Inhibition of mitochondrial carbonic anhydrases prevents Amyloid β‐induced cell death and blood brain barrier permeability in cerebrovascular endothelial cells

Abstract

AbstractBackgroundThe Neurovascular Unit (NVU) is an important multicellular structure of the central nervous system (CNS) which has been observed to become dysfunctional in Alzheimer Disease (AD) and cerebral amyloid angiopathy (CAA). Amyloidosis disrupts the function of cerebrovascular endothelial cells (ECs) through the activation of the intrinsic apoptotic pathway, and the induction of blood brain barrier (BBB) permeability. Previous findings in our lab demonstrate that Acetazolamide (ATZ) and methazolamide (MTZ), pan‐Carbonic Anhydrase (CA) inhibitors, prevent mitochondria mediated apoptosis in cerebrovascular ECs through the reduction of Aβ induced cytochrome C release from the mitochondria, caspase‐9 activation, and mitochondrial H2O2 production. Due to the reduction of mitochondria mediated cell death via pan‐CA inhibitors we hypothesize that the mitochondrial isoforms, CA‐VA/B, are involved in cell death and other deleterious mechanisms induced by Aβ. Thus, we anticipate using selective CA‐V inhibitors will reduce cerebrovascular EC apoptosis and prevent the loss of BBB resistance.MethodHuman cerebral microvascular ECs (hCMECs) were treated with Aβ40‐Q22 Dutch mutant alone and in combination with a CA‐V inhibitor. We measured outcomes of mitochondria mediated apoptosis and BBB integrity. Apoptosis was evaluated by measuring the changes in the protein expression of caspase‐9, BAX, and BCL‐2 with western blot. We used live‐cell imaging to measure active caspase‐3. The release of cytochrome C and the mitochondrial membrane potential was detected using immunocytochemistry (ICC). Mitochondria H2O2 production was measured by isolating mitochondria and using a commercially available kit, Amplex Red (Invitrogen). We assessed the permeability of hCMECs using the ECIS ZΘ technology. The changes in protein expression of EC activation markers, VCAM‐1 and ICAM‐1, were determined with western blot.ResultInhibition of CA‐VA/B prevented Aβ40‐Q22 induced DNA fragmentation, caspase‐9 and ‐3 activation, cytochrome C release from the mitochondria, loss of mitochondria membrane potential, and mitochondrial H2O2 production in human cerebrovascular ECs. The inhibition of CA‐VA/B also reduced Aβ40‐Q22 induced BBB permeability and increase in vascular adhesion protein VCAM‐1 in hCMECs.ConclusionThe inhibition of CA‐V is protective to cerebrovascular ECs in models of amyloidosis, through the prevention of mitochondria‐mediated cell death and loss of cerebral EC barrier function.