Study On Cerebrovascular And Blood-brain Barrier Injury In Mice With Homologous Point Mutation Of NOTCH3 Gene R544C

Background and ObjectivesCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL)is a kind of cerebrovascular disease caused by Notch3 gene mutation,which is the most common single-gene genetic small cerebral vascular disease.CADASIL is caused by the missense point mutation of Notch3 gene,which causes odd cysteine residues in a specific epidermal growth factor receptor(EGFR)in the extracellular domain of Notch3 protein.The protein conformation changes and deposits in the depression of vascular smooth muscle cell membrane.Osmiophilic granules(GOM)are generated by recruiting other proteins,which has toxic effect on small cerebral vessels.The clinical manifestations of CADASIL are recurrent stroke,cognitive decline,headache and emotional disorder.The MRI manifestations of CADASIL include extensive white matter changes,lacunar and intracerebral microbleeds(CMBs),etc.CADASIL is the most common single gene inherited cerebrovascular disease,and its animal model can be used as a good tool for the study of cerebrovascular disease.Previous studies have reported that endothelial cell damage,astrocyte and microglia activation were detected in the animal model of intracerebral hemorrhage,accompanied by peripheral cell loss,which led to the destruction of blood-brain barrier(BBB).These cells are neurovascular cells.It can be seen that cerebral micro hemorrhage is closely related to the destruction of NVU and BBB.The previous investigation of our research team found that the mutation hot spot of CADASIL patients in Fujian Province was R544 C in exon 11 of Notch3 gene.Compared with the patients without R544 C point mutation,the patients with R544 C point mutation were more likely to have cerebral microbleeding and the degree was more serious.We speculated that the patients with R544 C point mutation were more likely to have cerebral microbleeding,which was related to NVU and BBB destruction.The research team constructed the homologous point mutation mice with R544 C point mutation of Notch3 gene.By observing the brain neurovascular unit and blood-brain barrier of the mice,we preliminarily explored the possible pathogenesis of microbleeding caused by R544 C point mutation of Notch3 gene,in order to provide a new tool for the research of cerebrovascular disease and a new target for drug development.Methods1.Through using CRISPR/cas9 technology,the mice with R544 C point mutation of Notch3 gene were constructed.Hematoxylin eosin,Klüver-Barrera Luxol fast blue,immunohistochemistry and immunofluorescence staining were used to observe the brain white matter damage and cerebrovascular damage in the mutant mice.2.According to the damage of cerebral vessels in mice with R544 C homologous mutation of Notch3 gene,the damage of blood-brain barrier was further investigated by Western bolt,immunohistochemistry and immunofluorescence staining.A3.q-PCR was used to detect the changes of signal pathway related to BBB injury.Results1.The mice with homologous point mutation of NOTCH3 gene R544 C suffered from loss of white matter myelin sheath and enlarged perivascular space.2.Compared with wild-type mice,the mice with R544 C mutation showed proliferation of astrocytes,reduction of cerebrovascular smooth muscle cells and attenuation of endothelial cells in the brain,suggesting vascular injury in the neurovascular unit.3.Compared with wild-type mice,R544 C point mutation mice showed a decrease in brain tight junction protein(Claudin-5,Occludin),suggesting that the integrity of blood-brain barrier was damaged,and the expression of claudin-5 signaling pathway was decreased.4.Compared with wild-type mice,the m RNA level of MMP-9,TGFβ-1 in the brain of R544 C point mutation mice is increased.Conclusion1.The mice with homologous point mutation of NOTCH3 gene R544 C had white matter damage and cerebrovascular damage.2.The mice with homologous point mutation of NOTCH3 gene R544 C had neurovascular unit damage and blood-brain barrier damage.3.The blood brain barrier damage in the mice with homologous point mutation of NOTCH3 gene R544 C may be related to the increase of MMP-9 and TGFβ-1.