ELK1 Inhibition Alleviates Amyloid Pathology And Memory Decline By Promoting The SYVN1-Mediated Ubiquitination And Degradation Of PS1 In Alzheimer’s Disease

Objective:Alzheimer’s disease（AD）is a common neurodegenerative disease in the elderly.Extracellular signal-regulated kinase1/2（ERK1/2）is over-activated in AD and promotes the development of AD.However,ERK1/2 inhibitors cannot be applied clinically due to their toxic side effects and inability to cross the blood-brain barrier.Ets-like protein 1（ELK1）is the main target of ERK1/2,which is usually activated upon phosphorylation at Ser383 and Ser389 by ERK1/2.However,the role of ELK1 in AD pathogenesis remains to be elucidated.Our study aimed to explore the role and mechanism of ELK1 in the occurrence and development of AD,which might provide novel approaches and new target for the prevention and treatment of AD in clinical settings.Methods:（1）The expression of ELK1 in ADWestern blot and immunofluorescence of brain slices were used to detect the protein level of ELK1 in the hippocampus of AD patients,AD model mice and AD model cells.（2）The effect of knockdown of ELK1 on the cognitive function,synaptic plasticity and neuropathology in AD model miceThe APP23/PS45 double transgenic AD model mice received adeno-associated virus carrying ELK1 sh RNA(AAV_{sh ELK1})hippocampal microinjection（i.c.v.）at 2 months of age to knockdown ELK1.The cognitive function of mice was detected by Barnes maze and Morris water maze at 5 months of age.After the behavioral tests,hippocampal long-term potentiation（LTP）of some mice was recorded by electrophysiology in vitro.As for the remaining mice,half of the brain tissue was used for immunohistochemical analysis to detect the number of senile plaques in hippocampus.The hippocampus tissue was isolated from the other half of the brain tissue and the protein was extracted.The levels of Aβ40 and Aβ42were detected by ELISA,and the protein levels of APP,BACE1,PS1 andβ-CTF were detected by Western blot.（3）The effect of ELK1 on the expression of PS1Western blot was used to detect the protein levels of APP,BACE1,PS1,NCT,APH1A and PEN2 in N2A^APPand 2EB2 cells transfected with ELK1 or ELK1 small hairpin RNA(sh _ELK1)plasmid.RT-QPCR was used to detect the m RNA levels of APP,BACE1 and PS1 in 2EB2 cells transfected with ELK1 or sh _ELK1plasmid.The nuclear protein was extracted from HEK293 cells transfected with ELK1 and the ELK1 general sequence molecular probe labeled with infrared dye Alexa Fluor 700 was synthesized,and EMSA was used to detect the binding of ELK1 to the promoter of APP,BACE1 and PS1.CHX assay and Western blot were used to determine the effect of ELK1 on PS1 degradation in N2A^APPcells.Western blot was used to determine the degradation pathway of PS1 in N2A^APPcells treated with proteasome inhibitor MG132 or lysosome inhibitor chloroquine（CQ）.CO-IP and Western blot were used to verify PS1 was degradated by ubiquitin-proteasome pathway in HEK293 cells after treatement with or without MG132 and transfection with PS1 and Ub plasmids.CO-IP and Western blot were used to detect the effect of ELK1 on PS1 ubiquitination in HEK293 cells transfected with PS1 and Ub together with or without ELK1 plasmids.（4）The mechanism of ELK1 regulating ubiquitination and degradation of PS1Based on Ubi Browser prediction,the endogenous interaction between PS1 with SYVN1 or NEDD4L was detected by CO-IP and Western blot in N2A^APPcells.The exogenous interaction between SYVN1 and PS1 was detected by CO-IP and Western blot in HEK293 cells transfected with SYVN1 and PS1 plasmids.CHX assay and Western blot were used to determine the effect of SYVN1 on PS1 degradation in HEK293 cells.CO-IP and Western blot were used to detect the effect of SYVN1 on PS1ubiquitination in HEK293 cells transfected with PS1 and Ub together with or without SYVN1 plasmids.Western blot was used to detect the effect of ELK1 on the expression of SYVN1 in N2A^APPand 2EB2 cells transfected with ELK1 or sh _ELK1plasmid.The endogenous interaction between ELK1and PS1 was detected by CO-IP and Western blot in N2A^APPcells.The exogenous interaction between ELK1 and PS1 was detected by CO-IP and Western blot in HEK293 cells transfected with ELK1 and PS1 plasmids.CO-IP and Western blot were used to detect the effect of ELK1 on the interaction between SYVN1 and PS1 in HEK293 cells transfected with PS1and SYVN1 together with or without ELK1 plasmids.CO-IP and Western blot were used to detect the effect of ELK1 on the SYVN1-mediated PS1ubiquitination in HEK293 cells transfected with PS1,Ub and SYVN1together with or without ELK1 plasmids.（5）The effect of ELK1 phosphorylation on the SYVN1-mediated ubiquitination and degradation of PS1Western blot was used to detect the protein level of p-ELK1 in the hippocampus of AD model mice and AD model cells.Western blot was used to detect the effect of ELK1 phosphorylation inhibitor TAT-DEF-ELK1（TDE）on the expression of PS1 in N2A^APPcells.CO-IP and Western blot were used to detect the effect of ELK1 phosphorylation on the interaction between ELK1 and PS1 in HEK293 cells transfected with PS1 and ELK1 or p-ELK1 plasmids.CO-IP and Western blot were used to detect the effect of ELK1 phosphorylation on PS1 ubiquitination in HEK293 cells after transfection with PS1,Ub and ELK1 or p-ELK1plasmids followed by treatment with or without TDE.CO-IP and Western blot were used to detect the effect of ELK1 phosphorylation on the SYVN1-mediated PS1 ubiquitination in HEK293 cells after transfection with PS1,Ub,SYVN1 and ELK1 or p-ELK1 plasmids followed by treatment with or without TDE.（6）The effect of inhibition of ELK1 phosphorylation on the cognitive function,synaptic plasticity and neuropathology in AD model miceThe APP23/PS45 double transgenic AD model mice received intraperitoneal injections of TDE（i.p.,8mg/kg/day）starting at 2 months of age to inhibit ELK1 phosphorylation.The cognitive function of mice was detected by Barnes maze and Morris water maze at 5 months of age.After the behavioral tests,hippocampal long-term potentiation（LTP）of some mice was recorded by electrophysiology in vitro.As for the remaining mice,half of the brain tissue was used for immunohistochemical analysis to detect the number of senile plaques in hippocampus.The hippocampus tissue was isolated from the other half of the brain tissue and the protein was extracted.The levels of Aβ40 and Aβ42 were detected by ELISA,and the protein levels of APP,BACE1,PS1 andβ-CTF were detected by Western blot.Results:（1）The expression of ELK1 is increased in ADThe protein level of ELK1 was significantly increased in the hippocampus of AD patients,AD model mice and AD model cells.（2）Knockdown of ELK1 improves cognitive function,synaptic plasticity and neuropathology in AD model miceCompared with WT mice,AD mice showed significant cognitive and synaptic impairment,while knockdown of ELK1 markedly improved the impaired cognitive and synaptic function in AD mice.Compared with WT mice,the protein levels of AD-associated proteins including APP,BACE1,PS1 andβ-CTF,the levels of Aβ40 and Aβ42 as well as the number of senile plaques were significantly increased in the hippocampus of AD mice.However,knockdown of ELK1 significantly reduced the protein levels of PS1 and BACE1,decreased the levels of Aβ40 and Aβ42 as well as the number of senile plaques in the hippocampus of AD mice.（3）ELK1 promotes APP amyloidogenic processing by inhibiting the ubiquitination and degradation of PS1In N2A^APPcells,knockdown of ELK1 by sh _ELK1significantly reduced the protein level of PS1,but not APP,BACE1,NCT,APH1A or PEN2,however,overexpression of ELK1 had no effect on the protein levels of the above proteins.In 2EB2 cells,knockdown of ELK1 also reduced the protein level of PS1,but had no effect on the m RNA level of PS1,and there was no binding between ELK1 and the promoter of PS1 gene,indicating that ELK1 did not affect the synthesis of PS1.However,PS1 was mainly degraded by the ubiquitin-proteasome pathway and ELK1 inhibited the ubiquitination and degradation of PS1.（4）ELK1 inhibits the SYVN1-mediated ubiquitination and degradation of PS1According to Ubi Browser prediction,SYVN1 is the most likely E3ubiquitin ligase of PS1,followed by NEDD4L.SYVN1 had obvious endogenous and exogenous interactions with PS1,while NEDD4L had no interaction with PS1.Furthermore,SYVN1 significantly promoted the ubiquitination and degradation of PS1,indicating that SYVN1 was the E3ubiquitin ligase of PS1.Neither knockdown nor overexpression of ELK1affected the protein level of SYVN1,but there were obvious endogenous and exogenous interactions between ELK1 and PS1,and ELK1significantly inhibited the interaction between SYVN1 and PS1 as well as the SYVN1-mediated PS1 ubiquitination,indicating that ELK1 reduced the SYVN1-mediated ubiquitination and degradation of PS1 by competitively inhibiting the binding between PS1 and SYVN1.（5）Phosphorylation of ELK1 inhibits the SYVN1-mediated ubiquitination and degradation of PS1The protein level of p-ELK1 was significantly increased in the hippocampus of AD model mice and AD model cells.ELK1phosphorylation inhibitor TDE significantly reduced the protein level of PS1.Compared with ELK1,p-ELK1 showed stronger interaction with PS1,and p-ELK1 further inhibited the SYVN1-mediated PS1 ubiquitination.On the contrary,inhibition of ELK1 phosphorylation by TDE significantly reversed the inhibition of ELK1 on the SYVN1-mediated ubiquitination of PS1.Taken together,these results suggest that ELK1,particularly in its phosphorylated form,plays a crucial role in the SYVN1-mediated ubiquitination and subsequent degradation of PS1.（6）Inhibition of ELK1 phosphorylation improves cognitive function,synaptic plasticity and neuropathology in AD model miceInhibition of ELK1 phosphorylation by TDE markedly improved the impaired cognitive and synaptic function in AD mice.Inhibition of ELK1phosphorylation by TDE significantly reduced the protein level of PS1,decreased the levels of Aβ40 and Aβ42 as well as the number of senile plaques in the hippocampus of AD model mice.Conclusion:Our study demonstrates that ELK1 and its phosphorylated form p-ELK1 are aberrantly elevated and plays critical roles in the pathogenesis of AD.Both genetic knockdown and pharmacological inhibition of ELK1reduce APP amyloidogenic processing by promoting the SYVN1-mediated ubiquitination and degradation of PS1,thereby inhibiting Aβgeneration and improving the synaptic and cognitive impairments in a mouse model of AD.These findings reveal new insights into the role of ELK1 in AD pathogenesis and provide scientific basis for the development of ELK1inhibitor to treat the learning and memory deficits associated with both patients with AD and aged populations.