Gangliosides, abundant in the nervous system, are known to play crucial modulatory roles in cellular recognition, interaction, adhesion, and signal transduction, particularly during early developmental stages. The expression of gangliosides in the nervous system is cell specific and developmentally regulated, and closely related to the differentiation state of the cell. We hypothesize that the temporal and spatial expression of gangliosides in the nervous system is closely related to the neural differentiation, including neuronal migration and neuron/axon interaction, and that their expression is tightly regulated by their metabolism, in particular, biosynthesis. Taking advantage of the recent advances in the embryonic stem (ES) cell field, we will examine the differential expression, metabolism, and regulatory mechanisms of these important glycoconjugates during various stages of cellular differentiation. To better understand the metabolic basis of the ganglioside expression, we will focus on the subcellular localization, intracellular trafficking, and regulatory mechanisms of the expression of several key glycosyltransferases (GTs), particularly GM3-synthase (ST-I), GD3-synthase (ST-II), and GM2/GD2-synthase (GalNAcT), that regulate the synthesis of gangliosides along different pathways. To achieve these goals, three specific aims are proposed to examine the following aspects of their regulation.
Aim 1. Regulation of ganglioside expression during neural development, a. Characterization of stage- and cell-specific GSL markers in differentiating ES cells and ganglioside knockout ES cells; b. Characterization of the functional role of gangliosides in neuronal migration.
Aim 2. Transcriptional regulation of GTs in differentiating neural cells, a. Characterization of the promoter and enhancer elements the GT genes, b. Characterization of the transcription factors that interact with the promoter/enhancer elements of these genes during ES cell differentiation.
Aim 3. Post-translational modification of enzymes for ganglioside biosynthesis--Intracellular localization, trafficking, and complex formation of GTs in differentiating ES cells. An understanding of the molecular mechanisms underlying the differential expression of cell surface gangliosides should greatly enrich our knowledge of their functions in normal brain development and developmental disorders that result in mental retardation. ?
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