Targeted transport of biotherapeutics at the blood-brain barrier.

INTRODUCTION: The treatment of neurological diseases is significantly hampered by the lack of available therapeutics. A major restraint for the development of drugs is denoted by the presence of the blood-brain barrier (BBB), which precludes the transfer of biotherapeutics to the brain due to size restraints. AREAS COVERED: Novel optimism for transfer of biotherapeutics to the brain has been generated via development of targeted therapeutics to nutrient transporters expressed by brain capillary endothelial cells (BCECs). Targeting approaches with antibodies acting as biological drug carriers allow for proteins and genetic material to enter the brain, and qualified therapy using targeted proteins for protein replacement has been observed in preclinical models and now emerging in the clinic. Viral vectors denote an alternative for protein delivery to the brain by uptake and transduction of BCECs, or by transport through the BBB leading to neuronal transduction. EXPERT OPINION: The breaching of the BBB to large molecules has opened for treatment of diseases in the brain. A sturdier understanding of how biotherapeutics undergo transport through the BBB and how successful transport into the brain can be monitored is required to further improve the translation from successful preclinical studies to the clinic.

The Npc2Gt(LST105)BygNya mouse signifies pathological changes comparable to human Niemann-Pick type C2 disease.

INTRODUCTION: Niemann-Pick type C2 disease (NP-C2) is a fatal neurovisceral disorder caused by defects in the lysosomal cholesterol transporter protein NPC2. Consequently, cholesterol and other lipids accumulate within the lysosomes, causing a heterogeneous spectrum of clinical manifestations. Murine models are essential for increasing the understanding of the complex pathology of NP-C2. This study, therefore, aims to describe the neurovisceral pathology in the NPC2-deficient mouse model to evaluate its correlation to human NP-C2. METHODS: Npc2-/- mice holding the LST105 mutation were used in the present study (Npc2Gt(LST105)BygNya). Body and organ weight and histopathological evaluations were carried out in six and 12-week-old Npc2-/- mice, with a special emphasis on neuropathology. The Purkinje cell (PC) marker calbindin, the astrocytic marker GFAP, and the microglia marker IBA1 were included to assess PC degeneration and neuroinflammation, respectively. In addition, the pathology of the liver, lungs, and spleen was assessed using hematoxylin and eosin staining. RESULTS: Six weeks old pre-symptomatic Npc2-/- mice showed splenomegaly and obvious neuropathological changes, especially in the cerebellum, where initial PC loss and neuroinflammation were evident. The Npc2-/- mice developed neurological symptoms at eight weeks of age, severely progressing until the end-stage of the disease at 12 weeks. At the end-stage of the disease, Npc2-/- mice were characterized by growth retardation, tremor, cerebellar ataxia, splenomegaly, foam cell accumulation in the lungs, liver, and spleen, brain atrophy, pronounced PC degeneration, and severe neuroinflammation. CONCLUSION: The Npc2Gt(LST105)BygNya mouse model resembles the pathology seen in NP-C2 patients and denotes a valuable model for increasing the understanding of the complex disease manifestation and is relevant for testing the efficacies of new treatment strategies.

A novel strategy for delivering Niemann-Pick type C2 proteins across the blood-brain barrier using the brain endothelial-specific AAV-BR1 virus.

Treating central nervous system (CNS) diseases is complicated by the incapability of numerous therapeutics to cross the blood-brain barrier (BBB), mainly composed of brain endothelial cells (BECs). Genetically modifying BECs into protein factories that supply the CNS with recombinant proteins is a promising approach to overcome this hindrance, especially in genetic diseases, like Niemann Pick disease type C2 (NPC2), where both CNS and peripheral cells are affected. Here, we investigated the potential of the BEC-specific adeno-associated viral vector (AAV-BR1) encoding NPC2 for expression and secretion from primary BECs cultured in an in vitro BBB model with mixed glial cells, and in healthy BALB/c mice. Transduced primary BECs had significantly increased NPC2 gene expression and secreted NPC2 after viral transduction, which significantly reversed cholesterol deposition in NPC2 deficient fibroblasts. Mice receiving an intravenous injection with AAV-BR1-NCP2-eGFP were sacrificed 8 weeks later and examined for its biodistribution and transgene expression of eGFP and NPC2. AAV-BR1-NPC2-eGFP was distributed mainly to the brain and lightly to the heart and lung, but did not label other organs including the liver. eGFP expression was primarily found in BECs throughout the brain but occasionally also in neurons suggesting transport of the vector across the BBB, a phenomenon also confirmed in vitro. NPC2 gene expression was up-regulated in the brain, and recombinant NPC2 protein expression was observed in both transduced brain capillaries and neurons. Our findings show that AAV-BR1 transduction of BECs is possible and that it may denote a promising strategy for future treatment of NPC2.

Sortilin regulates blood-brain barrier integrity.

Brain homeostasis depends on the existence of the blood-brain barrier (BBB). Despite decades of research, the factors and signalling pathways for modulating and maintaining BBB integrity are not fully elucidated. Here, we characterise the expression and function of the multifunctional receptor, sortilin, in the cells of the BBB, in vivo and in vitro. We show that sortilin acts as an important regulatory protein of the BBB's tightness. In rats lacking sortilin, the BBB was leaky, which correlated well with relocated distribution of the localisation of zonula occludens-1, VE-cadherin and ß-catenin junctional proteins. Furthermore, the absence of sortilin in brain endothelial cells resulted in decreased phosphorylation of Akt signalling protein and increased the level of phospho-ERK1/2. As a putative result of MAPK/ERK pathway activity, the junctions between the brain endothelial cells were disintegrated and the integrity of the BBB became compromised. The identified barrier differences between wild-type and Sort1-/- brain endothelial cells can pave the way for a better understanding of sortilin's role in the healthy and diseased BBB.

The blood-brain barrier studied in vitro across species.

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BECs) supported by pericytes and astrocytes. The BBB maintains homeostasis and protects the brain against toxic substances circulating in the blood, meaning that only a few drugs can pass the BBB. Thus, for drug screening, understanding cell interactions, and pathology, in vitro BBB models have been developed using BECs from various animal sources. When comparing models of different species, differences exist especially in regards to the transendothelial electrical resistance (TEER). Thus, we compared primary mice, rat, and porcine BECs (mBECs, rBECs, and pBECs) cultured in mono- and co-culture with astrocytes, to identify species-dependent differences that could explain the variations in TEER and aid to the selection of models for future BBB studies. The BBB models based on primary mBECs, rBECs, and pBECs were evaluated and compared in regards to major BBB characteristics. The barrier integrity was evaluated by the expression of tight junction proteins and measurements of TEER and apparent permeability (Papp). Additionally, the cell size, the functionality of the P-glycoprotein (P-gp) efflux transporter, and the expression of the transferrin receptor were evaluated and compared. Expression and organization of tight junction proteins were in all three species influenced by co-culturing, supporting the findings, that TEER increases after co-culturing with astrocytes. All models had functional polarised P-gp efflux transporters and expressed the transferrin receptor. The most interesting discovery was that even though the pBECs had higher TEER than rBECs and mBECs, the Papp did not show the same variation between species, which could be explained by a significantly larger cell size of pBECs. In conclusion, our results imply that the choice of species for a given BBB study should be defined from its purpose, instead of aiming to reach the highest TEER, as the models studied here revealed similar BBB properties.

Gene therapy to the blood-brain barrier with resulting protein secretion as a strategy for treatment of Niemann Picks type C2 disease.

Treatment of many diseases affecting the central nervous system (CNS) is complicated by the inability of several therapeutics to cross the blood-brain barrier (BBB). Genetically modifying brain capillary endothelial cells (BCECs) denotes an approach to overcome the limitations of the BBB by turning BCECs into recombinant protein factories. This will result in protein secretion toward both the brain and peripheral circulation, which is particularly relevant in genetic diseases, like lysosomal storage diseases (LSD), where cells are ubiquitously affected both in the CNS and the periphery. Here we investigated transfection of primary rat brain capillary endothelial cells (rBCECs) for synthesis and secretion of recombinant NPC2, the protein deficient in the lysosomal cholesterol storage disease Niemann Pick type C2. We demonstrate prominent NPC2 gene induction and protein secretion in 21% of BCECs in non-mitotic monocultures with a biological effect on NPC2-deficient fibroblasts as verified from changes in filipin III staining of cholesterol deposits. By comparison the transfection efficiency was 75% in HeLa-cells, known to persist in a mitotic state. When co-cultured with primary rat astrocytes in conditions with maintained BBB properties 7% BCECs were transfected, clearly suggesting that induction of BBB properties with polarized conditions of the non-mitotic BCECs influences the transfection efficacy and secretion directionality. In conclusion, non-viral gene therapy to rBCECs leads to protein secretion and signifies a method for NPC2 to target cells inside the CNS otherwise inaccessible because of the presence of the BBB. However, obtaining high transfection efficiencies is crucial in order to achieve sufficient therapeutic effects. Cover Image for this issue: https://doi.org/10.1111/jnc.15050.

Epigenetic Regulation of Ferroportin in Primary Cultures of the Rat Blood-Brain Barrier.

Ferroportin plays an essential role for iron transport through the blood-brain barrier (BBB), which is formed by brain capillary endothelial cells (BCECs). To maintain the integrity of the BBB, the BCECs gain support from pericytes and astrocytes, which together with neurons form the neurovascular unit (NVU). The objectives of the present study were to investigate ferroportin expression in primary cells of the NVU and to determine if ferroportin mRNA (Fpn) expression is epigenetically regulated. Primary rat BCECs, pericytes, astrocytes, and neurons all expressed ferroportin mRNA at varying levels, with BCECs exhibiting the highest expression of Fpn, peaking when co-cultured but examined separately from astrocytes. Conversely, Fpn expression was lowest in isolated astrocytes, which correlated with high DNA methylation in their Slc40a1 promoter. To provide further evidence for epigenetic regulation, mono-cultured BCECs, pericytes, and astrocytes were treated with the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (SB), which significantly increased Fpn and ferroportin protein in BCECs and pericytes. Furthermore, 59Fe export from BCECs was elevated after treatment with VPA. In conclusion, we present first time evidence stating that Fpn expression is epigenetically regulated in BCECs, which may have implications for pharmacological induction of iron transport through the BBB.

Targeting the transferrin receptor for brain drug delivery.

Obtaining efficient drug delivery to the brain remains the biggest challenge for the development of therapeutics to treat diseases of the central nervous system. The main obstacle is the blood-brain barrier (BBB), which impedes the entrance of most molecules present in the systemic circulation, especially large molecule drugs and nanomedicines. To overcome this obstacle, targeting strategies binding to nutrient receptors present at the luminal membrane of the BBB are frequently employed. Amongst the numerous potential targets at the BBB, the transferrin receptor (TfR) remains the most common target used to ensure sufficient drug delivery to the brain. In this review, we provide a full account on the use of the TfR as a target for brain drug delivery by describing the function of the TfR in the BBB, the historical background of its use in drug delivery, and the most recent evidence suggesting TfR-targeted medicines to be efficient for brain drug delivery with a clear clinical potential.

Modulating the antibody density changes the uptake and transport at the blood-brain barrier of both transferrin receptor-targeted gold nanoparticles and liposomal cargo.

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 ∗ 103 antibodies/µm2), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration.

Hepcidin Mediates Transcriptional Changes in Ferroportin mRNA in Differentiated Neuronal-Like PC12 Cells Subjected to Iron Challenge.

Ferroportin is the only known iron exporter, and its regulation seems to be controlled at both transcriptional, post-transcriptional, and post-translational levels. The objective of the current work was to investigate how cellular iron status affects the expression of the ferroportin gene Fpn under the influence of hepcidin, known to post-translational lower the available ferroportin protein. Nerve growth factor-beta (NGF-ß)-differentiated PC12 cells, used as a model of neuronal cells, were evaluated in terms of their viability and expression of ferroportin after inducing cellular iron overload with ferric ammonium citrate (FAC) or hepcidin, iron deficiency with deferoxamine (DFO), or hepcidin in combination with FAC or DFO. Ferritin mRNA was significantly upregulated following treatment with 20 mM FAC. The viability of the differentiated PC12 cells was significantly reduced after treatment with 30 mM FAC or 1.0 µM hepcidin, but when combining FAC and hepcidin treatment, the cells remained unaffected. The expression of Fpn was concurrently upregulated after treatment with FAC in combination with hepcidin. Fifty millimolar DFO also increased Fpn. Together, these data point towards a transcriptional induction of Fpn in response to changes in cellular iron levels. Epigenetic regulation of Fpn may also occur as changes in genes associated with epigenetic regulation of Fpn were demonstrated.

Correction to: Hepcidin Mediates Transcriptional Changes in Ferroportin mRNA in Differentiated Neuronal-like PC12 Cells Subjected to Iron Challenge.

The original version of this article unfortunately contained mistakes on Figs. 1, 2, and 7 as some of the data were not visible. With this, the correct images are hereby published.

Antibody affinity and valency impact brain uptake of transferrin receptor-targeted gold nanoparticles.

Rationale: The ability to treat invalidating neurological diseases is impeded by the presence of the blood-brain barrier (BBB), which inhibits the transport of most blood-borne substances into the brain parenchyma. Targeting the transferrin receptor (TfR) on the surface of brain capillaries has been a popular strategy to give a preferential accumulation of drugs or nanomedicines, but several aspects of this targeting strategy remain elusive. Here we report that TfR-targeted gold nanoparticles (AuNPs) can accumulate in brain capillaries and further transport across the BBB to enter the brain parenchyma. Methods: We characterized our targeting strategy both in vitro using primary models of the BBB and in vivo using quantitative measurements of gold accumulation by inductively-coupled plasma-mass spectrometry together with morphological assessments using light microscopy after silver enhancement and transmission electron microscopy with energy-dispersive X-ray spectroscopy. Results: We find that the uptake capacity is significantly modulated by the affinity and valency of the AuNP-conjugated antibodies. Specifically, antibodies with high and low affinities mediate a low and intermediate uptake of AuNPs into the brain, respectively, whereas a monovalent (bi-specific) antibody improves the uptake capacity remarkably. Conclusion: Our findings indicate that monovalent ligands may be beneficial for obtaining transcytosis of TfR-targeted nanomedicines across the BBB, which is relevant for future design of nanomedicines for brain drug delivery.

Targeting transferrin receptors at the blood-brain barrier improves the uptake of immunoliposomes and subsequent cargo transport into the brain parenchyma.

Drug delivery to the brain is hampered by the presence of the blood-brain barrier, which excludes most molecules from freely diffusing into the brain, and tightly regulates the active transport mechanisms that ensure sufficient delivery of nutrients to the brain parenchyma. Harnessing the possibility of delivering neuroactive drugs by way of receptors already present on the brain endothelium has been of interest for many years. The transferrin receptor is of special interest since its expression is limited to the endothelium of the brain as opposed to peripheral endothelium. Here, we investigate the possibility of delivering immunoliposomes and their encapsulated cargo to the brain via targeting of the transferrin receptor. We find that transferrin receptor-targeting increases the association between the immunoliposomes and primary endothelial cells in vitro, but that this does not correlate with increased cargo transcytosis. Furthermore, we show that the transferrin receptor-targeted immunoliposomes accumulate along the microvessels of the brains of rats, but find no evidence for transcytosis of the immunoliposome. Conversely, the increased accumulation correlated both with increased cargo uptake in the brain endothelium and subsequent cargo transport into the brain. These findings suggest that transferrin receptor-targeting is a relevant strategy of increasing drug exposure to the brain.

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier.

The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)]2 α2(IV), agrin, perlecan, and nidogen 1 and 2 in vitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured in vitro coherently form a BBB and express basement membrane proteins as a feature of maturation. Cover Image for this issue: doi: 10.1111/jnc.13789.

Expression of Iron-Related Proteins at the Neurovascular Unit Supports Reduction and Reoxidation of Iron for Transport Through the Blood-Brain Barrier.

The mechanisms for iron transport through the blood-brain barrier (BBB) remain a controversy. We analyzed for expression of mRNA and proteins involved in oxidation and transport of iron in isolated brain capillaries from dietary normal, iron-deficient, and iron-reverted rats. The expression was also investigated in isolated rat brain endothelial cells (RBECs) and in immortalized rat brain endothelial (RBE4) cells grown as monoculture or in hanging culture inserts with defined BBB properties. Transferrin receptor 1, ferrireductases Steap 2 and 3, divalent metal transporter 1 (DMT1), ferroportin, soluble and glycosylphosphatidylinositol (GPI)-anchored ceruloplasmin, and hephaestin were all expressed in brain capillaries in vivo and in isolated RBECs and RBE4 cells. Gene expression of DMT1, ferroportin, and soluble and GPI-anchored ceruloplasmin were significantly higher in isolated RBECs with induced BBB properties. Primary pericytes and astrocytes both expressed ceruloplasmin and hephaestin, and RBECs, pericytes, and astrocytes all exhibited ferrous oxidase activity. The coherent protein expression of these genes was demonstrated by immunocytochemistry. The data show that brain endothelial cells provide the machinery for receptor-mediated uptake of ferric iron-containing transferrin. Ferric iron can then undergo reduction to ferrous iron by ferrireductases inside endosomes followed by DMT1-mediated pumping into the cytosol and subsequently cellular export by ferroportin. The expression of soluble ceruloplasmin by brain endothelial cells, pericytes, and astrocytes that together form the neurovascular unit (NVU) provides the ferroxidase activity necessary to reoxidize ferrous iron once released inside the brain.

Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

BACKGROUND: Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. METHODS: Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. RESULTS: The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer. CONCLUSIONS: The data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology.

Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail.