ABCB1 and ABCG2 Regulation at the Blood-Brain Barrier: Potential New Targets to Improve Brain Drug Delivery.

The drug efflux transporters ABCB1 and ABCG2 at the blood-brain barrier limit the delivery of drugs into the brain. Strategies to overcome ABCB1/ABCG2 have been largely unsuccessful, which poses a tremendous clinical problem to successfully treat central nervous system (CNS) diseases. Understanding basic transporter biology, including intracellular regulation mechanisms that control these transporters, is critical to solving this clinical problem.In this comprehensive review, we summarize current knowledge on signaling pathways that regulate ABCB1/ABCG2 at the blood-brain barrier. In Section I, we give a historical overview on blood-brain barrier research and introduce the role that ABCB1 and ABCG2 play in this context. In Section II, we summarize the most important strategies that have been tested to overcome the ABCB1/ABCG2 efflux system at the blood-brain barrier. In Section III, the main component of this review, we provide detailed information on the signaling pathways that have been identified to control ABCB1/ABCG2 at the blood-brain barrier and their potential clinical relevance. This is followed by Section IV, where we explain the clinical implications of ABCB1/ABCG2 regulation in the context of CNS disease. Lastly, in Section V, we conclude by highlighting examples of how transporter regulation could be targeted for therapeutic purposes in the clinic. SIGNIFICANCE STATEMENT: The ABCB1/ABCG2 drug efflux system at the blood-brain barrier poses a significant problem to successful drug delivery to the brain. The article reviews signaling pathways that regulate blood-brain barrier ABCB1/ABCG2 and could potentially be targeted for therapeutic purposes.

Characterization and comparison of human glioblastoma models.

Glioblastoma (GBM) is one of the deadliest cancers. Treatment options are limited, and median patient survival is only several months. Translation of new therapies is hindered by a lack of GBM models that fully recapitulate disease heterogeneity. Here, we characterize two human GBM models (U87-luc2, U251-RedFLuc). In vitro, both cell lines express similar levels of luciferase and show comparable sensitivity to temozolomide and lapatinib exposure. In vivo, however, the two GBM models recapitulate different aspects of the disease. U87-luc2 cells quickly grow into large, well-demarcated tumors; U251-RedFLuc cells form small, highly invasive tumors. Using a new method to assess GBM invasiveness based on detecting tumor-specific anti-luciferase staining in brain slices, we found that U251-RedFLuc cells are more invasive than U87-luc2 cells. Lastly, we determined expression levels of ABC transporters in both models. Our findings indicate that U87-luc2 and U251-RedFLuc GBM models recapitulate different aspects of GBM heterogeneity that need to be considered in preclinical research.

Participation of Monocarboxylate Transporter 8, But Not P-Glycoprotein, in Carrier-Mediated Cerebral Elimination of Phenytoin across the Blood-Brain Barrier.

PURPOSE: In this study, we investigated in detail the transport of phenytoin across the blood-brain barrier (BBB) to identify the transporter(s) involved in BBB-mediated phenytoin efflux from the brain. METHODS: We evaluated the brain-to-blood efflux transport of phenytoin in vivo by determining the brain efflux index (BEI) and uptake in brain slices. We additionally conducted brain perfusion experiments and BEI studies in P-glycoprotein (P-gp)-deficient mice. In addition, we determined the mRNA expression of monocarboxylate transporter (MCT) in isolated brain capillaries and performed phenytoin uptake studies in MCT-expressing Xenopus oocytes. RESULTS: [14C]Phenytoin brain efflux was time-dependent with a half-life of 17 min in rats and 31 min in mice. Intracerebral pre-administration of unlabeled phenytoin attenuated BBB-mediated phenytoin efflux transport, suggesting carrier-mediated phenytoin efflux transport across the BBB. Pre-administration of P-gp substrates in rats and genetic P-gp deficiency in mice did not affect BBB-mediated phenytoin efflux transport. In contrast, pre-administration of MCT8 inhibitors attenuated phenytoin efflux. Moreover, rat MCT8-expressing Xenopus oocytes exhibited [14C]phenytoin uptake, which was inhibited by unlabeled phenytoin. CONCLUSION: Our data suggest that MCT8 at the BBB participates in phenytoin efflux transport from the brain to the blood.

Inhibition of human microsomal PGE2 synthase-1 reduces seizure-induced increases of P-glycoprotein expression and activity at the blood-brain barrier.

The cause of antiseizure drug (ASD) resistance in epilepsy is poorly understood. Here, we focus on the transporter P-glycoprotein (P-gp) that is partly responsible for limited ASD brain uptake, which is thought to contribute to ASD resistance. We previously demonstrated that cyclooxygenase-2 (COX-2) and the prostaglandin E receptor, prostanoid E receptor subtype 1, are involved in seizure-mediated P-gp up-regulation. Thus, we hypothesized that inhibiting microsomal prostaglandin E2 (PGE2) synthase-1 (mPGES-1), the enzyme generating PGE2, prevents blood-brain barrier P-gp up-regulation after status epilepticus (SE). To test our hypothesis, we exposed isolated brain capillaries to glutamate ex vivo and used a combined in vivo-ex vivo approach by isolating brain capillaries from humanized mPGES-1 mice to study P-gp levels. We demonstrate that glutamate signaling through the NMDA receptor, cytosolic phospholipase A2, COX-2, and mPGES-1 increases P-gp protein expression and transport activity levels. We show that mPGES-1 is expressed in human, rat, and mouse brain capillaries. We show that BI1029539, an mPGES-1 inhibitor, prevented up-regulation of P-gp expression and transport activity in capillaries exposed to glutamate and in capillaries from humanized mPGES-1 mice after SE. Our data provide key signaling steps underlying seizure-induced P-gp up-regulation and suggest that mPGES-1 inhibitors could potentially prevent P-gp up-regulation in epilepsy.-Soldner, E. L. B., Hartz, A. M. S., Akanuma, S.-I., Pekcec, A., Doods, H., Kryscio, R. J., Hosoya, K.-I., Bauer, B. Inhibition of human microsomal PGE2 synthase-1 reduces seizure-induced increases of P-glycoprotein expression and activity at the blood-brain barrier.

Matrix Metalloproteinase-Mediated Blood-Brain Barrier Dysfunction in Epilepsy.

The blood-brain barrier is dysfunctional in epilepsy, thereby contributing to seizure genesis and resistance to antiseizure drugs. Previously, several groups reported that seizures increase brain glutamate levels, which leads to barrier dysfunction. One critical component of barrier dysfunction is brain capillary leakage. Based on our preliminary data, we hypothesized that glutamate released during seizures mediates an increase in matrix-metalloproteinase (MMP) expression and activity levels, thereby contributing to barrier leakage. To test this hypothesis, we exposed isolated brain capillaries from male Sprague Dawley rats to glutamate ex vivo and used an in vivo/ex vivo approach of isolated brain capillaries from female Wistar rats that experienced status epilepticus as an acute seizure model. We found that exposing isolated rat brain capillaries to glutamate increased MMP-2 and MMP-9 protein and activity levels, and decreased tight junction protein levels, which resulted in barrier leakage. We confirmed these findings in vivo in rats after status epilepticus and in brain capillaries from male mice lacking cytosolic phospholipase A2 Together, our data support the hypothesis that glutamate released during seizures signals an increase in MMP-2 and MMP-9 protein expression and activity levels, resulting in blood-brain barrier leakage.SIGNIFICANCE STATEMENT The mechanism leading to seizure-mediated blood-brain barrier dysfunction in epilepsy is poorly understood. In the present study, we focused on defining this mechanism in the brain capillary endothelium. We demonstrate that seizures trigger a pathway that involves glutamate signaling through cytosolic phospholipase A2, which increases MMP levels and decreases tight junction protein expression levels, resulting in barrier leakage. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit barrier dysfunction in epilepsy and decrease seizure burden.

The Activating C-type Lectin-like Receptor NKp65 Signals through a Hemi-immunoreceptor Tyrosine-based Activation Motif (hemITAM) and Spleen Tyrosine Kinase (Syk).

NKp65 is an activating human C-type lectin-like receptor (CTLR) triggering cellular cytotoxicity and cytokine secretion upon high-affinity interaction with the cognate CTLR keratinocyte-associated C-type lectin (KACL) selectively expressed by human keratinocytes. Previously, we demonstrated that NKp65-mediated cellular cytotoxicity depends on tyrosine 7, located in a cytoplasmic sequence motif of NKp65 resembling a hemi-immunoreceptor tyrosine-based activation motif (hemITAM). HemITAMs have been reported for a few activating myeloid-specific CTLRs, including Dectin-1 and CLEC-2, and consist of a single tyrosine signaling unit preceded by a triacidic motif. Upon receptor engagement, the hemITAM undergoes phosphotyrosinylation and specifically recruits spleen tyrosine kinase (Syk), initiating cellular activation. In this study, we addressed the functionality of the putative hemITAM of NKp65. We show that NKp65 forms homodimers and is phosphorylated at the hemITAM-embedded tyrosine 7 upon engagement by antibodies or KACL homodimers. HemITAM phosphotyrosinylation initiates a signaling pathway involving and depending on Syk, leading to cellular activation and natural killer (NK) cell degranulation. However, although NKp65 utilizes Syk for NK cell activation, a physical association of Syk with the NKp65 hemITAM could not be detected, unlike shown previously for the hemITAM of myeloid CTLR. Failure of NKp65 to recruit Syk is not due to an alteration of the triacidic motif, which rather affects the efficiency of hemITAM phosphotyrosinylation. In summary, NKp65 utilizes a hemITAM-like motif for cellular activation that requires Syk, although Syk appears not to be recruited to NKp65.

Role of l-Type Amino Acid Transporter 1 at the Inner Blood-Retinal Barrier in the Blood-to-Retina Transport of Gabapentin.

Gabapentin is an antiseizure drug that is known to also have beneficial effects on the retinal cells. To use gabapentin in retinal pharmacotherapy, it is critical to understand gabapentin distribution in the retina. The purpose of this study was to clarify the kinetics of gabapentin influx transport across the inner and outer blood-retinal barrier (BRB), which regulates the exchange of compounds/drugs between the circulating blood and the retina. In vivo blood-to-retina gabapentin transfer was evaluated by the rat carotid artery injection technique. In addition, gabapentin transport was examined using in vitro models of the inner (TR-iBRB2 cells) and outer BRB (RPE-J cells). The in vivo [3H]gabapentin transfer to the rat retina across the BRB was significantly reduced in the presence of unlabeled gabapentin, suggesting transporter-mediated blood-to-retina distribution of gabapentin. Substrates of the Na+-independent l-type amino acid transporter 1 (LAT1), such as 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), also significantly inhibited the in vivo [3H]gabapentin transfer. [3H]Gabapentin uptake in TR-iBRB2 and RPE-J cells exhibited Na+-independent and saturable kinetics with a Km of 735 and 507 µM, respectively. Regarding the effect of various transporter substrates/inhibitors on gabapentin transport in these cells, LAT1 substrates significantly inhibited [3H]gabapentin uptake in TR-iBRB2 and RPE-J cells. In addition, preloaded [3H]gabapentin release from TR-iBRB2 and RPE-J cells was trans-stimulated by LAT1 substrates through the obligatory exchange mechanism as LAT1. Immunoblot analysis indicates the protein expression of LAT1 in TR-iBRB2 and RPE-J cells. These results imply that LAT1 at the inner and outer BRB takes part in gabapentin transport between the circulating blood and retina. Moreover, treatment of LAT1-targeted small interfering RNA to TR-iBRB2 cells significantly reduced both the level of LAT1 protein expression and [3H]gabapentin uptake activities in TR-iBRB2 cells. In conclusion, data from the present study indicate that LAT1 at the inner BRB is involved in retinal gabapentin transfer, and also suggest that LAT1 mediates gabapentin transport in the RPE cells.

Aß40 Reduces P-Glycoprotein at the Blood-Brain Barrier through the Ubiquitin-Proteasome Pathway.

Failure to clear amyloid-ß (Aß) from the brain is in part responsible for Aß brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aß across the blood-brain barrier is the efflux transporter P-glycoprotein (P-gp) in the luminal plasma membrane of the brain capillary endothelium. P-gp is reduced at the blood-brain barrier in AD, which has been shown to be associated with Aß brain accumulation. However, the mechanism responsible for P-gp reduction in AD is not well understood. Here we focused on identifying critical mechanistic steps involved in reducing P-gp in AD. We exposed isolated rat brain capillaries to 100 nm Aß40, Aß40, aggregated Aß40, and Aß42. We observed that only Aß40 triggered reduction of P-gp protein expression and transport activity levels; this occurred in a dose- and time-dependent manner. To identify the steps involved in Aß-mediated P-gp reduction, we inhibited protein ubiquitination, protein trafficking, and the ubiquitin-proteasome system, and monitored P-gp protein expression, transport activity, and P-gp-ubiquitin levels. Thus, exposing brain capillaries to Aß40 triggers ubiquitination, internalization, and proteasomal degradation of P-gp. These findings may provide potential therapeutic targets within the blood-brain barrier to limit P-gp degradation in AD and improve Aß brain clearance. SIGNIFICANCE STATEMENT: The mechanism reducing blood-brain barrier P-glycoprotein (P-gp) in Alzheimer's disease is poorly understood. In the present study, we focused on defining this mechanism. We demonstrate that Aß40 drives P-gp ubiquitination, internalization, and proteasome-dependent degradation, reducing P-gp protein expression and transport activity in isolated brain capillaries. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit P-gp degradation in Alzheimer's disease and improve Aß brain clearance.

Engaging neuroscience to advance translational research in brain barrier biology.

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and--when barrier integrity is impaired--in the pathology of many common CNS disorders such as Alzheimer's disease, Parkinson's disease and stroke.

Key residues at the membrane-distal surface of KACL, but not glycosylation, determine the functional interaction of the keratinocyte-specific C-type lectin-like receptor KACL with its high-affinity receptor NKp65.

Keratinocyte-associated C-type lectin (KACL) is a peculiar C-type lectin-like receptor (CTLR) due to its selective expression by human keratinocytes and cognate interaction with the genetically coupled CTLR NKp65. KACL and NKp65 are members of the CLEC2 and NKRP1 subfamilies of natural killer gene complex (NKC)-encoded CTLR, respectively. Most NKRP1 molecules are expressed on NK cells and T cells and act as receptors of CLEC2 glycoproteins with their genes being intermingled in a certain sub-region of the mammalian NKC. The reasons for the tight genetic linkage of these dedicated receptor/ligand pairs are unknown, as is the physiological expression of NKp65. Recently, we reported that the CTLR NKp65 and KACL interact with high affinity, resulting in activation of NKp65-expressing NK-92MI cells. Here, we address the molecular basis of this high-affinity interaction by analysing KACL mutants with KACL-specific monoclonal antibodies (mAb), soluble NKp65 (sNKp65) and NK-92MI-NKp65 cells. We find that none of the three N-linked carbohydrates of KACL glycoproteins significantly contributes to KACL surface expression and NKp65 interaction. However, KACL mutants with non-conservative amino acid substitutions of arginine 158 or isoleucine 161 abrogated binding of both KACL-specific mAb OMA1 and sNKp65, well in line with the blockade of NKp65-KACL interaction by OMA1. Accordingly, functional recognition of these KACL mutants by NK-92M-NKp65 cells was completely abolished. Arginine 158 and isoleucine 161 located at the membrane-distal surface of KACL were defined as residues, decisively determining functional KACL-NKp65 interaction that is independent of KACL glycosylation.

Glioblastoma Standard of Care: Effects on Tumor Evolution and Reverse Translation in Preclinical Models.

Glioblastoma (GBM) presents a significant public health challenge as the deadliest and most common malignant brain tumor in adults. Despite standard-of-care treatment, which includes surgery, radiation, and chemotherapy, mortality rates are high, underscoring the critical need for advancing GBM therapy. Over the past two decades, numerous clinical trials have been performed, yet only a small fraction demonstrated a benefit, raising concerns about the predictability of current preclinical models. Traditionally, preclinical studies utilize treatment-naïve tumors, failing to model the clinical scenario where patients undergo standard-of-care treatment prior to recurrence. Recurrent GBM generally exhibits distinct molecular alterations influenced by treatment selection pressures. In this review, we discuss the impact of treatment-surgery, radiation, and chemotherapy-on GBM. We also provide a summary of treatments used in preclinical models, advocating for their integration to enhance the translation of novel strategies to improve therapeutic outcomes in GBM.

Optimization, Characterization, and Comparison of Two Luciferase-Expressing Mouse Glioblastoma Models.

Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.

Oxidative stress alters mitochondrial homeostasis in isolated brain capillaries.

BACKGROUND: Neurovascular deficits and blood-brain barrier (BBB) dysfunction are major hallmarks of brain trauma and neurodegenerative diseases. Oxidative stress is a prominent contributor to neurovascular unit (NVU) dysfunction and can propagate BBB disruption. Oxidative damage results in an imbalance of mitochondrial homeostasis, which can further drive functional impairment of brain capillaries. To this end, we developed a method to track mitochondrial-related changes after oxidative stress in the context of neurovascular pathophysiology as a critical endophenotype of neurodegenerative diseases. METHODS: To study brain capillary-specific mitochondrial function and dynamics in response to oxidative stress, we developed an ex vivo model in which we used isolated brain capillaries from transgenic mice that express dendra2 green specifically in mitochondria (mtD2g). Isolated brain capillaries were incubated with 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) or hydrogen peroxide (H2O2) to induce oxidative stress through lipid peroxidation. Following the oxidative insult, mitochondrial bioenergetics were measured using the Seahorse XFe96 flux analyzer, and mitochondrial dynamics were measured using confocal microscopy with Imaris software. RESULTS: We optimized brain capillary isolation with intact endothelial cell tight-junction and pericyte integrity. Further, we demonstrate consistency of the capillary isolation process and cellular enrichment of the isolated capillaries. Mitochondrial bioenergetics and morphology assessments were optimized in isolated brain capillaries. Finally, we found that oxidative stress significantly decreased mitochondrial respiration and altered mitochondrial morphology in brain capillaries, including mitochondrial volume and count. CONCLUSIONS: Following ex vivo isolation of brain capillaries, we confirmed the stability of mitochondrial parameters, demonstrating the feasibility of this newly developed platform. We also demonstrated that oxidative stress has profound effects on mitochondrial homeostasis in isolated brain capillaries. This novel method can be used to evaluate pharmacological interventions to target oxidative stress or mitochondrial dysfunction in cerebral small vessel disease and neurovascular pathophysiology as major players in neurodegenerative disease.

Proteasome inhibition protects blood-brain barrier P-glycoprotein and lowers Aß brain levels in an Alzheimer's disease model.

BACKGROUND: Loss of P-glycoprotein (P-gp) at the blood-brain barrier contributes to amyloid-ß (Aß) brain accumulation in Alzheimer's disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aß triggers P-gp loss by activating the ubiquitin-proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aß accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade®; BTZ), protects blood-brain barrier P-gp from degradation in hAPP mice in vivo. METHODS: We treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood-brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aß plasma and brain levels with ELISA. RESULTS: We found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood-brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aß40 and Aß42 brain levels compared to vehicle-treated hAPP mice. CONCLUSIONS: Our data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aß brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aß transport across the blood-brain barrier may prove an effective treatment for patients.

Amyloid-ß contributes to blood-brain barrier leakage in transgenic human amyloid precursor protein mice and in humans with cerebral amyloid angiopathy.

BACKGROUND AND PURPOSE: Cerebral amyloid angiopathy (CAA) is a degenerative disorder characterized by amyloid-ß (Aß) deposition in the blood-brain barrier (BBB). CAA contributes to injuries of the neurovasculature including lobar hemorrhages, cortical microbleeds, ischemia, and superficial hemosiderosis. We postulate that CAA pathology is partially due to Aß compromising the BBB. METHODS: We characterized 19 patients with acute stroke with "probable CAA" for neurovascular pathology based on MRI and clinical findings. Also, we studied the effect of Aß on the expression of tight junction proteins and matrix metalloproteases (MMPs) in isolated rat brain microvessels. RESULTS: Two of 19 patients with CAA had asymptomatic BBB leakage and posterior reversible encephalopathic syndrome indicating increased BBB permeability. In addition to white matter changes, diffusion abnormality suggesting lacunar ischemia was found in 4 of 19 patients with CAA; superficial hemosiderosis was observed in 7 of 9 patients. Aß(40) decreased expression of the tight junction proteins claudin-1 and claudin-5 and increased expression of MMP-2 and MMP-9. Analysis of brain microvessels from transgenic mice overexpressing human amyloid precursor protein revealed the same expression pattern for tight junction and MMP proteins. Consistent with reduced tight junction and increased MMP expression and activity, permeability was increased in brain microvessels from human amyloid precursor protein mice compared with microvessels from wild-type controls. CONCLUSIONS: Our findings indicate that Aß contributes to changes in brain microvessel tight junction and MMP expression, which compromises BBB integrity. We conclude that Aß causes BBB leakage and that assessing BBB permeability could potentially help characterize CAA progression and be a surrogate marker for treatment response.

Freshly isolated retinal capillaries to determine efflux transporter function at the inner BRB.

Since it has been known that in vitro cell lines for analyzing drug transport at the inner blood-retinal barrier (BRB) do not completely retain several in vivo functions, new ex vivo/in vitro methods to evaluate drug transport across the inner BRB help us understand the role of this barrier in maintaining the homeostasis of vision and regulating drug distribution to the retina. To expand the limitations of existing in vitro approaches, we established a protocol to isolate fresh rat retinal capillaries as ex vivo model of the inner BRB. Fresh retinal capillaries were prepared by applying serial filtration steps and using density gradient centrifugation. We performed mRNA and protein analyses by reverse transcription-polymerase chain reaction and immunostaining that indicated expression of marker proteins such as facilitative glucose transporter 1 and claudin-5 in freshly isolated rat retinal capillaries. We also used fluorescent transporter substrates to characterize functional activity of organic anion transporter (Oat) 3, P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein (Mrp) 4 in isolated retinal capillaries. Capillary luminal accumulation of fluorescent substrates of P-glycoprotein and Bcrp was decreased in the presence of transporter inhibitors. Moreover, luminal accumulation of the Oat3 and Mrp4 substrate, 8-(2-[fluoresceinyl]aminoethylthio) adenosine-3',5'-cyclic monophosphate (8-[fluo]-cAMP), was reduced by substrates/inhibitors of Oat3 and Mrp4. In conclusion, our study shows that freshly isolated retinal capillaries retain marker protein expression and transporter functional activity. It is suggested that isolated retinal capillaries are a useful tool to study transport across the inner BRB. Using freshly isolated retinal capillaries, we anticipate applying this approach to determine the role of transporters at the inner BRB during pathophysiological states of the eye and evaluate the drug delivery to the retina.

Synthesis and characterization of a BODIPY conjugate of the BCR-ABL kinase inhibitor Tasigna (nilotinib): evidence for transport of Tasigna and its fluorescent derivative by ABC drug transporters.

Tasigna (Nilotinib) is a BCR-ABL kinase inhibitor recently approved by the Food and Drug Administration, which is indicated for the treatment of drug-resistant chronic myelogenous leukemia (CML). The efflux of tyrosine kinase inhibitors by ATP-binding cassette (ABC) drug transporters, which actively pump these drugs out of cells utilizing ATP as an energy source, has been linked to the development of drug resistance in CML patients. We report here the synthesis and characterization of a fluorescent derivative of Tasigna to study its interaction with two major ABC transporters, P-glycoprotein (Pgp) and ABCG2, in in vitro and ex vivo assays. A fluorescent derivative of Tasigna, BODIPY FL Tasigna, inhibited the BCR-ABL kinase activity in K562 cells and was also effluxed by Pgp- and ABCG2-expressing cells in both cultured cells and rat brain capillaries expressing Pgp and ABCG2. In addition, [(3)H]-Tasigna was found to be transported by Pgp-expressing polarized LLC-PK1 cells in a transepithelial transport assay. Consistent with these results, both Tasigna and BODIPY FL Tasigna were less effective at inhibiting the phosphorylation of Crkl (a substrate of BCR-ABL kinase) in Pgp- and ABCG2-expressing K562 cells due to their reduced intracellular concentration. Taken together, these data provide evidence that BODIPY FL Tasigna is transported by Pgp and ABCG2, and Tasigna is transported by Pgp. Further, we propose that BODIPY FL Tasigna can potentially be used as a probe for functional analysis of Pgp and ABCG2 in cancer cells and in other preclinical studies.

Modulation of P-glycoprotein at the blood-brain barrier: opportunities to improve central nervous system pharmacotherapy.

Pharmacotherapy of central nervous system (CNS) disorders (e.g., neurodegenerative diseases, epilepsy, brain cancer, and neuro-AIDS) is limited by the blood-brain barrier. P-glycoprotein, an ATP-driven, drug efflux transporter, is a critical element of that barrier. High level of expression, luminal membrane location, multispecificity, and high transport potency make P-glycoprotein a selective gatekeeper of the blood-brain barrier and thus a primary obstacle to drug delivery into the brain. As such, P-glycoprotein limits entry into the CNS for a large number of prescribed drugs, contributes to the poor success rate of CNS drug candidates, and probably contributes to patient-to-patient variability in response to CNS pharmacotherapy. Modulating P-glycoprotein could therefore improve drug delivery into the brain. Here we review the current understanding of signaling mechanisms responsible for the modulation of P-glycoprotein activity/expression at the blood-brain barrier with an emphasis on recent studies from our laboratories. Using intact brain capillaries from rats and mice, we have identified multiple extracellular and intracellular signals that regulate this transporter; several signaling pathways have been mapped. Three pathways are triggered by elements of the brain's innate immune response, one by glutamate, one by xenobiotic-nuclear receptor (pregnane X receptor) interactions, and one by elevated beta-amyloid levels. Signaling is complex, with several pathways sharing common signaling elements [tumor necrosis factor (TNF) receptor 1, endothelin (ET) B receptor, protein kinase C, and nitric-oxide synthase), suggesting a regulatory network. Several pathways include autocrine/paracrine elements, involving release of the proinflammatory cytokine, TNF-alpha, and the polypeptide hormone, ET-1. Finally, several steps in signaling are potential therapeutic targets that could be used to modulate P-glycoprotein activity in the clinic.

Protecting P-glycoprotein at the blood-brain barrier from degradation in an Alzheimer's disease mouse model.

BACKGROUND: Failure to clear Aß from the brain is partly responsible for Aß brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aß across the blood-brain barrier is the efflux transporter P-glycoprotein (P-gp). In AD, P-gp levels are reduced, which contributes to impaired Aß brain clearance. However, the mechanism responsible for decreased P-gp levels is poorly understood and there are no strategies available to protect P-gp. We previously demonstrated in isolated brain capillaries ex vivo that human Aß40 (hAß40) triggers P-gp degradation by activating the ubiquitin-proteasome pathway. In this pathway, hAß40 initiates P-gp ubiquitination, leading to internalization and proteasomal degradation of P-gp, which then results in decreased P-gp protein expression and transport activity levels. Here, we extend this line of research and present results from an in vivo study using a transgenic mouse model of AD (human amyloid precursor protein (hAPP)-overexpressing mice; Tg2576). METHODS: In our study, hAPP mice were treated with vehicle, nocodazole (NCZ, microtubule inhibitor to block P-gp internalization), or a combination of NCZ and the P-gp inhibitor cyclosporin A (CSA). We determined P-gp protein expression and transport activity levels in isolated mouse brain capillaries and Aß levels in plasma and brain tissue. RESULTS: Treating hAPP mice with 5 mg/kg NCZ for 14 days increased P-gp levels to levels found in WT mice. Consistent with this, P-gp-mediated hAß42 transport in brain capillaries was increased in NCZ-treated hAPP mice compared to untreated hAPP mice. Importantly, NCZ treatment significantly lowered hAß40 and hAß42 brain levels in hAPP mice, whereas hAß40 and hAß42 levels in plasma remained unchanged. CONCLUSIONS: These findings provide in vivo evidence that microtubule inhibition maintains P-gp protein expression and transport activity levels, which in turn helps to lower hAß brain levels in hAPP mice. Thus, protecting P-gp at the blood-brain barrier may provide a novel therapeutic strategy for AD and other Aß-based pathologies.

Restoring blood-brain barrier P-glycoprotein reduces brain amyloid-beta in a mouse model of Alzheimer's disease.

Reduced clearance of amyloid-beta (Abeta) from brain partly underlies increased Abeta brain accumulation in Alzheimer's disease (AD). The mechanistic basis for this pathology is unknown, but recent evidence suggests a neurovascular component in AD etiology. We show here that the ATP-driven pump, P-glycoprotein, specifically mediates efflux transport of Abeta from mouse brain capillaries into the vascular space, thus identifying a critical component of the Abeta brain efflux mechanism. We demonstrate in a transgenic mouse model of AD [human amyloid precursor protein (hAPP)-overexpressing mice; Tg2576 strain] that brain capillary P-glycoprotein expression and transport activity are substantially reduced compared with wild-type control mice, suggesting a mechanism by which Abeta accumulates in the brain in AD. It is noteworthy that dosing 12-week-old, asymptomatic hAPP mice over 7 days with pregnenolone-16alpha-carbonitrile to activate the nuclear receptor pregnane X receptor restores P-glycoprotein expression and transport activity in brain capillaries and significantly reduces brain Abeta levels compared with untreated control mice. Thus, targeting intracellular signals that up-regulate blood-brain barrier P-glycoprotein in the early stages of AD has the potential to increase Abeta clearance from the brain and reduce Abeta brain accumulation. This mechanism suggests a new therapeutic strategy in AD.