Mechanisms Of BACE1 Contribute To Cerebral Small Vessel Injury

Cerebral small vessel disease(CSVD),which refers to aberrant processes affecting the cerebral small vessel bed initiated by vascular risk factors,contributes to a group of phenotypes of pathology,neuroimaging and clinical symptoms.Among various contributors,hypertension is the most important risk factor for CSVD.Endothelial injury and blood-brain barrier breakdown have been found to be associated with CSVD pathogenesis in multiple lines of evidence.Hypertensive vasculopathy and cerebral amyloid angiopathy(CAA)are the two most prevalent forms of sporadic CSVD.Although the two subtypes are categorized by different pathological,clinical and neuroimaging markers,they share the general pathogenesis in the prodromal stage,suggesting a common mechanism provoking CSVD onset.However,the exact upstream molecules inducing the downstream pathogenesis still remains unclear.Our previous studies and other groups have shown an elevation of β-secretase 1(BACE1)levels in the cerebral vessels from CAA patients,while any changes in vascular BACE1 expression under hypertension conditions lack investigations.In the present study,we detect a～3.71-fold increase in BACE1 expression in the isolated cerebral microvascular from hypertensive patients compared to that in normotensive patients using western blot.Meanwhile,immunofluorescence on human brain sections shows a～2.05-fold increase of the BACE1-positive area in the microvascular endothelium.These findings suggest that vascular BACE1 may generally involve in pathogenesis of different CSVDs.Notably,both experiments of western blot and immunofluorescence indicate～40.16%-47.59%lower occludin levels in cerebral microvessels from hypertensive patients with respect to normotensive controls.More importantly,data analysis shows a negative correlation between microvascular BACE1 and occludin levels in human brain samples,suggesting that vascular BACE1 may affect cerebral small vessels through an occludin-dependent mechanism.In the process to uncover the exact pathway,we identify that the endothelial tight junction protein,occludin,is a completely new substrate for BACE1.BACE1 is able to cleave occludin resulting in full-length occludin reductions with occludin N-and C-terminal fragment generations.Parallel results exhibit that BACE1 can not cleave other major tight junction proteins,such as ZO-1,JAM-A and claudin-1,however,BACE 1 elevation in endothelial cells elicits a decrease in these tight junction proteins.Further studies reveal that overprocessing of occludin by elevated endothelial BACE1 promotes caveolin-1 accumulation in the endothelial plasma membrane and subsequent caveolin-l-mediated endocytosis,resulting in lysosomal degradation of membrane tight junction proteins and clearance of cytosolic tight junction proteins by cytosolic proteases.Additionally,abundant caveolin-1 increases the binding of endothelial isoform of nitric oxide synthase(eNOS)to caveolin-1 in the endothelial membrane,leading to an attenuation of eNOS translocation into the cytosol and subsequent phosphorylated activation with resultant eNOS inactivation and endothelial dysfunction in association with deficient eNOS downstream signalling.In addition to occludin,β-site amyloid precursor protein(APP),a well known substrate for BACE 1,is also abundantly expressed in cerebrovascular endothelial cells.Endothelial APP processing by BACE1 exhibits fulllength APP reductions with the final product β-amyloid peptides(Aβ)secretion out of endothelial cells.Aβ has been found to exert regulatory effects on endothelial states and functions.To explore whether elevated endothelial BACE1 provokes CSVD via another APP-Aβ axis,we treat cerebral endothelial cell lines with Aβ1-40 or Aβ1-42,and observe both human and murine Aβ1-40 or Aβ1-42 as inhibitors on eNOS function in human or mouse endothelial cells.All of these in vitro results suggest that higher endothelial BACE1 contributes to endothelial tight junction disruption and eNOS dysfunction through the coincident BACE1-occludin and BACE1-APP-Aβ pathways.To investigate whether the two mechanisms could induce CSVD-like pathological and behavioral phenotypes,we developed a novel transgenic mouse model overexpressing human BACE1(～5.11-fold increase)in the vascular endothelium(VE)under the control of the endothelial-specific Tie2 promoter element(VE-BACE 1 mice).Compared to age-matched WT controls,9-month-old VE-BACE1 mice display decreased protein levels of major tight junction protein occludin,ZO-1,JAM-A and claudin-1,raised plasma Aβ concentrations and reduced eNOS activation.Furthermore,the initial endothelial damage provokes chronic reduction of cerebral blood flow(CBF)at 12 months of age and BBB leakage at 15 months of age in VE-B ACE 1 mice,while these abnormalities worsen with increasing age.Moreover,the pathological feature of microbleeds and behavioral changes comprising locomotor activity reduction and learning and memory impairment occur in VE-B ACE 1 mice at 15 months of age.Conversely,inhibition of aberrant BACE1 activity by BACE1 inhibitor MK-8931 ameliorates tight junction loss,eNOS dysfunction,and memory deficits in VE-BACE1 mouse brains.This treatment for three months safely functions under a low-dose treatment,and no adverse effects and death of mice occurred during the study period.Taken together,our findings establish a novel and direct relationship between endothelial BACE1 and cerebral small vessel damage,indicating that CSVD-related risk factors,such as hypertension,induced the upregulation of endothelial BACE1,which promoted the overprocessing of occludin and APP with subsequent degradation of tight junction proteins and endothelial dysfunction.The early shift of the cerebral endothelium from a normal phenotype to a damaged one further promotes small vessel injury and cognitive impairment at a later stage.Collectively,this study provides causal evidence of elevated endothelial BACE1 levels for CSVD onset and development,and targeting abnormal endothelial BACE1 as an early intervention may be a very important strategy to reduce CSVD progression.