Toxic amyloid-β assemblies, amylospheroids, inactivate eNOS through a NAKα3-mediated ROS/PKC pathway in human brain microvascular endothelial cells

[Speaker] Tomoya Sasahara:1,2
[Co-author] Kaori Satomura:1,2, Minako Hoshi:1,3
1:Center for Brain and Neurodegenerative Disease Research, Institute of Biomedical Research and Innovation, Japan, 2:TAO Health Life Pharma Co., Ltd., Japan, 3:Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Japan

 Cerebrovascular deposition of insoluble fibrous protein, mainly composed of amyloid-β protein (Aβ), known as cerebrovascular amyloid angiopathy, is associated with vascular dysfunctions. Aβ oligomers have been reported to cause endothelial dysfunctions but it has not been clarified what Aβ oligomers have endothelial toxicity. Amylospheroids (ASPD), 30-mer Aβ assemblies in average, were reported to bind to α3 subunit of Na+, K+-ATPase (NAKα3) and cause degeneration of mature neurons by impairing NAKα3 activity (Ohnishi T et al. PNAS2015). Here we elucidated whether ASPD were involved in endothelial dysfunctions.
 Since activity of endothelial nitric oxide synthase (eNOS) is linked to vascular functions, we first examined whether ASPD affect eNOS activity. The experiment using NO indicator diaminofluorescein-2-loaded human brain microvascular endothelial cells (hBMVEC) revealed that ASPD suppressed NO release. The activity of eNOS is regulated by phosphorylation, mainly at Thr495 and Ser1177, and the analysis of western blotting clarified that ASPD phosphorylated eNOS at Thr495 but not affect Ser1177, leading to eNOS inactivation. We then investigated the mechanisms of Thr495-eNOS phosphorylation and protein kinase C (PKC) inhibitor, bisindolylmaleimide I, blocked this phosphorylation. Activated PKC (phosphorylation at Ser660) was increased by ASPD-stimulation and this activation of PKC was inhibited by reactive oxygen species (ROS) scavenger, tempol. Additionally we found that ASPD increased ROS production in hBMVEC using ROS-fluorescence indicator, CellROX. Knockdown of ASPD-binding target NAKα3 using siRNA inhibited the ASPD-induced eNOS inactivation. ASPD suppressed NO release, which induce vasodilation, as a result of eNOS inactivation in hBMVEC, suggesting that ASPD suppress vasorelaxation. We then confirmed whether ASPD really inhibit vasorelaxation using rat thoracic aortic rings. The aortic rings were stimulated by muscarinic agonist carbachol in a dose-dependent manner. The vasorelaxation of ASPD-pretreated rings was suppressed compared with vehicle-treated rings.
 Summarizing the above, we elucidated the ASPD effects and those mechanisms in endothelium; ASPD inhibit NO release by the suppression of eNOS activity by phosphorylation at Thr495 through a NAKα3 binding-mediated ROS/PKC pathway in hBMVEC and additionally the suppression of NO release affects not only cell functions but also vascular functions. Our results suggested that ASPD play role on vascular dysfunctions by suppressing eNOS activity.

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