IP-LC-MSMS Enables Identification of Three Tau O-GlcNAcylation Sites as O-GlcNAcase Inhibition Pharmacodynamic Readout in Transgenic Mice Overexpressing Human Tau

O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates tau phosphorylation and aggregation: the pharmacological increase of tau O-GlcNAcylation upon treatment with inhibitors of O-GlcNAc hydrolase (OGA) constitutes a potential strategy to tackle neurodegenerative diseases. Analysis of tau O-GlcNAcylation could potentially be used as a pharmacodynamic biomarker both in preclinical and clinical studies. The goal of the current study was to confirm tau O-GlcNAcylation at S400 as a pharmacodynamic readout of OGA inhibition in P301S transgenic mice overexpressing human tau and treated with the OGA Inhibitor Thiamet G and to explore if additional O-GlcNAcylation sites on tau could be identified. As a first step, an immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) methodology was developed to monitor changes in O-GlcNAcylation around S400 of tau in mouse brain homogenate (BH) extracts. Second, additional O-GlcNAc sites were identified in in-house produced recombinant O-GlcNAcylated human tau at relatively high concentrations, thereby facilitating collection of informative LC-MS data for identification of low-concentration O-GlcNAc-tryptic tau peptides in human transgenic mouse BH extracts. This strategy enabled, for the first time, identification of three low abundant N-terminal and mid-domain O-GlcNAc sites of tau (at S208, S191, and S184 or S185) in human transgenic mouse BH. Data are openly available at data.mendeley.com (doi: 10.17632/jp57yk9469.1; doi: 10.17632/8n5j45dnd8.1; doi: 10.17632/h5vdrx4n3d.1).

Alzheimer’s disease; IP-LC-MSMS; O-GlcNAc hydrolase inhibition; P301S transgenic mouse brain homogenates; tau O-GlcNAcylation.