Quantitative Membrane Protein Expression at the Blood–Brain Barrier of Adult and Younger Cynomolgus Monkeys

Abstract Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.

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Methamphetamine Alters Blood Brain Barrier Protein Expression in Mice, Facilitating Central Nervous System Infection by Neurotropic Cryptococcus neoformans

Journal of Emergency Medicine ◽

10.1016/j.jemermed.2013.09.003 ◽

2013 ◽

Vol 45 (5) ◽

pp. 807-808

Author(s):

Benjamin C. Murphy

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Protein Expression ◽

Cryptococcus Neoformans ◽

Blood Brain Barrier ◽

Central Nervous System Infection ◽

Brain Barrier ◽

Blood Brain

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Expression of Neuronal CXCL10 Induced by Rabies Virus Infection Initiates Infiltration of Inflammatory Cells, Production of Chemokines and Cytokines, and Enhancement of Blood-Brain Barrier Permeability

Journal of Virology ◽

10.1128/jvi.02154-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 870-876 ◽

Cited By ~ 42

Author(s):

Qingqing Chai ◽

Ruiping She ◽

Ying Huang ◽

Zhen F. Fu

Keyword(s):

Protein Expression ◽

Blood Brain Barrier ◽

Rabies Virus ◽

Inflammatory Cells ◽

Brain Barrier ◽

Blood Brain ◽

Brain Barrier Permeability ◽

Rabies Virus Infection ◽

Ifn Γ ◽

Bbb Permeability

It has been shown that enhancement of blood-brain barrier (BBB) permeability is modulated by the expression of chemokines/cytokines and reduction of tight junction (TJ) proteins in the brains of mice infected with rabies virus (RABV). Since CXCL10 was found to be the most highly expressed chemokine, its temporal and spatial expression were determined in the present study. The expression of the chemokine CXCL10 was initially detected in neurons as early as 3 days postinfection (p.i.) in the brains of RABV-infected mice, after which it was detected in microglia (6 days p.i.) and astrocytes (9 days p.i.). Neutralization of CXCL10 by treatment with anti-CXCL10 antibodies reduced gamma interferon (IFN-γ) production and Th17 cell infiltration, as well as restoring TJ protein expression and BBB integrity. Together, these data suggest that it is the neuronal CXCL10 that initiates the cascade that leads to the activation of microglia/astrocytes, infiltration of inflammatory cells, expression of chemokines/cytokines, reduction of TJ protein expression, and enhancement of the BBB permeability.

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Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.3.h1241 ◽

2001 ◽

Vol 280 (3) ◽

pp. H1241-H1248 ◽

Cited By ~ 168

Author(s):

J. D. Huber ◽

K. A. Witt ◽

S. Hom ◽

R. D. Egleton ◽

K. S. Mark ◽

...

Keyword(s):

Protein Expression ◽

Western Blot ◽

Blood Brain Barrier ◽

Inflammatory Pain ◽

Molecular Properties ◽

Brain Barrier ◽

Peripheral Inflammation ◽

Blood Brain ◽

Junctional Protein

Effects of inflammatory pain states on functional and molecular properties of the rat blood-brain barrier (BBB) were investigated. Inflammation was produced by subcutaneous injection of formalin, λ-carrageenan, or complete Freund's adjuvant (CFA) into the right hind paw. In situ perfusion and Western blot analyses were performed to assess BBB integrity after inflammatory insult. In situ brain perfusion determined that peripheral inflammation significantly increased the uptake of sucrose into the cerebral hemispheres. Capillary depletion and cerebral blood flow analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Western blot analyses showed altered tight junctional protein expression during peripheral inflammation. Occludin significantly decreased in the λ-carrageenan- and CFA-treated groups. Zonula occluden-1 expression was significantly increased in all pain models. Claudin-1 protein expression was present at the BBB and remained unchanged during inflammation. Actin expression was significantly increased in the λ-carrageenan- and CFA-treated groups. We have shown that inflammatory-mediated pain alters both the functional and molecular properties of the BBB. Inflammatory-induced changes may significantly alter delivery of therapeutic agents to the brain, thus affecting dosing regimens during chronic pain.

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EXTH-21. DIFFERENTIAL PROTEIN EXPRESSION LEVELS OF ABC AND SOLUTE CARRIER TRANSPORTERS AT THE BLOOD-BRAIN BARRIER OF HUMAN BRAIN AND GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noz175.355 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi86-vi86

Author(s):

Xun Bao ◽

Jianmei Wu ◽

Youming Xie ◽

Seongho Kim ◽

Sharon Michelhaugh ◽

...

Keyword(s):

Protein Expression ◽

Human Brain ◽

Blood Brain Barrier ◽

Mouse Brain ◽

Brain Barrier ◽

Normal Brain ◽

Drug Penetration ◽

Expression Levels ◽

Transporter Protein ◽

Blood Brain

Abstract BACKGROUND Mechanistic understanding and quantitative prediction of drug penetration across the human blood-brain barrier (BBB) is critical to rational drug development and treatment for brain cancer especially glioblastoma. However, prediction of drug brain/tumor penetration has been significantly hindered mainly due to the lack of quantitation data on transporter protein expression levels at the human BBB. This study was to determine protein expression levels of major transporters and markers at the BBB of human brain and glioblastoma. METHOD The absolute protein expression levels of major transporters and markers were determined in isolated microvessels of human brain (N=30), glioblastoma (N=47), rat (N=10) and mouse brain (N=10), using liquid chromatography with tandem mass spectrometry (LC-MS/MS) based targeted proteomics. RESULTS In isolated microvessels of 30 human brain specimens, the median protein abundances for ABCB1, ABCG2, GLUT1, GLUT3, LAT1, MCT1, Na/K ATPase, and Claudin-5 were 3.38, 6.21, 54.51, 7.17, 3.42, 5.71, 32.14, and 1.15 fmol/µg protein, respectively. In glioblastoma microvessels, ABCB1, ABCG2, MCT1, GLUT1, Na/K ATPase, and Claudin-5 protein levels were significantly reduced, while LAT1 was increased and GLU1 remained the same. ABCC4, OATP1A2, OATP2B1, and OAT3 were undetectable in isolated microvessels of both human brain and glioblastoma. There was species difference in transporter protein expression levels in isolated microvessels of human, rat and mouse brain. Specifically, rodent BBB expressed significantly higher ABCB1 but similar ABCG2, as compared to human BBB. CONCLUSION The physical and biochemical barriers of the BBB in glioblastomas are largely disrupted, as indicated by the loss or significant reduction in protein expression of the tight junction marker (claudin-5), brain endothelial cell marker (GLUT1), and major efflux transporters (ABCB1 and ABCG2) as compared to normal human BBB. Differential BBB transporter protein expression levels provides mechanistic and quantitative basis for the prediction of heterogeneous drug penetration into human normal brain and glioblastoma.

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Protein Kinase C Activation Modulates Reversible Increase in Cortical Blood–Brain Barrier Permeability and Tight Junction Protein Expression during Hypoxia and Posthypoxic Reoxygenation

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2010.119 ◽

2010 ◽

Vol 30 (11) ◽

pp. 1847-1859 ◽

Cited By ~ 70

Author(s):

Colin L Willis ◽

Diana S Meske ◽

Thomas P Davis

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Expression ◽

Tight Junction ◽

Vascular Permeability ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Kinase C ◽

Blood Brain ◽

Junction Protein

Hypoxia (Hx) is a component of many disease states including stroke. Ischemic stroke occurs when there is a restriction of cerebral blood flow and oxygen to part of the brain. During the ischemic, and subsequent reperfusion phase of stroke, blood–brain barrier (BBB) integrity is lost with tight junction (TJ) protein disruption. However, the mechanisms of Hx and reoxygenation (HR)-induced loss of BBB integrity are not fully understood. We examined the role of protein kinase C (PKC) isozymes in modifying TJ protein expression in a rat model of global Hx. The Hx (6% O2) induced increased hippocampal and cortical vascular permeability to 4 and 10 kDa dextran fluorescein isothiocyanate (FITC) and endogenous rat-IgG. Cortical microvessels revealed morphologic changes in nPKC-θ distribution, increased nPKC-θ and aPKC-ζ protein expression, and activation by phosphorylation of nPKC-θ (Thr538) and aPKC-ζ (Thr410) residues after Hx treatment. Claudin-5, occludin, and ZO-1 showed disrupted organization at endothelial cell margins, whereas Western blot analysis showed increased TJ protein expression after Hx. The PKC inhibition with chelerythrine chloride (5 mg/kg intraperitoneally) attenuated Hx-induced hippocampal vascular permeability and claudin-5, PKC (θ and ζ) expression, and phosphorylation. This study supports the hypothesis that nPKC-θ and aPKC-ζ signaling mediates TJ protein disruption resulting in increased BBB permeability.

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Maternal glucocorticoid exposure alters tight junction protein expression in the brain of fetal sheep

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00828.2009 ◽

2010 ◽

Vol 298 (1) ◽

pp. H179-H188 ◽

Cited By ~ 26

Author(s):

Grazyna B. Sadowska ◽

Shadi N. Malaeb ◽

Barbara S. Stonestreet

Keyword(s):

Spinal Cord ◽

Cerebral Cortex ◽

Protein Expression ◽

Tight Junction ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Barrier Permeability ◽

Blood Brain Barrier Permeability ◽

Blood Brain ◽

Brain Barrier Permeability

We examined the expression of tight junction (TJ) proteins in the cerebral cortex, cerebellum, and spinal cord of fetuses after maternal treatment with single and multiple courses of dexamethasone. Ewes received either single courses of four 6-mg dexamethasone or placebo injections every 12 h for 48 h between 104 and 107 days or the same treatment once a week between 76–78 and 104–107 days of gestation. TJ protein expression was determined by Western immunoblot analysis on tissue harvested at 105–108 days of gestation. Blood-brain barrier permeability has been previously quantified with the blood-to-brain transfer constant ( Ki) with α-aminoisobutyric acid ( 39 ). After a single course of dexamethasone, claudin-5 increased ( P < 0.05) in the cerebral cortex, occludin and claudin-1 increased in the cerebellum, and occludin increased in the spinal cord. After multiple dexamethasone courses, occludin and zonula occludens (ZO)-1 increased in the cerebral cortex, and occludin and claudin-1 increased in the cerebellum. Junctional adhesion molecule-A and ZO-2 expressions did not change. Linear regression comparing Ki to TJ proteins showed inverse correlations with claudin-1 and claudin-5 in the cerebral cortex after a single course and ZO-2 in the spinal cord after multiple courses and direct correlations with ZO-1 in the cerebellum and spinal cord after multiple courses. We conclude that maternal glucocorticoid treatment increases the expression of specific TJ proteins in vivo, patterns of TJ protein expression vary after exposure to single and multiple glucocorticoid courses, and decreases in blood-brain barrier permeability are associated with increases in claudin-1, claudin-5, and ZO-2 expression and decreases in ZO-1 expression. In utero glucocorticoid exposure alters the molecular composition of the barrier and affects fetal blood-brain barrier function.

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