In the present study, a cell penetrating peptide (CPP)-amyloid conjugate was prepared (T-peptide), where the amyloid-forming sequence was homologous to a nucleating sequence from human Tau protein (306VQIVYK311). Kinetic and biophysical studies showed the peptide formed long-lived oligomers which were taken up by endocytosis and localized in perinuclear vesicles and in the cytoplasm of murine hippocampal neuroblastoma cells and human HeLa cells. Thioflavin S (ThS) staining of amyloid colocalized with pathological phosphorylated Tau, suggesting that the peptide was able to seed endogenous wild-type Tau. Subsequent experiments showed that aggregates present in the lysosomes mediated lysosome membrane permeability (LMP). We observed a decrease in total Tau, irrespective of phosphorylation state, consistent with Tau fragmentation by lysosomal proteases. We found cytotoxicity of T-peptide could be abrogated by inhibitors of lysosomal hydrolases and caspases, consistent with a model where Tau fragments processed by the lysosome leak into the cytoplasm and induce toxicity in subsequent downstream steps. It is our hope that the T-peptide system may prove amenable to the evaluation of small molecule inhibitors of cytotoxicity, especially those which target either Tau aggregation or the lysosomal/autophagy system.
Keywords: Alzheimer’s disease; amyloid; cell penetrating peptide; endosome; lysosome; tau protein; tauopathy.