Study Of Aβ₄₂ And P-tau^396,404 Sequence And Conformation-dependent Antibodies In The Diagnosis And Treatment Of Alzheimer’s Disease

Alzheimer’s disease（AD）is a progressive neurodegenerative disease whose core pathological features are Aβplaques formed by misfolding ofβ-amyloid and hyperphosphorylated tau aggregated into neurofibrillary tangles.Aducanumab and Lecanemab,both approved by the U.S.Food and Drug Administration（FDA）,can slow cognitive decline in the early stages of AD.The Aβ,tau,and neurodegeneration framework provides a way to diagnose and monitor AD and helps guide drug development and biomarker selection.Based on this framework,AD is considered to be diagnosed only when both Aβand pathological tau biomarkers are present.However,the lack of sequence-and conformation-dependent antibodies against neurotoxic Aβ₄₂ and p-tau limits the rapid development of blood diagnosis and immunotherapy for AD.Accordingly,this study screened sequence-specific and conformation-dependent monoclonal antibodies（m Abs）against Aβ₄₂ and tau phosphorylated at serine 396 and 404(p-tau^396,404),developed highly sensitive and specific detection method for Aβ₄₂ monomer(Aβ₄₂M),oligomer(Aβ₄₂O),and p-tau^396,404,as well as a dual-targeted immunotherapy strategy for Aβ₄₂ and reactive oxygen species（ROS）.The thesis mainly includes the following aspects:（1）Sensitive and specific detection of blood Aβ₄₂M and Aβ₄₂O.The conformation-dependent m Abs against different aggregates of Aβ₄₂were developed,and the detection methods were established for detecting Aβ₄₂M,Aβ₄₂O,and total Aβ₄₂.It was found that the blood Aβ₄₂O level increased significantly,but the Aβ₄₂M level decreased as the disease progressed in APP/PS1 mice.Therefore,a lateral flow immunosensor（LFIS）capable of simultaneously detecting both Aβ₄₂M and Aβ₄₂O was developed.The detection results in blood samples of 5×FAD mice and clinically diagnosed AD patients showed that the immunosensor could sensitively and accurately detect Aβ₄₂M and Aβ₄₂O,which can be applied in the blood diagnosis of AD.（2）Highly sensitive and specific detection of p-tau^396,404 level in whole blood samples.The m Abs against p-tau^396,404 were developed and the preferred antibody pair for the detection of p-tau^396,404 was selected.Blood p-tau^396,404 level of 5×FAD and htau mice increased significantly with the disease progression.Based on this finding,a colorimetric/surface-enhanced Raman dual-mode immunosensor adapted to complex whole blood samples was developed,with the lowest quantitative detection limit of 1.5 pg/m L.The immunosensor was used to detect blood samples of AD patients,and the results showed that blood p-tau^396,404 level can distinguish AD patients from normal elderly individuals（NEI）（AUC=0.86,sensitivity=86.7%,specificity=83.3%）,and has the potential to distinguish AD patients at different disease stages.（3）The combined detection of Aβ₄₂O and p-tau^396,404 improves the accuracy of AD blood diagnosis.A dual-target LFIS capable of simultaneous,visualized,and highly sensitive detection of Aβ₄₂O and p-tau^396,404 was developed with the lowest quantitative detection limit of 0.3 and 1 pg/m L,respectively.The results of blood samples from AD patients and NEI showed that simultaneous detection of Aβ₄₂O and p-tau^396,404（AUC=0.93,specificity=86.7%）was more effective than single Aβ₄₂O（AUC=0.85,specificity=73.3%）or p-tau^396,404（AUC=0.88,specificity=80.0%）detection,with higher accuracy.（4）Aβ₄₂/ROS dual-targeted immunotherapy improves AD pathology.An Aβ₄₂ and ROS dual-targeted silica nanoparticle（RVG29-b MSN@Ce-1F12）was synthesized with good biosafety and Aβ₄₂ different aggregates targeting.RVG29-b MSN@Ce-1F12 injected into the tail vein can cross the BBB and achieve in vivo labeling of brain Aβplaques.The treatment results of APP/PS1 mice showed that,compared with 1F12 and RVG29-b MSN@Ce,RVG29-b MSN@Ce-1F12 could more significantly reduce the burden of multiple core pathologies,and improve the olfactory and cognitive impairment of mice.The results of this study show that Aβ₄₂M,Aβ₄₂O,and p-tau^396,404 in the blood are valuable biomarkers for the diagnosis of AD.The detection of combined Aβ₄₂O and p-tau^396,404 has better diagnostic accuracy for AD.Aβ₄₂ and ROS dual-targeted therapeutic strategy can effectively improve AD pathology,and the therapeutic effect is better than a single Aβ₄₂ or ROS-targeted therapeutic strategy.The above research results provided new tools and strategies for AD diagnosis and prevention.