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Development of Mass Spectrometric Methods for Tissue Imaging and LC-based Quantification of Glycosphingolipids/Gangliosides including Tay-Sachs disease based Neuraminidase-deficient Mouse Models and Human Gut Microbiota

von Gerichten, Johanna

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Abstract

Tay-Sachs disease (TSD) is an autosomal-recessive genetic disorder which results in the dysfunction of the metabolic enzyme hexosaminidase A (HexA). It leads to severe lysosomal storage of acidic glycosphingolipid, namely ganglioside GM2, and early fatalities for humans with the infantile on-set form. Despite fifty years of research, to date there is no effective treatment beyond palliative care. It was found that mouse models of HexA deficiency display only moderate GM2 accumulation, which was connected with a late onset neuronal phenotype. Therefore neuraminidases were investigated as possible bypass enzymes for the degradation of GM2 and offer a new opportunity for therapeutic approaches in humans. However, to assess the extent of side effects for such a therapeutic bypass, the substrate specificity and ganglioside (GG) turnover has to be defined in detail. This work presents the development of an HILIC based LC MS2 method as well as mass spectrometry imaging (MSI) using DESI (QqQ)MS2 and MALDI TOF to monitor GG pattern changes in mouse brains. The HILIC MS2 analysis of mouse brain tissue with neuraminidase 3 or 4 deficiency in the background of TSD as well as combined knockouts of GG synthesis enzymes revealed an overlapping but distinct substrate processing for the neuraminidases Neu3 and Neu4. MSI of the same tissue samples displayed similar patterns in spatial neural GM2 accumulation that suggest rather a broad distribution of these sialidases in mouse brain. Proposed neuroinflammation and demyelination in mouse brains of TSD led to a modulated HILIC MS2 method with which hexosylceramide isomer separation of GG precursor β-glucosylceramide (β-GlcCer) and prominent myelin sheath component β-galactosylceramide (β-GalCer) was achieved. Decreased levels of β-GalCer as a marker for demyelination in brains of TSD combined with neuraminidase deficiency could not be observed at the age of 6 month. Furthermore, proof of concept study and screening of various WT mouse tissues revealed the adaptability of this method. Even α-anomeric HexCers could be separated from mammalian β-anomers. In contrast to the mentioned β-HexCers, invariant natural killer T cells are activated most effectively when recognizing galactosylceramide with an α-glycosidic linkage appearing on the cell surface receptor CD1d of antigen presenting cells. One natural bacterial source of this compound in contact with our body is Bacteroides fragilis, a bacterial member of the human gut microbiome. This work highlights the detection and separation of α-GalCer(d17:0;h17:0) in B.fragilis and three other bacteria of the human gut microbiome β-HexCers. Very recent preliminary studies indicate the identification of an α-glycosidic GalCer in the mouse microbiome with the proposed structure of BdS α-GalCer(d18:0;h16:0).

Item Type: Dissertation
Supervisor: Mayer, Prof. Dr. Matthias
Date of thesis defense: 13 November 2018
Date Deposited: 30 Jan 2019 09:02
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
540 Chemistry and allied sciences
570 Life sciences
600 Technology (Applied sciences)
620 Engineering and allied operations
Controlled Keywords: LC-MS, Elektrospray-Ionisation, Tandem-Massenspektrometrie
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