Uncovering the role of connexins and Yap in blood brain barrier hyperpermeability and microvascular injury in cerebral amyloid angiopathy

Authors: Muyu Situ 1  and Anuska V. Andjelkovic 2

Affiliations:  1 Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI
2 Department of Pathology and Neurosurgery, University of Michigan, Ann Arbor, MI   

Background (165)
Cerebral  Amyloid  Angiopathy  (CAA)  is  a  cerebral  small  vessels  disease  characterized  by accumulation  of  amyloid-β  (Aβ)  around  the  small  caliber  vessels  (arterioles  and  capillaries).  Structural alterations and loss of integrity in the neurovascular unit/blood-brain barrier (NVU/BBB) in CAA results in “leakier” microvessels, microhemorrhage, increased stroke risk, and cognitive impairment. Although a significant body of evidence has pinpointed several potential mechanisms (i.e.,  neuroinflammation  and  oxidative  metabolism)  to  drive  BBB  injury  in  CAA,  molecular mechanisms remain to be fully elucidated. To determine the profile of microvascular injury in CAA murine model (Tg-SwDI mice), we performed RNA sequencing  analysis on the isolated brain microvessels.  Transcriptome  profiling  revealed  alteration  in  genes  like  gap  junction  proteins, connexin 43 (Cx43) and Cx45, and  Yap1 (Yes-associated protein), a component of the Hippo signaling  pathway  involved  in  remodeling  of  actin  cytoskeleton,  extracellular  matrix,  and regulation of inflammation. Our present study aims to understand how changes and interactions between Cx43/Cx45, and Yap modifies barrier integrity and promotes cerebrovascular injury in the CAA condition.

Methods (57)
Using in vitro (Aβ 1-40  and Aβ 1-42  exposure) and in vivo models  (Tg-SwDI mice), we performed assessment of cell structural changes (immunocytochemistry, confocal microscopy), functional (optical  tweezers  method),  FRAP  assay,  in  vitro  permeability  assay)  and  signaling  events (western blotting and proximity ligation assay) associated with alteration of brain endothelial cells (BECs) mechanics and barrier permeability in CAA settings. 

Results (90)
Consequences of Aβ-afflicted brain microvessels and Aβ exposure in BECs promotes an overall increased protein expression and a redistribution of Yap expression from the cytoplasm to the nucleus, contributing to structural changes, such cellular stiffening and cell permeability. Similarly, Aβ promotes upregulation of Cx43 and Cx45 (i.e., GJs and HCs formation and activity) as well as loss  of  tight  junction  integrity  resulting  in  a  hyperpermeable  barrier.  Inhibition  (via  selective inhibitors) and/or modifications of each component  expression (via siRNA transfection) or whole Yap/Cx43/Cx45 axis alters the cell mechanics and consequently barrier permeability.  Conclusion (36) Collectively, our data suggest that vascular Aβ deposition in CAA pathology promotes injury and junctional  alterations  through  a  Cx43/Cx45/Yap  axis  in  the  brain  endothelium  contributing  to development of CAA associated pathology (i.e., BBB hyperpermeability, and microbleeds).