A Pilot Study Of Exosomes NOTCH3,NFL And Aβ42 In CADASIL

Background:Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL)is the most common single-gene hereditary cerebral small vessel disease.The main clinical manifestations are recurrent cerebral infarction,migraine,mood disorders and cognitive decline,which seriously affect the quality of life of patients.The main pathological changes of CADASIL include abnormal aggregation of mutant NOTCH3 protein and the deposition of granular osmiophilic material(GOM).At present,the specific pathogenesis is unknown.Exosomes are extracellular vesicles with a diameter of nano-meters,which can be secreted by a variety of cells in the body and can be found in a variety of body fluids.Several studies have shown that exosomal secretion and autophagy-lysosome pathways are synergistically involved in the elimination of abnormal proteins within cells.The latest study found that the vascular smooth muscle cells(VSMCs)of CADASIL patients have autophagy-lysosome dysfunction.However,it is unclear that whether the plasma exosomes of CADASIL patients have changed.Objective:In this study,we compare the morphology,quantity and pathology-related protein levels(NOTCH3,NFL and Aβ42)of plasma exosomes to find the differences between CADASIL patients and matched healthy controls,and we conduct a preliminary discussion on the possible role of exosomes in the pathogenesis of CADASIL.Methods:After September 1,2018,the plasma samples of 30 patients with CADASIL were collected from the Department of Neurology,Henan Provincial People’s Hospital,and the plasma samples of 30 controls were collected from the Physical Examination Center,Henan Provincial People’s Hospital.the age,gender,race,education level,etc.of the controls were matched with CADASIL patients.All subjects have undergone detailed clinical examinations,including Magnetic resonance imaging(MRI)examination,Montreal Cognitive Assessment(MOCA)scale and Mini-Mental State Examination(MMSE)scale,to judge the clinical severity of CADASIL patients.The plasma exosomes of CADASIL patients and normal controls were extracted by immunoprecipitation technology,and the morphology and quantity of exosomes were characterized and analyzed by Transmission Electron Microscopy(TEM)and Nanoparticle Tracking Analysis(NTA).Enzyme-linked immunosorbent assays(ELISA)was used to detect the levels of pathology-related proteins NOTCH3,NFL and Aβ42(normalized to CD63)in the plasma exosomes of CADASIL patients and matched normal controls.Results:After standardization by CD63,the level of NOTCH3 in plasma exosomes of CADASIL patients was much lower than that of healthy controls,and the difference was statistically significant(P=0.0002).The level of NFL in plasma exosomes of CADASIL patients was significantly higher than that of healthy controls(P=0.0112).The Aβ42 level in plasma exosomes of CADASIL patients was lower than that of healthy controls,but the difference was not statistically significant(P=0.9012).The level of NOTCH3 in plasma exosomes in CADASIL patients was positively correlated with the severity of white matter lesions(r=0.532,P=0.0051).Compared with matched healthy controls,the number of the plasma exosomes of CADASIL patients was significantly reduced(P=0.0409).Conclusions:The plasma exosomes NOTCH3 level of CADASIL patients is low,and the number of exosomes is relatively small,which may be related to the pathological changes of CADASIL,providing a new perspective for the study of the pathogenesis of CADASIL.The level of NFL in plasma exosomes is significantly increased,which helps to detect the level of NFL in peripheral blood.And the level of NOTCH3 is correlated with the severity of white matter lesions in CADASIL patients.Therefore,plasma exosomal NFL and NOTCH3 levels can be used as biomarkers for monitoring,predicting CADASIL disease progression and evaluating the efficacy of future clinical trials.