Inhibiting Phosphorylation of Tau (τ) Proteins at Ser262 Using Peptide-Based R1 Domain Mimetics

The microtubule-associated protein tau (τ) is a phosphoprotein that is crucial for regulating microtubule dynamics. Tau is highly enriched in neurons, where it functions by binding tubulin and stabilizing axonal microtubules. Phosphorylation of tau within its microtubule-binding repeat (R) domains significantly reduces its affinity for tubulin, leading to a loss in microtubule stability. In neurons, dysregulated kinase activity often results in the formation of hyper-phosphorylated tau isoforms that remain permanently detached from microtubules. If left untreated, hyper-phosphorylated tau can aggregate into insoluble, prion-like oligomers that contribute to the pathogenesis of neurodegenerative disease. Consequently, there is considerable interest in developing inhibitors that reduce levels of hyper-phosphorylated tau in neurons. In this study, we have generated a synthetic peptide mimetic (tR1) of the tau R1 domain as an inhibitor of microtubule-affinity regulating kinase 2 (MARK2). In vitro assays showed that tR1 inhibits the MARK2-mediated phosphorylation of tau within its R1 domain at Ser262, a residue critical for favorable tau-tubulin interactions. We also demonstrate that tR1 peptides are > 90% stable up to 24 h in neurobasal medium and RPMI media supplemented with human serum. Uptake experiments in cultured rat primary cortical neurons indicate that tR1 is internalized through an energy-dependent mechanism and can be delivered to the cytoplasm when co-treated with bafilomycin A1 or chloroquine. Furthermore, we show tR1 inhibits phosphorylation of endogenous tau at Ser262 in cultured neurons following activation of intracellular kinases. This inhibitory effect was selective for kinases that phosphorylate tau at Ser262, as tR1 did not inhibit tau phosphorylation at Thr231. Collectively, these results establish tR1 as a highly-stable, peptide-based kinase inhibitor that reduces the level of phosphorylated tau proteins in neurons.