Study Of USP10 On Tau Aggregation In Alzheimer’s Disease Pathogenesis

Background:Alzheimer’s disease（AD）is the most common neurodegenerative disease in the elderly.Intracellular hyperphosphorylated Tau aggregates known as neurofibrillary tangles（NFTs）in addition to senile plagues（SPs）formed by extracellular Aβdeposits are features of AD.Tau is a microtubule-associated protein that binds to microtubules（MTs）to maintain normal function of microtubules.In AD,hyperphosphorylated Tau is thought to cause detachment from MTs and subsequently aggregation into fibrils,leading to neuronal death.Tau aggregation and clearance are regulated by various posttranslational modifications,among which ubiquitination is the key modification that controls Tau to enter the degradation system,and phosphorylated Tau can enter the proteasome and lysosome for degradation by ubiquitination.The ubiquitination of protein is reversible and can be reversed by deubiquitinases（DUBs）.Ubiquitin-specific protease 10（USP10）is a highly conserved deubiquitinases that is widely expressed in the brain,but whether it is involved in the regulation of Tau aggregation and whether it is involved in the molecular mechanisms of AD process remains unclear.Objective:To clarify the involvement of USP10 in onset of AD,and to elucidate the mechanism by which USP10 promotes Tau accumulation,leading to neuronal degeneration.The results from this study are going to provide new clues for the pathogenesis of AD and new target for clinical drug development.Methods:The Gene Expression Omnibus（GEO）database was performed to analysis the gene expression levels of USP10.The distribution and the level of USP10 in AD postmortem,APP/PS1 and P301S mice were detected by immunofluorescence and western blotting（WB）.N2a cells were transfected with Tau441 plasmids or stimulated with Aβ₄₂oligomers（Aβo）,WB was performed to detect USP10 level.AAV-USP10 or Aβo were performed to treat with hippocampal primary neurons,and the level of USP10,Tau5,p S199,AT8,p-AMPK（Thr172）,AMPK,Tau-ubi and Tau-USP10 were detected by immunoblotting and immunohistochemical staining.0.1%Triton soluble and insoluble fractions were separated,and Tau5 as well as p-Tau levels in each fraction were measured.Cycloheximide（CHX）and WB were utilized to detect the level of Tau5.SH-SY5Y and N2a cells were overexpressed with USP10-OE or si-USP10 plasmids,and WCL-ubi,USP10,Tau5,p-Tau and ubi-Tau were detected by WB.Immunoprecipitation,IP-MS and fluorescence co-localization analysis were used to detect USP10-Tau binding.Molecular docking was used to show the USP10-Tau binding pattern,and interfering peptides were designed and synthesized.In vitro tube assay was performed to clarify the ability of peptides on interfering USP10-Tau binding.Incubation of primary neurons with FITC-labeled peptides could be taken up by neurons.CCK8 assay detects neuronal activity after peptide incubation.Overexpression of USP10 in HEK293Tau cells while incubating the peptides to find the proper administration in cells.Neurons overexpressing USP10 or treated with Aβo were administered with peptides incubation and immunoblotted for Tau5 and p-Tau levels.Mice were stereotactically injected with USP10 or Vector virus in hippocampus.The level of anxiety and cognitive function were assessed using behavioral experiments.Golgi staining,electrophysiology and WB were used to detect the synaptic functions.Tau5,p-Tau and Aβcontent were detected by WB and IF.Results:1.GSE5281 dataset showed an increase gene expression of USP10 in AD patients’hippocampi,and USP10 was elevated in AD postmortem brain,APP/PS1 mice and Aβo-treated N2a cells,but not in P301S mice and Tau overexpressing N2a cells.2.Addition of Aβo was observed to induce USP10 upregulation and an increase in the level of Tau5 and p-Tau,while a decrease in Tau-ubi levels.3.WB and CHX results showed that overexpression of USP10 directly caused an increase in the level of total and phosphorylated Tau,and a delay in Tau degradation,causing Tau aggregation.4.IP-MS,immunoprecipitation and immunofluorescence assays showed Tau interacted with USP10.Molecular docking displayed that USP10 had high binding affinity for Tau.Based on the docking results,interfering peptides were synthesized.WB and CCK8 assays showed interfering peptides competitively blocked the binding of USP10 to Tau with little cytotoxicity.5.WB showed the binding of Tau to USP10 was significantly inhibited in primary neurons using 307-326K+341-378K,while reversed upregulation of total and phosphorylated Tau caused by USP10 overexpression as well as Aβo incubation.6.The behavioral results showed that USP10 overexpression did not affect anxiety level in mice,but cognitive function was markedly reduced in mice.Golgi staining,electrophysiology and WB showed impaired synaptic function in mice.WB and IF showed a significant increase in the level of p-Tau and Tau5,along with an increase in Aβaggregation.Conclusion:In AD,Aβcauses upregulation of USP10,which deubiquitinates Tau and promotes Tau hyperphosphorylation,causing Tau aggregation and leading to cognitive impairment.Inhibiting the interaction of Tau with USP10 could reverse the above associated tauopathy and it provide new clues for AD treatment strategy.