Islet Amyloid Polypeptide Cross-seeds Tau And Drives The Neurofbrillary Pathology In Alzheimer’s Disease

Part I.IAPP deposition in the brains of AD patientsObjective: To determine the expression and deposition of pancreatic amyloid polypeptide(IAPP)in the brains of AD patients.Methods: Immunohistochemistry assay was used to detect the IAPP deposition in brain tissue of AD patients and healthy controls;Western blot was used to detect the content difference of IAPP protein in 1% TX-100 insoluble components of brain tissue between AD patients and healthy controls;The content difference of IAPP in cerebrospinal fluid(CSF)between AD patients and healthy controls was detected by ELISA;The interaction between IAPP protein and phosphorylated tau protein(AT8)in brain tissues of AD patients and healthy controls was detected by Co-IP method;Quantitative Real-Time PCR(RT-PCR)was used to detect the difference of IAPP gene expression between AD patients and healthy controls;Immunofluorescence was used to detect the cellular localization of IAPP in the brain of AD patients;GST pull-down test was used to detect the domain of IAPP binding to tau protein in vitro;Tau P301 S and wild-type(WT)mice were injected with FITC fluorescent labeled IAPP aggregates through the tail vein.The difference of the amount of FITC fluorescent labeled IAPP aggregates entering the central nervous system through the blood-brain barrier(BBB)was detected by immunofluorescence.Results: Immunohistochemical results showed that IAPP deposition existed in the brain tissue of AD patients,but no significant IAPP signal was found in the healthy controls;Western blot showed that IAPP was contained in the insoluble components of brain tissue of AD patients;The results of ELISA showed that the content of IAPP in CSF was higher in AD patients;Co-IP experiment showed that IAPP interacted with AT8;RT-PCR did not detect IAPP m RNA expression in AD patients and healthy controls;Immunofluorescence showed that IAPP existed in neurons and microglia of brain of AD patients,but IAPP was not detected in astrocytes;GST pull-down experiment showed that IAPP interacted with tau protein tandem repeat(tau RD);Tail vein injection of FITC-IAPP PFF in mice can enter into the central nervous system through the BBB,especially in tau P301 S mouse.Conclusion: IAPP was deposited in the brain of AD patients and was co-localized with tau protein aggregates.IAPP in blood can enter into the central nervous system through the BBB and be ingested by neurons and microglia.IAPP binds to the tau RD domain.Part II.IAPP promotes tau aggregation into more pathogenic IAPP-Tau aggregatesObjective: To investigate the effects of IAPP on tau protein aggregation and neurotoxic effect of IAPP-Tau PFF in vitro.Methods: 1.Thioflavin T(Th T)method was used to detect the effect of different IAPP concentrations on the aggregation rate of Tau protein;The morphological differences of IAPP PFF,Tau PFF and IAPP Tau PFF were detected by negative staining technique;The binding of IAPP to Tau protein was detected by immunoelectron microscopy(Immuno-EM);Protease K(PK)was used to detect the resistance of Tau PFF and IAPP Tau PFF to PK digestion;The aggregation ability of endogenous Tau RD induced by Tau PFF and IAPP-Tau PFF was detected by immunofluorescence;Western blot was used to detect the PFF amount in different experiment groups,and to test the ability of Tau PFF and IAPP-Tau PFF to aggregate endogenous Tau RD into 1% TX100 insoluble precipitation.2.Primary neurons of tau P301 S fetal mice were cultured in vitro and were treated with PBS,IAPP PFF,tau PFF and IAPP-tau PFF respectively.The phosphorylation levels of tau at different sites were detected by immunofluorescence(AT8/AT100/S202/S396);The expression of AT8 in neurons treated with different PFF was detected by Western blot;TUNEL and PI/Hoechst were used to detect neuronal apoptosis after different PFF treatment;DIL staining was used to detect the loss of dendritic spines after different PFF treatment;The primary neurons of tau KO mice were cultured in vitro and were treated with IAPP PFF.The apoptosis of neurons was further detected by TUNEL assay.Results: 1.ThT binding experiment showed that IAPP could significantly promote the aggregation rate of Tau protein in a concentration dependent manner;Immunoelectron microscopy(Immuno-EM)and Negative staining showed that IAPP interacted with Tau protein to form a new IAPP-Tau PFF strain,which was different from Tau PFF;PK digestion results indicated that Tau PFF and IAPP-Tau PFF showed different digestion patterns against PK;Compared with Tau PFF,immunofluorescence results showed that IAPP-Tau PFF seemed to be more easier to induce endogenous Tau aggregation and transmission;Western blot showed that IAPP-Tau PFF was more likely to induce endogenous Tau to aggregate into 1% TX100 insoluble precipitation;No difference in PFF amount between cells and animal models was found by Western blot assay.2.Compared to IAPP PFF and Tau PFF,IAPP-Tau PFF had stronger ability to induce Tau phosphorylation;Western blot showed more evident Tau phosphorylation(AT8)after IAPP-Tau PFF treatment;TUNEL and PI/Hoechst results showed that IAPP-Tau PFF had stronger potential to induce apoptosis of primary neurons;DIL method showed that the dendritic spines of primary neurons were significantly injured after IAPP-Tau PFF treatment;Compared with the primary neurons of Tau P301 S mice,the apoptosis of Tau KO primary neurons treated with IAPP PFF was significantly decreased.Conclusion: 1.IAPP interacts with Tau protein to form IAPP-Tau PFF strain,which has faster aggregation rate and shows different biochemical characteristics from Tau PFF.2.IAPP-Tau PFF has stronger neurotoxicity than Tau PFF.The neurotoxicity of IAPP PFF is Tau dependent.Part III.IAPP-Tau PFF promotes the formation and pathological transmission of p-Tau in Tau P301 S mouseObjective: To investigate the effect of IAPP-Tau PFF on the formation and pathological transmission of p-Tau in vivo.Methods: PBS,IAPP PFF,Tau PFF and IAPP-Tau PFF were injected into the unilateral hippocampus of Tau P301 S mice by stereotactic technique.The levels of p-Tau in different time(1 month and 3 months)and different brain areas(prefrontal cortex,hippocampus,entorhinal cortex,posterior pressor cortex and auditory cortex)were detected by immunohistochemistry;The expressions of p-Tau(AT8/AT100),total Tau protein,synapse associated protein and glial cell markers in hippocampus and cortex of different PFF injection groups were detected by Western blot;The activation differences of microglia and astrocytes in hippocampus of mice treated with different PFFs were detected by immunofluorescence.Results: Compared with PBS,IAPP PFF and Tau PFF group,immunohistochemical results showed that IAPP-Tau PFF group significantly increased p-Tau level in hippocampus and cortex alongside the injection site,which was more obvious 3 months post injection;Western blot showed that there was no difference in total Tau expression in different PFF groups.The p-Tau level increased significantly in the injection side,and similarly,the expression of microglia marker Iba1 and astrocyte marker GFAP increased significantly;Immunofluorescence showed that the activation of microglia and astrocytes in hippocampus was more obvious in IAPP-Tau PFF group.Conclusion: Compared with PBS,IAPP PFF and Tau PFF,IAPP-Tau PFF significantly increased the level of p-Tau(AT8/AT100)and the activation of microglia and astrocytes in Tau P301 S mouse model in a time-dependent manner.Part IV.IAPP-Tau PFF promotes cognitive dysfunction and synaptic impairment in Tau P301 S mouseObjective: To investigate the effects of IAPP-Tau PFF on cognitive learning and synaptic function of Tau P301 S mouse in vivo.Methods: PBS,IAPP PFF,Tau PFF and IAPP-Tau PFF were injected into the unilateral hippocampus of Tau P301 S mice by stereotactic technique.After 3 months,the changes of cognitive function of Tau P301 S model mice were detected by water mirror maze and Y maze;Patch clamp recording technique was used to detect the long-term enhancement of synaptic activity in hippocampal neurons;The ultrastructure of synapses of hippocampus of different PFF groups was detected by transmission electron microscope(TEM);Golgi staining was used to observe the maturity and density of dendritic spines in hippocampal neurons of different PFF groups;The expression of synapsin I,synaptophysin and PSD95 proteins in hippocampus were detected by Western blot.Results: Compared with PBS,IAPP PFF and Tau PFF group,behavioral results showed that the learning ability of water mirror maze and Y maze in IAPP-Tau PFF group was significantly reduced;Electrophysiological results showed that the long-term potentiation effect of synapses in hippocampal neurons of IAPP-Tau PFF group was much weakened;The results of transmission electron microscope(TEM) showed that the synaptic structure of hippocampal neurons in IAPP-Tau PFF group was much more injured;Golgi staining showed that the dendritic spines of hippocampal neurons decreased more significantly in IAPP-Tau PFF group;Western blot showed that the expression of synapsin I,synaptophysin and PSD95 proteins in IAPP-Tau PFF group significantly decreased.Conclusion: Compared with PBS,IAPP PFF and Tau PFF,IAPP-Tau PFF has more significant effects on neuronal synaptic dysfunction and cognitive impairment in Tau P301 S mice.