Study On The Mechanism Of Tau Protein Degradation In Alzheimer’s Disease Therapy Mediated By Self-assembled PROTAC Peptide Nano-carrier

【Background and objective】Alzheimer’s disease(AD)is a refractory neurodegenerative disease characterized by amyloid plaques and tau tangles.Its main symptoms are memory loss and progressive neurocognitive impairment.The main pathological features of AD are senile plaques and nerve fiber tangles(NFTs),which are composed of extracellular β-Amyloid protein(Aβ)Composed of tau with excessive phosphorylation within neurons.However,so far,many have targeted A β The "promising" candidate drugs related to pathology have all failed in clinical trials.Therefore,it may be time to consider alternative targets for AD treatment.Tau protein is an important microtubule related protein that serves as a stabilizer for neuronal microtubules.When tau is hyperphosphorylated,it migrates from microtubules and triggers a self proliferative cascade reaction of tau aggregation,leading to toxic NFT production,neuronal cell death,and ultimately AD.Due to the fact that tau protein is a key driver and potential target of AD,targeted clearance of tau protein may be an effective treatment for AD.Protein degradation targeted chimeras(PROTACs)technology is a fast and effective novel target protein clearance strategy,but its stability,half-life,and blood-brain barrier efficiency urgently need to be improved.This study aims to use the neurotransmitter like nanocarriers successfully constructed in previous studies to load PROTACs and prepare them as PROTACs neurotransmitter carriers.The study will systematically explore the nano characterization,in vivo and in vitro targeted clearance of tau protein activity,and anti AD activity of PROTACs neurotransmitter carriers,aiming to provide theoretical basis for the development and application of PROTACs neurotransmitter carriers as new anti AD drugs.【Methods and results】Firstly,PROTACs are chemically synthesized and purified,and then the molecules of PROTACs are identified using hydrogen spectroscopy analysis.Drug preparation was completed by using microinjection pump,Rotary evaporator and other instruments,and then the morphology,particle size,potential,entrapment efficiency,drug loading,stability,in vitro drug release,etc.of nanoparticles were characterized and analyzed by using Malvern light scattering instrument,microplate reader,etc;To investigate the cytotoxic effect of PROTACs NPs on N2 a cells(Neuro-2a cells)using the CCK-8 method.The results showed that the PROTACs NPs nanoparticles had uniform particle size(around200nm),good dispersibility,and good storage and in vivo stability,as well as drug release characteristics;twenty μ PROTACs NPs with concentrations of M and below have no significant toxic or side effects on N2 a cells(cell survival rates>80%),providing a basis for the safety of PROTACs NPs in subsequent in vitro and in vivo experiments.Next,FAM labeled PROTACs NPs(20 μ M)and FAM labeled PROTACs(20 μ M)were added to confocal dishes incubated with N2 a cells,respectively.After culturing for different times,observe the uptake of nanoparticles by cells under confocal microscopy at different action times.To investigate the clearance effect of PROTACs NPs nanoparticles on tau protein in N2 a cells,different concentrations of PROTACs NPs and PROTACs were used to treat successfully transfected N2 a cells for a certain period of time.Flow cytometry and Western Blot methods were used to verify the ability of PROTACs NPs to clear tau protein in vitro.At the same time,immunoprecipitation experiment was also carried out to study whether Keap1 and tau could produce immune coprecipitation.The results indicate that PROTACs NPs nanoparticles can significantly enhance the uptake of fluorescein labeled PROTACs molecules by N2 a cells,indicating that the nanoparticles have good membrane permeability at the cellular level.The experimental results of flow cytometry and Western Blot showed that compared with the PROTACs treatment group,the PROTACs NPs treatment group showed a more significant downregulation of tau protein,while the high-dose group showed a more significant decrease,indicating that the nanoparticle formulation can significantly improve the ability of PROTACs molecules to clear tau protein in vitro.In addition,under the action of PROTACs NPs,Keap1 and tau can produce immune coprecipitation.In order to investigate the in vivo targeting and tissue distribution of PROTACs NPs nanoparticles,Di R fluorescent dye was selected as the tracer for PROTACs NPs nanoparticles in vivo.Mice were injected with Di R loaded NPs nanoparticles or Di R/DMSO/PBS solution through the tail vein.After 2 hours,the mice were anesthetized and euthanized,and organs such as the brain,heart,liver,spleen,and kidney were taken.Fluorescence distribution in isolated organs was observed using a small animal live imaging device.In addition,PROTACs NPs were prepared using FAM fluorescein labeled PROTACs molecules and injected into wild-type adult mice through the tail vein.After 2hours,the brain tissue was taken out and frozen into sections,and the fluorescence distribution in the brain tissue was observed under a special microscope.The results showed that the fluorescence intensity of the Di R-NPs nanoparticles group was generally higher than that of the control group(Di R-DMSO-PBS group),and Di R-NPs nanoparticles could significantly enrich and penetrate the brain.Compared with the control group,the Di R-NPs nanoparticle group showed an approximately 10 fold increase in fluorescence intensity in the brain.Frozen brain slices also have the same effect.In order to investigate the anti AD activity of PROTACs NPs nanoparticles in vivo,we selected 3 × Tg AD model mice(APP/PS1/tau transgenic mice)were used as experimental mice.Different drugs(PBS,NPs,PROTACs,and PROTACs NPs)were injected into the tail vein of the mice every day.After 10 consecutive days of injection,cognitive behavioral changes were evaluated through Morris water maze experiments.Then the mice were euthanized and brain tissue was extracted,and brain homogenate and pathological sections were prepared.The clearance efficiency of PROTACs NPs nanoparticles on tau protein was evaluated through Western Blot and immunofluorescence staining experiments.The results of cognitive behavioral experiments showed that compared to the PBS and NPs groups,the mice in the PROTACs NPs group showed significant improvement in dementia symptoms,while the PROTACs group showed a certain degree of improvement in dementia symptoms,but the improvement was not as significant as the PROTACs NPs group.Immunofluorescence staining and Western Blot experiments on brain tissue sections showed a significant decrease in tau protein in the PROTACs NPs group compared to other groups.Finally,the in vivo biocompatibility and safety issues of PROTACs NPs nanoparticles were discussed.During drug treatment,regularly measure the weight changes of mice in each group.After drug treatment,the differences in liver and kidney function indicators in the blood of each treatment group of mice were detected and analyzed to evaluate whether PROTACs NPs have hepatorenal toxicity in vivo.After the end of drug treatment,important organs of each group of mice(such as brain,heart,liver,spleen,lung,kidney,etc.)were dissected and collected,and the organ coefficients of each group of mice were weighed,calculated,and compared.Then,the mouse organs were made into pathological sections and stained with hematoxylin&eosin(HE staining)to observe whether there were morphological changes or inflammatory damage in the pathological sections of the PROTACs-NPs treatment group.The results showed that no significant changes were observed in organ coefficient and liver and kidney function between the PBS,NPs,PROTACs,and PROTACs NPs groups.The pathological examination of the organs was consistent with the biochemical results,and no significant pathological changes were observed.【Conclusions】In conclusion,this study confirmed that PROTACs NPs nanoparticles have good particle size,uniformity and stability;PROTACs NPs nanoparticles can effectively induce the clearance of overexpressed tau protein in cells in vivo and in vitro;NPs,as nanocarriers,effectively play the role of a nanodelivery system targeting cells and brain lesion sites,ensuring the therapeutic effect of PROTAC molecules on AD in vivo;PROTACs NPs nanoparticles have good biocompatibility in vivo.