Expanding the MiniAp‐4 BBB‐shuttle family: Evaluation of proline cis‐trans ratio as tool to fine‐tune transport

Venoms have recently emerged as a promising field in drug discovery due to their good selectivity and affinity for a wide range of biological targets. Among their multiple potential applications, venoms are a rich source of blood‐brain barrier (BBB) peptide shuttles. We previously described a short nontoxic derivative of apamin, MiniAp‐4, which can transport a wide range of cargoes across the BBB. Here, we have studied the conformation of the proline residue of a range of MiniAp‐4 analogues by high‐field NMR techniques, with the aim to identify whether there is a direct relation between the cis/trans population and a range of features, such as the capacity to transport molecules across a human‐based cellular model and stability in various media. The most promising candidate showed improved transport properties for a relevant small fluorophore.     

Enhanced transport of plant‐produced rabies single‐chain antibody‐RVG peptide fusion protein across an in cellulo blood–brain barrier device

The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood–brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single‐chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv‐RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv‐RVG fusion. Both ScFv and ScFv‐RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv‐RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant‐produced ScFv‐RVG^P fusion could translocate across the cells. This study indicates that the plant‐produced ScFv‐RVG^P fusion protein was able to cross the in celluloBBB and neutralize RABV.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Age‐related ultrastructural changes of the basement membrane in the mouse blood‐brain barrier

The blood‐brain barrier (BBB) is essential for a functional neurovascular unit. Most studies focused on the cells forming the BBB, but very few studied the basement membrane (BM) of brain capillaries in ageing. We used transmission electron microscopy and electron tomography to investigate the BM of the BBB in ageing C57BL/6J mice. The thickness of the BM of the BBB from 24‐month‐old mice was double as compared with that of 6‐month‐old mice (107 nm vs 56 nm). The aged BBB showed lipid droplets gathering within the BM which further increased its thickness (up to 572 nm) and altered its structure. The lipids appeared to accumulate toward the glial side of the BM. Electron tomography showed that the lipid‐rich BM regions are located in small pockets formed by the end‐feet of astrocytes. These findings suggest an imbalance of the lipid metabolism and that may precede the structural alteration of the BM. These alterations may favour the accretion of abnormal proteins that lead to neurodegeneration in ageing. These findings warrant further investigation of the BM of brain capillaries and of adjoining cells as potential targets for future therapies.     

Dl‐3n‐butylphthalide improves traumatic brain injury recovery via inhibiting autophagy‐induced blood‐brain barrier disruption and cell apoptosis

Blood‐brain barrier (BBB) disruption and neuronal apoptosis are important pathophysiological processes after traumatic brain injury (TBI). In clinical stroke, Dl‐3n‐butylphthalide (Dl‐NBP) has a neuroprotective effect with anti‐inflammatory, anti‐oxidative, anti‐apoptotic and mitochondrion‐protective functions. However, the effect and molecular mechanism of Dl‐NBP for TBI need to be further investigated. Here, we had used an animal model of TBI and SH‐SY5Y/human brain microvascular endothelial cells to explore it. We found that Dl‐NBP administration exerts a neuroprotective effect in TBI/OGD and BBB disorder, which up‐regulates the expression of tight junction proteins and promotes neuronal survival via inhibiting mitochondrial apoptosis. The expressions of autophagy‐related proteins, including ATG7, Beclin1 and LC3II, were significantly increased after TBI/OGD, and which were reversed by Dl‐NBP treatment both in vivo and in vitro. Moreover, rapamycin treatment had abolished the effect of Dl‐NBP for TBI recovery. Collectively, our current studies indicate that Dl‐NBP treatment improved locomotor functional recovery after TBI by inhibiting the activation of autophagy and consequently blocking the junction protein loss and neuronal apoptosis. Dl‐NBP, as an anti‐inflammatory and anti‐oxidative drug, may act as an effective strategy for TBI recovery.     

Tumor necrosis factor receptor‐associated factor 5 is an essential mediator of ischemic brain infarction

Tumor necrosis factor receptor‐associated factor 5 (TRAF5) is an adaptor protein of the tumor necrosis factor (TNF) receptor superfamily and the interleukin‐1 receptor/Toll‐like receptor superfamily and plays important roles in regulating multiple signaling pathways. This study was conducted to investigate the role of TRAF5 in the context of brain ischemia/reperfusion (I/R) injury. Transient occlusion of the middle cerebral artery was performed on TRAF5 knockout mice (KO), neuron‐specific TRAF5 transgene (TG), and the appropriate controls. Compared with the WT mice, the TRAF5 KO mice showed lower infarct volumes and better outcomes in the neurological tests. A low neuronal apoptosis level, an attenuated blood‐brain barrier (BBB) disruption and an inhibited inflammatory response were exhibited in TRAF5 KO mice. TRAF5 TG mice exhibited an opposite phenotype. Moreover, the Akt/FoxO1 signaling pathway was enhanced in the ischemic brains of the TRAF5 KO mice. These results provide the first demonstration that TRAF5 is a critical mediator of I/R injury in an experimental stroke model. The Akt /FoxO1 signaling pathway probably plays an important role in the biological function of TRAF5 in this model.     

Glial‐cell‐mediated re‐induction of the blood‐brain barrier phenotype in brain capillary endothelial cells: A differential gel electrophoresis study

In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood‐brain barrier (BBB) models. Hence, the re‐induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re‐induction remain largely unknown. Here, we used a DIGE‐based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re‐induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine pathway in the BBB phenotype re‐induction process and we discuss asymmetric dimethylarginine's potential role in regulating endothelial function (in addition to its role as a by‐product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re‐induced BBB functions than in cells with limited BBB functions.     

Pharmacokinetics and distribution of fluvoxamine to the brain in rats under oxidative stress

Abstract

The effects of oxidative stress (OS) on the pharmacokinetics of fluvoxamine (FLV), particularly on FLV distribution in the plasma, were studied in ferric-nitrilotriacetate-induced OS rat models (OS rats). The study protocol involved a continuous FLV infusion (25.0 μg/kg/min). The resulting mean plasma FLV concentration measured in steady state OS rats was 0.13?±?0.01 μg/mL, which was significantly lower than plasma concentrations measured in control rats (0.19?±?0.01 μg/mL). Moreover, the mean FLV concentration in the OS rat brain (0.51?±?0.08 μg/g) was determined to be approximately half the concentration in control rat brains (0.95?±?0.11 μg/g). The FLV concentrations in both the unbound fraction of plasma and erythrocytes of OS rats were significantly greater than that of control rats. These results suggest the potential attenuation of FLV's pharmacological effects in patients under OS.     

Pathogen translocation across the blood–brain barrier

Neurological manifestations caused by neuroinvading pathogens are typically attributed to penetration of the blood–brain barrier (BBB) and invasion of the central nervous system. However, the mechanisms used by many pathogens (such as Borrelia ) to traverse the BBB are still unclear. Recent studies revealed that microbial translocation across the BBB must involve a repertoire of microbial–host interactions (receptor–ligand interactions). However, the array of interacting molecules responsible for the borrelial translocation is not yet clearly known. Pathogens bind several host molecules (plasminogen, glycosaminoglycans, factor H, etc.) that might mediate endothelial interactions in vivo . This review summarizes our current understanding of the pathogenic mechanisms involved in the translocation of the BBB by neuroinvasive pathogens.     

D1 receptor‐mediated endogenous tPA upregulation contributes to blood‐brain barrier injury after acute ischaemic stroke

Blood‐brain barrier (BBB) integrity injury within the thrombolytic time window is becoming a critical target to reduce haemorrhage transformation (HT). We have previously reported that BBB damage was initially damaged in non‐infarcted striatum after acute ischaemia stroke. However, the underlying mechanism is not clear. Since acute ischaemic stroke could induce a significant increase of dopamine release in striatum, in current study, our aim is to investigate the role of dopamine receptor signal pathway in BBB integrity injury after acute ischaemia using rat middle cerebral artery occlusion model. Our data showed that 2‐h ischaemia induced a significant increase of endogenous tissue plasminogen activator (tPA) in BBB injury area and intra‐striatum infusion of tPA inhibitor neuroserpin, significantly alleviated 2‐h ischaemia‐induced BBB injury. In addition, intra‐striatum infusion of D1 receptor antagonist SCH23390 significantly decreased ischaemia‐induced upregulation of endogenous tPA, accompanied by decrease of BBB injury and occludin degradation. More important, inhibition of hypoxia‐inducible factor‐1 alpha with inhibitor YC‐1 significantly decreased 2‐h ischaemia‐induced endogenous tPA upregulation and BBB injury. Taken together, our data demonstrate that acute ischaemia disrupted BBB through activation of endogenous tPA via HIF‐1α upregulation, thus representing a new therapeutic target for protecting BBB after acute ischaemic stroke.     

Claudin‐5a in developing zebrafish brain barriers: another brick in the wall

Claudins serve essential roles in regulating paracellular permeability properties within occluding junctions. Recent studies have begun to elucidate developmental roles of claudins within immature tissues. This work has uncovered an involvement of several claudins in determining tight junction properties that have an effect on embryonic morphogenesis and physiology. During zebrafish brain morphogenesis, Claudin‐5a determines the paracellular permeability of tight junctions within a transient neuroepithelial‐ventricular barrier that maintains the hydrostatic fluid pressure required for brain ventricular lumen expansion. However, the roles of Claudins in development may well extend beyond being mere junctional components. Several post‐translational modifications of Claudins have been characterized that indicate a direct regulation by developmental signals. This review focuses on the involvement of Claudin‐5a in cerebral barrier formation in the zebrafish embryo and includes some speculations about possible modes of regulation.     

Modulation of insulin transport in rat brain microvessel endothelial cells by an ecto-phosphatase activity.

The physiological function of alkaline phosphatase (ALP) remains controversial. It was recently suggested that this membrane-bound enzyme has a role in the modulation of transmembranar transport systems into hepatocytes and Caco-2 cells. ALP activity expressed on the apical surface of blood-brain barrier cells, and its relationship with (125)I-insulin internalization were investigated under physiological conditions using p-nitrophenylphosphate (p-NPP) as substrate. For this, an immortalized cell line of rat capillary cerebral endothelial cells (RBE4 cells) was used. ALP activity and (125)I-insulin internalization were evaluated in these cells. The results showed that RBE4 cells expressed ALP, characterized by an ecto-oriented active site which was functional at physiological pH. Orthovanadate (100 microM), an inhibitor of phosphatase activities, decreased both RBE4-ALP activity and (125)I-insulin internalization. In the presence of L-arginine (1 mM) or adenosine (100 microM) RBE4-ALP activity and (125)I-insulin, internalization were significantly reduced. However, D-arginine (1 mM) had no significant effect. Additionally, RBE4-ALP activity and (125)I-insulin internalization significantly increased in the presence of the bioflavonoid kaempferol (100 microM), of the phorbol ester PMA (80 nM), IBMX (1 mM), progesterone (200 microM and 100 microM), beta-estradiol (100 microM), iron (100 microM) or in the presence of all-trans retinoic acid (RA) (10 microM). The ALP inhibitor levamisole (500 microM) was able to reduce (125)I-insulin internalization to 69.1 +/- 7.1% of control. Our data showed a positive correlation between ecto-ALP activity and (125)I-insulin incorporation (r = 0.82; P < 0.0001) in cultured rat brain endothelial cells, suggesting that insulin entry into the blood-brain barrier may be modulated through ALP.     

Membrane mediated development of the vertebrate blood‐gas‐barrier

During embryonic lung development, establishment of the gas‐exchanging units is guided by epithelial tubes lined by columnar cells. Ultimately, a thin blood‐gas barrier (BGB) is established and forms the interface for efficient gas exchange. This thin BGB is achieved through processes, which entail lowering of tight junctions, stretching, and thinning in mammals. In birds the processes are termed peremerecytosis, if they involve cell squeezing and constriction, or secarecytosis, if they entail cutting cells to size. In peremerecytosis, cells constrict at a point below the protruding apical part, resulting in fusion of the opposing membranes and discharge of the aposome, or the cell may be squeezed by the more endowed cognate neighbors. Secarecytosis may entail formation of double membranes below the aposome, subsequent unzipping and discharge of the aposome, or vesicles form below the aposome, fuse in a bilateral manner, and release the aposome. These processes occur within limited developmental windows, and are mediated through cell membranes that appear to be of intracellular in origin. In addition, basement membranes (BM) play pivotal roles in differentiation of the epithelial and endothelial layers of the BGB. Laminins found in the BM are particularly important in the signaling pathways that result in formation of squamous pneumocytes and pulmonary capillaries, the two major components of the BGB. Some information exists on the contribution by BM to BGB formation, but little is known regarding the molecules that drive peremerecytosis, or even the origins and composition of the double and vesicular membranes involved in secarecytosis. Birth Defects Research (Part C) 108:85–97, 2016. © 2016 Wiley Periodicals, Inc.     

Afamin is synthesized by cerebrovascular endothelial cells and mediates α-tocopherol transport across an in vitro model of the blood–brain barrier

α-Tocopherol (αTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of αTocH transport into the CNS, transfer mechanisms across the blood–brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known αTocH-binding protein, contributes to αTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, αTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high-density lipoprotein-mediated transport, an essential and in vivo operating αTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to αTocH homeostasis at the BBB in vivo .     

The role of LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis in Memantine mediated protective effects on blood‐brain barrier in AD microenvironment

The dysfunction of the blood‐brain barrier (BBB) is one of the main pathological features of Alzheimer's disease (AD). Memantine (MEM), an N‐methyl‐d ‐aspartate (NMDA) receptor antagonist, has been reported that been used widely for AD therapy. This study was performed to demonstrate the role of the MEM in regulating BBB permeability in AD microenvironment as well as its possible mechanisms. The present study showed that LINC00094 was dramatically increased in Abeta_1‐42‐incubated microvascular endothelial cells (ECs) of BBB model in vitro. Besides, it was decreased in MEM‐incubated ECs. Silencing LINC00094 significantly decreased BBB permeability, meanwhile up‐regulating the expression of ZO‐1, occludin and claudin‐5. Furthermore, silencing LINC00094 enhance the effect of MEM on decreasing BBB permeability in AD microenvironment. The analysis of the mechanism demonstrated that reduction of LINC00094 inhibited Endophilin‐1 expression by up‐regulating miR‐224‐4p/miR‐497‐5p, promoted the expression of ZO‐1, occludin and claudin‐5, and ultimately alleviated BBB permeability in AD microenvironment. Taken together, the present study suggests that the MEM/LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis plays a crucial role in the regulation of BBB permeability in AD microenvironment. Silencing LINC00094 combined with MEM provides a novel target for the therapy of AD.     

Increase of tumor necrosis factor‐α in the blood induces early activation of matrix metalloproteinase‐9 in the brain

Increases of cytokine in the blood play important roles in the pathogenesis of influenza‐associated encephalopathy. TNF‐α was administered intravenously to wild‐type mice, after which blood, CSF and brain tissue were obtained, and changes in BBB permeability, the amounts of MMP‐9 and TIMP‐1, and the localization of activated MMP were assessed. There was a significant increase in BBB permeability after 6 and 12 hr. MMP‐9 was increased after 3 hr in the brain and cerebrospinal fluid, which was earlier than in the serum. TIMP‐1 protein in the brain increased significantly after MMP‐9 had increased. Activation of MMP‐9 was observed in neurons in the cerebral cortex and hippocampus, and in vascular endothelial cells. These findings suggest that an increase in blood TNF‐α promotes activation of MMP‐9 in the brain, and may also induce an increase in permeability of the BBB. Early activation of MMP‐9 in the brain may contribute to an early onset of neurological disorders and brain edema prior to multiple organ failure in those inflammatory diseases associated with highly increased concentrations of TNF‐α in the blood, such as sepsis, burns, trauma and influenza‐associated encephalopathy.     

Brain capillary endothelium and choroid plexus epithelium regulate transport of transferrin-bound and free iron into the rat brain

Iron transport into the CNS is still not completely understood. Using a brain perfusion technique in rats, we have shown a significant brain capillary uptake of circulating transferrin (Tf)-bound and free 59Fe (1 nm) at rates of 136 +/- 26 and 182 +/- 23 microL/g/min, respectively, while their respective transport rates into brain parenchyma were 1.68 +/- 0.56 and 1.52 +/- 0.48 microL/g/min. Regional Tf receptor density (Bmax) in brain endothelium determined with 125I-holo-Tf correlated well with 59Fe-Tf regional brain uptake rates reflecting significant vascular association of iron. Tf-bound and free circulating 59Fe were sequestered by the choroid plexus and transported into the CSF at low rates of 0.17 +/- 0.01 and 0.09 +/- 0.02 microL/min/g, respectively, consistent with a 10-fold brain-CSF concentration gradient for 59Fe, Tf-bound or free. We conclude that transport of circulating Tf-bound and free iron could be equally important for its delivery to the CNS. Moreover, data suggest that entry of Tf-bound and free iron into the CNS is determined by (i) its initial sequestration by brain capillaries and choroid plexus, and (ii) subsequent controlled and slow release from vascular structures into brain interstitial fluid and CSF.