at later time points without signs of peaking. This large animal natural history study for Sandhoff and Tay-Sachs is the first of its kind, providing insight into disease progression at the biochemical level. This report may help in the development and testing of new therapies to treat these disorders
Komplexní analýza glykosfingolipidů v biologických vzorcích pomocí HPLC/MS
No full textLipids are biomolecules found in all living organisms, where they have several vital functions. Lipids are the subject of lipidomics, a subgroup of metabolomics, which represents one of the so-called ?omics? disciplines that also includes genomics, proteomics, and glycomics. Dysregulation of lipid metabolism can reflect the onset of various diseases including cancer, therefore, lipidomic analysis can provide valuable information about ongoing pathophysiological processes in humans. The theoretical part provides an extensive overview of glycosphingolipids, including their biosynthesis, structure complexity, classification, nomenclature, and various biological functions in association with pathophysiological conditions as a hallmark of a variety of diseases, for example, cancer. Special attention is also paid to the sample preparation and key analytical methods for their identifications and quantitation. Qualitative and quantitative approaches are discussed as well, along with their advantages and limitations. The experimental part deals with the development, optimization, and application of analytical methods for the analysis of especially glycosphingolipids in biological samples, such as human plasma/serum and tissues using chromatographic techniques coupled to mass spectrometry. Special attention is devoted to in-depth structural characterization and qualitative glycosphingolipids profiling using a lipid class separation approach (HILIC). Correspondingly, glycosphingolipid profiling is used to expand the database of lipids that are routinely analyzed, and for the mutual comparison of healthy volunteers and cancer patients. Therefore, the analysis of glycosphingolipids has the potential to facilitate the discovery of novel biomarkers for the early detection of various diseases.Lipidy jsou biomolekuly, které se vyskytují ve všech živých organismech, kde plní několik životně důležitých funkcí. Lipidy jsou předmětem zkoumání vědního oboru zvaného lipidomika patřící do podskupiny metabolomiky, jež představuje jednu z omických disciplín, mezi které patří také genomika, proteomika a glykomika. Dysregulace metabolismu lipidů může odrážet nástup a rozvoj různých onemocnění včetně rakoviny, lipidomická analýza tedy může poskytnout cenné informace o probíhajících patofyziologických procesech u lidí. Teoretická část práce poskytuje rozsáhlý přehled o glykosfingolipidech, jejich biosyntéze, strukturní složitosti, klasifikaci, názvosloví a nejrůznějších biologických funkcí ve spojení s patofyziologickými stavy, které jsou charakteristickým znakem různých onemocnění zahrnující i rakovinu. Zvláštní pozornost je také věnována přípravě vzorků a klíčovým analytickým metodám umožňující jejich identifikaci a kvantifikaci. Rovněž jsou zde diskutovány kvalitativní a kvantitativní přístupy spolu s jejich výhodami a omezeními. Experimentální část se zabývá vývojem, optimalizací a aplikací analytických metod pro analýzu zejména glykosfingolipidů v biologických vzorcích jako je lidská plasma/sérum a tkáně, a to za použití chromatografických technik ve spojení s hmotnostní spektrometrií. Zvláštní pozornost je dedikována důkladné strukturní charakterizaci a profilování glykosfingolipidů za použití přístupu separace lipidových tříd (HILIC). V souladu s tím je profilování glykosfingolipidů použito k rozšíření databáze běžně analyzovaných lipidů a ke vzájemnému porovnání rozdílů mezi profily lipidů zzdravých dobrovolníků a pacientů s rakovinou. Lipidomická analýza glykosfingolipidů má tudíž potenciál umožnit a usnadnit objev nových biomarkerů pro včasnou detekci různých onemocnění včetně rakoviny.Fakulta chemicko-technologickáDokončená práce s úspěšnou obhajobo
Intracerebroventricular administration of a modified hexosaminidase ameliorates late-stage neurodegeneration in a GM2 mouse model.
No full textThe GM2 gangliosidoses, Tay-Sachs disease and Sandhoff disease, are devastating neurodegenerative disorders caused by β-hexosaminidase A (HexA) deficiency. In the Sandhoff disease mouse model, rescue potential was severely reduced when HexA was introduced after disease onset. Here, we assess the effect of recombinant HexA and HexD3, a newly engineered mimetic of HexA optimized for the treatment of Tay-Sachs disease and Sandhoff disease. Enzyme replacement therapy was administered by repeat intracerebroventricular injections in Sandhoff disease model mice with dosing beginning before and after signs of neurodegeneration. As previously observed, HexA effectively increased the lifespan of Sandhoff disease mice by 3.5-fold only when treatment was started before onset of neurodegeneration. In contrast, HexD3 halted motor decline and ameliorated late-stage disease severity even when dosing began late, after neurodegeneration onset. Additionally, HexD3 had advantages over HexA in enzyme stability, distribution potential, and homodimer activity. Overall, our data indicate that advanced therapeutics may widen the treatment window for neurodegenerative disorders
The SGLT2 Inhibitor Dapagliflozin Disrupts the Cell Cycle at High Concentrations Without Altering Glycosphingolipid (De Novo)Biosynthesis
No full textModern computational screening methods are valuable tools for repurposing approved drugs for novel therapeutic applications. They provide initial insights into alternative uses and may significantly shorten the lengthy process of drug development and regulatory approval. Treatment options for glycosphingolipidoses, lysosomal storage diseases involving glycosphingolipids (GSLs), are currently limited to a few drugs that inhibit de novo GSL biosynthesis, such as eliglustat and miglustat (Zavesca®). In the search for alternative drugs, dapagliflozin emerged as a promising candidate for off-target therapy. In the present study, we investigated whether dapagliflozin can indeed inhibit GSL synthesis, as predicted by previous computational analyses, and compared its effects with those of the glycosphingolipid synthesis inhibitor, the eliglustat analog Genz-123346, in murine 3T3 and Hepa 1-6 cell lines. While Genz-123346 significantly inhibited glycosphingolipid biosynthesis at concentrations as low as 1 µM, dapagliflozin, even up to 50 µM, had no effect on biosynthesis or de novo biosynthesis in either cell line. These results indicate that dapagliflozin, although assessing effects on the cell cycle, including proliferation at high concentrations, is not a suitable candidate for treating glycosphingolipid storage diseases by substrate reduction
Interaction of the GM2 Activator Protein with Sulfated and Sialylated Glycosphingolipids
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