| Objective: The β-amyloid(Aβ)hypothesis is very important in the pathogenesis of Alzheimer’s disease(AD).According to Aβ hypothesis,we had generated AD transgenic zebarfish model,and had found cerebral microvascular disorders in zebrafish brain.In order to find out the relationship between Aβ and cerebral microvascular disorder in AD progression,using APPsw transgenic zebrafish model and brain microvascular endothelial cell model to study the relationship between Aβ and cerebral microvascular disorder,as well as the important role in AD pathogenesis.As amyloid precursor protein is the only source of Aβ production,it is thought that the understanding of the role of APP in cerebral microvascular disorder and neuronal dysfunction is helpful to the understanding of AD pathogenesis,providing a new alert and treatment strategies of AD.Methods:(1)Generation of AD transgenic zebrafish model: using zebrafish appb promoter to regulate human APP Swedish mutant gene(APPsw)to construct Tol2 expression plasmid.Establishing AD transgenic zebrafish model by microinjecting the Tol2 plasmid into the yolk sac.Detecting APPsw expression in the m RNA and protein levels in transgenic zebrafish model.Verifying transgenic zebbrafish had AD-like pathological feature by observing AD-like behavior and pathological changes.(2)Observation of the brain pathological changes of different month aged APPsw transgenic zebrafish: pathological observation including neurons and cerebral vascular ultrastructural changes,the morphology and volume of neurons,Aβ content and the m RNA expression level changes of claudin family genes.(3)The APP expression and function in brain microvascular endothelial cells: using primary cultured rat brain microvascular endothelial cells(BMECs)and b End.3 microvascular endothelial cell line to detect APP expression.Knocking-in and knocking-down APP/ APPsw gene in BMECs to study APP function.Cultured BMECs in oxygen-glucose deprivation(OGD)environment and detected APP expression.Results:(1)Generation of AD transgenic zebrafish model: p Tol2-appb-APPsw-CMVEGFP expression plasmid was successfully constructed and APPsw transgenic zebrafish model successfully generated.The transgenic zebrafish expressed fluorescence mainly in microvascular endothelial cells,neurons,eyes,heart and blood vessels.Using PCR and Western-blot to detect APPsw expression in transgenic zebrafish,the results of whole embryo in-situ immunohistochemistry showed that the location of APPsw protein exprssion was same as that of the EGFP fluorescence.Using T-maze to test the learning and memory behavior of 6 months and 12 months transgenic zebrafish,results showed the latency of zebrafish in last trail were significantly shorter than that in the first trail(P<0.01)in both 6-month-old transgenic group and normal zebrafish group,but there was no significant difference between last trail and first trail latency in 12-month-old transgenic zebrafish group,while the latency of the control zebrafish in last trail were significantly shorter than that of 12-month-old transgenic zebrafish(P<0.01).Furthermore,observation the locomotor activity of 12 months transgenic zebrafish,the results showed that transgenic zebrafish behavioral abnormalities: the total distance(P<0.05),the time staying in upper area(P<0.05),total activity time(P<0.05),the frequency of exploring upper area(P<0.05),was increased in comparison with normal zebrafish.HE staining showed that granule cells loosely arranged,and neurons dropout severely in telencephalon and cortex in the 12-month-old transgenic zebrafish.Aβ1-42 immunohistochemistry showed Aβ1-42 deposition in the brain,especially in the cerebral parenchyma and microvasculature.All results showed that the transgenic zebrafish were successfully generated and processed AD-like behavior and pathological changes.(2)Observation of the brain pathological changes of different month aged APPsw transgenic zebrafish: the results of TEM showed cerebral microvascular structure changes in the 6-month-old transgenic zebrafish: the vascular endothelial cells swelled,chromatin margination,the basement membranes(BM)were not smooth.Vascular structural disorder became more serious in 9 and 12 months-old transgenic zebrafish: endothelial cell shrank,chromatin margination;BM appeared thickened,broken and loose,and mitochondria,synaptic vesicles and other organelles reduced.Neuronal structure began to change in 9-months-old transgenic zebrafish with irregular nucleus morphology,mitochondria and endoplasmic reticulum swelling,and shortened mitochondria crest.Neuronal structural in 12-month-old transgenic zebrafish brain were changed more severely: cell nucleus contents decayed,mitochondrial swelling and lack of mitochondrial cristae.Nissl staining showed that the numbers of neurons in telencephalon and cortexin were decreased in 9-month-old transgenic zebrafish and were significantly decreased in 12-month-old transgenic zebrafish in comparison with control zebrafish(P < 0.05).Aβ1-42 immunohistochemistry showed that 3-month-old transgenic zebrafish brain had few Aβ1-42 positive blood vessels in the cortex and the number of Aβ1-42 positive blood vessels in the cortex increased and began to deposition in the telencephalon in 6-month-old transgenic zebrafish.More blood vessels were positively stained with Aβ1-42 and small amount of Aβ1-42 plaque deposits in cortex and telencephalon in the 9-month-old transgenic zebrafish.There was a large number of Aβ1-42 positively stained plaques near the microvessels in telencephalon in 12-months-old transgenic zebrafish.The results of q RT-PCR showed the expression level of claudin family gene changed with age significantly: the expression of claudin-2,claudin-5b,claudin-k were increased while that of claudin-5a,claudin-11 a claudin-11 b,claudin-h were decreased in transgenic zebrafish.(3)The APP expression and function in brain microvascular endothelial cells: APP expression were detected by PCR,Western-blot and immunofluorescence assays,the results showed BMECs can express APP.APP/APPsw overexpression plasmid pc DNA3-APP/APPsw-IRES-EGFP and APP/APPsw knockdown sh RNA plasmid p GPU6/m RFP/Neo-sh APP were successfully constructed.Transfected b End.3 by lipofectamine 3000 and screened positive expression b End.3 cells by flow cytometer.q RT-PCR and Western-blot verified the intracellular APP expression.Cell viability was detected by MTT.It was found that the viability of APP knocked-down b End.3 cells was significantly decreased(P <0.01)compared to that of control group.Morphological changes of transfection b End.3 cells were observed by using an atomic force microscope(AFM).The results showed that the cell surface of of APP knocked-down b End.3 cells were irregular,and the height of cell surface was increased compared to that of control group.The viability of BMECs with OGD was measured by MTT.The results showed that with OGD time increasing,the BMECs cell activity was decreased.The results of q RT-PCR and Western blot showed APP expression in BMECs of OGD 16 h group and 24 h group was higher than OGD 4h group.All the results showed that BMECs had the ability of expressing APP and generating Aβ proteins,and APP gene deletion caused cerebral microvascular endothelial cell membrane structure changes,as well as APP expression level increased when the brain microvascular endothelial cells were damaged,which suggesting that APP play an important role in maintaining the integrity of cerebral microvascular endothelial cells.Conclusions: Aβ overexpression and deposition in vascular endothelial cells caused vascular endothelial cell structural damage and changed the permeability of BBB,thereby the organelles in the cells around blood vessels and neurovascular unit damage resulting in neuronal apoptosis and loss,cognitive impairment and dementia.Thus,cerebral microvascular disorders caused by excessive Aβ deposition in cerebral microvascular plays a key factor to cause for AD development,which suggesting reducing Aβ deposition in cerebral microvascular and improving and protecting microvascular function may prevent and slow down AD development. |
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