ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT financed … · 2016-09-07 · 2016. november 2. /...

TÁMOP-4.1.1.C-13/1/KONV-2014-0001 projekt

Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB 521. www.brc.hu

„Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére”

program keretében finanszírozott

ELŐADÁS KIVONAT

CLASSROOM LECTURE HANDOUT

financed by the program

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”

Dátum / Date:

2016. NOVEMBER 2. / NOVEMBER 2, 2016

Helyszín / Place:

MTA SZBK BIOFIZIKAI INTÉZET, TANÁCSTEREM / LECTURE ROOM, INST. OF BIOPHYSICS, BIOLOGICAL RESEARCH CENTRE

SZEGED, TEMESVÁRI KRT. 62.

Az előadás címe / Title of the presentation:

MOLECULAR STRUCTURE AND FUNCTIONS OF BIOLOGICAL BARRIERS

Előadó / Speaker:

IMOLA WILHELM

I. Wilhelm November 2, 2016

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MOLECULAR STRUCTURE AND FUNCTION OF

BIOLOGICAL BARRIERS Imola Wilhelm

Institute of BiophysicsNovember 2, 2016

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001

-These barriers are paracellular diffusion barriers and they separate internal and external fluid compartments, an indispensable prerequisite for every organ development and function.

-Compartimentalization is of vital importance for the function of multicellular organisms.

-The integrity and function of many vertebrate organs depend on cellular barriers that are mainly formed by intercellular protein complexes of the plasma membrane.

CELLULAR BARRIERS

CELL TYPES INVOLVED-epithelial cells-endothelial cells (tight only in the brain)

Intercellular junctions -tight junctions (most apical structures)-adherens junctions-desmosomes-gap junctions


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ORGANS PROTECTED BY BIOLOGICAL BARRIERS

SKIN

Protects the organism from the environment.

Localization:stratifying

epithelial tissue (keratinocytes)

http://mutagenetix.utsouthwestern.edu/phenotypic/phenotypic_rec.cfm?pk=359

KIDNEYGlomerulus

Loop of Henle

Proximal tubule

Distal tubule

Collecting duct

Separates urinefrom renal parenchyma

Localization:epithelial cells along the nephron

http://www.elcamino.edu/faculty/msteinberg/a&p_images_kidney%20_lobe.htm


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INTESTINE

Localization:epithelial cells covering the lumen of the intestine

Separates the content of the intestine from the rest of the body

http://www.nature.com/nri/journal/v10/n2/fig_tab/nri2707_F1.html

LIVER

Localization: hepatocytes and bile duct cells Separates blood from bile

http://jnm.snmjournals.org/content/51/2/301.figures-only

LUNG

Localization:bronchial and alveolar epithelial cells

Separates the airspace from blood

http://hyperphysics.phy-astr.gsu.edu/hbase/biology/respir.html


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TESTISbetween adjacent Sertoli cells of seminiferoustubule: blood testis barrier

BRAINBlood-CSF barrier: epithelial cells of

the choroid plexus

Blood brain barrier: endothelial cells of cerebral capillaries

PLACENTAbetween trophoblasts

MOLECULAR STRUCTUREOF PARACELLULAR BARRIERS:

TIGHT JUNCTIONS

ROLE OF TIGHT JUNCTIONS• Barrier: restrict paracellular movement of substances

•Fence: separate the apical membrane from the basolateral membrane: cell polarity

xx

xx x

x

apical basolateral


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TIGHT JUNCTION PROTEINS• TRANSMEMBRANE PROTEINS OF TIGHT JUNCTIONS •TETRASPAN PROTEINS

•Claudins (in CECs: claudin-5, claudin-1, -3, -12, claudin-8, -10, -15, -17, -19, -20, -22 or -23 •Marvel family

•Occludin (multiple isoforms)•Tricellulin (Marvel D2)•Marvel D3

•SINGLE SPAN•Immunoglobulin superfamily (IgSF) type (JAMs, CAR, ESAM) (in CECs: JAM-A JAM-B and JAM-C, ESAM (endothelial cell-selective adhesion molecule)•Non-IgSF type single span proteins (CRB3, Bves)

• PLAQUE PROTEINS OF THE TJ•PDZ proteins

•MAGUK (ZO-1, ZO-2, ZO-3)•MAGI (MAGI-1, 2, 3)•PAR (PAR-3, PAR-6)•MUPP1•AF-6/afadin

•non-PDZ proteins: cingulin, symplekin, 7H6, Pilt

TRANSMEMBRANE PROTEINS OF THE TIGHT JUNCTIONS


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(Furuse et al, J. Cell Biol. 123, 1993)

FOLDING MODEL OF OCCLUDIN

FUNCTION OF OCCLUDIN AT THE TIGHT JUNCTIONS

-Overexpression of occludin increases TEER by 30-40%.

-Blocking peptides against the second extracellular loop decreases TEER.

-Binding to ZO-1 is necessary for TJ localization.

-There are tight junctions in occludin deficient cells: redundant function.

REGULATION OF OCCLUDIN

-posttranslational modifications (phosphorylation)

-internalization

-proteolytic degradation (metalloproteinases, proteasome)


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TISSUE BARRIERS eLocation ID e24782Doi 10.4161/tisb.24782

CLAUDINS

FUNCTIONS OF CLAUDINS-claudin family: more than 20 members (currently 27)

-20-27 kDa

-claudins constitute the backbone of the tight junctions.

-claudin-5 loss opens the barrier for molecules smaller than 800 Da

-depending on the composition of the extracellular loops claudins

have distinct functions: some of them are tightening the

paracellular barrier, while others are pore forming.

-regulation of ion homeostasis

-homo- and heterophylic binding

HOMO- AND HETEROPHYLIC BINDING OF CLAUDINS

Biochim Biophys Acta. 2008 Mar;1778(3):631-45. Structure and function of claudins.Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J, Blasig IE.


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http://php.med.unsw.edu.au/cellbiology/index.php?title=File:Tricellulin.jpg

J Cell Biol. 1998 Jul 13;142(1):117-27Martin-Padura et al.

-immunglobulin superfamily-JAM-A, JAM-B, JAM-C, JAM-4, JAM-L-molecular masses of 30–40 kDa-extracellular domain, a transmembrane segment and a short cytoplasmic tail

- homophilic binding, except JAM-C.

-heterophylic bindingJAM-A binds the leukocyte integrin αLß2, JAM-B binds the α4β1 integrin

-JAM-A associates with occludin and PDZ proteins: ZO-1, AF-6, CASK, PAR-3 and MUPP-1


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CYTOPLASMIC (PLAQUE) PROTEINS OF THE TIGHT JUNCTIONS

ZONULA OCCLUDENS (ZO) FAMILY

PDZ

SH3

GUK

PROLINE RICH

80-90 amino-acids. Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (ZO-1) n general PDZ domains bind to a short region of the C-terminus of other specific proteins.

SRC Homology 3 Domain of about 60 amino acids. Regulate the activity state of adaptor proteins and other tyrosine kinases

Guanylate kinase domain, enzymatically inactive.Phospho-protein-binding modules.

INTERACTIONS OF ZO PROTEINS

TJ proteins (transmembrane, plaque), AJ proteinsactin-cytoskeleton, signaling molecules

Prog Biophys Mol Biol. 2003 Jan;81(1):1-44.Tight junction proteins.Gonzalez-Mariscal et al.


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FUNCTIONS OF ZO PROTEINS

-scaffold for the junctional complex

-expressed in cells lacking TJs

-role in signal transduction (binding of ZO-1 to ZONAB and binding of ZO-2 to SAF-B)

-SH-3 (src-homology 3) binding of a protein kinase

-KO animals are not viable but formation of TJ strands in the presence of either ZO-1 or ZO-2 is possible.

SIGNALING MOLECULES LOCALIZED TO THETIGHT JUNCTIONS

-protein kinase C isoforms (atypical isoforms)

-other protein kinases and phosphatases (c-Yes, PP-2A)

-G-proteins-Gsα (colocalizes with ZO-1)-Gi2α (colocalize with ZO-1 and ZO-2 and bind

to the Src homology 3 domain of ZO-1)-Goα (localized in the subapical region, overlapping

with ZO-1)

-small GTP binding proteins (Rho)

SIGNALING AT THE TIGHT JUNCTION

-Calcium: absence of Ca++leads to a desintegration of tight junctions

-Cyclic nucleotides: cAMP improves barrier properties, cGMP decreases barrier properties

-small G-proteins: heterotrimeic G-proteins important elements of signaling pathways, Rho regultes cytoskeleton

--Phosphorylation: multiple roles (example - occudin Ser/Thr phosphorylation is required for TJs, Tyr phosphorylation increase s permeability.


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ADHERENS JUNCTIONS

-N, -E, -VE, -P cadherins-homophylic Ca2+-dependent binding-intracytoplasmic domain binds to catenins-very important in cell adhesion and migration (metastasisformation)

-alpha, -beta, -gamma catenins-beta and gamma catenin binds directly to cadherins-beta-catenin participates directly in signal transduction

-nectins immunglobulin like transmembrane proteins-afadin, ponsin

MOLECULAR STRUCTURE AND FUNCTION OF BIOLOGICAL BARRIERS

Part I - SummaryINTRODUCTION

compartmentalization; epithelial, endothelial cells; tight junctions

ORGANS PRTOTECTED BY BIOLOGICAL BARRIERSskin, kidney, intestine, liver, lung, testis, brain

THE JUNCTIONAL COMPLEXTight junctions: role, TJ strands, molecular compositionProteins of the TJs: occludin, claudins, ZO proteinsSignaling at the tight junctionsAdherens junctions


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Paul Ehrlich

THE BLOOD-BRAIN BARRIER

•~ 2000 – neurovascular unit

MILESTONES OF BBB RESEARCH

• ~1880 (1985) Paul Erlich: certain dyes, when injected into thevascular system, were rapidly taken up by all organs with theexception of the brain (explanation: lack of affinity)

•1900 Lewandowski: first use of the word blood-brain barrier (Bluthirnschranke)

•1913 Goldmann: concept of the blood-brain barrier location: glial limiting membrane•1967 Reese and Karnowski: endothelial barrier

•~ 1980 (1987) Janzer and Raff: role of astrocytes

•~ 1990 - molecular dissection of the tight junction

•Human brain:2% of the body weight

20% of the O2 consumption

- 100 billion neurons

- glial cells: ratio in the cerebral cortex gray matter is ~ 1,5

- Endothelial cells:-0,1-1% of the brain volume-involved in approx. 70% of CNS diseases -total area: 10 m2

-total lenght: >600 km


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BBB

Where is it located?

What are the constituents?

What is the role?

How does it work?

CELLULAR COMPONENTS OF THE BBB

Microglia

Astrocyte endfeet

Pericyte

Tight junctions

Nerve endingEndothelial cell

Basement membrane


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CELLULAR COMPONENTS OF THE BBB

CEREBRAL ENDOTHELIAL CELLBASEMENT MEMBRANE

PERICYTEASTROCYTE ENDFEET

RBC WBCCAPILLARY LUMEN

EPITHELIAL CHARACTERISTICS- Presence of a high number of tight junctions- Low rate of pinocytosis-High density of mitochondria

CEREBRAL ENDOTHELIAL CELLS

ENDOTHELIAL CHARACTERISTICS-Factor VIII (von Willebrand factor)-Alkaline phosphatse-Specific lectin binding-Uptake of acetylated LDL

Morphology: elongated

Origin: neuroectodermMorphology: depends on location and activation state –round/oval cell body with fine processesSpecialized end feet on vessel wall (coverage up to 90%)Markers: GFAPLocationIn capillaries – share basal lamina with endothelial cells and pericytesIn post-capillary venules – their basal lamina forms a distinct layer leaving intervening perivascular spaceIn arterioles, arteries and veins - separated from endothelial/pericyte layer by smooth muscle layers

ASTROCYTES


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- Rich in mitochondria

-Caveolae, coated pits, vesicles: active material exchange

-PROTEINS OF THE ENDFEETAQP4 (OAP- Orthogonal Array of intramembraneous Particles)Kir4.1 potassium channelsCx-43P2Y4

ASTROCYTIC ENDFEET

-TGF-β (transforming growth factor-β)-GDNF (glial-derived neurotrophic factor)-bFGF (basic fibroblast growth factor) -IL-6 (interleukin-6)-Sonic hedgehog -extracellular matrix produced by astrocytes can induce BBB characteristics in brain endothelial cells

Recent proteomic analysis revealed changes in 55 proteins –involved mainly in cell structure and motility and protein metabolism and modification

ASTROCYTE-ENDOTHELIAL INTERACTIONS

Astrocytes are able to synthesize a large number of biologically active molecules which can contribute to the induction of BBB phenotype.

- CECs were shown to induce the expression of plasminogen activator inhibitor-1 mRNA in astrocytes

ASTROCYTE-ENDOTHELIAL INTERACTIONS II

Astrocytes and brain endothelial cells mutually influence each other

- CECs increase the number of OAPs in the astrocytic endfeet

- CECs-derived LIF has been shown to induce astrocytic differentiation


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ASTROCYE-ENDOTHELIAL CELL INTERACTIONS

Morphology: round/oval cell body, fine processes closely associated with endotheliumMarkers: alpha-actin, NG2Location: between two layers of basal lamina

PERICYTES

PERICYTES

- Described in 1873 by the French scientist Charles-Marie Benjamin Rouget

- Originally called Rouget cells.

-The Rouget cell was renamed some years later due to its anatomical location abluminal to the endothelial cell (EC) and luminal to parenchymal cells.

-The ratio of pericytes to EC varies from species to species and organ to organ and varies even within the capillary bed. In the brain the average ratio of pericytes to EC in the rat capillary is 1:5. In the mouse the ratio is 1:4 and in humans 1:3–4.


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PERICYTE SIGNALING

PDGF-beta

Notch-1

Notch-ligandNotch-ligand

TGF-beta

TGF-beta

Inhibition of proliferationECM production

TGF-beta R

N-cadherin

TGF-beta

PERICYTE

CEC

BASAL MEMBRANE

RBCPericyte recruitmentPDGF-beta R

PDGF-beta

PDGF-beta

Endothelial maturationBBB formation

Ang-1

Tie-2

Ang-1

Pericyte loss by targeting the PDGF-beta signaling pathway

- Pericyte deficiency increases the permeability of the BBB to water and arange of low-molecular-mass and high-molecular-mass tracers. The increasedpermeability occurs by endothelial transcytosis

-Pericytes function at the BBB in at least two ways:- by regulating BBB-specific gene expression patterns in endothelial cells- by inducing polarization of astrocyte end-feet surrounding CNS bloodvessels.

PERICYTE LOSS

BASEMENT MEMBRANE

Collagen IVFibronectinProteoglycans (heparan sulfate and glycosaminoglycans) LamininIntegrin


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Barrier: restricts the transport from blood to the brain of potentially toxic or harmful substances

Carrier: transport of nutrients to the brain and removal of metabolites

- transcellular barrier (low level of endocytosis and transcytosis)

-efflux pumps

- Fourfold defense line:

- enzymatic barrier (acetylcholinesterase, alkalinephosphatase, γ-glutamyl transpeptidase, monoamine oxidases, and drug metabolizing enzymes).

.. ...

- paracellular barrier (interendothelial junctions)

DUAL ROLE OF THE BBB

EFFLUX TRANSPORTERS

-ABCB1 (P-gp, MDR)

-ABCC (MRPs): ABCC4, ABCC5

-ABCG2 (BCRP)

ATP-binding cassette transporters (ABC transporters)transport a wide variety of substrates across cellular membranes, including metabolic products, lipids and sterols, and drugs


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DOES ENTER THE BRAIN:

EtanolNicotine

Lipid soluble substances:

glucose, amino acids:

DO NOT CROSS THE BBB:

Water soluble substances:

LIPID SOLUBILITY AND BRAIN UPTAKE

TRANSPORTERSUPTAKE TRANSPORTERSSolute carrier family, SLC transporters, >50 families

- facilitative transporters (allow solutes to flow downhill with their electrochemical gradients)- secondary active transport (allow solutes to flow uphill against their electrochemical gradient by coupling to transport of a second solute that flows downhill with its gradient)

EFFLUX TRANSPORTERS (ABC transporters)


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GLUCOSE TRANSPORT (SLC2)-12 transmembrane protein -facilitative transport (blood: 5 mM, extracellular fluid: 0.5-3.5 mM)-Km: 2-5 mM

GLUT1 (SLC2A1): endothelium and erythrocytesGLUT2: liver, pancreasGLUT3: neuronsGLUT4: muscle, addipose tissue

SLC3, SLC7 -cationic and neutral amino acids (SLC7A5=LAT1, SLC7A1=y+

system Bo+ (basic amino acid preferring, SLC7A3) )

SLC15 -proton/oligopeptide transporters (PEPT1, 2)

SLC16 -monocarboxylate transporters (MCT: lactate, pyruvate, hydroxybutyrate)

SLC21 -organic anion transporters (OATPs)

SLC22 -organic anion, zwitterion, cation transporters

SLC38 -sodium-coupled neutral amino acid transporter(system A (alanine preferring, SLC38A2))

OTHER SLC TRANSPORTERS

SPECIAL TRANSPORT SYSTEMS

-serotonin transporter SLC6A4

-glutamate transporters:EAAT1 (SLC1A3)EAAT2 (SLC1A2)

-histamine transporter: OCT3 (SLC22A3)


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ION TRANSPORT

-Na+/K+ ATP-ase

-Na+/K+/2Cl- transporter (SLC12A)

-Na+/H+ antiporter

-H+ ATP-ase

-saturable Cl- transport

CELLULAR TRANSMIGRATIONTHROUGH THE BBB

- rolling: selectins- adhesion: integrins (alpha4 beta1)-migration: ICAM (CD54), LFA1, JAM

-reorganization of the actin cytoskeleton-protein phosphorylation

-transcellular: through individual EC-paracellular: through the junctions

LEUKOCYTE MIGRATION

Only in activated CECs (inflammation)

http://dc352.4shared.com/doc/-4JNWl_7/preview.html


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RECEPTORS ON ENDOTHELIAL CELLS

-adrenergic-histamine (H1, H2)-bradykinin-endothelin -opiate-glutamate-serotonin-adenosine-insulin-transferrin-LDL receptor

receptor-mediated uptake, drug delivery

Abbott NJ et al. (2006) Astrocyte–endothelial interactions at the blood–brain barrierNat. Rev. Neuro. 7: 41–53 doi:10.1038/nrn1824

PATHWAYS ACROSS THE BLOOD–BRAIN BARRIER

*CNS diseases → BBB permeabilityinitiation or aggravation of the disease

-mechanisms of permeability change – affected:-pinocytosis-intrerendothelial junctions-endothelial cell surface charge-cytoskeleton (actin, microtubular system)

*relative impermeability of the BBB → low penetration of drugs

CLINICAL ASPECTS OF THE BBB


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PATHOLOGICAL CONDITIONS WITH INCRESED BBB PERMEABILITY

- Stroke- Brain trauma- Epilepsy- Neurodegenerative disorders- Brain tumors- Inflammatory disorders- CNS infections- Cellular transmigration through the barriers

METHODS OF INVESTIGATION

- Morphological analysis: immunohistochemistry, EM, permeability measurements- In vitro:

- Isolated capillaries: biochemical and molecular biological studies- Cell culture:

-primary-cell lines-coculture (astrocytes, pericytes)

- In vivo studies: brain uptake, two-photon microscopy

MORPHOLOGICAL ANALYSIS: IMMUNOHISTOCHEMISTRY

CONTROL DECOMPENSATED SHOCK

EXPRESSION OF OCCLUDIN IN CEREBRAL MICROVESSELS


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MORPHOLOGICAL ANALYSIS: ELECTRON MICROSCOPY

10 µm

-isolated capillaries

-endothelial cell culture

-in vitro BBB

-in vivo

Models: BRAIN ENDOTHELIAL CELLS

ASTROCYTES

METHODS OF INVESTIGATION

IN VITRO MODEL OF THE BBB


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MICROFLUIDIC MODEL OF THE BBB

0

20

40

60

80

100

120

0 20 40 60 80 100 120 140 160

min

MEASUREMENT OF THE TRANS-ENDOTHELIAL ELECTRICAL RESISTANCE

TAKE HOME MESSAGE


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Cell types involved in barrier formation:epithelial cellsendothelial cells (BBB)

Epithelial barriers are found in:kidney, intestine, liver, lungs, etc.

Endothelial barrier:blood-brain barrier (BBB)

BIOLOGICAL BARRIERS

Molecular basis of paracellular barriers:

tight junctions-transmembrane proteins: occludin, claudins, JAMs-plaque proteins: ZO proteins, etc.

adherens junctionscadherins and catenins

BIOLOGICAL BARRIERS

BBB location:

BBB constituents:

Involved in the majoritity of neurological disorders (cerebral ischemia tumors, neurodegenerative disorders, trauma....)

at the level of brain capillaries

endothelial cells, astrocytes, pericytes



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BBB functions:

Barrier: restricts the transport from blood to the brain of potentially toxic or harmful substances

-paracellular: tight junctions-transcellular-enzymatic-efflux pumps: ABC-B1, C, G2

Carrier: transport of nutrients to the brainremoval of metabolites

-SLC transporters


CELLS OF THE BBB:-endothelial cells-pericytes-astrocytes

TIGHT JUNCTIONS-transmembrane-plaque proteins TRANSPORTERS:

-efflux-influx

Thank you for your attention!

This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-

oriented, student-friendly modernization of the biomedical education for strengthening the international

competitiveness of the rural Hungarian universities " TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.

ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT financed … · 2016-09-07 · 2016. november 2. /...

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