Role And Mechanism Of Abnormal Activation Of Chk1 In Inducing Alzheimer’s Disease Through CIP2A-PP2A Signaling

Background:Alzheimer’s disease（AD）is a common neurodegenerative disorder accompanied by progressive development of cognitive impairments and synaptic dysfunction,with dementia as the ultimate clinical symptom.The two characteristic pathological hallmarks in AD are neurofibrillary tangles（NFTs）and senile plaques（SPs）in the brain.NFTs and SPs are respectively formed by abnormal hyperphosphorylated microtubule-associated protein Tau and the aggregation of Aβgenerated byβ-amyloid precursor protein（APP）through cleavage byβ-andγ-secretase.Hyperphosphorylated and aggregated Tau and Aβoligomers have toxic effects on synapses and induce neuronal damage and loss in AD brain.Cell cycle checkpoint protein kinase 1（Chk1）is a Ser/Thr protein kinase which is activated in response to DNA damage,the latter which is an early event in AD development.Whether Chk1 is activated in AD brain and participates in AD pathogenesis remain unclarified.Objective:To explore the role and mechanism of Chk1 activation in mediating AD-like Tau/APP hyperphosphorylation,synaptic damage and cognitive impairment through CIP2A-PP2A signaling,and to explore the potential effect of Chk1 inhibitor in AD therapy.Methods:Western blotting was performed to detect the protein levels of total and active phosphorylated forms of Chk1,and Chk1 downstream target CIP2A in AD human brains,APP/PS1 transgenic mice and primary neurons with Aβtreatment.Primary neurons were cultured with 2μM Aβoligomers for 48 h with or without pre-incubation of Chk1 inhibitor SB218078（1μM）or PF477736（1μM）for 48 h.Cytotoxicity was detected by LDH assay kit,PP2A activity was detected by PP2A activity assay kit.DNA damage,Chk1,active phosphorylated forms of Chk1,CIP2A,Tau protein phosphorylation（Tau-S396,）and APP phosphorylation（APP-T668）were detected by Western blotting.AAV-Chk1 virus was injected into the lateral ventricle of C57 mice to induce Chk1 overexpression in vivo,and the mice were tested for cognitive function with behavioral tests.Synaptic impairment was evaluated by Gogli staining.The phosphorylation levels of Tau/APP were detected by Western blotting.Chk1 inhibitor GDC-0575（0,0.2,0.5,1μM）was used to treat AD cell models（HEK293/tau cells and N2a/APP cells）.Tau and APP phosphorylation were detected by Western blotting,PP2A activity assay and ELISA were employed to detect the PP2A activity and the level of Aβ_40/42 respectively.Chk1 inhibitor GDC-0575 was used to treat APP/PS1 mice by oral gavage for 3 weeks.Western blotting,PP2A activity assay and ELISA were employed to verify the effect of Chk1 inhibitor on PP2A activity,Tau phosphorylation and Aβproduction.The cognitive function of the mice was assessed by a suite of behavior tests.Nissl staining and Golgi staining were used to evaluate the neuron loss and synaptic impairment respectively.Results:1.Chk1 activity and the levels of CIP2A are increased in the brains of AD patients,APP/PS1transgenic mice and primary neurons with Aβtreatment.2.Chk1 inhibition reverses CIP2A overexpression,PP2A inhibition,Tau hyperphosphorylation in Aβ-treated primary neurons.3.Chk1 overexpression in neurons of the mouse brain induces CIP2A upregulation,Tau and APP-T668 hyperphosphorylation,synaptic impairments and cognitive deficits.4.Chk1 inhibitor（GDC-0575）reduces CIP2A expression,activates PP2A,decreases Tau phosphorylation and Aβlevels in AD cell models.5.Chk1 inhibitor（GDC-0575）decreases Tau hyperphosphorylation,Aβoverproduction and reversed AD-like cognitive deficits and synaptic impairments in APP/PS1 mice.Conclusion:Our study uncovers a mechanism by which DNA damage-induced Chk1activation mediates Tau and APP-T668 hyperphosphorylation through CIP2A-PP2A signaling pathway,leading to synaptic impairment and cognitive memory dysfunction and highlight the therapeutic potential of Chk1 inhibitors in AD.