The O-GlcNAc post-translational modification involves the attachment of single N-acetyl-D-glucosamine residues to serine and threonine of nucleocytoplasmic proteins of multicellular eukaryotes. The microtubule associated-protein tau has been suggested to be extensively O-GlcNAc modified and to compete with tau phosphorylation. Tau becomes hyperphosphorylated in Alzheimer’s disease (AD) which leads to the formation of the characteristic aggregates seen in the brains of AD patients. The reciprocal relationship between tau O-GlcNAcylation and tau phosphorylation suggests that if O-GlcNAc levels rise then phosphorylation levels should decrease. If O-GlcNAc levels can be increased in vivo, hyperphosphorylation of tau might be, therefore, antagonized by the increased levels of O-GlcNAc. One way to increase O-GlcNAcylation of tau is to inhibit the enzyme which removes O-GlcNAc, a glycoside hydrolase referred to as OGA. Existing OGA inhibitors were unsuitable for use in vivo. For this reason, a potent, selective, and stable inhibitor, termed Thiamet-G, was developed in the laboratory. Treatment with Thiamet-G increased O-GlcNAc levels in both cells as well as in the mouse brain. These substantial increases in O-GlcNAc, resulted in decreased levels of tau phosphorylation at sites of phosphorylation that are implicated in AD. Collaboratively, I also found that Thiamet-G treatment results in prevention of neurodegeneration in the JNPL3 mouse model and further observed that this effect does not appear to arise by prevention of hyperphosphorylation but more likely by blocking the aggregation of tau. This proposal is further supported by experiments showing that O-GlcNAc modification of tau impairs the aggregation of truncated and full length human tau in vitro. In order to study the site-specific effects of tau O-GlcNAcylation several sites of tau O-GlcNAc were mapped collaboratively and the major site of tau O-GlcNAc was found to be Ser400. I developed a site-specific Ser400 O-GlcNAc tau antibody and find that this site of glycosylation plays a key role in the aggregation of tau in vitro. The tools described herein should prove useful in further clarifying the role of O-GlcNAc in tau biology and also may serve to clarify OGA as a target with therapeutic potential.
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Thesis advisor: Vocadlo, David
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