Effects Of Tau Mutations On Self-aggregation And Microtubule Assembly

Frontotemporal dementia(FTD)is the second most common dementia besides Alzheimer’s disease(AD).One kind of FTD named FTDP-17 links to chromosome 17 which is caused by the mutations in the MAPT gene located on chromosome 17q21.31.Up to now,about 80 pathogenic missense mutations of MAPT gene have been found gradually in the case of familial frontotemporal dementia,most of which occur in the coding region where tau protein binds microtubules.Tau is microtubule associated protein.It promotes microtubule assembly in neurons,but the pathological tau protein detaches from microtubule and self-aggregates,resulting neuron death.Most studies of tau protein focus on the pathological mechanism caused by hyperphosphorylation instead of its mutation of tau.It is reported that the mutation of Tau gene in FTDP-17 can also induce the same neurofibrillary tangles(NFT)as Alzheimer’s disease.Therefore,focusing on the mutation of Tau protein,especially in the microtubule binding region,is a new method to study the effects of different mutations correlated with Tau protein fibrillation and microtubule assembly function.In this study,we chose five tau mutantswhich are clinically discovered in FTDP-17,K257 T,N279K,P301 L,K317M,and V337 M,which are located in the microtubule bingding repeat regions(R1-R4).We used the Tau k18 fragment as experimental objects.First,different mutants were successfully obtained by performing single point mutations at different sites on the Tau k18 sequence.Then we used ThT fluorescence and Transmission Electron Microscopy to detect the difference of mutants’ fibrillation process induced by heparin.We used the Light Scattering Experiment to study the kinetics of microtubule assembly promoted by each Tau k18 mutant and compare its effect to wildtype.To analyze which amino acids will affect the binding of Tubulin and Tau k18,we used Homologous Modeling and Molecular Docking of Tau k18 and Tubulin heterodimers.After that we speculate the effects of each mutation on microtubule assembling.By comparing the fibrillation ability and the promotion of microtubule assembling between these five Tau k18 mutants,we show that relative to wild-type Tau k18,the V337 M and P301 L mutants can significantly accelerate theiraggregation,N279 K and K317 M mutants delay their fibrillation while K257 T affects and delays the nucleation state of fibrillation.In addition,all the mutants affect the assembly kinetics of microtubules and reduce microtubule assembly efficiency in different degrees compared to wild-type.In particular,K317 M,K257T mutant has the greatest effect,while V337 M has the least.