The Mechanism Of Sleep Deprivation Aggravating Cognitive Dysfunction In AD Mouse

BackgroundAlzheimer’s disease（AD）is the most common dementia disease,accounting for 50%-70%of senile dementia.Research shows that about45%of AD patients have sleep disorders.Before complete dementia,the pathological progress of AD can last for about 20 years,and the deposition of amyloid protein in the early clinical stage of AD is related to sleep disorders,so sleep disorders may affect the early and progressive stages of AD,so it is of great practical significance to block or delay the impact of sleep disorders on AD progress.Sleep disorder can lead to the disorder of rhythm gene,which can regulate autophagy and microglia’s response to AβIngestion of.In addition,in AD patients,rhythmic gene expression is disturbed.These studies indicate that the rhythm gene is closely related to AD,which may be the mechanism factor of sleep disorder aggravating AD.Cryptochromes-2（CRY2）is the core component of the biological clock and an important factor for generating and maintaining circadian rhythm.Its changes can affect the expression of other major rhythm genes.CRY2plays a key role in DNA damage checkpoint control and regulates important cell cycle progression genes.Based on the above research background,we propose a scientific hypothesis:sleep deprivation will aggravate the cognitive decline of AD and Aβdeposition,CRY2 may participate in the molecular mechanism of protein deposition.ObjectiveTo explore the molecular mechanism of sleep deprivation aggravating cognitive dysfunction and Aβdeposition in AD mice,and the mechanism of dexmedetomidine in improving cognitive impairment in AD miceMethod1.Verify that sleep deprivation aggravates cognitive dysfunction and Aβdeposition in AD mice,and explore the expression changes of CRY2protein.5x FAD（AD）female mice at the age of 7 weeks and wild type（WT）female mice in the same nest were divided into four groups:WT group,WT mice sleep deprivation group（WT+SD）,AD group,AD mice sleep deprivation group（AD+SD）.The sleep deprivation model of mice was established in the sleep deprivation group after 21 consecutive days of sleep deprivation.After the establishment of the model,PET-CT glucose metabolism test and cognitive behavior test were performed on each group of mice to evaluate the impact of sleep deprivation on the cognitive function of mice.Detect Aβdeposition in hippocampus in AD mouse by immunofluorescence.Detect the expression and cellular localization of CRY2.2.Explore the effect of CRY2 overexpression on cognitive function and Aβdeposition in AD mice.BV-2 cells of mouse microglial cell line were transfected with lentivirus overexpression CRY2.BV-2 cells were divided into control group（Control）,lentivirus control group（LV-NC）,lentivirus overexpression CRY2 group（LV-CRY2）.The expression level of CRY2 was detected by RT-PCR,and the apoptosis of cells in each group was detected by flow cytometry.AD mice were randomly divided into AD+SD group,AD group,AD lentivirus control group（AD+LV-NC）,AD lentivirus CRY2 overexpression group（AD+LV-CRY2）.After the SD model is completed,the mice were tested for cognitive behavior,evaluated the effect of overexpression of CRY2 on the cognitive function,and detected Aβdeposition in hippocampus in AD mice with immunofluorescence3.Explore the effect of CRY2 knockdown on cognitive function and Aβdeposition in AD mice with sleep deprivation.BV-2 cells were transfected with CRY2 knocked down small interfering RNA（CRY2si RNA）or control small interfering RNA（NC si RNA）,and H₂O₂ was added to establish apoptosis model.BV-2 cells were divided into NC-si RNA group,NC-si RNA+H₂O₂ group,CRY2-si RNA group,CRY2-si RNA+H₂O₂ group.The expression level of CRY2 was detected by RT-PCR,and the apoptosis of cells in each group was detected by flow cytometry.AD mice were randomly divided into AD group,AD+SD group,sleep deprivation group（AD+SD+NC-si RNA）with stereotactic injection of control interfering RNA into DG area of hippocampus of AD mice,and sleep deprivation group（AD+SD+CRY2 si RNA）with stereotactic injection of knock down CRY2 interfering RNA into DG area of hippocampus of AD mice.After modeling,mice were tested for cognitive behavior and immunofluorescence to evaluate the cognitive function and Aβdeposition.4.Explore the role of CRY2 in sleep deprivation aggravating AD cognitive dysfunction and Aβdeposition.AD mice were randomly divided into AD group and AD+SD group.The expression of SOCS family m RNA and STAT1,STAT3,STAT5,STAT6 protein phosphorylation were detected by RT-PCR.AD mice were randomly divided into AD+SD group,AD group,AD+LV-NC group and AD+LV-CRY2 group.Western blot was used to detect the expression of CISH and p-STAT1 protein.AD mice were randomly divided into AD group,AD+SD group,AD+SD+NC si RNA group,AD+SD+CRY2 si RNA group,and the expression of CISH and p-STAT1 protein was detected.AD mice were randomly divided into AD group,AD+SD group,sleep deprivation group with stereotactic injection of control adeno-associated virus（AD+SD+AAV-NC）into the DG area of the hippocampus of AD mice,and sleep deprivation group with stereotactic injection of CISH overexpression adeno-associated virus（AD+SD+AAV-CISH）into the DG area of the hippocampus of AD mice.After modeling,mice were tested for cognitive behavior and immunofluorescence to evaluate the cognitive function and Aβdeposition.5.Effect of anesthetic drug dexmedetomidine on cognitive function and Aβdeposition in AD mice.AD mice and WT mice in the same litter were randomly divided into four groups:wild type mouse with normal saline group（WT+NS）,5x FAD mice with normal saline group（AD+NS）,5x FAD mouse with dextrmedetomidine group（AD+Dex）,and 5x FAD mouse with dextrmedetomidine and yohimbine group（AD+Dex+Y）.After14 days of continuous gavage,mice were tested for cognitive behavior and immunofluorescence to evaluate the cognitive function and Aβdeposition Result1.Sleep deprivation for 21 days has no effect on the cognitive function of WT mice,but it can lead to the cognitive function worse in AD mice,reduce the number and time proportion of entering the new arm in the test of Y maze,prolong the latency of finding the platform in the water maze,reduce the number of crossing the platform,reduce the time proportion in the target quadrant,and lead to more Aβdeposition in DG area of hippocampus in AD mouse.CRY2 is expressed in the cytoplasm of microglia.Sleep deprivation increases the expression of CRY2 protein in the hippocampus of AD mice.2.The expression of CRY2 in BV-2 cells transfected with LV-CRY2was significantly increased,and the apoptosis of cells was increased.The cognitive function of AD mice injected with lentivirus LV-CRY2 in hippocampus was worse than that of AD mice injected with lentivirus LV-NC,and Aβdeposition increases.3.The expression of CRY2 in BV-2 cells transfected with CRY2si RNA decreased significantly,and the knockdown of CRY2 could improve H₂O₂ induced apoptosis.Intrahippocampal injection of CRY2-si RNA can improve the cognitive function and Aβdeposition of sleep deprivation induced in AD mice.4.Compared with AD group,CISH expression in AD+SD group decreased and p-STAT1 expression increased.Compared with AD+LV-NV group,CISH expression in AD+LV-CRY2 group decreased and p-STAT1expression increased.Compared with AD+SD+NC si RNA group,CISH expression in AD+SD+CRY2 si RNA group increased and p-STAT1expression decreased.CISH is overexpressed in the hippocampus of AD mice.Compared with the control group,the water maze and Y maze of AD mice overexpressing CISH are significantly improved,and Aβdeposition decreased.5.Compared with AD+NS group and AD+Dex+Y group,the cognitive function of AD+Dex group mice is better and less Aβdeposition.ConclusionSleep deprivation aggravates the cognitive dysfunction in AD mouse,the up-regulated expression of CRY2 in hippocampus of AD mouse leads to increased apoptosis of microglia through CISH mediated p-STAT1phosphorylation,reducing microglia’s phagocytosis on Aβ,leading to increase deposition of Aβ.Dextrmedetomidine can reduce the deposition of Aβ,and improve the cognitive dysfunction of AD mouse.