Role Of KEAP1-NRF2 Signaling In Tau-Induced Inhibition Of Synaptic Protein Expression And The Underlying Molecular Mechanisms

Background: Tauopathies are a subset of neurodegenerative diseases characterized by the aggregation of human Tau(hTau)in neurons as the main pathological feature,such as Alzheimer’s disease(AD),frontotemporal dementia and parkinsonism linked to chromosome 17(FTDP-17),etc.Cognitive impairment is the main clinical manifestation of the tauopathies patients and synapse is fundermental for learning and memory functions.However,the mechanism underlying Tau-induced synapse impairment and cognitive dysfunction is unclear.Objective: This study was to investigate the effects of human-derived frontotemporal dementia mutant hTau protein(P301S)on synaptic morphology and synaptic protein expression,and the underlying mechanism.Methods: Animal and cell models of Tauopathy were established by overexpressing P301S-hTau in mouse hippocampus,primary hippocampal neurons and mouse neuroblastoma 2a(N2a)cells,respectively.Western blotting,quantitative polymerase chain reaction(q PCR),immunohistochemistry and immunofluorescence were used to detect protein and m RNA of synaptic proteins.The cognition of mice was assessed by Morris water maze,fear conditioning test,novel object recognition and other behavioral assays.The transcriptional activity of nuclear factor erythroid-2 related factor 2(NRF2)was measured by dual luciferase reporter gene assay.The binding of NRF2 to the anti-oxidative response element(ARE)motif was examined by electrophoretic mobility shift assay(EMSA)and chromosome immunoprecipitation.Results: 1.Overexpressing P301S-hTau significantly increased oxidative stress in mouse hippocampus,primary hippocampal neurons and N2 a cells,accompanied by decreased NRF2 protein level and increased KEAP1 protein level.2.Downregulation of NRF2 inhibited the expression of synaptic proteins PSD93,PSD95 and SYN1,decreased dendritic spine density,impaired neuronal morphological complexity,and led to cognitive impairment.3.NRF2 transcriptionally regulated synaptic protein expression by binding to promoter region of the genes DLG2(encoding synaptic protein PSD93),DLG4(encoding synaptic protein PSD95)and SYN1;overexpressing P301S-hTau inhibited the transcriptional regulation of NRF2 on the synaptic protein genes,leading to reduced synaptic protein expression.4.KEAP1 inhibited NRF2 protein level and reduced NRF2 level in the nucleus through a ubiquitin-proteasome degradation system.5.P301S-hTau could directly bind to and acetylates KEAP1,and thus inhibited its ubiquitination and protein degradation rate with an elevated KEAP1 protein level.6.Downregulation of KEAP1 ameliorated synaptic injury caused by P301S-hTau aggregation and decreased NRF2.7.KEAP1 could bind to P301S-hTau via the K312 site,and K312 R mutations or the peptide targeting the K312 site significantly blocked KEAP1’s binding to P301S-hTau,with amelioration of the downregulated synaptic proteins expression,the decreased dendrtic spines,the reduced neuronal complexity and cognitive dysfunction caused by P301S-hTau.Conclusion: P301S-hTau protein could function as an acetylase to acetylate KEAP1 protein,which in turn reduced NRF2 protein level and the transcriptional activity,leading to the decreased synaptic protein expression,dendritic spines and neuronal complexity,and eventually impaired cognitive function.Targeting the KEAP1/NRF2 pathway,i.e.,blocking KEAP1 binding to P301S-hTau,or down-regulating KEAP1 expression,or overexpressing NRF2 could improve synaptotoxicity and cognitive dysfunction caused by P301S-hTau.These findings revealed novel mechanisms underlying hTau-induced synaptic and cognitive deficits,which also provided new molecular markers for arresting the Tauopthies.