Role And Mechanism Of Mfsd2a In Blood-brain Barrier Injury And Cognitive Impairment Caused By Chronic Cerebral Hypoperfusion

Background and aims: Chronic cerebral hypoperfusion(CCH)is the common pathological basis of Alzheimer’s Disease(AD)and Vascular Dementia(VaD),which can cause a series of pathophysiological changes and eventually lead to cognitive impairment.In recent years,a number of new drugs against β-amyloid protein and tau protein deposition have failed.Therefore,it is very important to pay attention to the role of vascular factors including chronic hypoperfusion in AD and VaD.Blood-brain barrier(BBB)injury occurs in the early stage of CCH and then lead to brain structural and functional damage.And the amelioration of BBB damage has been proved to improve CCH-induced VCI.Therefore,BBB may be a potential target for the treatment of cognitive impairment caused by CCH.Major facilitator superfamily domain-containing protein2a(Mfsd2a)is a newly confirmed protein which is important for BBB integrity.And it is also the only one molecule that can inhibit the vesicular transcytosis of BBB endothelial cells(ECs),which is crucial for maintaining the permeability of the BBB.Intervention against this target may be a promising novel treatment for AD and VaD.However,the role and mechanism of Mfsd2 a in the pathophysiological process of BBB injury under the CCH conditions have not yet been reported.In this study,we examined the expression of Mfsd2 a in the hippocampus of CCH rats.Then,the expression of Mfsd2 a was up-regulated via adeno-associated virus transfection to explore its role in CCH-induced BBB injury and cognitive impairment.In addition,we also evaluated the changes in the structure and function of the BBB and in the expression level of Mfsd2 a in hippocampus of CCH rats after the intervention with enriched environments(EEs),and explored the role and underlying mechanisms of both in improving the cognitive function of CCH rats in EEs.We hope that these will provide new targets and strategies for the prevention and treatment of VCI-related cognitive disorders such as VaD and AD.Methods: 1.A CCH model was constructed via producing permanent bilateral common carotid artery occlusion(2VO)in rats.On days 1,3,7,14,21,and 28 after 2VO or sham surgery,Evans blue(EB)leakage in the hippocampus was measured by chemical colorimetry to dynamically evaluate BBB permeability.On postoperative days 3,7,14,and 28,western blot was performed to measure the expression of Mfsd2 a protein.At the 14 th day,the expression and localization of Mfsd2a(green)and CD31(red)were evaluated by using dual immunofluorescence technique.From the 29 th day,the spatial learning and memory abilities of rats were assessed using the Morris water maze(MWM)test and the novel object recognition(NOR)test.2.Rats were randomly divided into 4 groups: Sham group,2VO group,2VO+control AAV group,and 2VO+Mfsd2a AAV group.The recombinant adeno-associated virus(AAV2/9-CMVr-Mfsd2a-3xflag-GFP virus)overexpressing Mfsd2 a was delivered via stereotaxic injection to the 2VO+Mfsd2a AAV group and an empty vector(AAV2/9-CMV-GFP control virus)to the2VO+control AAV group.And on days 3,7,14,28,and 56 after transfection,western blot was used to evaluate the expression of Mfsd2 a protein in the rat hippocampus to validate the effect of viral transfection.Fourteen days after virus transfection,the rats in the respective groups received either 2VO surgery or sham surgery.On days 1,3,7,14,and 28 after surgery,the amount of EB in the hippocampus of rats from the four groups was measured using colorimetric analysis.Western blot was used to detect the expression levels of Mfsd2 a,the BBB caveolae vesicles inhibitory protein,and the tight junction related proteins,including Zonula Occluden-1(ZO-1),claudin-5,and occludin.Moreover,the ultrastructure of the hippocampal BBB was observed by transmission electron microscopy.From the 29 th day,the spatial learning and memory abilities of rats were assessed using the MWM test and the NOR test.3.The rats were randomly divided into four groups: Sham+SE group,2VO+SE group,Sham+EE group,and 2VO+EE group.Rats in the enriched environment(EE)intervention group(Sham+EE and 2VO+EE)were exposed to an EE,while the standard environment(SE)intervention group(Sham+SE and 2VO+SE)rats were housed in a standard cage.After 14 days,the 2VO surgery or sham operation was performed.After the operation,the rats were returned to the corresponding environment according to their group.The amount of EB in the hippocampus of rats from the four groups was measured using colorimetric analysis.The ultrastructural changes of the hippocampal BBB were observed through transmission electron microscopy.Moreover,the expression levels of the BBB permeability-related proteins(Mfsd2a,ZO-1 and claudin-3),MMP-2/-9,and the classical Wnt/β-catenin signaling pathway-related proteins(Wnt3α,β-catenin,p-GSK-3β,and total-GSK-3β)in the hippocampus were detected using western blot.The effects of EE intervention on the cognitive function of rats in the four groups were evaluated by NOR and MWM tests four weeks after surgery.Results:1.The results of the MWM test showed that compared with the sham rats,rats in the2 VO group required longer to locate the platform,but spent significantly less time in the target quadrant.And the results of the NOR test showed that the 2VO rats had a lower Discrimination Index(DI)scores than the sham rats.In addition,EB leakage in the 2VO group increased,which gradually decreased after reaching its peak on postoperative day 7,and was still higher than that of sham rats until the 28 th day.The expression of Mfsd2 a protein decreased from postoperative day 3 and reached the lowest level on day 7.Moreover,the co-expression of Mfsd2 a and CD31 was observed by dual immunofluorescence,and the fluorescence intensity of Mfsd2 a in hippocampus of rats in 2VO group was significantly lower than that in Sham group.2.The expression of Mfsd2 a in the 2VO+Mfsd2a AAV group remained at a high level from day14 to day 56 post-transfection.Overexpression of Mfsd2 a attenuated the EB leakage,alleviated the high permeability,and reduced the number of caveolae vesicles in the hippocampal BBB in CCH rats.In addition,compared with the 2VO+control AAV rats,the rats in the 2VO+Mfsd2a AAV group required substantially less time to locate the platform,but spent significantly more time in the target quadrant in the MWM test.Moreover,the results of the NOR test showed that rats in the 2VO+Mfsd2a AAV group had a higher DI scores than the rats in the 2VO+control AAV group.3.The results of the MWM and NOR tests indicated that CCH rats exposed to the SE developed obvious cognitive impairment and BBB damage which was manifested through increased EB leakage and ultrastructural destruction.These changes were significantly alleviated after the EE intervention.Meanwhile,the expression of proteins Mfsd2 a,ZO-1 and claudin-3,which were beneficial to maintaining normal BBB permeability,was up-regulated,while the expression of proteins MMP-2 and MMP-9,which were related to the destruction of BBB integrity,was decreased after the EE intervention.In addition,EEs significantly up-regulated the expression levels of Wnt/β-catenin signaling pathway proteins Wnt3α and p-GSK-3β in the hippocampus of CCH rats,and inhibited the degradation of β-catenin protein,while there was no statistical difference in the expression of total-GSK-3β protein between the SE intervention group and the EE intervention group.Conclusions: 1.Permanent bilateral common carotid artery occlusion surgery successfully constructed a rat model of CCH.The CCH rats developed cognitive dysfunction,as well as increased BBB permeability and decreased Mfsd2 a expression in the hippocampus.2.Decreased Mfsd2 a expression increased BBB permeability by enhancing vesicular endocytosis in ECs.The overexpression of Mfsd2 a inhibited the abnormally enhanced vesicle endocytosis in the BBB of CCH rats,which alleviated the CCH induced BBB injury and thus ameliorated the spatial learning and memory impairment of CCH rats.3.An EE intervention reduced the CCH induced BBB destruction via increasing the expression of Mfsd2 a.Moreover,EEs also ameliorated BBB injury by activating the Wnt/β-catenin signaling pathway,up-regulating the expression of BBB tight junction proteins,and down-regulating the expression of BBB hydrolytic proteins in the hippocampus of CCH rats.These findings suggest that Mfsd2 a may be a novel target for improving CCH induced BBB injury and cognitive impairment,while BBB protection may be a new mechanism for EEs to ameliorate cognitive dysfunction related to CCH.