In vitro blood-brain barrier (BBB) models using primary rat brain microvessel endothelial cells (BMEC) are often hampered by a lack of culture purity and poor barrier properties. To address these problems, the translation inhibitor puromycin was used to purify rat BMEC cultures. BMEC purities of 99.8% were routinely attained using puromycin treatment, and this technique proved to be far superior to other purification methods of similar difficulty. In contrast to cultures without puromycin treatment, purity of puromycin-treated cultures was unaffected by initial seeding density. Next, rat BMEC monolayer transendothelial electrical resistance (TEER) was increased by glucocorticoid treatment with either corticosterone (CORT) or hydrocortisone (HC), and a corresponding decrease in monolayer permeability to small molecules was observed. Importantly, cultures treated with both puromycin and glucocorticoid attained significantly higher TEER values (CORT 168 ± 13 W · cm 2 ; HC 218 ± 66 W · cm 2 ) than those treated by the glucocorticoid alone (CORT 57 ± 5 W · cm 2 ;HC 70 ± 2 W · cm 2 ). Glucocorticoid induction resulted in BMEC morphological changes that accompanied the increases in TEER, and BMEC tight junctions exhibited improved integrity as visualized by the localization of tight junction proteins zonula occluden-1, occludin and claudin-5. The combined use of puromycin and glucocorticoid therefore provides an in vitro system that is well suited for molecular level BBB investigations. The cerebral microvasculature separates the brain interior from the bloodstream and has been termed the blood-brain barrier (BBB) as a result of its impermeable properties. The BBB assists in maintaining brain homeostasis and protects the brain against harmful blood-borne substances. A single layer of brain microvascular endothelial cells (BMEC) is responsible for the limited solute transfer between blood and brain, and these specialized endothelial cells (EC) display distinctive attributes when compared with peripheral endothelium. Low BMEC permeability results from continuous tight junctions between adjoining ECs (Reese and Karnovsky 1967), low levels of pinocytosis and a general lack of fenestrae (Brightman and Reese 1969;Joo 1971). Because of the impermeable phenotype, the BBB plays major roles in disease pathology and hinders drug delivery efforts. Because of the inherent difficulties in performing molecular level studies of disease pathology in vivo, and the fact that prediction of BBB drug permeability prior to animal studies would be highly advantageous, a representative in vitro model would be of high utility. Unfortunately, when

Glutamate transporters are thought to be assembled as trimers of identical subunits that line a central hole, possibly the permeation pathway for anions. Here, we have tested the effect of multimerization on transporter function. To do so, we coexpressed EAAC1 WT with the mutant transporter EAAC1 R446Q , which transports glutamine, but not glutamate. Application of 50 μM glutamate or 50 μM glutamine to cells coexpressing similar numbers of both transporters resulted in anion currents of 165 pA and 130 pA, respectively. Application of both substrates at the same time generated an anion current of 297 pA, demonstrating that the currents catalyzed by the wild-type and mutant transporter subunits are purely additive. This result is unexpected for anion permeation through a central pore, but could be explained by anion permeation through independently-functioning subunits. To further test the subunit independence, we coexpressed EAAC1 WT and EAAC1 H295K , a transporter with a 90-fold reduced glutamate affinity as compared to EAAC1 WT , and determined the glutamate concentration dependence of currents of the mixed transporter population. The data were consistent with two independent populations of transporters with apparent glutamate affinities similar to those of EAAC1 H295K and EAAC1 WT , respectively. Finally, we coexpressed EAAC1 WT with the pH-independent mutant transporter EAAC1 E373Q , showing two independent populations of transporters, one being pH dependent, the other being pH-independent. In conclusion, we propose that EAAC1 assembles as trimers of identical subunits, but that the individual subunits in the trimer function independently of each other.Plasma membrane glutamate transporters actively remove glutamate from the synaptic cleft after excitatory neurotransmission is complete. Uptake into the cells surrounding the synapse against a glutamate concentration gradient is achieved by these transporters by coupling transmembrane glutamate movement to the cotransport of three sodium ions and one proton, and the countertransport of one potassium ion (1,2). In addition to the movement of ions across the membrane being directly coupled to glutamate transport, glutamate transporters also catalyze uncoupled transmembrane flux of anions (3). This anion conductance is thought to be an integral property of the transporters and is not mediated by indirect coupling of transport to a secondary anion channel (3-5).Address correspondence to: Christof Grewer, PhD, Department of Physiology and Biophysics, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136; Phone: (305) 243-1021; Fax: (305) The mammalian glutamate transporters belong to a large family of membrane transport proteins that comprise also neutral amino acid transporters, such as the alanine serine cysteine transporters (ASCTs (6,7)), and dicarboxylate transporters (8,9). A large number of biochemical data from both mammalian (10,11) and bacterial glutamate transporters (12,13), as well as recent crystallographic evidence f...

No abstract

The blood-brain barrier (BBB) is a multifunctional endothelial interface separating the bloodstream from the brain interior. Although the mature BBB is well characterized, the embryonic development of this complex system remains poorly understood. Embryonic neural progenitor cells (NPC) are a potential inductive cell type populating the developing brain, and their ability to influence BBB properties was therefore examined. When puromycin-purified brain microvascular endothelial cells (BMEC) were co-cultured with embryonic NPC in a two-compartment Transwell system, the BMEC exhibited enhanced barrier properties in the form of increased transendothelial electrical resistance (TEER) and decreased permeability to the small molecule tracer, sodium fluorescein. These changes required the presence of NPC in the early stages of differentiation and were accompanied by alterations in the fidelity of BMEC tight junctions as indicated by occludin, claudin 5, and zonula occluden-1 redistribution at cell-cell borders. In contrast to the findings with NPC, post-natal astrocytes elicited a delayed, but longer duration response in BMEC TEER. BMEC co-culture also suppressed neuronal differentiation of NPC indicating a reciprocal signaling between the two cell populations. This study demonstrates that NPC-BMEC interactions are prevalent and for the first time demonstrates that NPC are capable of inducing BBB properties. Keywords: blood-brain barrier, brain microvasculature, endothelial cell, neural progenitor cell, neural stem cell. The blood-brain barrier (BBB) is comprised of a specialized class of endothelium that forms a cellular barrier between the bloodstream and the interstices of the adult brain. By restricting non-specific flux of blood-borne constituents, the BBB plays an important role in maintaining parenchymal homeostasis, and strictly regulates transport of ions, small molecules, proteins, and cells into and out of the brain. The BBB accomplishes these tasks because its unique endothelium is endowed by epithelial-like tight junctions joining adjacent endothelial cells (ECs), lacks fenestrae, and possesses a rich array of molecular transport systems. Although the endothelium is the principle determinant of barrier function, perivascular non-endothelial cells in the local microenvironment have been shown to make significant contributions. Astrocytes (Stewart and Wiley 1981;Risau et al. 1986b;Janzer and Raff 1987), neurons (Tontsch andBauer 1991), and pericytes (Balabanov and Dore-Duffy 1998;Ramsauer et al. 2002) have all been demonstrated to provide cues that result in the unique BBB endothelial phenotype.Although the inductive properties of the aforementioned brain cell types have been confirmed through a multitude of in vivo and in vitro studies, the cell type(s) responsible for early embryonic BBB induction have not been distinguished. The developmental timecourse of embryonic BBB formation differs between species, but it is generally well accepted that the onset of BBB development begins pre-natally and is

No abstract

In vitro blood-brain barrier (BBB) models often consist of brain microvascular endothelial cells (BMECs) that are co-cultured with other cells of the neurovascular unit, such as astrocytes and neurons, in order to enhance BBB properties. Obtaining primary astrocytes and neurons for co-culture models can be laborious, while yield and heterogeneity of primary isolations can also be limiting. Neural progenitor cells (NPCs), due to their self-renewal capacity and ability to reproducibly differentiate into tunable mixtures of neurons and astrocytes, represent a facile, readily scalable alternative. To this end, differentiated rat NPCs were co-cultured with rat BMECs and shown to induce BBB properties such as elevated trans-endothelial electrical resistance (TEER), improved tight junction continuity, polarized p-glycoprotein efflux, and low passive permeability at levels indistinguishable from those induced by primary rat astrocyte co-culture. An NPC differentiation time of 12 days, with the presence of 10% fetal bovine serum, was found to be crucial for generating NPC-derived progeny capable of inducing the optimal response. This approach could also be extended to human NPC-derived astrocytes and neurons which similarly regulated BBB induction. The distribution of rat or human NPC-derived progeny under these conditions was found to be a roughly 3:1 mixture of astrocytes to neurons with varying degrees of cellular maturity. BMEC gene expression analysis was conducted using a BBB gene panel, and it was determined that 23 of 26 genes were similarly regulated by either differentiated rat NPC or rat astrocyte co-culture while 3 genes were differentially altered by the rat NPC-derived progeny. Taken together, these results demonstrate that NPCs are an attractive alternative to primary neural cells for use in BBB co-culture models.

No abstract

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial “Excitatory Amino Acid Transporters” (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a β-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in “Myelin Oligodendrocyte Glycoprotein” (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFγ and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a β-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.