The Study Of The Role And Mechanism Of Liver X Receptor β In SD-induced Cognitive Impairment

Background and AimsIt is known that many factors can cause cognitive impairment,among which sleep deprivation(SD)is one of the most common clinical causes of cognitive impairment and has gradually become an important clinical issue that has attracted much attention.Studies have shown that SD reduction in cognitive function is related to the reduction of hippocampal neuroplasticity-related proteins and impaired hippocampal neurogenesis.However,the specific mechanism and potential intervention targets of SD-induced cognitive impairment have not yet been clarified.Therefore,in-depth exploration of this mechanism is of great scientific value for finding drug therapeutic targets to prevent or treat cognitive impairment.Studies have shown that in rodents,sleep deprivation affects synaptic functional and structural plasticity in different ways.Hippocampal plasticity plays an important role in cognitive impairment caused by sleep deprivation.It has been confirmed that the protein expression level of the postsynaptic glutamate receptor AMPAR subunit Glu A1 is significantly reduced in the SD model,leading to immature dendritic spine density and reduced hippocampal synaptic efficiency,which in turn causes behavioral manifestations of cognitive impairment in SD animals.Previous studies have found that the maintenance of AMPAR homeostasis is regulated by ubiquitination.Neural precursor cell expressed developmentally downregulated 4-1(Nedd4-1)is an E3 ubiquitin ligase that has been reported to interact with AMPAR,and Nedd4-1 is significantly increased after SD.On the other hand,abnormal function of Liver function and neuronal survival.This receptor includes two subtypes,LXRα and LXRβ,of which LXRβ is closely related to central degenerative diseases.Studies have found that Lxrβ-deficient mice develop autism-like behaviors and hippocampal hypoplasia;while LXRs agonists have beneficial effects on Alzheimer disease,Parkinson disease and stroke.GW3965 is a synthetic LXRs agonist that can activate LXRα and LXRβ isoforms at the same time and easily penetrates the blood-brain barrier to exert its effects.However,the role of LXRβ in cognitive impairment caused by SD remains largely unknown.This study intends to establish a mouse model of SD-induced cognitive impairment,and comprehensively use experimental methods such as animal behavior,electrophysiology,molecular biology,and epigenetics to conduct the following studies:(1)Evaluate the impact of activated LXRβ on SD-induced cognitive impairment Behavioral changes;(2)Study the effect of activating LXRβ on synaptic plasticity in SD mice;(3)Elucidate the effect of Nedd4-1 overexpression on the ubiquitination level of AMPAR subunit Glu A1;(4)Verify that LXRβ mediates the transcriptional regulation of Nedd4-1 and inhibits Ubiquitination of AMPAR is a key regulatory mechanism that affects cognitive impairment caused by SD.The above studies revealed that the expression of LXRβ is down-regulated and the expression of negative transcriptional regulator Nedd4-1 is increased,thereby mediating the excessive ubiquitination and degradation of AMPAR,which may be an important mechanism by which SD causes cognitive impairment.This study will explore potential new targets for drug treatment and provide new ideas and theoretical basis for the treatment of cognitive impairment.Methods1.A sleep deprivation instrument was used to continuously deprive mice of sleep for 5days,evaluate and analyze whether SD mice develop cognitive impairment through new object recognition,Y maze and morris water maze;detect two LXRs in the hippocampus of mice in the Control group and SD group Changes in protein expression levels of receptor subtypes.After intraperitoneal injection of the LXRs agonist GW3965 for 5 days,the effect of GW3965 on the cognitive impairment behavior of SD mice was evaluated.2.Determine the cell specificity and neuron type of LXRβ expression in CA1 through immunofluorescence technology.3.Construct sh RNA adeno-associated virus to down-regulate the expression of LXRαand LXRβ in the CA1 region of mice.After intervention with GW3965,observe the behavioral changes of mice in different groups to determine the key LXRs subtypes for GW3965 to improve cognitive impairment in SD mice.4.Stereotaxically inject the adeno-associated overexpression virus LXRβ into the brain,establish the SD model through the above method,and observe the behavioral changes of mice.5.Establish an SD mouse model,and after treatment with GW3965.MED64,whole-cell patch clamp and Golgi staining techniques were used to detect its effects on LTP,excitatory synaptic transmission and dendritic spine density in the CA1 area of SD mice to prove GW3965 corrects abnormal synaptic plasticity.6.Detect the ubiquitination level of AMPAR subunit Glu A1,AMPAR subunit Glu A1 and Glu A2 membrane proteins and total AMPAR subunit Glu A1 in SD mice before and after GW3965 treatment through co-immunoprecipitation(Co-IP)and Western blot techniques.7.Stereotaxically inject the protease inhibitor MG132 into the brain to observe the behavior of SD mice,excitatory synaptic transmission of neurons in the CA1 area,and changes in AMPAR subunit Glu A1 membrane protein and total protein to determine the cognition caused by SD.The disorder may be due to proteasomedependent degradation of glutamate receptors in the CA1 region of the hippocampus.8.Through Co-IP and Cleavage under targets and tagmentation(CUT&Tag)technology,it was determined that the E3 ubiquitin ligase Nedd4-1 may be the target gene of the nuclear receptor LXRβ.9.Establish an SD mouse model,and detect Nedd4-1 m RNA levels in RT-q PCR experiments after GW3965 treatment.Primary hippocampal neurons were cultured in vitro,and changes in Nedd4-1 m RNA levels were detected after the cells were treated with GW3965 for 24 hours.10.Lentiviral overexpression vector upregulates the expression of Nedd4-1 in the CA1 region of SD mice,and observes the behavior of the mice,the changes in AMPAR subunit Glu A1 membrane protein and total protein in the hippocampus,to determine the possibility of activating LXRβ to improve cognitive impairment caused by SD.It is due to the regulation of Nedd4-1 in hippocampal CA1.Results1.An acute SD mouse model was successfully constructed,and behavioral testing revealed that SD can induce cognitive impairment.Western blot found that the expression level of LXRβ in the hippocampus of SD mice was significantly reduced,but the expression level of LXRα had no significant change.In addition,GW3956 treatment can significantly improve the cognitive impairment of SD mice.2.LXRβ is mainly expressed in excitatory neurons,and a small amount of LXRβ is expressed in inhibitory neurons,astrocytes and microglia.3.LXRβ is the key LXRs subtype that mediates GW3965’s improvement of cognitive impairment in SD mice.4.Overexpression of LXRβ in hippocampal CA1 can significantly improve cognitive impairment in SD mice.5.The agonist GW3965 can promote the increase in m EPSCs frequency and amplitude,Glu R1 membrane protein and total protein expression,restore damaged LTP,and increase dendritic spine density in CA1 pyramidal neurons of SD mice.It is suggested that activation of LXRβ can correct synaptic plasticity abnormalities.6.GW3965 can reduce the ubiquitination level of AMPAR subunit Glu A1 in SD mice and promote the expression of membrane proteins and total proteins of AMPAR subunits Glu A1 and Glu A2.7.Stereotactic injection of the protease inhibitor MG132 into the hippocampal CA1 area can improve the cognitive impairment caused by SD,suggesting that the cognitive impairment caused by SD is due to the proteasome-dependent degradation of glutamate receptors in the hippocampal CA1 area.8.Through Co-IP and CUT&Tag technology,the E3 ubiquitin ligase Nedd4-1 was identified as the target gene of the nuclear receptor LXRβ.9.In vivo and in vitro results demonstrate that GW3965 can reduce Nedd4-1 m RNA levels.10.Overexpression of Nedd4-1 in hippocampal CA1 region significantly reverses the improvement effect of GW3965 on cognitive impairment in SD mice and inhibits the up-regulation effect of GW3965 on Glu A1 protein expression.It is suggested that activating LXRβ can improve the cognitive impairment caused by SD by inhibiting the transcription and expression of Nedd4-1 and reducing the ubiquitination and degradation of Glu A1.ConclusionsOur study reveals that LXRβ dysfunction is involved in SD-induced cognitive impairment;GW3965 improves cognitive impairment in SD mice by activating LXRβ;it is first clear that LXRβ regulates Nedd4-1 transcription,mediates AMPAR ubiquitination and is involved in sleep deprivation role in learning and memory impairment.The summary is as follows:(1)Abnormal expression of LXRβ in the hippocampus were involved in the occurrence and development of SD-induced cognitive impairment.(2)The activation of LXRβ played a role in improving SD-induced cognitive impairment by correcting abnormal synaptic plasticity in the hippocampus.(3)Activating LXRβ reduces the transcription of Nedd4-1 and inhibits the ubiquitination and degradation of AMPAR subunit Glu A1 to improve cognitive impairment induced by SD.