IDENTIFICATION OF NOVEL GANGLIOSIDE-ASSOCIATED PROTEINS AND ADVANCES IN SINGLE-CELL GANGLIOSIDE METABOLOMICS IN NERVE CELLS
Prendergast, Jillian Marie
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Gangliosides, glycosphingolipids that carry at least one sialic acid residue, are enriched in the plasma membranes of neurons. Despite their abundance, little is known about the molecules they interact with in the plasma membrane and how their metabolism differs from cell-to-cell. Rare human disorders of ganglioside biosynthesis and catabolism have devastating neurological defects and currently have no treatments. My thesis project aims to take a two pronged approach to help understand ganglioside function and ganglioside metabolic disorders. The first aim was to identify novel ganglioside interacting proteins by utilizing an unbiased screening method, mass spectrometric interaction analyses (iTRAQ) mass spectrometry. Primary rat cerebellar granule neuron cell surface proteins were isolated and ganglioside specific interacting proteins were collected via affinity chromatography on GT1b- and GM1-derivatized beads. iTRAQ and subsequent validation studies revealed reproducible differences between GT1b- and GM1-bound proteins from biological replicates. Rigorous statistical analyses revealed six GT1b-selective binding proteins, three of which suggested that GT1b participates in the regulation of glutamate-receptor surface expression. The second aim was to develop methods to quantify ganglioside metabolism in single cells. Fluorescently labeled gangliosides and various precursor glycosphingolipids were added to primary neuronal cultures. Separation of metabolites and ultrasensitive quantification was achieved using capillary electrophoresis coupled to a laser-induced fluorescence detection system with a sub-zeptomole (10e-21 mole) detection limit. Addition of labeled glycosphingolipids to primary cerebellar cultures allowed determination of anabolic and catabolic products at the single cell level. Neuronal and glial cells could be differentiated with this method and heterogeneity was distinguishable among cells when analyzed on the single cell level. Analysis of metabolism in culture homogenates (thousands of cells) was consistent with metabolism averaged from a large number of single cells. Through understanding both ganglioside metabolism and the molecules that gangliosides interact with treatments could be developed for these rare human disorders.