Prion-like Tau Aggregation:Driving Forces And Intervention Research

Objective Abnormal hyperphosphorylation and aggregation of the microtubule-associated protein tau(tau)play a key role in Alzheimer’s disease(AD)and other related neurodegenerative diseases.Recent studies suggest that the prion-like propagation characteristics of pathological tau in the AD brain may be the molecular basis for its development.In this study,we explored whether tau truncation and phosphorylation contribute to its prion-like propagation and whether activation of autophagy could inhibit the progression of tau pathology.The research is divided into five parts.Part I: Hyperphosphorylation and truncation were the biochemical characteristics of tau aggregates in AD brainMethods In the present study,we investigated the phosphorylation level and truncation of total tau and SDS-and reducing agent-resistant high-molecular weight tau(HMW-tau)in control and AD brains by Western blots.Results(1)We found that tau in AD brain appears as a smear from low molecular weight(LMW)to HMW in western blots developed with pan-tau antibodies.Similar levels of LMW-tau were presented in AD and control brain,while HMW-tau only expressed in AD brain.(2)The phosphorylation levels of tau in the brain of AD patients were significantly increased.HMW-tau is hyperphosphorylated at multiple sites and does not contain unphosphorylated Ser46 and Ser198/199/202.(3)The N-terminal antibodies 43D(recognizes 6-18 a.a.of full length tau)and HT7(recognizes 159-163 a.a.)could only detect weak signals in HMW-tau,while the C-terminal antibodies tau46 and tau46.1 showed strong immunoreactivity.(4)The ratio of HMW-tau to LMW-tau detected by tau antibodies was increasing as the epitope of the antibodies ranges from N-terminal to C-terminal.(5)Tau truncation at Asp421 was significantly increased in AD brain,but is poorly correlated with the levels of HMW-tau.Conclusions These findings suggest that HMW-tau in AD brain was hyperphosphorylated at multiple sites and was mostly truncated at the Nterminus.Part II: Spatiotemporal evolution of phosphorylation during induced prion-like aggregation of tauMethods AD oligomeric tau(AD O-tau)was extracted from AD brains(Braak stage VI)and added into cell culutre or injected into the hippocampus of mouse brain.Western blots and immunofluorescence were used to analyze the temporal and spatial evolution of tau phosphorylation and aggregation during AD O-tau-induced prion-like propagation of tau pathology.Results(1)The phosphorylation levels of AD O-tau at Ser199,Thr205 and Thr214 sites were relatively low,but the phosphorylation levels at Ser262,Ser396/404(PHF-1)and Ser422 sites were relatively high,especially at Ser422.(2)AD O-tau effectively induced prion-like aggregation of tau in cultured cell model.(3)In cell model,tau hyperphosphorylation and prion-like aggregation induced by AD O-tau were time-dependent.(4)The prion-like aggregation induced by AD O-tau in the brain of C57BL/6wild-type mouse was time-dependent and age-related.Conclusions AD O-tau is abnormally hyperphosphorylated,which can induce tau prion-like aggregation and hyperphosphorylation in SHSY5 Y cells and wild-type mice brain in a time-dependent manner.Part III: The truncation of tau determines its self-aggregation and prion-like activityMethods We constructed a series of tau truncations and studied their self-aggregation activity in cell model.As a representative,tau151-391 or tau1-441(full-length tau)were expressed in eukaryotic cells.Tau151-391 and tau1-441 aggregates were then extracted from the cells and injected into the hippocampus of mouse brain.The activity of truncated tau(tau151-391)aggregates and full-length tau aggregates in inducing the prion-like propagation of pathological tau in vivo was compared.Results(1)The self-aggregation activity of tau151-391 was the strongest among all the truncations expressed in HEK-293 FT cells.(2)Tau151-391 not only has stronger self-aggregation activity,but also is easier to induce prion-like aggregation.(3)The tau151-391 protein aggregates have a prion-like spreading effect similar to AD O-tau in the mouse brain,which is significantly stronger than the full-length tau aggregates.Conclusions Tau151-391 is more prone to self-aggregate and be seeded to aggregate by AD O-tau than the full-length tau.Tau151-391 insoluble aggregates isolated from cultured eukaryotic cells can induce tau prion-like aggregation in mouse brain,similar to that of AD O-tau.Part IV: Hyperphosphorylation promotes tau prion-like propagationMethods The phosphorylation levels of endogenous tau were up-regulated or down-regulated by okadaic acid treatment or protein phosphatase 2A(PP2A)knockdown or overexpression.Tau ofdifferent phosphorylation levels were analyzed for their characteristics in prionlike aggregation induced by AD O-tau.Results(1)Hyperphosphorylated tau was more easily captured by AD O-tau,and to aggregate when induced by AD O-tau.(2)When HEK-293 FT cells expressing tau151-391 were treated with sh PP2 Ac to interfere the expression of protein phosphatase 2A catalytic subunit(PP2Ac),more prion-like aggregation occurred after being induced by AD O-tau.On the contrary,when overexpressing PP2 Ac,tau prion-like aggregation induced by AD O-tau decreased.(3)Knockdown of PP2 Ac promoted the aggregation of endogenous tau induced by AD O-tau in C57BL/6 mice,but knockdown of PP2 Ac itself was not enough to induce the autoaggregation of endogenous tau.Overexpression of PP2 Ac significantly reduced the aggregation of endogenous tau induced by AD O-tau.Conclusions The phosphorylation level of endogenous tau affects the pathological aggregation of tau and prion-like propagation induced by AD O-tau.The higher the phosphorylation level of tau,the easier it is to form aggregation.Part V: The effect of autophagy activator on tau prion-like aggregationMethods Rapamycin and trehalose,known as autophagy activators,were used to study the effect on tau prion-like aggregation and propagation through cell culture model and mouse hippocampus injection model.Results(1)Rapamycin could activate autophagy in the process of tau aggregation induced by AD O-tau.(2)In the cell culture model,rapamycin treatment significantly inhibited the aggregation of sarkosylinsoluble total tau induced by AD O-tau.(3)The autophagy activators rapamycin and trehalose can inhibit the aggregation and hyperphosphorylation of tau induced by AD O-tau in the brain of C57BL/6 mice.Conclusions Autophagy activators rapamycin and trehalose inhibit the prion-like aggregation and site-specific phosphorylation of tau induced by AD O-tau.