The primary focus of this project is to understand regulation of the ATP-driven xenobiotic efflux pumps, e.g., P-glycoprotein, at the blood-brain barrier. This focus is now expanded to include other blood-brain barrier efflux pumps, i.e., breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (Mrp2) and the blood-spinal cord barrier. To map the extracellular and intracellular signals that regulate these transporters, we use 1) pharmacological tools, 2) intact brain and spinal cord capillaries from rats and mice (including transgenics and knockouts), 2) fluorescent substrates, 3) confocal imaging to measure transport function, 4) Western blotting to measure transporter expression, and 5) brain perfusion in rats and mice in vivo to validate signaling-based changes in transporter function. Recent progress has been primarily in three areas: identification of signals that rapidly reduce basal P-glycoprotein activity without changing expression (non-genomic signaling), identification of ligand-activated nuclear receptors that upregulate transporter expression, and evaluation of blood-brain barrier function in a animal model of folate deficiency. 
 Non-Genomic Signaling: P-glycoprotein is a major obstacle to the delivery of small molecule drugs across the blood-brain barrier and into the CNS. We have tested a novel, signaling-based strategy to overcome this obstacle. We mapped an extended, non-genomic signaling pathway that rapidly (minutes) and reversibly reduced P-glycoprotein transport activity without altering transporter protein expression;the defined pathway encompasses elements of proinflammatory, sphingolipid and protein kinase-based signaling. Central to this pathway is signaling through sphingosine-1-phosphate receptor 1 (S1PR1). S1P, the S1P analog, fingolimod (FTY720), currently in clinical trials for treatment of multiple sclerosis, and its active, phosphorylated metabolite (FTY720P) acted through S1PR1 to reduce P-glycoprotein transport activity. We validated these findings in vivo using in situ brain perfusion in rats. Administration of S1P, FTY720 or FTY729P increased brain uptake of three radiolabeled P-glycoprotein substrate (including one chemotherapeutic) without altering tight junctional permeability;blocking S1PR1 abolished this effect. We also identified MRP! As the efflux transporter that move S1P out of the endothelial cells so it can interact with the extracellular binding site of S1PR1. 
 Nuclear Receptor of Transporter Expression: we examined whether the Glucocorticoid receptor (GR), a ligand-activated nuclear receptor targeted by both natural and synthetic glucocorticoids, regulates P-gp at CNS barriers. Naturally occurring glucocorticoids, cortisol in the human and corticosterone in the rat, regulate a wide range of physiological effects including gluconeogenesis, homeostasis, and apoptosis. However, the role of these hormones in maintenance of CNS barriers remains unresolved. Furthermore, the effects of synthetic glucocorticoids, a broad class of widely prescribed anti-inflammatory drugs, on CNS barriers are also poorly understood. However, these potent anti-inflammatory synthetic glucocorticoids are a mainstay in the treatment of cerebral edema and spinal cord injury. We hypothesize that both natural and synthetic glucocorticoids alter the expression and activity of P-gp at CNS barriers, thereby modifying drug delivery to the CNS. We have confirmed the basal expression of GR in both CNS barriers by qPCR and immunoblotting and further found that the depletion of corticosterone via adrenalectomy significantly decreased the expression and activity of P-gp in both the BBB and the BSCB. In-vivo treatment of both intact and adrenalectomized (ADX) rats with the synthetic glucocorticoid dexamethasone significantly increased P-gp activity and protein expression in both CNS barriers. In-vitro treatment of BBB and BSCB capillaries with dexamethasone also increased P-gp expression and activity and co-treatment with the GR antagonist, RU-486, abolished these increases. These results demonstrate that the endogenous glucocorticoid, corticosterone, maintains P-gp expression and activity at both the BBB and BSCB while the synthetic glucocorticoid, dexamethasone, in the presence or absence of endogenous glucocorticoids, increases the activity and expression of P-gp in a GR-dependent manner. Thus, natural glucocorticoids have a protective role in maintaining CNS barriers, while synthetic glucocorticoids may hinder delivery of therapeutic drugs to the CNS. 

 Folate Deficiency: In collaboration with Dr. Richard Finnells laboratory at the University of Texas, Austin, we examined the effect of folate deficiency on blood-brain barrier function. Folate is an essential nutrient involved in multiple cellular functions. Folate deficiency is associated with multiple clinical phenotypes, including neural tube defects during embryogenesis and neurological disorders in children and adults. For this project we used the proton-coupled folate transporter (PCFT)-null mouse, previously described as a model for systemic folate deficiency. PCFT is one of three folate transporters expressed in mammals. Brain capillaries from PCFT-null mice exhibited reduced expression of P-glycoprotein and the tight junction proteins, ZO-1, claudin-1 and occludin (66). Capillaries from PCFT-null mice did not increase P-glycoprotein expression in response to TCDD (AhR ligand) or valproate, an AED, CAR and PXR ligand and histone deacetylase inhibitor. Brain capillaries from S-folinic acid (SFA;40 mg/kg)-treated PCFT-null mice exhibited increased P-glycoprotein transport following VPA exposure. Thus, SFA supplementation restored one element of normal blood-brain barrier function. Taken together, these findings strongly suggest that folate deficiency disrupts blood-brain barrier function by targeting transporters and tight junctions. This may contribute to the development of neurological disorders in folate deficient individuals.

Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2013
Total Cost
$1,153,051
Indirect Cost
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State
Country
Zip Code
Emmert, Dana; Campos, Christopher R; Ward, David et al. (2014) Reversible dimers of the atypical antipsychotic quetiapine inhibit p-glycoprotein-mediated efflux in vitro with increased binding affinity and in situ at the blood-brain barrier. ACS Chem Neurosci 5:305-17
Miller, David S (2014) Sphingolipid signaling reduces basal P-glycoprotein activity in renal proximal tubule. J Pharmacol Exp Ther 348:459-64
Wang, Xueqian; Campos, Christopher R; Peart, John C et al. (2014) Nrf2 upregulates ATP binding cassette transporter expression and activity at the blood-brain and blood-spinal cord barriers. J Neurosci 34:8585-93
Cartwright, Tara A; Campos, Christopher R; Cannon, Ronald E et al. (2013) Mrp1 is essential for sphingolipid signaling to p-glycoprotein in mouse blood-brain and blood-spinal cord barriers. J Cereb Blood Flow Metab 33:381-8
Dallas, Shannon; Block, Michelle L; Thompson, Deborah M et al. (2013) Microglial activation decreases retention of the protease inhibitor saquinavir: implications for HIV treatment. J Neuroinflammation 10:58
Miller, David S; Cannon, Ronald E (2013) Signaling Pathways that Regulate Basal ABC Transporter Activity at the Blood-Brain Barrier. Curr Pharm Des :
Zibell, Guido; Unkruer, Bernadette; Pekcec, Anton et al. (2009) Prevention of seizure-induced up-regulation of endothelial P-glycoprotein by COX-2 inhibition. Neuropharmacology 56:849-55
Reichel, Valeska; Miller, David S; Fricker, Gert (2008) Texas Red transport across rat and dogfish shark (Squalus acanthias) choroid plexus. Am J Physiol Regul Integr Comp Physiol 295:R1311-9