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Alzheimer's disease (AD) is the most common form of dementia, marked by progressive brain degeneration and cognitive decline. A major pathological feature of AD is the accumulation of hyperphosphorylated tau (p-tau) in the form of neurofibrillary tangles (NFTs), which leads to neuronal death and neurodegeneration. P-tau also induces endoplasmic reticulum (ER) stress and activates the unfolded protein response, causing inflammation and apoptosis. Additionally, p-tau spreads in the brain through interactions with heparan sulfate (HS) proteoglycans, promoting aggregation and internalization. Targeting the tau-HS interaction offers a potential therapeutic strategy for AD. We present a novel HS mimetic with a lipophilic oleanolic acid linker and a sulfated trisaccharide, which shows strong cytoprotective effects against p-tau. Moreover, this compound alleviates p-tau-induced ER stress and inflammation. Molecular docking studies indicate that the conjugation of oleanolic acid enhances binding between the ligand and tau protofilament cores, facilitating protective interactions. These findings provide a foundation for the development of novel HS mimetics, enabling further investigation of tau-HS interactions in AD and other tauopathies.

Abnormal accumulation of tau protein in the brain is one pathological hallmark of Alzheimer's disease (AD). Many tau protein post-translational modifications (PTMs) are associated with the development of AD, such as phosphorylation, acetylation, and methylation. Therefore, a complete picture of the PTM landscape of tau is critical for understanding the molecular mechanisms of AD progression. Here, we offered a pilot study of combining two complementary analytical techniques, capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS, for bottom-up proteomics of recombinant human tau-0N3R. We identified 50 phosphorylation sites of tau-0N3R in total, which is about 25% higher than that from RPLC-MS/MS alone. CZE-MS/MS provided more PTM sites (i.e., phosphorylation) and modified peptides of tau-0N3R than RPLC-MS/MS, and its predicted electrophoretic mobility helped improve the confidence of the identified modified peptides. We developed a highly efficient capillary isoelectric focusing (cIEF)-MS technique to offer a bird's-eye view of tau-0N3R proteoforms, with 11 putative tau-0N3R proteoforms carrying up to nine phosphorylation sites and lower pI values from more phosphorylated proteoforms detected. Interestingly, under native-like cIEF-MS conditions, we observed three putative tau-0N3R dimers carrying phosphate groups. The findings demonstrate that CE-MS is a valuable analytical technique for the characterization of tau PTMs, proteoforms, and even oligomerization.