Study On The Neuropeptide S Improves Sleep Disturbance In APP/PS1 Mice By Reducing Aβ Deposition

Alzheimer’s disease（AD）is a common progressive neurodegenerative disease,which typical pathological features include amyloid-β（Aβ）deposition.AD has developed into a major public health problem with high prevalence and great damage to public health and happiness index.In the clinical manifestations of AD,sleep disorder is one of the main neurobehavioral disorders.About 25～66%of AD patients have sleep disorders such as insomnia,sleep fragmentation and excessive daytime sleepiness.Conversely,the occurrence of sleep disorder leads to the increase of Aβcontent in the brain and aggravates the deposition of Aβ,which is an important risk factor for the occurrence and development of AD.Therefore,AD and sleep disorder are a bidirectional relationship,discovering and identifying of key molecules that can mediate the bidirectional relationship of sleep disorder and AD is of great significance for the prevention and treatment of AD.Neuropeptide S（NPS）is an important central neuropeptide,which can regulate sleep-wake behavior.In addition,exogenous NPS can reduce the deposition of Aβ.Therefore,in this study,we will explore whether NPS can improve the sleep-wake behavior of APPswe/PS1d E9（APP/PS1,classical animal model of AD）mice by regulating Aβdeposition,and to providing a new strategy for the treatment and prevention of sleep disorders in AD.Objective:To explore the effects of NPS on sleep wake behavior and related molecular pathological mechanisms in APP/PS1 mice.The aim is providing a new theoretical basis and prevention strategies for the prevention and treatment of AD sleep disorders.Method:Taking 8-month-old APP/PS1 double transgenic mice as the research object,（1）To detect the effect of NPS on the sleep-wake cycle of APP/PS1 mice:single injection of and chronic injection of NPS were used.For the single NPS injection,the mice were divided into three groups:APP/PS1+Sal（control group）,APP/PS1+0.1nmol NPS,APP/PS1+1 nmol NPS;For the chronic injection of NPS,saline（APP/PS1+Sal-14 days group）and 1nmol NPS（APP/PS1+NPS-14 days group）were injected for 14 days,and WT+Sal-14 days group was used as the basic control group.The electrophysiological signals of electroencephalogram（EEG）and electromyography（EMG）were monitored and analyzed.（2）To detect the deposition of Aβand its precursor protein in the brain of APP/PS1 mice after NPS administration:The brain tissues after single NPS injection and chronic NPS injection were collected.Immunofluorescence was used to detect the changes of Aβdeposition.The protein levels of amyloid precursor protein（APP）,phosphorylated amyloid precursor protein（p-APP）and NPS receptor（NPSR）were detected by Western blot;（3）To detect whether the effect of NPS on sleep in APP/PS1 mice can be reversed after intracerebroventricular injection of Aβ:APP/PS1 mice injected with 1 nmol NPS for 14 days were divided into two groups:APP/PS1+NPS-14+Sal（control group）and APP/PS1+NPS-14+Aβ_1-42Os（experimental group）,the electrophysiological signals of EEG and EMG were analyzed by multichannel sleep monitoring.（4）To detect the activation of microglia in mouse brain tissue after single and chronic administration of Sal or NPS:immunofluorescence co-labeling IBA1（labeled icroglia）and 4G8（labeled 4G8 experiments were used.（5）To investigate the effect of NPS on microglia phagocytosis of Aβin vitro:cells were divided into six groups:（1）Aβ-,NPS-;（2）Aβ+,NPS-;（3）Aβ+,NPS 50 n M;（4）Aβ+,NPS 100 n M;（5）Aβ+,NPS 200 n M;（6）Aβ+,NPS 300 n M,then the content of Aβin cells was detected by Western blot and immunofluorescence co-labeling.Results:（1）Regulation of NPS on sleep-wake behavior of APP/PS1 mice:1）For the effect of single injection:the results showed that compared with the Sal group,the wakeness（W）in APP/PS1 mice which injected with 0.1 nmol NPS and 1nmol NPS group during the first 3 h increased by 43%and 105%,slow-wave sleep（SWS）decreased by 27%and 60%respectively;paradoxical sleep（PS）decreased at 1 nmol concentration（P<0.05）,but there was no statistical difference at 0.1 nmol（P>0.05）.2)For the effects of chronic NPS injection:the SWS results showed that compared with APP/PS1+Sal-14 days group,the W in APP/PS1+NPS-14 days group decreased significantly in 12 h light period and 24 h total time（both P<0.01）,the increased correspondingly（P<0.05）,and the PS did not change significantly（P<0.05）.In APP/PS1+NPS-14 days group,there was no significant difference in W and SWS during 12 h light period and 24 h total time compared with WT+Sal-14 days（P>0.05）,while PS decreased significantly（P<0.01）.（2）Effect of NPS on Aβpathological in APP/PS1 mouse:1)For the effect of single injection:compared with Sal group,there was no significant difference in the contents of APP,p-APP,NPSR and Aβ（both P>0.05）.2)For the effect of chronic NPS injection:compared with APP/PS1+Sal-14 days group,the content of APP and p-APP in APP/PS1+NPS-14 days group decreased（both P<0.01）,and there was no significant difference in the expression of NPSR（P>0.05）.Immunofluorescence results showed that the levels of Aβdeposition was decresed（P<0.01）;（3）For injection Aβ_1-42Os experiment:compared with APP/PS1+NPS-14+Sal group,W increased in 12 h light period,12 h dark period and total 24 h（P<0.05,P<0.01,P<0.01,respectively）in APP/PS1+NPS-14+Aβ_1-42Os group,SWS decreased in the corresponding time（P<0.05,P<0.01,P<0.01,respectively）,and PS had no significant change（both P>0.05）.（4）Experimental results of immunofluorescence co-labeling IBA1 and 4G8 in brain tissue sections:after chronic NPS treatment,the microglia of APP/PS1 mice were activated,and the Aβplaque co-located with microglia was decreased;（5）To investigate the effect of NPS on microglia phagocytosis in vitro:NPSR is abundantly expressed in microglia,and NPS could significantly promote the phagocytosis of microglia to Aβ（P<0.001）,but there was no significant difference between the concentrations of NPS（P>0.05）.Conclusion:Our results showed that exogenous NPS can improve the disorder of sleep-wake cycle in APP/PS1 mice.Specifically,NPS can reduce W duration,increase SWS time and reduce fragmented sleep,so as to reduce the sleep disturbance symptoms of APP/PS1 mice.This process was realized by inhibiting the formation of Aβand promoting the phagocytosis and clearance of Aβby microglia,to reduce the deposition of Aβin the brain.