title: The γ-secretase blocker DAPT reduces the permeability of the blood-brain barrier by decreasing the ubiquitination and degradation of occludin during permanent brain ischemia.
abstract: BACKGROUND
  Tight junction protein degradation is a principal characteristic of the blood-brain barrier (BBB) damage that occurs during brain ischemia.
  
  
  OBJECTIVE
  We investigated the mechanisms of occludin degradation that underlie permanent middle cerebral artery occlusion (pMCAO) in rats.
  
  
  RESULTS
  Western blot and Co-immunoprecipitation data indicated ubiquitination and degradation of occludin in brain after pMCAO, which was consistent with ZO-1 degradation in penumbra regions as observed at 24 h after pMCAO. We further investigated candidate protease(s) responsible for the degradation of occludin during pMCAO. The intraventricular administration of γ-secretase blocker DAPT significantly inhibited the pMCAO-induced neurovascular damage, whereas ALLM and Batimastat, which are inhibitors of calpain and metalloproteinase proteases, respectively, were less effective. Notably, we found that DAPT significantly inhibited BBB disruption in comparison with vehicle treatment, as assessed by Evans blue excretion. Interestingly, the confocal immunostaining revealed that activation of the E3 ubiquitin ligase Itch is associated with degradation of occludin in brain microvessels following ischemia. Furthermore, our data demonstrate that the inhibition of γ-secretase signaling and the itch-mediated ubiquitination of occludin likely underlie the vasoprotective effect of DAPT after pMCAO.
  
  
  CONCLUSIONS
  The γ-secretase blocker DAPT reduces the permeability of the BBB by decreasing the ubiquitination and degradation of occludin during permanent brain ischemia, suggesting that γ-secretase may represent a novel therapeutic target for preventing neurovascular damage.
authors.affiliation: Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, Zhejiang University, Hangzhou, China.
authors.forename: Gen-Sheng
authors.initials: GS
authors.lastname: Zhang
authors.forename: Yun
authors.initials: Y
authors.lastname: Tian
authors.forename: Ji-Yun
authors.initials: JY
authors.lastname: Huang
authors.forename: Rong-Rong
authors.initials: RR
authors.lastname: Tao
authors.forename: Mei-Hua
authors.initials: MH
authors.lastname: Liao
authors.forename: Ying-Mei
authors.initials: YM
authors.lastname: Lu
authors.forename: Wei-Feng
authors.initials: WF
authors.lastname: Ye
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.forename: Kohji
authors.initials: K
authors.lastname: Fukunaga
authors.forename: Yi-Jia
authors.initials: YJ
authors.lastname: Lou
authors.forename: Feng
authors.initials: F
authors.lastname: Han
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects*; D004151:Dipeptides / Q000627:therapeutic use*; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000627:therapeutic use*; D005786:Gene Expression Regulation / Q000187:drug effects; D047468:Immunoprecipitation; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D008297:Male; D062793:Occludin / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D019108:Tight Junctions / Q000187:drug effects; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects*; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: D004151:Dipeptides; D004791:Enzyme Inhibitors; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; D062793:Occludin; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D044767:Ubiquitin-Protein Ligases; D053829:Amyloid Precursor Protein Secretases