Cells Of Blood-brain Barrier Research Articles

Peptide-Drug Conjugates across the Blood-Brain Barrier: Using Viral Protein Domains to Shuttle Small Drugs to the Central Nervous System

Targeting the brain in chemotherapy is one of the hardest challenges of pharmacology. Peptides whose sequence is derived from specific domains of Dengue virus proteins are a valid tool to shuttle proteins to the brain. These peptides translocate the blood-brain barrier (BBB) by adsorption-mediated transcytosis, a mechanism that is reversible and not limited by saturation, in contrast to receptor- or transporter-mediated transcytosis.The use of peptides inspired in viral protein sequences to shuttle cargo across the BBB was extended to the transport of small molecule drugs. Peptide were conjugated to polycyclic antiviral drugs and tested for their efficacy to translocate a cellular in vitro model of the BBB. In addition to efficacy, the cytotoxicity of these conjugates was also tested. We have found conjugates that retain the antiviral efficacy of the drug, preserve the BBB-translocating capacity of the peptide, and are safe to BBB and neural cells.

Steroid Hormone Entry into the Brain Requires a Membrane Transporter in Drosophila.

Steroid hormones control various aspects of brain development and behavior in metazoans, but how they enter the central nervous system (CNS) through the blood-brain barrier (BBB) remains poorly understood. It is generally believed that steroid hormones freely diffuse through the plasma membrane of the BBB cells to reach the brain [1], because of the predominant "simple diffusion" model of steroid hormone transport across cell membranes. Recently, however, we challenged the simple diffusion model by showing that a Drosophila organic anion-transporting polypeptide (OATP), which we named Ecdysone Importer (EcI), is required for cellular uptake of the primary insect steroid hormone ecdysone [2]. As ecdysone is first secreted into the hemolymph before reaching the CNS [3], our finding raised the question of how ecdysone enters the CNS through the BBB to exert its diverse role in Drosophila braindevelopment. Here, we demonstrate in the Drosophila BBB that EcI is indispensable for ecdysone entry into the CNS to facilitate brain development. EcI is highly expressed in surface glial cells that form the BBB, and EcI knockdown in the BBB suppresses ecdysone signaling within the CNS and blocks ecdysone-mediated neuronal events during development. In an exvivo culture system, the CNS requires EcI in the BBB to incorporate ecdysone from the culture medium. Our results suggest a transporter-mediated mechanism of steroid hormone entry into the CNS, which may provide important implications in controlling brain development and behavior by regulating steroid hormone permeability across the BBB.

Target delivering paclitaxel by ferritin heavy chain nanocages for glioma treatment

Glioma is one of the most common aggressive brain malignancies, but the treatment of glioma is still far from satisfying. The efficiency of chemotherapy – the major choice of glioma treatment – is severely limited by low chemotherapeutic agents delivery across blood-brain barrier (BBB) and low tumor retention. Therefore, a safe and effective drug delivery system to help chemotherapy agents traverse the BBB and accumulate at tumor sites is urgently needed. Paclitaxel (PTX) is one of the most common and effective chemotherapy agent, however, it suffers from extremely low solubility and BBB penetration ability. Herein, we developed endogenous human ferritin heavy chain nanocage (HFn) as PTX carrier, which could specifically bind to widely overexpressed transferrin receptor 1 (TfR1) on BBB and glioma cells. We propose that this binding may help PTX cross the BBB and enhance its tumor accumulation. PTX-loaded HFn (HFn-PTX) was prepared and exhibited satisfactory targeting effect in Bend.3 and C6 cells in vitro. In vivo tissue biodistribution assay also demonstrated that HFn-PTX could indeed promote the accumulation of PTX in the brain. HFn-PTX exhibited the best anti-tumor effect with median survival time of 30 days, significantly longer than that of free PTX (14 days) and physiological saline group (13 days). In summary, we have designed and fabricated an effective delivery system for PTX to treat glioma. We also provided evidences that Transferrin (Tf) could not prevent the binding of HFn to TfR1 nor consequent TfR1-mediated HFn uptake, providing a clue for future research.

An iRGD-conjugated prodrug micelle with blood-brain-barrier penetrability for anti-glioma therapy

Various obstacles impede the chemotherapy efficiency of glioma in clinic, such as blood brain barrier (BBB) and blood brain tumor barrier (BBTB). Ligand-mediated polymeric micelles have shown great potential for improving the efficiency of glioma treatment. Herein, we developed a disulfide bond-conjugated prodrug polymer consisted of camptothecin (CPT) and polyethylene glycol (PEG) with further modification of iRGD peptide. The polymer of CPT-S-S-PEG-COOH could self-assemble into nanosized polymeric micelles with diameter around 100 nm, and loaded with photosensitizer IR780 for combination therapy. The micelles displayed good stability with controlled drug release under physiological environment. Importantly, the iRGD modified polymeric micelles demonstrated favorable ability to cross the BBB and target glioma cells via αv β integrin and neuropilin-1-mediated ligand transportation in vitro and in vivo. The whole synthesis process is simple and the drug loading content of CPT in the CPT-S-S-PEG-iRGD@IR780 micelles was higher than 10%. Moreover, CPT-S-S-PEG-iRGD@IR780 micelles combined chemotherapy with photodynamic therapy (PDT) displayed more excellent tumor-killing capability than the other groups. Thus, both in vitro and in vivo studies suggested that the targeting prodrug system could not only effectively cross various barriers to reach at glioma site, but also significantly enhance the antitumor effect with laser irradiation. Our findings consequently suggested that CPT-S-S-PEG-iRGD@IR780 micelles with laser irradiation are a promising drug delivery system for glioma therapy.

Functionalised LRP1 targeted carbon nanotubes across the blood-brain barrier in vitro and in vivo after intravenous injection

Abstract Despite extensive research in drug development, brain cancer is still lacking an efficacious cure due to the inability to deliver current therapeutics to the brain across the blood-brain barrier (BBB). Chemically functionalized carbon nanotubes (f-CNT) constitute a novel class of nanomaterials with attractive physical, chemical and electronic properties. The key advantage of f-CNTs is the extremely high surface area to size ratio allowing a high degree of chemical functionalization making them invaluable tools for designing drug delivery systems to the brain. One of the most interesting characteristics of f-CNTs is their ability to translocate across plasma membranes and enter the cells either passively by direct translocation across membranes or actively via endocytosis. Herein, we confirmed the ability of f-CNTs to cross the BBB and reach the brain in in vitro using a co-culture model of PBEC and primary rat astrocytes and in vivo after intravenous injection. Thanks to their unique optical properties, the uptake of f-CNT in brain was confirmed using state-of-the-art spectroscopic imaging techniques such as multi-photon luminescence imaging, fluorescence lifetime microscopy and Raman spectroscopy. Conjugation with angiopep-2 (ANG), a small peptide targeting the LRP1 receptor overexpressed in the BBB and glioma cells, further enhanced brain parenchyma accumulation in healthy brains. Higher uptake in glioma than brain parenchyma was also observed in glioma-bearing mice after intravenous administration. The inherent brain accumulation ability of f-CNTs coupled with improved brain-targeting by ANG favours the future clinical applications of f-CNTs-ANG to deliver active therapeutics for brain glioma therapy.

2287Endothelial SIRT6 exerts a beneficial role in cerebral ischemia/reperfusion injury by preserving blood-brain barrier integrity

Abstract Introduction Stroke is a major cause of mortality and morbidity worldwide. Yet, therapeutic strategies are limited to the early reperfusion which can, on the other hand, worsen the brain damage trough ischemia/reperfusion (I/R) injury. Post-stroke blood-brain barrier (BBB) impairment is associated with worsened outcome. Aging is a major risk factor for stroke and genes regulating lifespan also contribute to the determination of cerebral damage during I/R injury. Purpose Given the pivotal role of endothelial cells in BBB, we hypothesized that the endothelial-specific expression of the longevity gene SIRT6 may protect the BBB from ischemia/reperfusion damage thus having a beneficial role on stroke outcome. Methods Endothelial-specific SIRT6 knockout (eSIRT6−/−) mice and control littermates (CTRL) underwent transient middle cerebral artery occlusion (tMCAO) for 45 min followed by 48 hours of reperfusion. Immunohistochemistry (IHC) was used to investigate BBB permeability by IgG extravasation and molecular mechanisms. Primary human brain microvascular endothelial cells (HBMVECs) transfected with either SIRT6 (siSIRT6) or scrambled (siSCR) small interfering RNA were subjected to hypoxia/reoxygenation (H/R). An in vitro BBB model consisting of a monolayer of siRNA-treated HBMVECs was established and barrier function was assessed by 48 h-lasting transendothelial electrical resistance measurement. SIRT6 expression in monocytes from stroke patients was correlated with the short-term neurological outcome [ΔNIHSS% = (NIHSS discharge-NIHSS admission)/ NIHSS admission*100]. Results eSIRT6−/− displayed higher infarct volumes and lower survival rate compared to WT mice 48 h after tMCAO. The increased infarct volume was functionally relevant as eSIRT6−/− also showed worse post-stroke neurological impairment. Analysis of brain sections revealed increased BBB damage and increased endothelial expression of cleaved caspase-3 in eSIRT6−/− as compared to control littermates. In vitro, H/R reduced SIRT6 expression in HBMVECs. Mirroring the animal results, SIRT6 silencing impaired the barrier function of HBMVECs 48 h after exposure to H/R. In line with this, SIRT6-silenced HBMVECs showed reduced viability, increased cleaved caspase-3 expression and reduced activation of the anti-apoptotic survival pathway Akt as compared to control cells after H/R. The direct interaction between SIRT6 and Akt was confirmed by co-immunoprecipitation. In ischemic stroke patients, SIRT6 expression was higher in those with short-term neurological improvement (ΔNIHSS% >0) and negatively correlated with ΔNIHSS%. Conclusion Endothelial SIRT6 exerts a beneficial role in ischemic stroke by blunting I/R-mediated BBB damage. Specifically, SIRT6 reduces endothelial I/R-induced apoptotic death through activation of the protective Akt pathway. The longevity gene SIRT6 may represent a novel therapeutic target for the treatment of ischemic stroke. Acknowledgement/Funding Swiss National Science Foundation

Dual-Targeting Temozolomide Loaded in Folate-Conjugated Magnetic Triblock Copolymer Nanoparticles to Improve the Therapeutic Efficiency of Rat Brain Gliomas

In order to conduct an effective chemotherapy session as a treatment modality for glioblastoma tumors, a nanocarrier platform is required for the drug to cross the blood brain barrier (BBB) successfully and properly target glioma cells. Dual-targeting Temozolomide (TMZ) loaded triblock polymer coated magnetic nanoparticles (MNPs) were synthesized with a SPION core and by conjugating the surface with folic acid (FA), which were shown to effectively pass the BBB and target tumor cells. Two principal methods, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were employed for characterization of the synthesized nanoparticles. TMZ-loaded MNP-FA nanoparticles presented with a size of 58.61 nm, a zeta potential of -29.85 ± 0.47 mV, and a drug loading content of 6.85 ± 0.46%. Data gathered from inductively coupled plasma optical emission spectrometry (ICP-OES) and Prussian blue staining indicated effective dual-targeting, which subsequently led to an appreciably enhanced penetration through the BBB and accumulation of MNPs-FA in rat glioma cells. The anticancer effect of the dual-targeting MNPs-FA was also indicated by the increased survival time (>100%, p < 0.001) and decreased tumor volume (p < 0.001). In conclusion, the dual-targeting TMZ-loaded MNPs-FA are able to improve therapeutic efficiency toward brain gliomas in rats.

Unraveling In Vivo Brain Transport of Protein-Coated Fluorescent Nanodiamonds.

Nanotheranostics, combining diagnostics and therapy, has the potential to revolutionize treatment of neurological disorders. But one of the major obstacles for treating central nervous system diseases is the blood-brain barrier (BBB) preventing systemic delivery of drugs and optical probes into the brain. To overcome these limitations, nanodiamonds (NDs) are investigated in this study as they are a powerful sensing and imaging platform for various biological applications and possess outstanding stable far-red fluorescence, do not photobleach, and are highly biocompatible. Herein, fluorescent NDs encapsulated by a customized human serum albumin-based biopolymer (polyethylene glycol) coating (dcHSA-PEG) are taken up by target brain cells. In vitro BBB models reveal transcytosis and an additional direct cell-cell transport via tunneling nanotubes. Systemic application of dcHSA-NDs confirms their ability to cross the BBB in a mouse model. Tracking of dcHSA-NDs is possible at the single cell level and reveals their uptake into neurons and astrocytes in vivo. This study shows for the first time systemic NDs brain delivery and suggests transport mechanisms across the BBB and direct cell-cell transport. Fluorescent NDs are envisioned as traceable transporters for in vivo brain imaging, sensing, and drug delivery.

Preparation of Cholera Toxin Subunit B Functionalized Nanoparticles for Targeted Therapy of Glioblastoma.

Cholera toxin subunit B (CTB) is the nontoxic moiety of cholera toxin. It can target the glycosphingolipid GM1 expressed in the blood-brain barrier (BBB), neovasculature, and glioblastoma cells. Thus, CTB has been utilized as a multifunctional molecule for targeted therapy of glioblastoma. Here, we describe a detailed method for preparation of CTB functionalized paclitaxel (PTX)-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles. This unique modification can guide nanoparticles across the BBB and target glioblastoma cells. The characterization of nanoparticles such as size, zeta potential, morphology, drug loading, and encapsulation efficiency is shown in this chapter.

Amyloid Beta 25-35 induces blood-brain barrier disruption in vitro.

The amyloid β-peptide (Aβ) is transported across the blood-brain barrier (BBB) by binding with the receptor for advanced glycation end products (RAGE). Previously, we demonstrated that the Aβ fraction 25-35 (Aβ25-35) increases RAGE expression in the rat hippocampus, likely contributing to its neurotoxic effects. However, it is still debated if the interaction of Aβ with RAGE compromises the BBB function in Alzheimer' disease (AD). Here, we evaluated the effects of Aβ25-35 in an established in vitro model of the BBB. Rat brain microvascular endothelial cells (rBMVECs) were treated with 20μM active Aβ25-35 or the inactive Aβ35-25 (control), for 24h. Exposure to Aβ25-35 significantly decreased cell viability, increased cellular necrosis, and increased the production of reactive oxygen species (ROS), which triggered a decrease in the enzyme glutathione peroxidase when compared to the control condition. Aβ25-35 also increased BBB permeability by altering the expression of tight junction proteins (decreasing zonula occludens-1 and increasing occludin). Aβ25-35 induced monolayer disruption and cellular disarrangement of the BBB, with RAGE being highly expressed in the zones of disarrangement. Together, these data suggest that Aβ25-35-induces toxicity by compromising the functionality and integrity of the BBB in vitro. Graphical abstract Aβ25-35 induces BBB dysfunction in vitro, wich is likely mediated by OS and ultimately leads to disruption of BBB integrity and cell death.

Dengue Virus Infection of Blood-Brain Barrier Cells: Consequences of Severe Disease.

More than 500 million people worldwide are infected each year by any of the four-dengue virus (DENV) serotypes. The clinical spectrum caused during these infections is wide and some patients may develop neurological alterations during or after the infection, which could be explained by the cryptic neurotropic and neurovirulent features of flaviviruses like DENV. Using in vivo and in vitro models, researchers have demonstrated that DENV can affect the cells from the blood–brain barrier (BBB) in several ways, which could result in brain tissue damage, neuronal loss, glial activation, tissue inflammation and hemorrhages. The latter suggests that BBB may be compromised during infection; however, it is not clear whether the damage is due to the infection per se or to the local and/or systemic inflammatory response established or activated by the BBB cells. Similarly, the kinetics and cascade of events that trigger tissue damage, and the cells that initiate it, are unknown. This review presents evidence of the BBB cell infection with DENV and the response established toward it by these cells; it also describes the consequences of this response on the nervous tissue, compares these evidence with the one reported with neurotropic viruses of the Flaviviridae family, and shows the complexity and unpredictability of dengue and the neurological alterations induced by it. Clinical evidence and in vitro and in vivo models suggest that this virus uses the bloodstream to enter nerve tissue where it infects the different cells of the neurovascular unit. Each of the cell populations respond individually and collectively and control infection and inflammation, in other cases this response exacerbates the damage leaving irreversible sequelae or causing death. This information will allow us to understand more about the complex disease known as dengue, and its impact on a specialized and delicate tissue like is the nervous tissue.

Validation of Thiosemicarbazone Compounds as P-Glycoprotein Inhibitors in Human Primary Brain-Blood Barrier and Glioblastoma Stem Cells.

P-glycoprotein (Pgp) is highly expressed on blood-brain barrier (BBB) and glioblastoma (GB) cells, particularly on cancer stem cells (SC). Pgp recognizes a broad spectrum of substrates, limiting the therapeutic efficacy of several chemotherapeutic drugs in eradicating GB SC. Finding effective and safe inhibitors of Pgp that improve drug delivery across the BBB and target GB SC is open to investigation. We previously identified a series of thiosemicarbazone compounds that inhibit Pgp with an EC50 in the nanomolar range, and herein, we investigate the efficacy of three of them in bypassing Pgp-mediated drug efflux in primary human BBB and GB cells. At 10 nM, the compounds were not cytotoxic for the brain microvascular endothelial hCMEC/D3 cell line, but they markedly enhanced the permeability of the Pgp-substrate doxorubicin through the BBB. Thiosemicarbazone derivatives increased doxorubicin uptake in GB, with greater effects in the Pgp-rich SC clones than in the differentiated clones derived from the same tumor. All compounds increased intratumor doxorubicin accumulation and consequent toxicity in GB growing under competent BBB, producing significant killing of GB SC. The compounds crossed the BBB monolayer. The most stable derivative, 10a, had a half-life in serum of 4.2 h. The coadministration of doxorubicin plus 10a significantly reduced the growth of orthotopic GB-SC xenografts, without eliciting toxic side effects. Our work suggests that the thiosemicarbazone compounds are able to transform doxorubicin, a prototype BBB-impermeable drug, into a BBB-permeable drug. Bypassing Pgp-mediated drug efflux in both BBB and GB SC, thiosemicarbazones might increase the success of chemotherapy in targeting GB SC, which represent the most aggressive and difficult components to eradicate.

Impact of astrocyte and lymphocyte interactions on the blood-brain barrier in multiple sclerosis

ObjectiveThis study was designed to investigate the impact of astrocyte and lymphocyte (LC) interactions in the blood–brain barrier (BBB) on the pathogenesis of multiple sclerosis (MS). MethodsPrimary rat brain microvascular endothelial cells (rBMECs) and astrocytes isolated from Sprague–Dawley rats were used to establish in vitro BBB models. Transendothelial electrical resistance (TEER) and permeability were compared between rBMEC monocultures and rBMEC/astrocyte co-cultures to evaluate the validity of each as a BBB cell model. rBMEC/LC co-cultures and rBMEC/astrocyte/LC tri-cultures were established to evaluate inflammatory responses in MS by measuring the gene expression levels of nerve growth factor (NGF), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), interleukin 17 (IL-17), interferon γ (IFN-γ), and forkhead box P3 (Foxp3). ResultsThe rBMEC/astrocyte co-cultures exhibited higher TEER values and lower lymphocyte permeabilities than those of rBMEC monocultures. Compared to the rBMEC mono-cultures, the rBMEC/astrocyte/LC tri-cultures showed significantly decreased NGF, IL-17, and IFN-γ and increased MMP-2 and Foxp3 expression. Furthermore, the tri-cultures exhibited decreased NGF, IL-17, and IFN-γ expression compared to the rBMEC/astrocyte co-cultures, and increased MMP-2 expression compared to that shown by the rBMEC/LC co-cultures. MMP-9 expression did not vary significantly between the four established BBB cell models. ConclusionThese results suggest that the synergistic effect between astrocytes and LCs may increase the expression of MMP-2 and decrease that of IL-17 and IFN-γ at the BBB. Furthermore, the use of rBMEC/astrocytes/LC tri-cultures enabled us to test the synergistic effect between astrocytes and LCs and their roles in MS pathogenesis.

Targeting human brain cancer stem cells by curcumin-loaded nanoparticles grafted with anti-aldehyde dehydrogenase and sialic acid: Colocalization of ALDH and CD44

The use of chemotherapy against brain tumors faces various limitations to achieving its therapeutic effect, due to both the inability of anticancer agents to cross the blood–brain barrier (BBB) and the formation of brain cancer stem cells (BCSCs). Without adequate exposure, these chemotherapeutic drugs cannot have an antiproliferative effect on the tumors. Here, we developed curcumin (CCM)-loaded chitosan-poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) modified with sialic acid (SA) to permeate the BBB and with anti-aldehyde dehydrogenase (anti-ALDH) to target BCSCs. An increased chitosan concentration plays a pivotal role in maintaining a steady release of CCM from NPs. The viability of BBB cells and transendothelial electrical resistance were maintained after treatment with NPs for 4 h. Immunochemical staining of human brain microvascular endothelial cells confirmed that modification of SA on the surface of NPs greatly helped in permeation of the BBB through the use of N-acetylglucosamine. In addition, immunofluorescence images evidenced the assistance of anti-ALDH in inhibiting U87MG cells and BCSCs through targeting ALDH. ALDH was colocalized with CD44 in U87MG cells and BCSCs. The cell viability assay of U87MG cells and BCSCs supported the high level of inhibition after treatment with anti-ALDH-modified NPs. The drug delivery system in this study was designed in such a way to deliver CCM into the brain and subsequently inhibit the proliferation of glioblastoma cells and BCSCs.

IL-17A is associated with the breakdown of the blood-brain barrier in relapsing-remitting multiple sclerosis

IL-17 has been implicated in the pathogenesis of multiple sclerosis (MS). Here, we show that blockade of IL-17A, but not IL-17F, attenuated experimental autoimmune encephalomyelitis (EAE). We further show that IL-17A levels were elevated in the CSF of relapsing-remitting MS (RRMS) patients and that they correlated with the CSF/serum albumin quotient (Qalb), a measure of blood-brain barrier (BBB) dysfunction. We then demonstrated that the combination of IL-17A and IL-6 reduced the expression of tight junction (TJ)-associated genes and disrupted monolayer integrity in the BBB cell line hCMEC/D3. However, unlike IL-17A, IL-6 in the CSF from RRMS patients did not correlate with Qalb. These data highlight the potential importance of targeting IL-17A in preserving BBB integrity in RRMS.

Increased MDR1 Transporter Expression in Human Brain Endothelial Cells Through Enhanced Histone Acetylation and Activation of Aryl Hydrocarbon Receptor Signaling.

Multidrug resistance protein 1 (MDR1, ABCB1, P-glycoprotein) is a critical efflux transporter that extrudes chemicals from the blood-brain barrier (BBB) and limits neuronal exposure to xenobiotics. Prior studies in malignant cells demonstrated that MDR1 expression can be altered by inhibition of histone deacetylases (HDAC), enzymes that modify histone structure and influence transcription factor binding to DNA. Here, we sought to identify the mechanisms responsible for the up-regulation of MDR1 by HDAC inhibitors in human BBB cells. Immortalized human brain capillary endothelial (hCMEC/D3) cells were treated with HDAC inhibitors and assessed for MDR1 expression and function. Of the HDAC inhibitors profiled, valproic acid (VPA), apicidin, and suberoylanilide hydroxamic acid (SAHA) increased MDR1 mRNA and protein levels by 30-200%, which corresponded with reduced intracellular accumulation of the MDR1 substrate rhodamine 123. Interestingly, induction of MDR1 mRNA by HDAC inhibitors mirrored increases in the expression of the aryl hydrocarbon receptor (AHR) and its target gene cytochrome P450 1A1. To explore the role of AHR in HDAC inhibitor-mediated regulation of MDR1, a pharmacological activator (β-naphthoflavone, βNF) and inhibitor (CH-223191, CH) of AHR were tested. The induction of MDR1 in cells treated with SAHA was amplified by βNF and attenuated by CH. Furthermore, SAHA increased the binding of acetylated histone H3K9/K14 and AHR proteins to regions of the MDR1 promoter that contain AHR response elements. In conclusion, HDAC inhibitors up-regulate the expression and activity of the MDR1 transporter in human brain endothelial cells by increasing histone acetylation and facilitating AHR binding at the MDR1 promoter.

Circadian Control of Animal Physiology

We are interested in the mechanisms that generate circadian (~24 hour) cycles of physiology and behavior. Our studies use primarily a Drosophila model, which was invaluable for dissecting molecular mechanisms of the clock, but they also include mammalian models where appropriate. We are interested in the control of behavior by brain clocks and in the control of physiology by brain and peripheral clocks. We recently identified a clock in the Drosophila blood brain barrier (BBB) that confers a 24 hour rhythm on to permeability of the BBB. Specifically, the BBB clock regulates the activity of efflux transporters, such that these transporters pump xenobiotics out of the brain during the day, but are less active at night, thereby increasing BBB permeability at night. The underlying mechanism involves rhythmic expression of gap junctions that serve to maintain 24 hour cycles of magnesium, a positive regulator of efflux transporters, in BBB cells. As many drugs are targets of efflux transporters, we asked if permeability of such drugs is different at different times of day. Indeed, we found that phenytoin/Dilantin has time‐of‐day specific effects in a drosophila seizure model. In recent work, we have characterized rhythms of the BBB in mammalian cells and are addressing the relevant mechanisms.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Involvement of a Novel Organic Cation Transporter in Paeonol Transport Across the Blood-Brain Barrier.

Paeonol has neuroprotective function, which could be useful for improving central nervous system disorder. The purpose of this study was to characterize the functional mechanism involved in brain transport of paeonol through blood-brain barrier (BBB). Brain transport of paeonol was characterized by internal carotid artery perfusion (ICAP), carotid artery single injection technique (brain uptake index, BUI) and intravenous (IV) injection technique in vivo. The transport mechanism of paeonol was examined using conditionally immortalized rat brain capillary endothelial cell line (TR-BBB) as an in vitro model of BBB. Brain volume of distribution (VD) of [3H]paeonol in rat brain was about 6-fold higher than that of [14C]sucrose, the vascular space marker of BBB. The uptake of [3H]paeonol was concentration-dependent. Brain volume of distribution of paeonol and BUI as in vivo and inhibition of analog as in vitro studies presented significant reduction effect in the presence of unlabeled lipophilic compounds such as paeonol, imperatorin, diphenhydramine, pyrilamine, tramadol and ALC during the uptake of [3H]paeonol. In addition, the uptake significantly decreased and increased at the acidic and alkaline pH in both extracellular and intracellular study, respectively. In the presence of metabolic inhibitor, the uptake reduced significantly but not affected by sodium free or membrane potential disruption. Similarly, paeonol uptake was not affected on OCTN2 or rPMAT siRNA transfection BBB cells. Interestingly. Paeonol is actively transported from the blood to brain across the BBB by a carrier mediated transporter system.

SU31 - ESTABLISHING A BLOOD-BRAIN BARRIER MODEL FOR THE ANALYSIS OF ALZHEIMER'S DISEASE USING PATIENT DERIVED INDUCED PLURIPOTENT STEM CELLS

Background Alzheimer's Disease (AD) is the most common form of dementia. One characteristic of AD is the accumulation of amyloid-β (Aβ) peptides in different regions of the brain caused by the sequential cleavage of the transmembrane amyloid precursor protein. These extracellular amyloid plaques are a result of a dysregulated Blood-Brain Barrier (BBB) and emerge either due to an augmented production or a decreased excretion through brain barriers. The BBB with its several transporter systems shows age dependent expression levels of unspecific Aβ transporters. Recently, 19 loci including ABCA7, an ABC transporter responsible for Aβ efflux at the BBB, were discovered showing genome-wide significant association with the sporadic, late-onset form of AD (LOAD). Together with other research groups we identified several mutations in ABCA7 in AD patients, which is why an analysis of mutated ABCA7 in AD-related BBB models should be considered. We established patient-specific pluripotent stem cells from AD patients and healthy donors to differentiate endothelial cells and other related BBB cells for the investigation of disease mechanisms related to ABCA7 in AD patients. Methods Episomal vectors were used for the generation of induced Pluripotent Stem (iPS) cell lines from AD-specific B-lymphoblastoid cell lines (B-LCLs) and control B-LCLs of healthy donors. Alkaline phosphatase staining, immunofluorescence analysis, transcript analysis, western blot analysis, and the induction of three germ layers verified pluripotency. Successfully established lines were applied for the generation of BBB cells and neighboring cells. Endothelial cells and astrocytes were characterized by transcript analysis and flow cytometry. The differentiation process was analyzed for the altered expression of ABCA7. Results AD-specific iPS cells were successfully generated from B-LCLs. We could show alkaline phosphatase activity and the nuclear localization of pluripotency-related transcription factors including OCT4, SOX2, and NANOG. Transcript analysis and western blot analysis verified permanent induction of pluripotency marker genes including OCT4. The spontaneous differentiation of iPS cells into derivatives of the three germ layers was demonstrated. The efficient differentiation of high quality endothelial cells was shown by the expression of endothelial marker genes including von Willebrand factor. Astrocyte-specific markers including EAAT1 demonstrated efficient astrocyte differentiation. Discussion Human iPS cells provide a powerful tool for the analysis of AD-associated and patient-specific mutations in ABCA7. We successfully established endothelial cells and astrocytes representing suitable to study disease mechanisms of the BBB. Accordingly, this approach is an excellent opportunity to analyze pathogenic phenotypes of AD in vitro towards the identification of potential therapeutic targets.

Activated Microglia Disrupt the Blood-Brain Barrier and Induce Chemokines and Cytokines in a Rat in vitro Model.

Severe neuroinflammation is associated with blood brain barrier (BBB) disruption in CNS diseases. Although microglial activation and the subsequent changes in cytokine/chemokine (C/C) concentrations are thought to be key steps in the development of neuroinflammation, little data are available concerning the interaction of microglia with BBB cells. In this study, we investigated this interaction by adding LPS-activated microglia (LPS-MG) to the abluminal side of a BBB model composed of endothelial cells (EC), pericytes (Peri) and astrocytes (Ast). We then examined the abluminal concentrations of 27 C/Cs and the interactions between the LPS-MG and BBB cells. LPS-MG caused collapse of the BBB, revealed by decreases in the trans-endothelial electrical resistance (TEER) and by changes in the expression levels of tight junction (TJ) proteins. Under these conditions, 19 C/Cs were markedly increased on the abluminal side. Unexpectedly, although LPS-MG alone released 10 of the 19 C/Cs, their concentrations were much lower than those detected on the abluminal side of the BBB model supplemented with LPS-MG. Co-culture of LPS-MG with Ast caused marked increases in 12 of the 19 C/Cs, while co-culture of LPS-MG with EC and Peri resulted in a significant increase in only 1 of the 19 C/Cs (fractalkine). These results suggest that C/C dynamics in this system are not only caused by activated microglia but also are due to the interaction between activated microglia and astrocytes.