O-GlcNAc modification differentially regulates microtubule binding and pathological conformations of tau isoforms in vitro

Tau proteins undergo several posttranslational modifications in physiological and disease conditions. In Alzheimer's disease, O-GlcNAcylation modification of serine/threonine (S/T) residues in tau is reduced. In mouse models of tauopathy, O-GlcNAcase inhibitors lead to increased O-GlcNAcylation and decreased filamentous aggregates of tau. However, various nonfilamentous tau conformations, linked to toxicity and neurodegeneration in tauopathies, involve processes like oligomerization, misfolding, and greater exposure of the phosphatase-activating domain in the amino terminus of tau. Additionally, it is becoming clearer that posttranslational modifications may differently regulate tau pathobiology in an isoform-dependent manner. Therefore, it is crucial to investigate the effects of O-GlcNAcylation on nonfilamentous conformations of both the four-repeat (4R, e.g., hT40) and three-repeat (3R, e.g., hT39) tau isoforms. In this study, we assessed how O-GlcNAcylation impacts pathological tau conformations of the longest 4R and 3R tau isoforms (hT40 and hT39, respectively) using recombinant proteins. Mass spectrometry showed that tau is modified with O-GlcNAc at multiple S/T residues, primarily in the proline-rich domain and the C-terminal region. O-GlcNAcylation of hT40 and hT39 does not affect microtubule polymerization but has opposite effects on hT40 (increases) and hT39 (decreases) binding to preformed microtubules. Although O-GlcNAcylation interferes with forming filamentous hT40 aggregates, it does not alter the formation of pathological nonfilamentous tau conformations. On the other hand, O-GlcNAcylation increases the formation of pathological nonfilamentous hT39 conformations. These findings suggest that O-GlcNAcylation differentially modulates microtubule binding and the adoption of pathological tau conformations in the longest 4R and 3R tau isoforms.