| BackgroundAbout 25%ischemic stroke globally are caused by cerebral small vessel disease(SVD).More than half of people over 60 years old are suffering from SVD SVD affects vessels with a diameter ranging from 30 to 800?m,including arterioles,capillary vessels and venules.SVD is characteristic of ischemic or hemorrhagic stroke and causes impairment of cognitive function,gait and balance.About 5%of SVD was hereditary,of which CADASIL is the most common monogenic inherited disease.Although the general prevalence of CADASIL is unknown,4.15 in 100,000 adults in Scotland are diagnosed with CADASIL and 10.7 in 100,000 are estimated to carry the pathogenic mutation.However,the actual prevalence may be much higher because sporadic cases occur.It is reported CADASIL accounts for 2%of cases of lacunar stroke with leucoaraiosis in patients younger than 65 years old and for 11%of cases in those younger than 50 years old.CADASIL,the leading heritable cause of stroke and vascular dementia in adults,is characterized by adult-onset migraine,recurrent subcortical transient ischemic attacks or strokes,progressive dementia and psychiatric disturbances.Migraine and stroke are common first symptoms.The clinical manifestations of CADASIL varies substantially between and within families.These symptoms also vary in frequency with age and duration of disease.The average age of onset is approximately 41 years(±9.2 years),the age at death is around 65 years averagely and rangeing from 30 to 80 years depending on phenotype.The typical MRI abnormalities include lacunar infarcts,dilated perivascular spaces and symmetric hyperintense within the cerebral white matter especially external capsule and the anterior temporal lobes on T2-wieghted images.Hyperintensities in White matter on T2-weigted images are the most frequent finding and often located in periventricular area and deep white matter.The frequency and severity of lesions increase significantly with age.Hyperintensities in the temporal lobe,external capsule appear to differentiate CADASIL from sporadic SVD.MRI abnormalities are detectable from the age of 20 and are present in all mutation carriers after the age of 35 years.Treatment options of CADASIL are quite limited and include symptomatic treatment and secondary prevention.CADASIL is caused by mutations in the NOTCH3 gene on chromosome 19p13 which is mainly expressed in arterial smooth muscle cells(VSMCs)and pericytes yet only deficiency in cerebral arteries causes symptoms.Defects in NOTCH3 result in abnormalities of the arterial structure and myogenic responses.The vascular lesions are characterized by degeneration,loss of vascular smooth muscle cells,the abnormal extracellular accumulation of Notch3 extracellular domain(Notch3ECD)and granular osmiophilic material(GOM)and fibrosis of the media.NOTCH3 encodes a single pass trans-membrane heterodimeric receptor with an extracellular domain(ECD)composed of 34 epidermal growth factor(EGF)-like repeats,a transmembrane domain,and an intracellular domain(ICD).Each EGF-like repeat contains six highly conserved cysteine residues to form intramolecular disulfide bond pairs between C1-C3,C2-C4 and C5-C6,which are essential for ligand binding.Notch3 is first synthesized as a 280 kDa precursor and forms into a heterodimer receptor at membrane via S1 proteolytic cleavage by furin.After DSL(Delta/Serrate/Lag-2)ligand binding and S2 proteolytic cleavages by ADAM the heterodimer releases the 210 kDa extracellular domain.The shed ECD is subsequently endocytosed by DSL ligand cells and receiving cells.The ICD is then liberated after S3 proteolytic cleavages by y-secretase and translocates to the nucleus regulating target gene expression.To date,more than 300 mutations of NOTCH3 have been recorded in CADASIL patients.The majority are located in exons 2-24 encoding the 34 EGF-like repeats in all 33 exons.A total of 95%are missense mutations that change the number of cysteines and further result in misfolding and aggregation of ECD.Although the formation of abnormal disulfide bridges has been believed to affect ligand binding and signal transduction,the role of Notch3 signaling activity in the pathogenic mechanism is controversial.Many mutations were found unrelated with Notch3 signaling.Furthermore,total absence of Notch3 did not lead to CADASIL pathologic changes in knock-out-mice.It was suggested that CADASIL mutations might have a neomorphic effect of toxicity rather than compromising the canonical Notch3 function.The Notch3ECD aggregation is thus considered to be the main pathogenic mechanism of the disease similar to amyloid plaques in Alzheimer's disease.There is also some evidence for it.First,Notch3ECD deposits appears prior to other pathologic abnormalities in patients and transgenic mice.Moreover,studies established that mutant Notch3 tends to form more aggregates than the wild type,which also shows a distinct resistance to degradation.A previous study using cultivated VSMCs from a CADASIL patient found different expression levels of proteins involved in protein degradation and folding,which implied that mutant Notch3 causes endoplasmic reticulum(ER)stress and activates the unfolded protein response(UPR).Another consistent in vitro study observed that prolonged retention of mutant Notch3 aggregates in the ER decreases cell growth and increases sensitivity to stresses such as proteasome inhibition.Therefore,it is indicated that the mutation induces misfolding and multimerization of Notch3 which thus leads to cytotoxic effects.To date,some cysteine-sparing NOTCH3 mutations have been documented.A previous study found that some cysteine-sparing mutations were associated with structural changes in Notch3 similar to cysteine-affecting mutations.The study of some cysteine-sparing mutations also showed significantly enhanced aggregate properties in vitro.ObjectiveThe aim of this study was to identify the pathogenicity of the c.218G>C NOTCH3 mutation and discuss the mechanisms under CADASIL.Materials and methods1.Molecular genetics analysis of p.G73A1.1 conservativeness of the glycine at position 73 was analyzed between EGF-like repeats of Notch3.1.2 The conservativeness of the glycine at position 73 was analyzed between species.2.Prepare wild type Notch3ECD and c.218G>C mutant Notch3ECD plasmid2.1 pNOTCH3-bio-His was obtained from Gavin Wright(Addgene plasmid#53343).The plasmid was amplificated,extracted and purificated and confirmed by digestion with restriction enzyme and sequencing.2.2.The c.218G>C site-directed mutagenesis was constructed by Vigene Corporation.The mutant plasmid was amplificated,extracted and purificated and confirmed by sequencing.3.Detect the cellular location of Notch3ECD in HEK293T cell3.1 HEK293 cells were transiently transfected with wild-type or c.218G>C Notch3ECD plasmid respectively and cultured for 48h.3.2 The cells were immunostained with Notch3 antibody against Notch3ECD and Alexa Fluor 488-labeled secondary antibody.Labelled cells were captured by confocal microscopy.4.Investigate the effect of mutant Notch3ECD on cells survival4.1 HEK293 cells was transfected pNOTCH3-bio-His,c.218G>C mutant one and pcDNA4 respectively and cell viability was determined by CCK8 assay.4.2 HEK293 cells was transfected pNOTCH3-bio-His,c.218G>C mutant one and pcDNA4 respectively and cell viability was determined by ATP bioluminescence assay.5.Indentify the influence of mutant Notch3ECD on cell apoptosisHEK293 cells was transfected pNOTCH3-bio-His,c.218G>C mutant one and pcDNA4 respectively.Cell apoptosis rate of each group was identified via Annexin V-FITC and PI double-stained flow cytometry assay 48h after tranfection.6.Statistical analysisStatistical analysis were performed with SPSS version 23(SPSS Inc.,Chicago,IL,USA).Data are presented as means+SD.Unpaired Student' s t test and ANOVA were performed in compare.P-values<0.05(two-tailed)were considered significantResult1.The glycine at position 73 is highly conserved between EGF-like repeatsThe Gly at position 73 is highly conserved in nearly all the EGF-like repeats of human NOTCH3 except domain 17.2.The glycine at position 73 is highly conserved among different speciesThis location of glycine in C-loop shows high conservation through evolution from human to zebra fish,where it locates in C5-C6 in human,mouse,rat and in between the only disulfide bond of EGF-like domain 1 in the remaining species.3.p.G73A Notch3ECD decreased cell viability3.1 In CCK8 assays,Cells expressing p.G73A Notch3ECD showed significant reduction in cell viability compared with wild-type Notch3 ECD and control.There was no significant difference between the other two groups.3.2 In ATP bioluminescence assays,HEK293T cells expressing p.G73A Notch3ECD showed significant reduction in cell viability compared with wild-type Notch3ECD and control.There was no significant difference between the other two groups.4.p.G73A Notch3ECD promoted cell apoptosisOverexpressing of mutant Notch3ECD increased cell apoptosis rate compared with wild-type Notch3ECD and control 48h after transfection.No significant difference was found between wild-type Notch3ECD and control group.Conclusions1.The glycine at position 73 shows high conservation between EGF-like repeats and among diverse species.2.p.G73A Notch3ECD decreased the cell viability via promotion of cell apoptosis. |
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