Engaging Neuroscience to Advance Translational Research in Brain Barrier Biology

PeRSPeCTiveS

for interdisciplinary approaches to expand oPiNioN our knowledge and improve strategies for treatments of neurological disorders. Engaging neuroscience to advance Many factors have contributed to the lack of interaction between neuroscientists and translational research in brain brain barrier scientists, including the com plexities of each field and the numerous gaps in our understanding of the BBB. This, in barrier biology turn, has resulted in relatively little emphasis on brain barrier science as an interdisci Edward A. Neuwelt, Björn Bauer, Christoph Fahlke, Gert Fricker, plinary topic or an educational objective, Constantino Iadecola, Damir Janigro, Luc Leybaert, Zoltán Molnár, whereas greater emphasis might facilitate Martha E. O’Donnell, John T. Povlishock, Norman R. Saunders, Frank Sharp, such communication. Recent advances have increasingly demonstrated the common Danica Stanimirovic, Ryan J. Watts and Lester R. Drewes ground between the two fields of study and Abstract | The delivery of many potentially therapeutic and diagnostic compounds the urgency for crosstalk. The present article to specific areas of the brain is restricted by brain barriers, of which the most provides a vision for future study that inte grates both disciplines, highlighting areas of well known are the blood–brain barrier (BBB) and the blood–cerebrospinal fluid relevance and convergence (BOX 1). (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood Physiology of the NVU flow, and — when barrier integrity is impaired — in the pathology of many common The NVU consists of an endothelial cell CNS disorders such as Alzheimer’s disease, Parkinson’s disease and stroke. monolayer (connected by tight junctions and resting on the basal lamina), integral neighbouring cells (including pericytes and There is a commonly held notion that the BBB has many other roles and is not simply smooth muscle cells) and astrocytic endfeet blood–brain barrier (BBB) is a simple ana a control point for molecular trafficking in covering >98% of the vascular wall and tomical structure that restricts the traffic of and out of the brain. occasional neuronal terminals. The astro molecules in and out of the CNS but other The common wisdom has been that the cytes also extend processes that surround wise is not very relevant to neuroscience. BBB consists of endothelial cells and is either synapses and can thereby link neuronal This view is flawed however, as brain barrier open or closed depending on the status of activity with the oxygen and nutrient supply. sciences and neuroscience are inextricably tight junction proteins that create a restric Finally, components of the NVU include the linked in many areas of neurophysiology tive, fixed barrier. We now know that the circulating blood cells, such as polymor and neuropathology. BBB is in fact dynamic, with a wide perme phonuclear (PMN) cells, lymphocytes and The BBB is one of a number of blood– ability range that is controlled by intra monocytes that adhere and roll along the CNS interfaces, which also include the and intercellular signalling events among vascular lumen and perform surveillance blood–cerebrospinal fluid (CSF) barrier, endothelial cells, astrocytes and neurons in of neural signalling and cellular activity20 the blood–retinal barrier, the blood–nerve the BBB (and other cells that are in contact (FIG. 1a). barrier and the blood–labyrinth barrier — all with the BBB), as well as by paracellular The endothelial cells of the NVU are of which are important for the physiologi changes at the BBB. A further key concep highly polarized, with different integral cal functions of the CNS (FIG. 1). Among tual advance has been the discovery that the membrane proteins at the luminal and ablumi- these interfaces, the BBB occupies by far BBB is an integral part of the neurovascular nal surfaces. These include various receptors, the largest surface area. A wide range of unit (NVU)19 (FIG. 1a). enzymes and transporters that support the neurological conditions such as Alzheimer’s The complex regulation of barrier prop functions of this cellular barrier within the disease1–3, Parkinson’s disease4, multiple erties is far from understood. Gaining better NVU. For example, the endothelial barrier sclerosis5,6, trauma7,8, brain tumours9,10, insight into the physiological and patho performs vectorial transport of solutes — stroke11–14 and epilepsy15 are associated physiological processes that alter intra and including ions, nutrients and drugs — at with perturbations in the normal BBB that intercellular junction protein distribution the blood–brain interface. It also engages in contribute to their pathology (TABLE 1). and function is important for understand highly specialized interactions with blood Furthermore, the cells that constitute the ing how the barrier can be fixed when it cells, through specific luminal receptors, and BBB play a part in the control of cerebral does not function properly and how it can with elements of the basal lamina and under blood flow16,17 and neuronal development18. be manipulated for therapeutic purposes. lying cells (for example, astrocytic endfeet Thus, it is important to recognize that the Suffice it to say, herein lies the opportunity and neuron terminals) at the abluminal

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- CPFCNDWOKP +PVTCGPFQVJGNKCN GPVT[ $$$ UKIPCNNKPI F[UHWPEVKQP Figure 1 | The extended neurovascular unit. a | The blood–brain barrier therapeutic proteins to the CNS44,128. c | Pathological signalling in the extended (BBB) is an essential part of the neurovascular unit (NvU). A classical view of NvU. The proposed sequence order is based0CVWT onG4G dataXKGYU available^0GWT fromQUEKGPEG the epi- the NvU incorporates neurons, glial cells such as astrocytes and microglial lepsy field144 and requires further exploration in the context of other brain cells closely juxtaposed with vascular endothelial cells, pericytes and smooth diseases, including stroke and Alzheimer’s disease. The cycle starts with muscle cells. Blood cells, particularly polymorphonuclear (PMN) cells, lym- altered expression of vascular cell adhesion molecules and interactions of phocytes and monocytes, also interact with the BBB endothelium and are leucocytes with the endothelium, initiating intraendothelial signals that alter therefore an integral part of this unit. The interactions between these cellular BBB function and lead to neural tissue dysfunction as a consequence of K+ and components and inter- and intracellular signalling regulate NvU function to albumin entry into the brain interstitium. Astrocytes detect the altered neu- maintain homeostasis, or to respond to inflammation and disease. ronal activity and transmit signals back to the BBB, thereby facilitating interac- b | Receptor-mediated transcytosis of proteins at the BBB. Transcytosis is a tions with leucocytes and turning the sequence into a vicious circle that receptor-mediated transport mechanism by which proteins that are targeted maintains and exacerbates the pathological state. The activated endothelium to the CNS bind extracellular receptors in vascular lumen, are transported may, as an integral part of the extended NvU, disturb neuron–astrocyte inter- across the BBB endothelial cells, and are released in brain parenchyma. The actions, thereby adding an additional layer of pathological signalling to the presence of specific receptors (for example, the insulin receptor) on the sur- process. Astrocytes emerge from this cascade as a primary target for face of BBB endothelial cells has allowed targeting and transport of some interventions that aim to interrupt the proposed cycle. surface, through specific abluminal plasma composed of pre and postsynaptic endings, NVU and are defined below as part of the membrane proteins. Furthermore, astrocytes together with their related glia, are structur extended NVU. and pericytes possess their own complement ally and functionally related to the brain’s The NVU, together with the basal of transporters, channels, receptors and sig capillary bed, and together form the NVU lamina and extracellular matrix com nalling mechanisms with which they (FIG. 1a). The role of the NVU interface in ponents, engages in complex signal coordinate the role of the NVU in the context of the tripartite synapse is just ling processes (fast and slow; active and supporting nervous system function. beginning to be understood. The cellular trophic). Documented examples include components include the endothelium (form the propagation of Ca2+ waves through the The tripartite synapse and the NVU. A ing the barrier proper at the capillary level), NVU24, neuro–metabolic coupling25, neuro– major notion that has emerged from neuro astrocytic endfeet, pericytes and circulating haemodynamic coupling, neuro–angiogenic science over the past few years is the concept immune cells, which are adjoined at some coupling, and neuro–trophic coupling26,27 and of the ‘tripartite synapse’, which has com distance by nerve endings and vascular cell adhesionbased signalling networks28. pelled neuroscientists to consider the influ smooth muscle cells found at the arterial These NVU signalling processes are also ence of glia in synaptic function21,22. In the level19,23 (FIG. 1a,b). It should be noted linked to membrane transport29–33, regula cerebral microcirculation, tripartite synapses that immune cells indeed sneak into the tion of cellular permeability (specific or

Table 1 | Diseases that affect, or that are affected by, the blood–brain barrier (BBB) Disease or process BBB proteins and mechanisms affected refs Neurodegenerative diseases Alzheimer’s disease RAGe products — influx of amyloid-β 163 LRP1 multi-ligand lipoprotein receptor — efflux of amyloid-β 163 P-glycoprotein (also known as ABCB1) is reduced at the BBB and seems to play a 60,164 crucial part in clearing amyloid-β from the brain 165 Changes in ABCG2 are related to cerebral amyloid angiopathy and control BBB 166 transfer of amyloid-β Parkinson’s disease Polymorphism in P-glycoprotein drug transporter MDR1 gene association and 135,141 ABCB1 gene encoding the P-glycoprotein Cerebrovascular diseases tPA- and reperfusion-induced haemorrhage MMPs released by neutrophils and possibly endothelial cells degrade tight 167–170 junction proteins and basement membrane — increased risk of haemorrhage veGF-mediated BBB breakdown Occludin and claudin 5 — downregulation of mRNA and protein 171 Familial cerebral cavernous malformations CCM1 (also known as KRiT1), CCM2 or CCM3 (also known as PDCD10) localized 172,173 in endothelial cells and perhaps astrocyte endfeet — venous malformations with bleeding ischaemic brain oedema BBB breakdown due to MMP9 release by neutrophils —degradation of occludin, 174–177 claudins, junctional adhesion molecule (JAM) family proteins and basement membrane; SUR1 (also known as ATP-binding cassette subfamily C member 8)- regulated non-selective cation channel NC(Ca-ATP) mediates ischaemic cerebral oedema Acute mountain sickness and high-altitude cerebral vasogenic oedema 120 oedema Epilepsy and seizures epilepsy and exercise-induced dystonia GLUT1 mutations in brain endothelial cells 136,137, 139 Resistance to pharmacotherapy in some patients Multidrug efflux pumps from the ABC superfamily (for example, P-glycoprotein) 79,178 with epilepsy at the BBB Alexander’s disease — large brain, seizures and GFAP mutations — BBB abnormalities 179 retardation Leukoencephalopathy with epilepsy ClC2 is a broadly expressed plasma membrane chloride channel — epilepsy, 143 white matter degeneration and retinal degeneration in mice Infections Hiv entry into brain Hiv activation of STAT and RHO kinase downregulate claudin 5, ZO1 and ZO2 in 180–182 endothelial cells, which may increase Hiv entry Susceptibility to certain types of brain infections (for P-glycoprotein (also known as MDR1A andABCB1) deficiency at BBB — 183–185 example, malaria and CNS listeria moncytogenes) susceptibility to cerebral malaria; opc gene in Meningococcus produces protein that binds HBMeCs via α5β1 integrin receptors on fibronectin CNS infections in general Pathogens hijack BBB cellular machinery to enter the brain 185 NeuroAiDS in Hiv BBB efflux systems keep out antivirals from thebrain, fostering neuroAiDS 186 Malaria effects protein expression and permeability of human endothelial cells 125 selectively in the brain Neuroinflammation and brain tumours White blood cell oxidative stress causes adhesion to White blood cell proteins (such as selectins, vLA4, CD44 and α4β7 integrin) and 32,187, endothelium and transmigration across BBB brain endothelial proteins (such as selectin ligands, iCAM1, vCAM1 and CD44) 188 mediate migration across BBB. CD8+ cytotoxic T cell-mediated BBB breakdown and Perforin release degrades tight junction proteins 189 oedema Brain oedema — tumour, inflammation and others Aquaporins (astrocyte endfeet) 190,191 Brain oedema — role of steroids. Prednisone and Steroids act on glucocorticoid response elements on promoters of tight junction 192–194 dexamethasone decrease BBB leakage in acute genes (occludin, claudins and cadherin) to increase tight junction proteins and multiple sclerosis plaques, tumours and other increase BBB tightness pathologies

Table 1 (cont.) | Diseases that affect, or that are affected by, the blood–brain barrier (BBB) Disease or process BBB proteins and mechanisms affected refs BBB breakdown in multiple sclerosis: monocyte– tPA induction of eRK1 and eRK2 in endothelial cells mediates monocyte 195 endothelial interactions induce tPA in endothelial cells transmigration across BBB and control breakdown of occludin

BBB breakdown in multiple sclerosis: role of iL-17 and Helper T lymphocytes (TH17 cells) release iL-17 and iL-22 that act on receptors 196,197 iL-22 on brain endothelial cells that results in degradation of tight junction proteins and opening of the BBB Prevent leukocyte trafficking across the BBB — Monoclonal antibody to α4 integrin (Natalizumab) — adhesion molecule on 198–200 decreased multiple sclerosis relapses leukocytes necessary to attach to and cross the BBB in eAe inflammatory pain — cytokines mediate BBB Downregulation of occludin and claudin 5 201 breakdown Metabolic and psychiatric diseases Brain oedema — associated with diabetic ketoacidosis Na-K-Cl cotransporter, and Na–H exchanger at BBB 202–207 and cerebral ischaemia Adrenoleukodystrophy — abnormal white matter ABCD1 gene mutation — ATP binding cassette disorder; ABC superfamily 140,142 in brain with a wide range of neurological findings; retinal degeneration Obesity — leptin released from adipose tissue and Deficient BBB transporter protein function — reduced leptin transport across 208–210 binds to leptin receptor to modulate food intake the BBB imerslund–Gräsbeck syndrome familial vitamin Amnionless mutations — possible vitamin B12 transport into brain 211 B12 malabsorption — dementia and white matter abnormalities Canavan’s disease — large brain, seizures, retardation, Mutations in aspartoacetylase lead to accumulation of N-acetylaspartate 212 white matter degeneration and other signs Mucopolysaccharidosis Loss of the GUSB transporter with maturation underlies difficulty in treatment 213 Depression Polymorphisms in the drug transporter gene ABCB1 predict antidepressant 214 treatment response in depression Hepatic encephalopathy Affects potassium homeostasis in astrocytes, produces swelling and disrupts 215–217 control of extracellular potassium ABCB1, ATP-binding cassette, subfamily B, member 1; ABCG2, ABC transporter G family member 2; CCM1, mitochondrial group i intron splicing factor CCM1; CiC2, chloride channel protein 2; eAe, experimental autoimmune encephalomyelitis; eRK1, mitogen-activated protein kinase 3 (also known as extracellular signal-regulated kinase 1); GFAP, glial fibrillary acidic protein; GLUT1, solute carrier family 2, facilitated glucose transporter member 1; GUSB, beta-glucuronidase; HBMeC, human brain microvascular endothelial cell; iCAM1, intercellular adhesion molecule 1; iL-17, interleukin-17; LRP1, low-density lipoprotein receptor-related protein 1; MDR1, multidrug resistance protein 1; MMP, matrix metalloproteinase; opc, class 5 outer membrane protein; RAGe, advanced glycosylation end product-specific receptor; STAT, signal transducer and activator of transcription protein family; SUR1, sulfonylurea receptor 1; vCAM1, vascular cell adhesion protein 1; veGF, vascular endothelial growth factor; vLA4, integrin alpha 4; ZO1, tight junction protein ZO1. selective regulation, or through paracellular forms the blood–CSF barrier). The identity movement among cells of the NVU has pathways)32,34,35 and intracellular metabolic and cellular location of multiple — gener recently been expanded. These studies largely cascades25,36,37. ally efflux — transporters that are present in focused on whole brain and/or hypoxia in the NVU and that function possibly in drug neurons and astrocytes, and led to the dis FIG. 2 – Dynamic regulation of brain barrier perme- transport are shown in . New neuro coveries of a family of hCo3 transporters, ability by the NVU. Cells of the NVU form a science discoveries include structural and the electrogenic sodium bicarbonate cotrans complex and finetuned transport machine mechanistic insights into coupled transport porter (NBCe) and electroneutral sodium that balances the influx of nutrients and the ers (for example, GABA and no repinephrine bicarbonate cotransporter 3 (NBCn) families, efflux of wastes, toxins and drugs to maintain transporters (sodium and chloride and electroneutral sodiumdriven chloride/ CNS homeostasis38. Numerous factors regulate dependent GABA transporter (GAT) and bicarbonate exchanger 1 (NDCBe)47–49. the barrier permeability of the NVU, including sodiumdependent noradrenaline trans expression levels of these transporters vary modulation of membrane transporters and porter (NeT) family proteins), excitatory with brain region and cell type, with promi transcytotic vesicles, and modulation of amino acid transporters (eAATs)41 and nent expression of both NBC and NDCBe transcellular permeability34 (FIG. 1b). ATPbinding cassette (ABC) transporters42. transporters reported for neurons and The importance of investigating NVU Improved understanding of the physiologi choroid plexus, and little or no expression in transport proteins is underscored by the cal and biophysical mechanisms underlying astrocytes. little is known about expression recent finding that 10–15% of all proteins transport function43–46 should be applicable and function of the NBC and NDCBe trans in the NVU are transporters39. In 2003 it to both general brain function and porters in cells of the cerebrovasculature. was estimated that only about 50% of brain dysfunction in disease. Recent studies have provided important barrier transporter proteins had been identi Ion transporter proteins in cells of the new insights regarding the role of aquapor fied39,40. Since then, several new transporters NVU play an important part in maintaining ins in astrocytic endfeet50 and the control of have been detected and localized in the brain fluid balance in the brain, and our under water distribution within the brain51–55, and endothelium and the choroid plexus (which standing of their role in water and electrolyte have shown that abnormal fluid dynamics

60–62 Box 1 | A meeting of minds disease . Thus, understanding the signal ling pathways that regulate efflux trans In an effort to bring the two fields of neuroscience and brain barrier science closer, an international porter expression and activity is likely to panel of experts was assembled in March 2009 to discuss current areas of overlapping interests in be useful for improving CNS drug delivery, which the expertise and observations of one group might advance the research progress of the protecting the brain during systemic treat other. Leaders in the fields of neuroscience and brain barrier science identified five topics as central to advancing the treatment of CNS disorders; these included molecular physiology of the brain and ment and preventing pathogenesis or slow brain barriers; intercellular communication within the neurovascular unit (NVU); transport biology ing the progression of several CNS diseases. in the brain and brain barriers; neurodevelopment and the brain barriers; and imaging the structure, For example, three major pathways have function and dynamics of the brain and brain barriers. been identified that regulate Pglycoprotein Within each topic, four main questions were addressed: what are the key scientific opportunities (Pgp), a major efflux transporter at the BBB in the neuroscience field that may be applied to the brain barriers field and vice versa? What is the that limits brain penetration of therapeutic status of the science in the topic, including key scientific advances made in the respective fields drugs63. one pathway is triggered by the over the past 4 years, and are they relevant to the other field? What are the barriers to progress in inflammatory mediator tumour necrosis the topic? What are the highest-priority recommendations for developing and advancing factor α (TNFα), which signals through knowledge in the topic, including the key resources and approaches needed? tumour necrosis factor receptor superfamily For each of the five key topics, a panel of experts, co-chaired by renowned neuroscientists and brain barrier scientists, drafted reports answering the four primary questions as well as addressing member 1A (TNFR1), resulting in release the key question, ‘What is the single most important issue that would advance research in each of endothelin 1 (eT1). This in turn signals topic area?’ The draft reports were discussed among approximately 150 neuroscientists and brain through the endothelin B receptor (eTBR), barrier scientists at the 2009 Annual Blood–Brain Barrier Consortium Meeting in Oregon, USA (see resulting in signalling through nitric oxide Supplementary information S1,S2 (boxes)). The co-chairs and working groups incorporated into synthase and protein kinase Cβ (PKCβ) to their final reports the discussion and input from the combined group of scientists (see alter Pgp expression and function58,64–66. Supplementary information S3 (box) for the final reports from each of the five topics). Indeed, activating PKCβ reduced Pgp activ ity and enhanced delivery of small molecule therapeutics into the brain58. or aquaporin malfunctions may have patho function is mainly achieved by two compo A second pathway involves the neu logical consequences56. Several aquaporins nents in the brain capillary endothelium — rotransmitter glutamate, which signals are found in the brain including aquaporin ATPdriven membrane transporters known through the NMDA receptor, cyclooxyge 1 (AQP1), AQP3, AQP4, AQP5, AQP8 and as ‘efflux transporters’ and tight junctions nase 2 (CoX2) and the prostaglandin e2 AQP9, with AQP1, AQP4 and AQP9 most that ‘seal’ spaces between endothelial cells. receptor eP1 to upregulate Pgp expression heavily studied. Whereas AQP9 is found in and activity67–70. Inhibiting this pathway pre the astrocyte cell body, AQP4 is abundant Signalling pathways that regulate efflux vents seizureinduced Pgp upregulation and in perivascular astrocyte endfeet and also transporters.Transportermediated export improves brain penetration of antiepileptic where the astrocyte is in close apposition of xenobiotics can affect the pharmacoki drugs and reduces epileptic seizures71. to neurons. Choroid plexus exhibits AQP1 netics and pharmacodynamics of a large The third pathway involves activation of and AQP4, and endothelial cells of the NVU number of therapeutics, and poses a chal xenobiotic-sensing nuclear receptors, such as appear to have minor amounts of AQP4 lenge for the ability to deliver drugs into the the aryl hydrocarbon receptor (AhR), the at best. Pathways by which water moves CNS. Direct transporter inhibition has been glucocorticoid receptor, the pregnane xeno through the endothelial cells of the NVU pursued as one strategy, but it leaves little biotic receptor (PXR) and the constitutive are largely understudied. A relatively recent control over the extent and duration of the androstane receptor (CAR)72–78, to regulate finding is that that Co2 and Nh3 conduct inhibition. Accordingly, transporter inhibi transporter expression. In a recent study, ances are regulated by AQP1 and AQP4 tors are currently not in clinical use. Recent for example, activation of PXR has been (REF. 57). This has created a paradigm shift in efforts have therefore focused on targeting used to restore brain endothelial Pgp in an the way that we think about how metaboli the intracellular signalling pathways and Alzheimer’s disease mouse model, which cally relevant gases move through the NVU molecular switches that control efflux trans resulted in enhanced amyloidβ clearance — that is, that diffusion of the gases across porter regulation, for several reasons. First, from the brain60. In addition, several of the plasma membrane is not by simple diffusion modulating these pathways and switches signalling pathways have common elements but rather, by facilitated diffusion via the would allow finetuning of transporter (for example, TNFα, nuclear factor κB aquaporins. activity so that transporters can be turned (NFκB) and CoX2) that may be potential It has long been accepted that the NVU off for controlled periods, thus providing therapeutic targets. functions as a selective barrier to various a time window to deliver drugs58. Second, Thus, findings from studies using physi substances passing between blood and brain, such strategies could be used to upregulate ological and pathophysiological modulators, but these new discoveries have led to a more expression and activity of efflux transporters pharmacologic inhibitors and activators of developed understanding of the NVU, which in the NVU to minimize brain side effects efflux transporters may be useful for improv recognizes the NVU as a functionally com associated with the treatment of a disease in ing the delivery of drugs into the brain, pro plex blood–brain interface with multiple, the periphery (for example, ‘chemobrain’ in tecting the brain from harmful xenobiotics interacting roles. cancer patients)59. Third, efflux transporters and alleviating CNS disorders79,80. In addi are affected by — and likely contribute to — tion to studying brain barrier transporters, The NVU as a barrier to xenobiotics disease pathology of CNS disorders that are understanding the molecular regulation one of the most important roles of the accompanied by inflammation, oxidative of tight junctions may provide therapeutic NVU is to limit xenobiotics, including CNS stress and neurotransmitter release, and that opportunities in diseases in which endothe drugs, from entering the brain. This barrier include cancer, epilepsy and Alzheimer’s lial barrier integrity is disrupted81.

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As astrocytes .42 ).76 0GWTQP make extensive contacts with smooth mus cle cells at the arteriolar level93, vasotropic 4#)' 2IR /KETQINKC $%42 %JQTQKFRNGZWU /%6 actions of astrocytic calcium signals may ).76 /42 also affect smooth muscle cells directly or /42 /42 2IR /42 ).76 1#62 indirectly via brain endothelial cells (which /42 /42 $ in turn secrete vasoactive substances such as /%6 1#6 $NQQF nitric oxide)89. 1#62 $%42 /42 1#62 % The observation that calcium can act as #UVTQE[VG a signal in astrocytic endfeet located at the $ ).76 $CUQNCVGTCN /42 blood–brain interface suggests that these /42 /42 /%6 /42 .42 4#)' signals may influence endothelial cells at /42 $TCKP distances of less than 0.1 μm and thus, could 2IR 1#62 #RKECN 25 1#6 1#62 cause dynamic alterations in BBB function . # % ).76 /42 /%6 Clearly, the astrocyte is a major communica #DNWOKPCN /42 'PFQVJGNKCNEGNN /%6 tion link between the multiple parts of the .42 NVU. 4.+2 1#62 /42 $%42 1#62 1#62 /42 $ % $ ).76 /42 /%6 Neurodevelopment of the brain barriers /42 /%6 2IR Research on vascular and neuronal devel $NQQF 4#)' .WOKPCN opment has been converging over the past decade — for example, in the study of angio Figure 2 | Primary transporters in the neurovascular unit. The spatial and cellular relationships genesis in fetal brain. There are at least two of the transporters are shown. Only proteins detected at the protein0CVWT level G4Gare depicted.XKGYU^0GWT ForQUEKGPEG a more major reasons for this: first, there is evidence complete listing of carrier-mediated transport systems at the blood–brain interface see Ohtsuki and Terasaki38. BCRP, breast cancer resistance protein (also known as ABC transporter G family member 2); for shared molecules and coordinated cellular GLUT, solute carrier family 2, facilitated glucose transporter member; LRP, low-density lipoprotein mechanisms during the development of these 94,95 receptor-related protein family member; MCT, monocarboxylic acid transporter family member; MRP, systems ; and second, there is evidence multidrug resistance-associated protein family member; OAT, organic anion transporter family mem- that neurogenesis and angiogenesis are co ber; OATP, organic anion transporter polypeptide family member; Pgp, P-glycoprotein; RAGe, regulated in embryonic and adult brains94,96,97. advanced glycosylation end product-specific receptor; RLiP76, Ral-binding protein 1. Figure is The CNS vasculature develops by angiob modified, with permission, from REF. 161 © (2011) Bentham Science. lastic invasion of the head region that occurs in early phases of embryogenesis, and this vasculogenic process establishes the extrac Role of tight junctions in NVU function. NVU and the control of cerebral blood flow erebral vascular plexus that eventually covers In the brain capillary endothelium, tight Communication between neurons and glial the entire surface of the neural tube98–100. junctions that seal the spaces between neigh cells — especially astrocytes84 — in response After the primary vascular plexus is formed, bouring endothelial cells represent a passive to electrical and synaptic activity can influ further vascularization of the CNS is exclu barrier that restricts paracellular diffusion ence cerebral blood flow. This occurs under sively achieved by angiogenesis from the of watersoluble solutes, including drugs, conditions of physiological levels of neuro perineural vascular complex. Driven by from blood to brain. The role of tight junc nal activity85, strong or pathological stimuli86 metabolic demands of the expanding neu tions and their key constituent proteins, and spontaneous activation87. Astrocytic roectoderm, capillary sprouts invade from claudins and occludins, in the regulation calcium signals propagate to astrocytic the extracerebral vascular plexus toward the of barrier function is beginning to be elu endfoot extensions that are in contact with periventricular zone101. once formed, the cidated. however, it is not yet known how blood vessels and also extend to neighbour nascent brain vasculature is further stabi these and other proteins interact to create ing endfeet88, thereby triggering the release lized by the recruitment of mural cells and the highly effective and precisely regulated of vasoactive messengers89–91 and altering the formation of the extracellular matrix, tight junction. For example, although local cerebral blood flow. Neuroimaging and is finetuned by microenvironmen genetic ablation of claudin 5 has shown techniques such as functional MRI use these tal cues from the neighbouring cells102,103. that this tight junction protein is necessary changes in cerebral blood flow as an indica Through this process of maturation all the to limit movement of small molecules into tor of CNS activity. components of the brain vascular network the brain82, other studies have shown that A key finding is that astrocytic endfeet acquire the phenotype that allows them to claudin 5 is expressed in all endothelial release vasodilatory as well as vasoconstric form a fully differentiated NVU. cells, not just those in the NVU. Conversely, tive messengers and this depends on the It has been known for decades that there occludin is brain endothelial cellspecific availability of oxygen: if oxygen availability is is a functional NVU well before the middle but is not required for barrier function83. low, vasodilators prevail, whereas if oxygen of the 150day gestation of sheep104–106, and Thus, the molecular basis for the tightness availability is high, vasoconstrictors predom the existence of tight junctions during brain of the cerebrovascular endothelium com inate92. Vasoactive messengers released by development has also been noted in various pared to endothelia in most other tissues astrocytes include arachidonic acid, prostag other species, including humans, as sum remains unknown. landin members of the epoxyeicosatrienoic marized elsewhere107. over the past 5 years,

unequivocal evidence has been published also known as lAT1), but not tight junc vascular development of CNS barriers, of both structural and functional barriers in tion molecule, including occludin and tight but this has so far remained unexplored. the developing brain. In fact, studies using junction protein Zo1 or panendothelial Combining vascular and neuronal devel small molecular weight markers have shown molecules including platelet endothelial cell opmental approaches to tackle questions that functionally effective tight junctions adhesion molecule (PeCAM) and vascular that relate to brain barrier development are present as soon as blood vessels begin to endothelial cadherin27. The finding that promises to unravel the poorly understood penetrate the early CNS parenchyma and Wnt regulates CNSspecific angiogenesis mechanisms of barrier development, as as soon as epithelial cells of the choroid and induces specific NVU properties, such has recently been reviewed94. The blood– plexuses begin to differentiate108,109. as gene expression and restricted perme CSF barrier seems to be especially impor These tight junctions provide the basis ability27,94, suggests that CNS angiogenesis tant during development as the choroid for selectivity of barrier interfaces. efflux and brain barrier formation are linked by plexuses are functional, possess protein transporters (for example, Pgp, breast can Wnt regulation and mutual interactions. specific transport mechanisms and restrict cer resistance protein (also known as ABC The similarity of some immune and neural paracellular passage at a time in develop transporter G family member 2) and multi molecular mechanisms during develop ment when the brain parenchyma has low drug resistance proteins), which can reduce ment might also have implications for levels of vascularization108,116,117. the accumulation of drugs and toxins in the brain, are expressed in cerebral endothelial and choroid plexus epithelial cells early in Glossary 110–112 development . A recent study reported Abluminal Luminal that pericytes are required for BBB integrity Facing the neural cells or brain. Facing the capillary lumen. during embryogenesis113. Specifically, the data indicated that pericyte–endothelial cell Basal lamina Neuro–angiogenic coupling A thin, continuous layer of extracellular matrix surrounding The coupling of the development of neurons (neurogenesis) interactions regulate some properties of the the brain endothelial cells and pericytes. with new blood vessel formation (angiogenesis and BBB during development, and disruption of vasculogenesis). these interactions may lead to BBB dysfunc Blood–cerebrospinal fluid (CSF) barrier tion and thus, to neuroinflammation as part The blood–CSF barrier is at the choroid plexus epithelial Neuroependyma of the response to CNS injury and disease113. cells, which are joined together by tight junctions. The (Also known as neuroepithelium or ventricular zone.) A capillaries in the choroid plexus differ from those of the deep pseudostratified layer of cells lining the embryonic The functional role of the brain barriers blood–brain barrier in that there is free movement of ventricular system that proliferate into radial glial cells and during development is to provide the brain molecules between endothelial cells via fenestrations and neurons in the embryo, and into glial cells later in with a specialized internal environment. As intercellular gaps. development. The cells of the neuroependyma are linked shown in FIG. 3, one major barrier difference by strap junctions, which limit intercellular movement of Blood–labyrinth barrier molecules — particularly proteins — from cerebrospinal neuroependyma is that the lining the cerebral The cochlea is a structure of the inner ear involved in sound fluid to brain interstitial space in the embryo. By adulthood ventricles constitutes a barrier during early transduction and is vascularized by a dense set of these cells have transformed to the layer of thin generally development but not at later times, when it capillaries that are essential for delivering the nutrients and non-dividing ependymal cells lining the ventricular system has become the adult ependyma. The molec ions necessary for producing the fluids (endolymph and of the mature brain. ular properties114 and specific functions of perilymph) present in the cochlea. These capillaries are lined with endothelial cells that are joined by tight junctions Neuro–haemodynamic coupling the brain barriers alter as the brain matures, and physiologically form the blood–labyrinth barrier that is The coupling of neuronal firing and synaptic activity with to reflect its changing role, influenced by essential for sensitive auditory function. haemodynamic changes (for example, blood volume and the surrounding neural environment and its blood flow). intrinsic developmentally regulated proper Blood–nerve barrier The endothelial lining of blood vessels in peripheral nerves Neuro–metabolic coupling ties. In addition, the vasculature interacts is formed by continuous, non-fenestrated endothelia in The coupling of neural activity, an energy consuming with the neural environment — this includes which individual cells are linked by tight junctions, rendering process, with the energy producing metabolic processes to shared molecular processes that influence them impermeable to intravascular macromolecules. This maintain cellular homeostasis. the growth and maturation of the brain at blood–nerve barrier, and a similar mechanism in the specific stages of its development94. Several innermost perineurial sheath, isolate the endoneurial Neuro–trophic coupling interstitium, in much the same way as the blood–brain The coupling of neuronal production of key studies have identified important CNS barrier. Other factors, such as the absence of lymphatics, activity-dependent signals such as growth factors (for parenchymal cellderived molecular signals, are also analogous to the central nervous system. example, brain-derived neurotrophic factor (BDNF)) with including angiotensinogen and Wnt, that control of neurogenesis. seem to regulate the formation and function Blood–retinal barrier The blood–retinal barrier has two components: the retinal Paracellular of the cerebrovasculature and thereby the vascular endothelium and the retinal pigment epithelium. Paracellular is used here to refer to the transfer of 18,27,94,104,115 NVU . The retinal vascular endothelium is non-fenestrated and has substances between cells of an endothelium or epithelium. In addition to being essential for angio anatomical properties similar to those of cerebral vascular It is in contrast to ‘transcellular transport’, in which the genesis, Wnt and βcatenin signalling seems endothelium. The retinal pigment epithelium consists of a substances are transported through the cell. to be essential for expression of cerebral layer of epithelial cells, joined by tight junctions, that forms a barrier between the neuroretina and the choroid. Tripartite synapse endothelial cellspecific transporters such A tripartite (three-part) synapse consists of a as solute carrier family 2, facilitated glu Ependyma presynapse, a postsynapse and a glial cell functioning cose transporter member 1 (SlC2A1; also A thin cellular layer lining the ventricular system of the as a single unit. known as GlUT1), high affinity cationic brain. The cells of the ependyma are called ependymal cells and are a type of glia. They are linked by gap Xenobiotic-sensing nuclear receptor amino acid transporter 1 (SlC7A1; also junctions, which do not provide an impediment to diffusion A xenobiotic-activated transcription factor that controls known as CAT1) and large neutral amino of molecules, even against large proteins between the expression of proteins involved in xenobiotic acids transporter small subunit 1 (SlC7A5; cerebrospinal fluid and brain interstitial fluid. metabolism and efflux transport.

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Figure 3 | Barrier interfaces. a | endothelial cells (endo) in the neurovascular the physical barrier between the CSF-filled subarachnoid space (SAS) and unit have luminal tight junctions (shown by the arrow) that form the physical overlying structures. The blood vessels between0CVWT G4Gthe arachnoidXKGYU^0GWT membraneQUEKGPEG barrier of the interendothelial cleft. Outside the endothelial cell is a basement and the pial surface (PiA) have tight junctions (not shown). d | in early develop- membrane (bm) which also surrounds the pericytes (Peri). Around all of these ment the neuroependymal cells are connected to each other by strap junc- structures are the astrocyctic endfeet processes from nearby astrocytes. b | tions (shown by arrows) that are believed to form the physical barrier The endothelial cells of choroid plexus blood vessels are fenestrated and form restricting the passage of larger molecules, such as proteins, but not smaller a non-restrictive barrier (shown by dashed arrows) between the cerebrospinal molecules, such as sucrose. e | The mature adult ventricular ependyma does fluid (CSF) and blood vessel (bv). The epithelial cells (ep) have apical tight junc- not restrict the exchange of molecules (shown by dotted arrows). The neuro- tions (shown by arrows) that restrict intercellular passage of molecules. c | in vascular unit (a), blood–CSF barrier (b) and arachnoid barrier (c) are common the meninges, the blood vessels of the dura are fenestrated and provide little between developing and adult brain, whereas fetal neuroependyma (d) differs barrier function (not shown). However, the outer cells of the arachnoid mem- from adult ependyma (e). Figure is reproduced, with permission, from REF. 162 brane (Arach) have tight junctions (shown by arrows) and this cell layer forms © (2008) Cell Press.

The NVU in disease and NVU function, with consequent The complexity of the cellular interac The NVU is usually considered in the con oedema that may be life threatening. NVU tions in the NVU offers numerous potential text of its role in preventing CNS access of abnormalities themselves can result in dis targets for treatment. For example, one of drugs and proteins with neurotherapeutic tinct disease entities (see TABLE 1). These dis the newest treatments for multiple sclerosis potential. The NVU is also affected in many orders range from acute mountain sickness is a monoclonal antibody that binds the α4 CNS conditions and plays a part in their causing vasogenic oedema118–120 and hepatic integrin receptor found on leukocytes and pathology. Brain abscess, trauma, multiple encephalopathy causing astrocyte swelling that prevents adhesion of the leukocytes sclerosis, diabetic ketoacidosis and stroke in the NVU121,122, to malaria impacting the to brain endothelial cells126. This reduces can all alter brain endothelial cell function cerebral endothelium123–125. the effects of the leukocytes that lead to

the demyelination and brain injury seen in term ‘extended NVU’ (FIG. 1a) to underscore approaches154 will allow investigators to acute multiple sclerosis plaques. In addi the importance of blood cells and inflam examine discrete neuronal signalling events tion, molecules found on the luminal side of matory signals as key players in disturbed in the context of the NVU, providing a brain endothelial cells are now recognized as NVU and barrier function. We further degree of in vivo analytical power previously receptors or ligands for proteins associated anticipate that astrocytes may play an active achievable only using in vitro systems. As with various infectious microorganisms that part to maintain disturbed NVU function, brain vasculature is functionally implicated have a predisposition to invade the brain, for by feeding back pathological signals from in many brain diseases (TABLE 1), molecular example, malaria (see TABLE 1). Furthermore, the disturbed NVU to the BBB (FIG. 1c). changes in the NVU could be exploited as the large number of transporters and Indeed, astrocytes as well as microglial cells imageable biomarkers for early diagnosis receptors on the luminal and abluminal are physiological sensors of brain function or monitoring of the disease using targeted membranes of brain endothelial cells and and pathology145. The work by Appel and molecular imaging agents155; the added astrocyte endfeet provide numerous poten colleagues146, in which neuroprotective sig advantage of such biomarkers is their acces tial therapeutic targets for treatment of CNS nals may cause microglia to become protec sibility from the systemic circulation. diseases127. one example is coopting insu tive in axonal injury models, in Parkinson’s At a macroscopic level, analysis of drug lin receptors for transport of drugs across disease and in amyotrophic lateral sclerosis delivery to the CNS will be advanced by the NVU128, a possibility that is now being (in contrast to the majority of the literature, imaging technologies. For example, studies examined129,130. in which microglia are damaging) further in animals using positron emission tom Cerebral vascular abnormalities have emphasizes the need for greater communi ography (PeT) indicate that it is possible been reported in brains from patients with cation between neuroscientists and brain to assess endothelial Pgp function, and its Alzheimer’s disease and Parkinson’s dis barrier scientists. role in the uptake and binding of drugs ease131, and human genetics studies have in the intact CNS, by using suitable Pgp linked vascular phenotypes to amyloid imaging brain barrier function modulators that are labelled with a positron precursor protein (APP)132. Specifically, a At the microscopic level, new imaging tech emitting isotopes156 (FIG. 4). In fMRI, signal particular point mutation associated with niques, including confocal and timelapse intensity changes are detected as changes hereditary cerebral haemorrhage with microscopy, which allow simultaneous tag in local blood flow and oxygenation, pre amyloidosis of the Dutch type (hChWAD) ging and visualization of multiple molecular sumably linked to changes in neural activ leads to cerebral amyloid angiopathy targets, molecular imaging of brain cells ity. The opportunity exists to apply this (CAA) in both humans and transgenic in vitro and brain tissues ex vivo, have made technology (as well as other methods for mouse models133. CAA induces intracranial tremendous advances in recent years147. imaging cerebral perfusion, for example, haemorrhages, with cognitive decline and Although imaging techniques at the atomic dynamic magnetic resonance) with nano seizures, which may also contribute to the level and ‘labelless’ techniques such as particlebased brain mapping methods to pathology and symptoms of Alzheimer’s Raman spectroscopy148 have much improved advance our understanding of neuro–glio– disease. Patients who are amyloidβ A4 resolution, they can only be used to detect vascular coupling and BBB pathophysiol protein (APoe4)positive, who carry the one or a few molecular species simultane ogy157. As no single method can cover the most common lateonset genetic risk factor ously. With imaging mass spectroscopy several orders of magnitude in temporal for Alzheimer’s disease, also have a higher (IMS), tissue sections can be directly ana and spatial resolutions and at the same time rate of CAA134. Recent advances have also lysed for the spatial distribution of multiple capture cellular and vascular events, one described an influx and efflux mechanism molecular markers149. IMS is a powerful of the key opportunities to be harnessed in for amyloidβ, via advanced glycosylation method for ex vivo imaging biomarkers the future is a combination and integration end productspecific receptor (RAGe), that define particular regions, or following of data and knowledge obtained through lowdensity lipoprotein receptorrelated ‘biomarker’ responses to disease, pharmaco multimodal imaging techniques, such as protein (lRP1) and, more recently, Pgp20,60. logical treatment, electrical stimulation and MRI and PeT. These membrane proteins might be targets so on149. for modulating movement of amyloidβ out Further, the field of bioimaging relying Barriers to progress of the brain of patients with Alzheimer’s on confocal, multiphoton and spinning disk The most important barrier to progress disease to slow or reverse plaque formation. confocal microscopy has been enhanced in our understanding of the role of brain Although barrierspecific genes have not through the use of fluorescent murine trans barriers in brain functioning is the lack of been implicated in directly inducing CNS genic reporter systems, mostly using green communication between neuroscientists disease, there are genes associated with fluorescent protein (GFP) as a reporter. and brain barrier scientists. This lack of com barrier function that have been linked to These techniques have made great contri munication contributes to the omission of disease135–143. butions to in vivo tracking of exogenously the brain barrier sciences in interdisciplinary Recent data from the epilepsy field indi added cells (that is, tumour cells, immune education programs, thus perpetuating the cate a prominent role of interactions of leu cells and progenitor cells) and have gained gap between the fields. If the relationships cocytes, in particular PMN cells, with brain popularity as proxy reporters for endog among neurons, astrocytes and cells of the endothelium in the initiation of seizure enous genes (that is, transgenic mice)150–152. NVU are to be fully appreciated, it is essential activity144. This is a key observation that not These approaches have greatly enhanced our for researchers in both fields to expand their only points to the importance of the BBB understanding of the trafficking of inflam knowledge of the cellular and molecular in initiating pathology but also stresses the matory cells across the BBB in models of mechanisms at play in all cells of the NVU, need to consider the blood cells (leucocytes ischaemic brain injury and autoimmune not just those of the endothelial cell (cur in this case) as members of the family of demyelination, among others153. In addi rently studied by brain barrier scientists) or NVU cells. We therefore propose to use the tion, the recent introduction of optogenetic neurons and astrocytes (currently studied

=%?XGTCRCOKNKOCIKPIYKVJOKETQ2'6 models of the BBB in human diseases such as stroke is difficult since our clinical knowl %QPVTQN %[ENQURQTKP# edge in patients over time is so limited. Improved models may open up new avenues to define opportunities and time windows for therapeutic interventions.

Conclusions and future directions In order to further the science in both fields, it is important that understanding what con stitutes the functional blood–CNS interface under various physiological and pathophysi ological conditions is paramount for develop ing appropriate therapies to address different Figure 4 | MicroPeT images of the head biodistribution of a calcium channel blocker. Micro disease states. New and improved animal positron emission tomography (PeT) images of the head of a Wistar rat, showing the biodistribution models, including transgenic rodents, will be 11 of the calcium channel blocker [ C]verapamil, injected systemically,0CVWT eitherG4G aloneXKGYU (control)^0GWTQUEKGPEG or after beneficial in achieving this aim. The use of 11 pre-treatment of the animal with the P-glycoprotein (Pgp) inhibitor cyclosporin A. [ C]verapamil, a zebrafish as an easily accessible comparative substrate for the blood–brain barrier efflux transporter Pgp, gains access to the brain only after Pgp model for mechanistic in vivo studies should inhibition by cyclosporin A. images courtesy of P. elsinga, University Medical Center Groningen, The 160 Netherlands. be further explored . Investigations on the contribution of blood cells and inflamma tory signals as part of the NVU are needed. by neuroscientists). This broader approach based on twophoton excitation that have Although blood cells and inflammatory should include, for example, attention to all been employed to study dendritic functions signals are generally not considered part of classes of membrane proteins involved in and dynamics for investigating the func the NVU proper, they must be considered transport across the barriers and among cells tional interactions at the BBB interface. part of the extended NVU and are important of the NVU — such as ion transporters and Finally, there are misconceptions that mediators of CNS pathophysiology and need channels, nutrient transporters, drug trans need to be overcome, particularly with further investigation. Furthermore, animal porters — and to proteins that are involved in regard to the status of the NVU during models will be useful with application of transport mechanisms (including receptor development. A perception persists for advanced microscopy tools for realtime and mediated endocytosis) as well as the recep some researchers in the field of brain barrier spatial resolution of cellular interactions, tors and transduction pathways signalling to physiology, and therefore in the wider area signalling events and metabolism. these proteins. of neurobiology, that the barrier systems are Transporters, receptors and their signal Because the endothelial cells are thin immature in the developing brain in both ling pathways in the NVU are important and tightly embedded within the brain their structure and function. This misunder targets for improving CNS drug delivery and parenchyma, they are not easily isolated for standing of barrier function during neural brain protection, and in preventing CNS routine biochemical, molecular or cellular development represents an impediment to disease. Advancing knowledge of transporter analysis. This has posed substantial techni a full understanding of the biological proc function, expression, localization and regu cal difficulties that have delayed progress esses involved in barrier development and lation in the brain vasculature and CNS tis in the study of blood–brain interfaces. This the contribution of barrier functions to sues will surely aid progress. interface is a highly interactive structure, in neural development. The emphasis that is Further consideration of the role of the which endothelial cells engage with multi currently placed on understanding the role NVU in research into nervous system devel ple neighbouring cells including pericytes, of the NVU in neuronal development, and opment will likely continue to lend insight astrocytes, neurons and blood cells such as recent evidence that vascular and neuronal into both developmental neuroscience and leukocytes. Brain endothelial cells are very development have common mechanisms, the brain barrier sciences. There is also a flat cells, with a thickness of less than 0.5 μm make a fruitful merger of fields possible. need for improved animal models that are outside the nuclear region, comparable in Similarly, there are misconceptions that appropriate for studies investigating the links size to dendritic spines. The major technical the blood–brain interface is either open or between barrier versus neural development. difficulty here is the fact that the numer closed in brain tumours158, that is, opened Furthermore, neuroscientists should con ous cellular partners at the barrier interface as in systemic tissues or closed as in normal sider the possible contributions of the NVU interact with each other in a very thin brain. In truth, cerebral microvessels cours to the interpretation of their neuroscience compartment. As such, it is very inacces ing through most malignant brain tumours data, including analysis of genetically engi sible. In addition, barrier function can only have intermediate paracellular permeability neered mouse models, drug efficacy studies be studied in vivo because blood cells and so that some drugs and proteins can move and so forth. Acceptance of the recent evi blood flow are now considered as essential from blood into tumour158. however, recent dence in support of barrier function in the elements for its normal function. The pres evidence has shown that enhanced delivery developing brain, along with the advent of ence of an intact circulation is a technical of antitumour agents by further opening the an increased research focus on the develop difficulty for microscopic imaging studies blood–brain interface may improve survival ment of brain barriers will help to overcome because of the presence of mechanical vas in malignant brain tumour patients159. one impediments to progress in these fields. cular pulsations. We propose to apply highly key obstacle is the lack of efficacious yet The simultaneous and remarkable specialized microscope imaging techniques noninvasive methods. Modelling animal advancements in neuroscience and brain

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Gastroenterology 121, 1109–1119 (2001). D. A. Glucocorticoids induce transactivation of tight 205. O’Donnell, M. E., Duong, V., Suvatne, J., Foroutan, S. junction genes occludin and claudin-5 in retinal & Johnson, D. M. Arginine vasopressin stimulation of Acknowledgements endothelial cells via a novel cis-element. Exp. Eye Res. cerebral microvascular endothelial cell Na-K-Cl The meeting on which this report was based was partially 86, 867–878 (2008). cotransporter activity is V1 receptor and [Ca] funded by an R13 grant from the US National Institutes of 194. Harke, N., Leers, J., Kietz, S., Drenckhahn, D. & dependent. Am. J. Physiol. Cell Physiol. 289, Health (Grant 5 R13 CA086959-10). We would like to thank Forster, C. Glucocorticoids regulate the human C283–292 (2005). all of the people who attended the Engaging Neuroscience to occludin gene through a single imperfect palindromic 206. O’Donnell, M. E., Lam, T. I., Tran, L. & Anderson, S. E. 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