AAV9-mediated Cdk5 Inhibitory Peptide (CIP) Reverses Pathological Changes And Behavioral Deficits In The Alzheimer's Disease Model Mice

BackgroundAlzheimer' s Disease(AD)is one of the most popular degenerative diseases.The incidence of AD is rising with the aging of the population.Currently,there is no effective medicine to treat AD.Therefore,to deeply elucidate the pathological mechanism of AD and explore new therapeutic strategy is urgent.Cdk5(cyclin-dependent kinase 5)widely expresses in all tissures cells.It is able to phosphorylate substrats containing serine/threonine(Ser/Thr).However,the activity of Cdk5 is prominently high in the central nervous system,due to the enrichment of its activator p35 in nurons.By binding and activation of Cdk5,p35 forms Cdk5/p35 complex,which phosphorylates a series of substrates and plays an important role in physiological function of neruons,including adhesion,cell migration,synaptic plasticity,and so on.However,proteolytic production of p25 from p35 by calpain under stress conditions leads to forming of Cdk5/p25.Compared to Cdk5/p35,complex of Cdk5/p25 has higer activity,which phosphorylates a series of substrates related to neurodegenerative diseases.Cdk5/p25 involves in the pathogenosis of neurodegenerative diseases,e.g.AD.Cdk5 inhibitory peptide(CIP)is the first identified peptide to inhibit the activity of p25/Cdk5,However,its application was limitied due to its difficulty to pass to blood and brain barrier.In this study,we evaluate the possibility to use adeno associated virus 9(AAV9)as the delivery system for CIP in the brain of AD mice.AAV9 is the most appropriate vector of gene therapy for CNS due to its safety,long-term expression and low immunogenicity.Purpose1.To construct AAV9-GFP-CIP system,packing and purification of AAV virus.2.To check the time and location of virus expression after intracerebroventricular injection of virus into mice.3.In AD mice model or control mice,AAV9-GFP-CIP or control virus were injected,the effects of AAV9-GFP-CIP on pathological changes and behavioral changes in AD mice was observed 3 or 7 months after virus injection.MethodAD mouse model:APP/PS1 double transgenic miceVirus injection:intracerebroventricular injectionTime of injection:3 months old,after the occurrence of the pathologic changes of AD miceThe mice were divided into 4 groups with 8 in each group(all mice received injection at 3 months old):1.WT group(wide type),C57BL/6J mice receive injection of PBS(phosphate-buffered saline);2.AD/PBS group,APP/PS1 model mice receive injection of PBS;3.AD/GFP group,APP/PS1 model mce receive injection of AAV9-GFP virus solution;4.AD/CIP group,APP/PS1 model mice receive injection of AAV9-GFP-CIP virus solution.Animal behavioral observation and pathological analysis:Behaviral study were tested at 6 month older:by the Morris water maze test(testing memory and cognitive ability),shuttle box active avoidance test(testing memory and cognitive ability),the elevated plus maze test(testing anxiety state).In vivo(whole animal)and ex vivo(organ)imaging were conducted to detect the virus expression at 6-month-old.Paraffin embedded specimen and slices were prepared,and IHC was performed for A ? deposit,GFAP staining and Ibal staining for astrocytosis and microcytosis respectively.Western blotting anayisis of brian protein to measure levels of hyperphosphorylation of tau,Ibal and cleaved caspase 3,marker of apoptosis Nissl staining was conducted for neuron counting.Paraffin embedded sections of mice at 10-month-old were prepared to observe A ? deposit,GFAP staining and Ibal staining.Results1.AAV9-GFP-CIP and AAV9-GFP were produced successfully.GFP was expressed in the brian when one week and 3 months after injection of virus.2.Tau hyperphosphorylation of AD model mice were alleviated after AAV9-GFP-CIP treatment.3.A ? deposit were reduced after AAV9-GFP-CIP treatment in AD model mice at 6-month-old or and 10-month-old check points.4.Aastrocytosis and microcytosis were decreased after AAV9-GFP-CIP treatment in AD model mice at 6-month-old or and 10-month-old check points.5.CIP treatment reverse memory and cognitive deficit and reduced anxiety of AD model mice.ConclusionsIntracerebroventricular injection of AAV9-GFP-CIP leads to reverse in the memory and cognitive deficit and reduction in the anxiety of AD model mice.One the molecluar basis,hyperphosphorylation of tau,A ? deposit,inflammation and apoptosis of brian cells were significantly ameliorated.Therefore,AAV9-mediated CIP is a promosing targeting strategy for the treatment of AD.