A Mechanistic Study Of The Effect Of Circadian Rhythms On Brain Perfusion Via PTau-mediated NNOS-PSD95 Uncoupling

Objective:Acute ischemic stroke is a common neurological disease with a high disability and mortality rate,which imposes a heavy economic burden on families and the society.Because the circadian rhythm of rodents is opposite to that of humans,and studies on cerebral infarction have shown that there is a circadian rhvthm in the infarct volume of the rodent treated with middle cerebral artery occlusion and reperfusion(MCAO/R)model,i.e.,the infarct volume during the daytime(inactive period;Zeitgeber Time,ZT0-12)is larger than that at night(active period;ZT12-24).So there is a clear diurnal pattern of infarct volume variation,but the underlying mechanism remains unclear.Recently,it has been shown that pTau competitively binds to postsynaptic density 95(PSD95)in the Alzheimer’s disease model,resulting in uncoupling of neuronal nitric oxide synthase(nNOS),which can lead to neurovascular changes and then affect cerebral perfusion.The aim of this study was to demonstrate that pTau affects cerebral perfusion in mice under physiological conditions through the above mechanism in the MCAO/R model,resulting in differences in cerebral infarct volume between day and night.Methods:1.Wild-type male C57/BL mice(22-25 g)were selected and housed for 3 weeks in a circadian environment with 12 hours lights on+12 hours lights off.The mice were divided into two groups,ZT5-7 and ZT17-19,with 6 mice in each group.The expression level of pTau(AT8)protein in the mouse brain tissues was measured by Western blot(WB).The binding of nNOS to PSD95 was measured by Co-Immunoprecipitation(COIP).The nitric oxide(NO)assay kit(nitrate reductase method)was used to measure the NO content of whole brain in mouse.2.Male Per1 knockout mice(22-25 g)were selected and housed for 3 weeks in a circadian environment with 12 hours lights on+12 hours lights off.Two groups were divided into ZT5-7 and ZT17-19,with 6 mice in each group.The expression level of pTau(AT8)protein in mouse brain tissues was measured by using WB.The binding of nNOS to PSD95 was measured by COIP.The nitric oxide(NO)assay kit(nitrate reductase method)was used to measure the NO content of whole brain in mouse.By using 2,3,5-triphenyltetrazolium chloride(TTC)staining to observe and study the diurnal changes of infarct volume in wild-type mice as well as Per1-/-mice.Cerebral perfusion analysis was performed using laser speckle imaging in wild-type mice and Per1-/-mice treated with the MCAO/R model.3.Patients with acute ischemic stroke who attended the emergency neurology department of the first affiliated hospital of Soochow University and received intravenous thrombolysis therapy from August 2018 to May 2021 were collected.The patients were divided into good prognosis group(mRS of 0-2)and poor prognosis group(mRS of 3-6)according to the Modified Rankin Scale(mRS)score at 3 months after treatment.Clinical information,time of onset,blood tests,and imaging data were collected from the included patients.SPSS 26.0 was used to compare component differences and multivariate logistic regression analysis was performed on the data.Variables with p-value<0.05 were used to construct the risk prediction model.Moreover,receiver operating characteristic(ROC)analysis was performed to evaluate the clinical value of predicting prognostic outcomes at 3 months about diurnal difference in time of onset.Finally,the comparison of these differences in imaging-related data between day and night was also investigated.Results:1.The pTau(AT8)protein expression level was higher in the ZT5-7 group compared to the ZT17-19 group in wild-type mice,while there was no significant diurnal difference in total tau.The nNOS-PSD95 binding was weaker and the NO content was lower.2.There were no significant diurnal differences in pTau(AT8)protein expression,total tau expression,nNOS-PSD95 binding,NO content,cerebral perfusion and cerebral infarct volume in the ZT5-7 group compared with the ZT17-19 group of Per1-/-mice.In contrast,there were significant diurnal differences in infarct volume in the wild-type mice treated with MCAO/R model as well as in cerebral perfusion as revealed by laser speckle imaging,i.e.,the infarct area was larger in inactive mice(ZT5-7)than in active mice(ZT17-19),while the opposite was true for cerebral perfusion.3.A total of 321 patients with acute ischemic stroke who underwent intravenous thrombolysis were included in this clinical study,with 206 patients in the good prognosis group and 115 patients in the poor prognosis group.Analysis of baseline data showed statistical differences in age,sex,diurnal differences in time of onset,atrial fibrillation,diabetes mellitus,white blood cell,glycosylated hemoglobin,homocysteine,NIHSS score,and the TOAST between the two groups.Age,time of onset,diabetes mellitus,white blood cell,the NIHSS score,and the trail of org 10172 in acute stroke treatment(TOAST)were statistically different between the two groups after multivariate logistic regression model analysis.And then a risk prediction model was constructed based on variables above.ROC analysis revealed an area under the curve of 0.646,0.597,0.561,0.661,0.657,0.575,and 0.81 for age,diurnal differences in time of onset,diabetes mellitus,white blood cell,NIHSS score,the TOAST and risk prediction model,respectively.Under CTA+CTP imaging assessment,significant diurnal differences were found in the CTP ischemic volume,infarct core volume,and ischemic penumbra volume,i.e.,the CTP ischemic volume,infarct core volume,and ischemic penumbra volume were larger in patients with onset time at night than in patients with onset time during the day.Conclusion:In a circadian rhythm model of wild-type mice,pTau was indirectly shown to uncouple nNOS-PSD95 by competitively binding PSD95,i.e.,a decrease in nNOS-PSD95 binding,resulting in decreased nNOS activity and reduced NO production leading to cerebral perfusion deficit and increased infarct volume.It was found that the above products and the circadian variation in infarct volume were lost in the Per1-/-mice.Clinical studies have shown that circadian differences in onset time are closely related to the prognosis of patients with acute ischemic stroke.Thus,it is hypothesized that pTau may be one of the mechanisms affecting the circadian variation in infarct volume,thus providing a new therapeutic strategy for the treatment of cerebral infarction in the future.