Abstract
The expression of contactin-associated protein 1 (Caspr1) in brain microvascular endothelial cells (BMECs), one of the major cellular components of the neurovascular unit (NVU), has been revealed recently. However, the physiological role of Caspr1 in BMECs remains unclear. We previously reported the nonamyloidogenic processing of amyloid protein precursor (APP) pathway in the human BMECs (HBMECs). In this study, we found Caspr1 depletion reduced the levels of soluble amyloid protein precursor α (sAPPα) in the supernatant of HBMECs, which could be rescued by expression of full-length Caspr1. Our further results showed that ADAM9, the α-secretase essential for processing of APP to generate sAPPα, was decreased in Caspr1-depleted HBMECs. The reduced sAPPα secretion in Caspr1-depleted HBMECs was recovered by expression of exogenous ADAM9. Then, we identified that Caspr1 specifically regulates the expression of ADAM9, but not ADAM10 and ADAM17, at transcriptional level by nuclear factor-κB (NF-κB) signaling pathway. Caspr1 knockout attenuated the activation of NF-κB and prevented the nuclear translocation of p65 in brain endothelial cells, which was reversed by expression of full-length Caspr1. The reduced sAPPα production and ADAM9 expression upon Caspr1 depletion were effectively recovered by NF-κB agonist. The results of luciferase assays indicated that the NF-κB binding sites are located at −859 bp to −571 bp of ADAM9 promoter. Taken together, our results demonstrated that Caspr1 facilitates sAPPα production by transcriptional regulation of α-secretase ADAM9 in brain endothelial cells.
Highlights
The blood–brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) sheathed by perivascular astrocytes and pericytes, which is critical for maintaining brain homeostasis (Zhao et al, 2015; Sweeney et al, 2016)
We further found that the concentration of soluble amyloid protein precursor α (sAPPα) in the culture supernatant was significantly decreased compared to the control (top panel, transfected with contactin-associated protein 1 (Caspr1)-specific siRNA compared to the controls (Figure 2B)
The Caspr1 knockout human BMECs (HBMECs) were transfected with lentivirus containing the full-length A disintegrin and metalloprotease 9 (ADAM9) cDNA to restore the expression of ADAM9 (Figure 2C), and the concentrations of sAPPα in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA)
Summary
The blood–brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) sheathed by perivascular astrocytes and pericytes, which is critical for maintaining brain homeostasis (Zhao et al, 2015; Sweeney et al, 2016). The amyloid protein precursor (APP) is a transmembrane protein that is primarily processed by two distinct pathways. Caspr Regulates ADAM9-Dependent sAPPα Production cleaved by β-site APP cleavage enzyme 1 (β-secretase) and γ-secretase complex (De Strooper, 2003), producing amyloid β peptide (Aβ), which is the major constituent of amyloid plaques in brains of patients with Alzheimer disease. The alternative nonamyloidogenic pathway involves the cleavage of APP within the Aβ sequence by α-secretase, generating a soluble N-terminal fragments called soluble amyloid protein precursor α (sAPPα; Thinakaran and Koo, 2008). We and others identified that nonamyloidogenic pathway is present in brain endothelial cells (Allinson et al, 2003; Kitazume et al, 2010; Wang et al, 2016); the mechanism regulating sAPPα production in brain endothelial cells remains incompletely understood
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