DAPK1 Induce Nerve Degeneration In Alzheimer's Disease

Background Alzheimer's disease(AD)is a progressive neurodegenerative disease characterized by memory deficits and cognitive decline.The main two major features of the disease are neurofibrillary tangles formed by intracellular tau hyperphosphorylation and senile plaques formed by extracellular A? short peptides.However,A? plaques and neurofibrillary tangles are not sufficient to explain all the features of AD,and there is no real correlation between the level of cortical plaques and the cognitive impairment associated with AD,the use of immunotherapeutic methods to reduce brain A? levels did not improve the cognitive function of AD patients,although evidence suggests clearance of the A? plaques.In early AD,degeneration takes place in Basal forebrain cholinergic neurons,eventually leading to choline neurotransmitter decrease in its target nucleus such as hippocampus,cortex,amygdala,with the loss of these projections,cognitive function gradually decreased.If we can find the molecular mechanism of cholinergic neuron degeneration in early AD,it will provide new treatment ideas and measures for AD patients.In our earlier study,DAPK1(death-associated protein kinase 1)was found to bind with different substrates(eg,tau,NR2 B,p53),induce synaptic injury of neurons and activation of multiple cell death pathways in cerebral ischemia,leading to neuronal apoptosis or necrosis,the important regions of DAPK1 binding to the substrate are mainly kinase domain,death domain,calmodulin binding domain,and DAPK1 through the kinase domain binding with Tau mediate ischemic injury has been confirmed,based on the previous work,we use the transgenic means to interfere cholinergic neurons DAPK1 activity,combined with behavioral,molecular biology and electrophysiological methods,found that inhibition of DAPK1 can effectively treat neurodegeneration and memory loss early in the AD.Suggest DAPK1 plays an important role in the treatment of Alzheimer's disease.Objective(1)Clear that DAPK1 involvement in early Alzheimer's disease mediates the degeneration of cholinergic neurons;(2)Conform the molecular mechanism of DAPK1-mediated apoptosis of cholinergic neurons in early dementia;(3)Explore the new strategy for the treatment of senile dementia with DAPK1 as a target.Methods First,we imitated the Alzheimer's disease by injecting synthetic human A?1-42 into the bilateral lateral ventricles of mice,then we use the Western Blotting technique to detect the levels of tau phosphorylation,synaptic-associated protein and ChAT,the content of A? in hippocampus of mice was detected by enzyme-linked immunosorbent assay(ELISA),the spatial learning and memory of mice were examined by Water Morris,the Fear Conditioning experiment to test the fear memory of the mice,detection of spontaneous activity and anxiety in mice respectively by open field and elevated plus maze,by means of electrophysiological patch clamp,long-term potentiation(LTP),spontaneous excitatory postsynaptic currents(s EPSC),spontaneous inhibitory postsynaptic currents(s IPSCs)were detected,the brain structure of the mice was examined by hematoxylin staining,the mouse genotype was identified by PCR,the amount of DAPK1 m RNA was detected by fluorescence quantitative PCR,in order to obtain cholinergic neuron-specific knockout kinase domain of death – associated protein kinase in mice,we crossed conditionally induced knockout DAPK1-KD mice with ChAT-IRES-Cre ER transgenic mice expressing cre recombinase in cholinergic neurons,double transgenic mice can be induced by Tamoxifen.Results 1.Construction mouse model of Alzheimer's Disease.Alzheimer's disease model was successfully constructed,at the time point for 7d,14 d,28d we got on some relevant test,found that the deficit of fear memory occurred 7d after A?1-42 injection,but the increase of tau protein phosphorylation and the endogenous A? content occurred 14 days after injection.2.Synaptic dysfunction occurred 7 days after the injection.The synaptic function was observed 7 days after A? injection by electrophysiological methods,mainly in the spontaneous excitatory postsynaptic current frequency increases,the amplitude was unchanged,inhibition of spontaneous postsynaptic current frequency was unchanged,but the amplitude decreased,by recording CA1 pyramidal neuronal field potential,found that LTP is difficult to induce and maintain.3.A?1-42 injection into the lateral ventricle leads to cholinergic system damage.There was no significant change in the number of cholinergic neurons in the basal forebrain compared with the PBS control group mice 7 days after A?1-42 injection into the lateral ventricle,but the content of ChAT,as a cholinergic neuron marker was significantly lower than that in control group mice in the hippocampus,meanwhile the activity of Ach E in hippocampus was significantly increased when compared with the control group.4.Successful construction of cholinergic neurons specific knockout the kinase domain of death – associated protein kinase 1 transgenic mice.The conditionally induced knockout DAPK1-KD mice were crossed with ChAT-IRES-Cre ER transgenic mice expressing cre recombinase in cholinergic neurons,double transgenes were induced by tamoxifen then we will obtain cholinergic neurons specifically knockout the kinase domain of death-associated protein kinase 1 of transgenic mice,the results of q PCR showed that the m RNA content of knockout DAPK1 was significantly lower than that of the control group,and knocking out the kinase domain in DAPK1 does not affect the growth of mice,the structure of the mouse brain,the spontaneous activity of mice and mood.5.Knockout of DAPK1 kinase domain protects cholinergic system damage.The activity of acetylcholinesterase and the expression of ChAT protein in hippocampus of mice were detected 7 days after A?1-42 injection in DAPK1-KD transgenic mice,and the results showed that knockout of the kinase domain in DAPK1 could protect cholinergic system damage.6.Knockout of DAPK1 kinase domain protects synaptic injury.After 7 days of injection of A?1-42,the electrophysiological results showed that the injury of s EPSC and s IPSC in hippocampal CA1 area returned to normal level,the long-term potentiation of CA3-CA1 was also reversed.7.Knockout of DAPK1 kinase domain improves mouse learning and memory behavior performance.After 7 days of A?1-42 injection,the water maze showed a significant increase in spatial learning ability of DAPK1 kinase domain knockout mice,and returned to a similar level to that of the control group,the Fear Conditioning experimental results indicate that delete DAPK1 kinase domain can rescue the emotional memory disorder.Conclusions In the A?1-42 simulated Alzheimer's model,cholinergic system damage and synaptic loss occurred in the early stage,and specific knockout the kinase domain of death-associated protein kinase 1 in cholinergic neurons inhibits the hippocampus synaptic function damage and learning memory reduction.