Tau phosphorylation at Alzheimer's disease biomarker sites impairs its cleavage by lysosomal proteases

INTRODUCTION: Phospho-tau peptides from the proline-rich domain (PRD) of tau are sensitive biomarkers for Alzheimer's disease (AD). The PRD is known to be relatively resistant to lysosomal proteolytic cleavage, but the effects of phosphorylation on cleavage are unknown. METHODS: Using in silico modeling and in vitro protease assays, we quantified the effects of phosphorylation on lysosomal proteolysis of tau. We further assessed levels of lysosomal proteases in patient-derived cerebrospinal fluid (CSF) relative to phosphorylated tau-181 (p-tau181). RESULTS: Phosphorylation renders the PRD significantly resistant to cleavage by the lysosome, especially at less acidic pH setpoints. In Alzheimer's disease subjects, CSF levels of lysosomal proteases correlate with p-tau181, suggesting that p-tau peptides are released with lysosomal contents. DISCUSSION: Loss of lysosomal acidity may contribute to the release of phospho-tau biomarkers. This study shows that phosphorylation of tau impairs its cleavage by proteases in a pH-dependent manner and provides a novel molecular basis for p-tau biomarker accumulation in AD. HIGHLIGHTS: Phosphorylated tau-181 (p-tau181) and p-tau217 originate from tau regions that are poorly cleaved by lysosomal proteases. Phosphorylation further impairs the proteolytic cleavage of AD biomarker peptides. Impaired proteolytic cleavage of phosphorylated tau is pH dependent. Levels of p-tau181 are correlated with lysosomal proteases in Alzheimer's disease (AD) cerebrospinal fluid samples. AD-associated lysosomal dysfunction may contribute to presence of disease biomarkers.