| Part1 Mechanism of chronic sleep restriction inducing cognitive deficits and cortical A? deposition in miceObjectives:Chronic sleep restriction could affect human body health in many aspects,especially cognitive function.By using chronic sleep restriction,we tried to study the cognitive impairments and A? deposition in frontal cortex.Furthermore,APP protein,BACE1-AS,BACE1 m RNA,BACE1 protein,C99 protein and other APP processing key points were detected,in order to reveal the possible mechanism of A? concentration in mice frontal cortex caused by chronic sleep restriction.Methods:8-months-old C57 mice divided into 2 groups: 1.C57 sleep restriction group(C57SR),2.C57 control group(C57CO),were chosen to put into the slowly rotating wheels(40 cm in diameter)driven by an designed engine for 2 months to achieve the chronic sleep restriction.Experimental group wheels were designed as 12:00(on)~+8:00(off),at constant speed(0.4 m/min),control group wheels were designed as 20:00(on)~+8:00(off),at constant speed(0.66 m/min).Light-dark cycle was 12 hours,from 8:00 to 20:00.Morris water maze was chosen to compare the behavioral changes.Enzyme-Linked Immunosorbent Assay(ELISA)and Immuno Fluorescence were chosen to compare A?accumulation level,Western Blot was chosen to detect APP,BACE1 protein and C99 protein level,Realtime PCR was used to evaluate BACE1-AS and BACE1 m RNA expression.Results:1.Morris water maze showed that C57 SR mice spent more time finding the pl atform in spatial acquisition in Day2-5(p<0.001),and stayed less time in goal quad rant(45.13±14.98 s vs 66.95±15.67 s,p=0.013<0.05),and crossed less times the platf orm area(1.25±0.88 vs 4.12±0.83,p<0.001)in probe trial in Day5 than C57 CO m ice;2.Compared with C57 CO mice,ELISA showed that A? accumulated more sig nificantly in frontal cortex of C57 SR mice(35.11±5.55pg/mg vs 25.27±1.15pg/mg,p=0.002<0.05),Immuno Fluorescence showed that A? deposited more significantly in frontal cortex of C57 SR mice(0.177±0.0145/pixel vs 0.122±0.010/pixel,p<0.001);3.3.Western Blot revealed an equal level of C57 SR and C57 CO APP(24.24±1.34 vs 24.37±1.33,p>0.05),otherwise,we found significantly more BACE1 and C99 pr otein expressed in mice of C57 SR than that of C57 CO,relative expression were 37.57±3.04 vs 21.53±2.80,p<0.001 and 26.72±1.23 vs 16.17±1.90,p<0.001,respectiv ely;4.BACE1-AS level was found at a higher level in mice of C57 SR compared with that of C57CO(518.31±63.46 vs 242.24±187.22,p=0.034<0.05).The findings of BACE1 m RNA was similar to that of BACE1-AS(1217.71±590.60 vs 447.56±329.00,p=0.043<0.05).Conclusion:2-months-chronic sleep restriction could cause cognitive impairments and A?deposition in adult wild type C57 mice,paralleled with the increasing of BACE1-AS,BACE1 m RNA level and beta-site cleavage of APP.Chronic sleep restriction resulted in A? elevation by way of bete-secretase,independent of basic APP concentration.Our research also indicated that BACE1-AS and BACE1 m RNA were key factors of A?increase in frontal cortex,BACE1-AS upregulating BACE1 protein and A? harmed cognitive function of mice under circumstances of chronic sleep restriction.Part2 Cortical mitochondrial dysfunction and accumulation of mitochondria-related A? caused by chronic sleep restriction in miceObjectives:By detecting the function and morphology of mitochondria isolated from cortex of mice after chronic sleep restriction,and the concentration of mitochondria-related A?,we tried to research the relationship between mitochondrial dysfunction/morphological changes and elevation of cortical neuronal mitochondria-related A? in chronic sleep restriction model mice,and the relationship between mitochondria-related A? level and total A? level/mitochondrial APP in frontal cortex.Methods:C57 mice were put into the slowly rotating wheels(40 cm in diameter)fitted as shown in Part 1.8-months-old mice were divided into 2 groups--sleep restriction group(C57SR)and control group(C57CO).Mitochondrial electron transport chain IV concentration,ATP activity and mitochondrial membrane potential were examined,electron microscopy examination of cortex was carried out.ELISA was used to detect cortical mitichondria-related A? accumulation in mice of both groups.Results:1.Electron microscopy examination,showed obvious injuries in C57 SR mice m itochondria;2.Compared with C57 CO mice,C57 SR mice had significant lower mit ochondrial electron transport chain IV concentration(12.691±1.58pg/mg vs 8.722±0.89pg/mg,p<0.001),lower ATP activity(52.965±2.89?mol/L vs 15.416±6.90?mol/L,p<0.001)and lower mitochondrial membrane potential(3.23±0.38 vs 3.90±0.49,p=0.043<0.05,dyed by JC-1);3.Mitochondria-related A? accumulation was found more se vere in the frontal cortex of C57 SR mice than that of C57 CO mice significantly.3.In chronic sleep restriction model,mice mitochondrial dysfunction(reflected by cort ical ATP activity)correlated with mitochondria-related A? level closely(r2=0.7874,p<0.001).In chronic sleep restriction model mice,cortical mitochondria-related A? l evel didn't show significant relationship with cortical total A? concentration(p=0.066>0.05)and mitochondrial APP(p=0.084>0.05).Conclusion:1.Chronic sleep restriction could cause the mitochondrial dysfunction and morphological injuries in the frontal cortex of adult C57 mice;2.Chronic sleep restriction could lead to significant decrease in mitochondrial electron transport chain IV concentration,ATP activity and mitochondrial membrane potential decrease in cortical neuron.3.After chronic sleep restriction,A? accumulated in frontal cortical mitochondrial matrix showed significantly close relationship with mitochondrial dysfunction.4.After2-months-chronic sleep restriction,cortical mitochondria-related A? level didn't correlate with cortical total A? level and mitochondrial APP concentration.Part3 The function of IGF-1R signaling pathway in the A? deposition induced by chronic sleep restrictionObjectives:By evaluating of body weight,length,Lee's index change and plasma total IGF-1level of mice under the circumstance of chronic sleep restriction,to reveal the relationshipbetween plasma total IGF-1 and kinds of physiological indices.By investigating the A?deposition,meanwhile,identifying APP,BACE1 and C99 in both wild type mice and IGF1R+/-mice after chronic sleep restriction,to study the protective function of reduced IGF-1R signal in A? deposition in frontal cortex of mice.Methods:8-months-old mice were divided into 4 groups: 1.wild type sleep restriction group(C57SR),2.wild type control group(C57CO),3.IGF1R+/-sleep restriction group(MUSR),4.IGF1R+/-sleep control group(MUCO).Mice weight,length and Lee's index were measured and calculated.ELISA was used to detect the A? deposition in all the mice,and Western Blot was chosen to compare the APP,BACE1 and C99 protein level in each group.Results:1.Compared with wild type mice,mutant mice showed lower body weight and Lees' s index(26.77±2.13 g vs 23.18±0.69 g,p<0.05;303.90±6.39 vs 295.12±7.07,p<0.05),the IGF-1R+/-mice showed weight and Lee's index gain,but the results d idn't reach significant level;in wild type mice,C57 SR showed less plasma IGF-1 l evel than that of C57 CO significantly(596.40± 33.20ng/ml vs 510.40±24.35ng/ml,p<0.001);2.ELISA detected that the A? levels of MUSR,MUC,C57 SR,C57CO mice were 22.51±2.53pg/mg,19.24±1.65pg/mg,35.08± 4.81pg/mg,25.23±1.00pg/mg under the circumstance of sleep restriction,respectively.3.Under the equal level of APP(MUSR:MUCO:C57SR:C57CO= 28.58±1.52 vs 26.86±2.16 vs 24.24±1.33 vs 24.37±1.32),frontal cortical BACE1/C99 protein in mice of MUSR,MUCO,C57 SR,C57CO were 23.56±1.44 vs 21.59±1.80 vs 37.57±3.03 vs 21.53±2.82(p<0.001),and 19.83± 1.85 vs 20.91±2.49 vs 26.72±1.23 vs 17.61±1.89(p<0.001),respect ively after 2-months chronic sleep restriction.Conclusion:2-months-chronic sleep restriction led to mild weight gain and obesity(result didn't reached significance),IGF-1R+/-mice showed natural low body weight and low fat ratio.Reduced the IGF-1R signal could ameliorate the A? deposition by way of decreasing the level BACE1 protein and beta-site cleavage of APP. |
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