Protein Expression and Functional Relevance of Efflux and Uptake Drug Transporters at the Blood-Brain Barrier of Human Brain and Glioblastoma.
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ABSTRACT:
SUBMITTER: Bao X
PROVIDER: S-EPMC7167337 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Seizure-provoking factors circulate late in gestation during normal pregnancy, but do not readily gain access to the brain due to the protective nature of the blood-brain barrier. In particular, efflux transporters are powerful ATP-driven pumps that actively prevent unwanted compounds from entering the brain. We hypothesized that acute inhibition of efflux transporters at the blood-brain barrier would result in spontaneous seizures in pregnant rats. We further hypothesized that the blood-brain barrier protects the maternal brain from seizure by increasing expression and/or activity of p-glycoprotein (P-gp), a major efflux transporter. Main blood-brain barrier efflux transporters were inhibited in-vivo in nonpregnant (Nonpreg) and pregnant (Preg; d19) Sprague Dawley rats (n=8/group). Seizures were monitored in conscious animals for 8h via chronically implanted electroencephalography (EEG) electrodes in the hippocampus and motor cortex and time-synced video. P-gp activity was measured via a calcein accumulation assay in freshly isolated cortical and hippocampal capillaries from Preg (d20) and Nonpreg rats (n=8-16/group), to assess regional susceptibility to transporter inhibition. P-gp expression, capillary density, and microglial activation as a measure of neuroinflammation were quantified using immunohistochemistry (n=4-6/group). Efflux transporter inhibition elicited hippocampal seizures within 1h in 100% of Preg rats that was not associated with neuroinflammation or elevated tumor necrosis factor alpha (TNF?) or vascular endothelial growth factor (VEGF), but negatively correlated with levels of estradiol. Hippocampal seizures were considerably less prevalent in Nonpreg rats. However, behavioral seizures in the motor cortex developed of similar severity in both groups of rats, demonstrating regional heterogeneity in response to efflux transporter inhibition. Basal P-gp activity was similar between groups, however, exposure to serum from Preg rats significantly decreased P-gp activity in the hippocampus, but not cortex, compared to serum from Nonpreg rats (0.29±0.1units/s in Preg vs. 0.06±0.02units/s in Nonpreg rats; p<0.05) that was not associated with elevated TNF? or VEGF. Thus, pregnancy differentially increased the susceptibility of the hippocampus to seizures in response to blood-brain barrier efflux transporter inhibition that may be due to the inhibitory effect of circulating factors in pregnancy on P-gp activity in the hippocampus.
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Project description:The blood--testis barrier (BTB) creates an immunological barrier that segregates the seminiferous epithelium into the basal and apical compartment. Thus, meiosis I/II and post-meiotic germ cell development take place in a specialized microenvironment in the apical compartment behind the BTB and these events are being shielded from the host immune system. If unwanted drugs and/or chemicals enter the apical compartment from the microvessels in the interstitium via the basal compartment, efflux pumps (e.g. P-glycoprotein) located in Sertoli cells and/or spermatids can actively transport these molecules out of the apical compartment. However, the mechanism(s) by which influx pumps regulate the entry of drugs/chemicals into the apical compartment is not known. In this study, a solute carrier (SLC) transporter organic anion transporting polypeptide 3 (Oatp3, Slco1a5) was shown to be an integrated component of the N-cadherin-based adhesion complex at the BTB. However, a knockdown of Oatp3 alone or in combination with three other major Sertoli cell drug influx pumps, namely Slc22a5, Slco6b1, and Slco6c1, by RNAi using corresponding specific siRNA duplexes failed to perturb the Sertoli cell tight junction (TJ) permeability barrier function. Yet, the transport of [(3)H]adjudin, a potential male contraceptive that is considered a toxicant to spermatogenesis, across the BTB was impeded following the knockdown of either Oatp3 or all the four SLC transporters. In short, even though drug transporters (e.g. influx pumps) are integrated components of the adhesion protein complexes at the BTB, they are not involved in regulating the Sertoli cell TJ permeability barrier function, instead they are only involved in the transport of drugs, such as adjudin, across the immunological barrier at the BTB.
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Project description:ATP-driven efflux transporters at the blood-brain barrier both protect against neurotoxicants and limit drug delivery to the brain. In other barrier and excretory tissues, efflux transporter expression is regulated by certain ligand-activated nuclear receptors. Here we identified constitutive androstane receptor (CAR) as a positive regulator of P-glycoprotein, multidrug resistance-associated protein 2 (Mrp2), and breast cancer resistance protein (BCRP) expression in rat and mouse brain capillaries. Exposing rat brain capillaries to the CAR activator, phenobarbital (PB), increased the transport activity and protein expression (Western blots) of P-glycoprotein, Mrp2, and BCRP. Induction of transport was abolished by the protein phosphatase 2A inhibitor, OA. Similar effects on transporter activity and expression were found when mouse brain capillaries were exposed to the mouse-specific CAR ligand, 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). In brain capillaries from CAR-null mice, TCPOBOP did not increase transporter activity. Finally, treating mice with 0.33 mg/kg TCPOBOP or rats with 80 mg/kg PB increased P-glycoprotein-, Mrp2-, and BCRP-mediated transport and protein expression in brain capillaries assayed ex vivo. Thus, CAR activation selectively tightens the blood-brain barrier by increasing transport activity and protein expression of three xenobiotic efflux pumps.
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Project description:Disruption of blood-brain barrier integrity and dramatic failure of brain ion homeostasis including fluctuations of pH occurs during cortical spreading depression (CSD) events associated with several neurological disorders, including migraine with aura, traumatic brain injury and stroke. NHE1 is the primary regulator of pH in the central nervous system. The goal of the current study was to investigate the role of sodium-hydrogen exchanger type 1 (NHE1) in blood brain barrier (BBB) integrity during CSD events and the contributions of this antiporter on xenobiotic uptake. Using immortalized cell lines, pharmacologic inhibition and genetic knockdown of NHE1 mitigated the paracellular uptake of radiolabeled sucrose implicating functional NHE1 in BBB maintenance. In contrast, loss of functional NHE1 in endothelial cells facilitated uptake of the anti-migraine therapeutic, sumatriptan. In female rats, cortical KCl but not aCSF selectively reduced total expression of NHE1 in cortex and PAG but increased expression in trigeminal ganglia; no changes were seen in trigeminal nucleus caudalis. Thus, in vitro observations may have a significance in vivo to increase brain sumatriptan levels. Pharmacological inhibition of NHE1 prior to cortical manipulations enhanced the efficacy of sumatriptan at early time-points but induced facial sensitivity alone. Overall, our results suggest that dysregulation of NHE1 contributes to breaches in BBB integrity, drug penetrance, and the behavioral sensitivity to the antimigraine agent, sumatriptan.
| S-EPMC7259629 | biostudies-literature
Project description:The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer's disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology.
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Project description:ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood-brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that D-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter.
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Project description:ATP-binding cassette (ABC) drug efflux transporters in the CNS are predominantly localized to the luminal surface of endothelial cells in capillaries to impede CNS accumulation of xenobiotics. Inflammatory mediators and cellular stressors regulate their activity. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of upper and lower motor neurons characterized by extensive neuroinflammation. Here we tested the hypothesis that disease-driven changes in ABC transporter expression and function occur in ALS. Given the multitude of ABC transporters with their widespread substrate recognition, we began by examining expression levels of several ABC transporters. We found a selective increase in only two transporters: P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) both at mRNA and protein levels, in the SOD1-G93A mouse model of ALS, specifically in disease-affected CNS regions. Detailed analysis revealed a similar disease-driven increase in P-gp and BCRP levels in spinal cord microvessels, indicating that their altered expression occurs at the blood spinal cord barrier. Transport activity of P-gp and BCRP increased with disease progression in spinal cord and cerebral cortex capillaries. Finally, P-gp and BCRP protein expression also increased in spinal cords of ALS patients. Preclinical drug trials in the mouse model of ALS have failed to decisively slow or arrest disease progression; pharmacoresistance imparted by ABC transporters is one possible explanation for these failures. Our observations have large implications for ALS therapeutics in humans and suggest that the obstacle provided by these transporters to drug treatments must be overcome to develop effective ALS pharmacotherapies.
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Project description:Glioblastoma (GBM) is one of the most common, deadly, and difficult-to-treat adult brain tumors. Surgical removal of the tumor, followed by radiotherapy (RT) and temozolomide (TMZ) administration, is the current treatment modality, but this regimen only modestly improves overall patient survival. Invasion of cells into the surrounding healthy brain tissue prevents complete surgical resection and complicates treatment strategies with the goal of preserving neurological function. Despite significant efforts to increase our understanding of GBM, there have been relatively few therapeutic advances since 2005 and even fewer treatments designed to effectively treat recurrent tumors that are resistant to therapy. Thus, while there is a pressing need to move new treatments into the clinic, emerging evidence suggests that key features unique to GBM location and biology, the blood-brain barrier (BBB) and intratumoral molecular heterogeneity, respectively, stand as critical unresolved hurdles to effective therapy. Notably, genomic analyses of GBM tissues has led to the identification of numerous gene alterations that govern cell growth, invasion and survival signaling pathways; however, the drugs that show pre-clinical potential against signaling pathways mediated by these gene alterations cannot achieve effective concentrations at the tumor site. As a result, identifying BBB-penetrating drugs and utilizing new and safer methods to enhance drug delivery past the BBB has become an area of intensive research. Repurposing and combining FDA-approved drugs with evidence of penetration into the central nervous system (CNS) has also seen new interest for the treatment of both primary and recurrent GBM. In this review, we discuss emerging methods to strategically enhance drug delivery to GBM and repurpose currently-approved and previously-studied drugs using rational combination strategies.
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Project description:The blood-brain barrier (BBB) is necessary for maintaining brain homeostasis, but it also represents a major challenge for drug delivery to the brain tumors. A suitable in vivo Glioblastoma Multiforme (GBM) model is needed for efficient testing of BBB crossable pharmaceuticals. In this study, we firstly confirmed the BBB functionality in 3dpf zebrafish embryos by Lucifer Yellow, Evans Blue and DAPI microinjection. We then transplanted human GBM tumor cells into the zebrafish brain, in which implanted GBM cells (U87 and U251) were highly mitotic and invasive, mimicking their malignancy features in rodents' brain. Interestingly, we found that, although extensive endothelial proliferation and vessel dilation were observed in GBM xenografts, the BBB was still not disturbed. Next, using the zebrafish orthotopic GBM xenograft model as an in vivo visual readout, we successfully identified a promising small compound named TNB, which could efficiently cross the zebrafish BBB and inhibit the progression of orthotopic GBM xenografts. These results indicate that TNB is a promising BBB crossable GBM drug worth to be further characterized in human BBB setting, also suggest the zebrafish orthotopic GBM model as an efficient visual readout for the BBB penetrating anti-GBM drugs.
| S-EPMC5662771 | biostudies-literature
Project description:The subarachnoid space, where cerebrospinal fluid (CSF) flows over the brain and spinal cord, is lined on one side by arachnoid barrier (AB) cells that form part of the blood-CSF barrier. However, despite the fact that drugs are administered into the CSF and CSF drug concentrations are used as a surrogate for brain drug concentration following systemic drug administration, the tight-junctioned AB cells have never been examined for whether they express drug transporters that would influence CSF and central nervous system drug disposition. Hence, we characterized drug transporter expression and function in AB cells. Immunohistochemical analysis showed P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in mouse AB cells but not other meningeal tissue. The Gene Expression Nervous System Atlas (GENSAT) database and the Allen Mouse Brain Atlas confirmed these observations. Microarray analysis of mouse and human arachnoidal tissue revealed expression of many drug transporters and some drug-metabolizing enzymes. Immortalized mouse AB cells express functional P-gp on the apical (dura-facing) membrane and BCRP on both apical and basal (CSF-facing) membranes. Thus, like blood-brain barrier cells and choroid plexus cells, AB cells highly express drug transport proteins and likely contribute to the blood-CSF drug permeation barrier.
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