Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells
- 171 Downloads
Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown.
Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking.
A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 μM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold).
Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.
KEY WORDSblood-brain barrier clioquinol ionophore P-glycoprotein transporter
Amyloid beta peptide
Brain microvascular endothelial cells
Central nervous system
Endothelial basal medium 2
Hank’s balanced salt solution
Immortalised human cerebral microvascular endothelial cell line
Inductively coupled plasma mass spectrometry
Phosphate buffered saline
Sodium dodecyl sulfate
Acknowledgments and Disclosures
The studies completed within this publication were funded by the following sources; NHMRC Project APP1048855; the Mason Foundation and the Bethlehem Griffiths Research Foundation. Mitchell P. McInerney is supported by an Australian Government Research Training Program Scholarship.
- 8.Selkoe DJ. Alzheimer's Disease. Cold Spring Harb Perspect Biol. 2011;3(7):a004457–7.Google Scholar
- 9.Roberts KF, Elbert DL, Kasten TP, Patterson BW, Sigurdson WC, Connors RE, et al. Amyloid-beta efflux from the central nervous system into the plasma. Ann Neurol. 2014;76(6):837–44.Google Scholar
- 10.Shibata M, Yamada S, Kumar SR, Calero M, Bading J, Frangione B, et al. Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest. 2000;106(12):1489–99.Google Scholar
- 11.Qosa H, Abuasal BS, Romero IA, Weksler B, Couraud PO, Keller JN, et al. Differences in amyloid-beta clearance across mouse and human blood-brain barrier models: Kinetic analysis and mechanistic modeling. Neuropharmacology. 2014;79C:668–78.Google Scholar
- 12.Cirrito JR, Deane R, Fagan AM, Spinner ML, Parsadanian M, Finn MB, et al. P-glycoprotein deficiency at the blood-brain barrier increases amyloid-beta deposition in an Alzheimer disease mouse model. J Clin Invest. 2005;115(11):3285–90.Google Scholar
- 14.Vogelgesang S, Cascorbi I, Schroeder E, Pahnke J, Kroemer HK, Siegmund W, et al. Deposition of Alzheimer's beta-amyloid is inversely correlated with P-glycoprotein expression in the brains of elderly non-demented humans. Pharmacogenetics. 2002;12(7):535–41.Google Scholar
- 15.Deo AK, Borson S, Link JM, Domino K, Eary JF, Ke B, et al. Activity of P-glycoprotein, a beta-Amyloid transporter at the blood-brain barrier, is compromised in patients with mild Alzheimer disease. J Nucl Med. 2014;55(7):1106–11.Google Scholar
- 22.Crouch PJ, Savva MS, Hung LW, Donnelly PS, Mot AI, Parker SJ, et al. The Alzheimer's therapeutic PBT2 promotes amyloid-beta degradation and GSK3 phosphorylation via a metal chaperone activity. J Neurochem. 2011;119(1):220–30.Google Scholar
- 23.Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, et al. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron. 2001;30(3):665–76.Google Scholar
- 26.Liebner S, Corada M, Bangsow T, Babbage J, Taddei A, Czupalla CJ, Reis M, Felici A, Wolburg H, Fruttiger M, Taketo MM, von Melchner H, Plate KH, Gerhardt H, Dejana E. Wnt/beta-catenin signaling controls development of the blood-brain barrier. J Cell Biol 2008;183(3):409–417.Google Scholar
- 27.Tran KA, Zhang X, Predescu D, Huang X, Machado RF, Gothert JR, et al. Endothelial beta-catenin signaling is required for maintaining adult blood-brain barrier integrity and central nervous system homeostasis. Circulation. 2016;133(2):177–86.Google Scholar
- 31.Adlard PA, Cherny RA, Finkelstein DI, Gautier E, Robb E, Cortes M, et al. Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxy quinoline analogs is associated with decreased interstitial Abeta. Neuron. 2008;59(1):43–55.Google Scholar
- 34.Lim JC, Kania KD, Wijesuriya H, Chawla S, Sethi JK, Pulaski L, et al. Activation of beta-catenin signalling by GSK-3 inhibition increases P-glycoprotein expression in brain endothelial cells. J Neurochem. 2008;106(4):1855–65.Google Scholar
- 36.Grossi C, Francese S, Casini A, Rosi MC, Luccarini I, Fiorentini A, et al. Clioquinol decreases amyloid-beta burden and reduces working memory impairment in a transgenic mouse model of Alzheimer's disease. J Alzheimers Dis. 2009;17(2):423–40.Google Scholar