Inhibition of mitochondrial carbonic anhydrases reduces Amyloid β induced cell death in cerebral 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). Cerebrovascular amyloid disrupts the function of brain endothelial cells (ECs). Previous findings from our lab demonstrated that Acetazolamide (ATZ) and methazolamide (MTZ), both pan‐carbonic anhydrase (CA) inhibitors, prevent NVU cell death in models of AD and CAA. Although these FDA‐approved pan‐CA inhibitors are protective, a more specific targeting of the isoforms involved in AD pathology may not only reduce side effects, but also increase the efficacy of pan‐CA inhibition. We have shown that CAIs reduce Aβ‐induced cytochrome C release from the mitochondria, caspase‐9 activation, and mitochondrial H2O2 production in NVU cells. Therefore, we hypothesize that mitochondrial CA‐VA/B are the main isoforms involved in these mechanisms and we anticipate that using inhibitors selective for CA‐VA/B will prevent cerebral EC apoptosis, thereby reducing NVU dysfunction and AD progression.MethodHuman cerebral microvascular EC were treated with the vasculotropic Aβ40‐Q22 Dutch mutant, alone and in combination with a number of CA‐V inhibitors. Cell viability was measured with WST‐1 reagent and apoptosis was measured as the amount of fragmented nucleosomes. To assess mitochondria‐mediated apoptotic pathways, the expression of BAX BCL‐2 and caspase 9 activation were evaluated. The levels of different CA enzymes in cerebral EC was also assessed following Aβ treatment.ResultInhibition of CA‐VA/B significantly increased cerebral EC viability in the presence of Aβ40‐Q22. CA‐VA/B inhibitors also significantly reduced Aβ40‐Q22 induced DNA fragmentation in a dose dependent manner. The inhibition of CA‐VA/B attenuated Aβ40‐Q22 induced caspase‐9 activation as well as the increase in expression of the pro‐apoptotic protein BAX in cerebrovascular ECs. Interestingly, we detected changes in the expression of mitochondrial isoform, CA‐VB, after treating cerebral ECs with Aβ.ConclusionThese results indicate that the targeted inhibition of CA‐V is protective against Aβ‐challenge in cerebral ECs through the inhibition of mitochondria‐mediated cell death and suggest that the efficacy of these new compounds should be explored in animal models of cerebrovascular amyloidosis.