la barrera hematoencefálica en patologías del sistema nervioso … · la barrera...

La barrera hematoencefálica en patologías del sistema nervioso

central: ¿una barrera impenetrable?

Ignacio (Nacho) A. RomeroDepartment of Life Sciences

The Open UniversityMilton Keynes, UK

Outline of the talkThe blood-brain barrier (BBB) in health:

•

Overview of BBB structure and function

•

Establishment of an in vitro human BBB model

•

Role of the BBB in CNS availability of drugs

The blood-brain barrier (BBB) in disease:

•

Understanding the molecular mechanisms of BBB dysfunction in CNS pathologies

The Blood-brain barrier (BBB) in health:

Structure and function

The barrier(s) of the central nervous system (CNS)

http://www.bodyworlds.com/en.html

SITES OF CNS BARRIERS

1.

Brain parenchyma capillary endothelium

2.

Arachnoid epithelium

3.

Choroid plexus epithelium1

2

3Exceptions:Circumventricular

organs (CVO)

Abbott NJ et al.

(2006) Astrocyte–endothelial interactions at the blood–brain barrier

Nat. Rev. Neuro.

7:

41–53 doi:10.1038/nrn1824

1. Control molecular traffic → supply nutrients, remove waste products & keep out toxins

2. Ion homeostasis → optimal neural signalling

3. Low protein environment → limit proliferation

4. Separate CNS and peripheral neurotransmitter pools → allows non-synaptic signalling

5. Immune surveillance with minimal inflammation or cell damage

FUNCTIONS OF CNS BARRIERS

Structure of the BBB: Neurovascular unit

+ other cell types: microglia, neurones

Basal laminaTight junction

EndotheliumPericyte

Astrocyte foot process

Perivascular macrophage

Molecular composition of CNS endothelial junctions

ZO-1ZO-2Z0-3

7H6 C

N

cingulin

srcp120

Adherens Junction

-catenin

-catenin

Actin

ZO-1 ZO-2ZO-3

7H6C

N

cingulin

src p120

VE-cadherinActin

Occludin

Claudins 3, 5, 12

PECAMPECAM

JAMs, ESAM

Tight Junction

The BBB in health

• Physical Barrier

• Transport Barrier

• Metabolic Barrier


In vitro human BBB model

Development of an in vitro human blood- brain barrier model

Brain endothelial cellsAstrocytes

Primary cultures

Didier et al., Cytokines and the BBB, J. Neurochem, 2003

Maru et al., Chemokines and glial tumours, JNI, 2008

Subileau et al., Chemokines and the BBB, JNEN, 2009

Tai et al., P-glycoprotein and the BBB, Brain Research, 2009

Endothelial cells

Astrocytes

• Individuals with intractable epilepsy →

temporal lobectomy(King’s College Hospital)

• Individuals with multiple sclerosis →

post-mortem cortical grey matter (UK MS Tissue Bank)

Immortalization of primary human adult brain microvascular endothelial cells

Lentiviral hTERT

supernatants

Primary human brain endothelium

~ 3 weeks post isolation

Lentiviral SV40 large T supernatants

5 days

5 days

Isolation of clones by limited dilution

~ 3-4 weeks post infection

~100 clones, one of which was selected and termed hCMEC/D3

Weksler et al., FASEB J, 2005

US Patent no. WO0066149

hCMEC/D3 brain endothelial cell line

Distributed to >120 research groups> 30 research articlesP.O. Couraud, ICGM, Paris B.B. Weksler, Cornell Uni, NY

PECAM-1 catenin

Expression of tight and adherens junction-associated

endothelial markers by hCMEC/D3 cells

-cateninVE-cadherin F-actin ZO1

Claudin-5 JAM-1

Adherens Junctions

Tight Junctions

Permeability to paracellular tracers: Comparison between hCMEC/D3 cells and other in vitro BBB models

Permeability Coefficient (x10-3

cm/min)

sucrose inulin FITC-dextran 4 kDa FITC-dextran 70 kDa Refs

Bovine primary

+astrocytes 0.8 0.4 Cecchelli et al., 1999

primary 0.5 Mar&Davis, 2002

Porcine primary 0.3 Franke et al., 1999

Rat RBE4 4.8 1.3 Rist et al, 1997

GPNT 7.4 2.6 1.0 0.05 Romero et al, 2003

Mouse b.END3 1.2 Omidi et al. 2003

Human primary 0.3 0.02 Unpublished results

+astrocytes

hCMEC/D3 1.7 0.4 0.3 0.01 Weksler et al. 2005

HUVEC 4.2 0.12 Unpublished results

CN

S m

icro

vasc

ular

EC

NON-CNS


Transcriptional control of TJ proteins

Minimal promoter (endothelium) 1505-1853

Activity of minimal occludin promoter-GFP reporter in endothelia

1 400 800 1200 1600 2000

Lung endotheliumBrain endothelium

>1,000 putative binding sites in the occludin promoter (TESS)

Sade H, et al. (2009) Biochim Biophys

Acta

1789(3):175

Sp-3 binds to occludin minimal promoter in hCMEC/D3 cells (EMSA)

Cold probe inhibition

Antibody supershift

1 -400 -800 -1200 -1600 2000

F8

Mithramycin (Sp-inhibitor) decreases occludin

promoter-GFP reporter expression in hCMEC/D3 cells

0

20

40

60

80

100

120

Occludin Claudin 5 Z01

ControlMM treated

Med

ian

fluor

esce

nce

Control

Occ

Cl-5

ZO-1

200nM Mithramycin

*

F8

1 -400 -800 -1200 -1600 2000

WT-min. promoterMut 1798-1804minimal promoter

Mut 1820-1825minimal promoter

Mutation of Sp-sites in occludin promoter inhibits its activity in hCMEC/D3 cells


Role of the BBB in CNS availability of drugs

CNS penetration of drugs: Diffusion across BBBParameters limiting diffusion:

↓lipophilicity

H-bonds>8 MW>700 Da

vinblastine

vincristine

Abbott NJ, Romero IA.Mol Med Today. 1996

1 2 3 4TJ TJ

Rc +ve

(a) Passivediffusion

Lipidsolublenon-polarmolecules

(b) ABCTransporterEfflux

Lipidsolublenon-polarmoleculesandconjugates

(c) SoluteCarriersSLC

GlucoseAmino acidsNucleosidesMonocarboxylatesSmall peptidesFFAsOrganic anionsOrganic cations

(d) TranscytosisReceptor-mediatedAdsorptive-mediated

RMTTransferrinMelanotransferrinLipoproteinsAmyloid βGlycosylated

proteins

IgGInsulinLeptinTNFEGF

(e) MononuclearCellMigration

AMTCationised

albumin

HistoneAvidinTATSynB1(Cell penetrating peptides)

PgpBCRP

MRP1-5*

MRP1-5*

RMT AMT

Molecular and Cellular traffic Across the BBB

Correlation between in vivo (adult rats) and in vitro (hCMEC/D3 cells) permeability of several standard drugs

vincristine

colchicine

R = 0.938

0.01

0.1

1

10

100

0 1 2 3 4

Pe

(10-3 cm·min-1)

Kin

(10-3

ml·s

-1·g

-1)

Morphine-6--d- glucuronide

prazosin

diazepam

Weksler et al., FASEB J, 2005

WB α-BCRP

Expression of the efflux transporter P-glycoprotein (MDR1; ABCB1) by hCMEC/D3 cells

WB α-Sp3

WB α-Actin

WB α-P-gp

130 kDa

42 kDa

160 kDa

70 kDa

Cyt

osol

Mem

bran

e

Nuc

leus

Cyt

oske

leto

n

Apical expression

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

Luminal Abluminal

Ave

rage

num

ber

gold

par

ticle

s (pe

r μm

) rIsotype controlAnti-P-gp

** **

**

Apical Basolateral

P-gp Isotype control

P-gp activity is inhibited by vinblastine and tariquidar: rh123 efflux and permeability assays

t1/2

:

Control ~ 13 min

Vinblastine ~ 112 min

0

20

40

60

80

100

120

0 100 200 300 400

Time (min)

Med

ian

fluor

esce

nce

inte

nsity

(%

4o C

)

DMSOVinblastine

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Control Tariquidar Vinblastine

rh12

3 C

Q

Apical-to-basolateral

Basolateral-to-apical

* *

Tai et alBrain Res. 2009

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Control Tariquidar Vinblastine FTC

Aβ

clea

ranc

e qu

otie

nt

The apical-to-basolateral permeability of 125I Aβ 1-40

is increased by P-gp and BCRP inhibition in BECs

* **

P-gp inhibitors BCRP inhibitor

Basolateral to apical Apical to basolateral

Tai et al (2009) J Cereb Blood Flow Metab

29(6):1079-83.

Basolateral

Apical

Aβ 1-40

or inulin

0.1 nM 125I Aβ 1-40 30 min

Aβ CQ = 125IAP

/ 125I total14CAP

/14Ctotal

The Blood-brain barrier (BBB) in disease

BBB dysfunction is a common feature of CNS pathologies

Cytokines/chemokines

-amyloidHTLV-1

BLOOD-BRAIN BARRIER BREAKDOWN

LEUKOCYTE INFILTRATION

ASTROCYTE

ENDOTHELIUM

BLOOD

BRAIN

What are the molecular mechanisms underlying BBB dysfunction in CNS pathologies?

• Multiple sclerosis

• HTLV-1-associated

myelopathy

• Alzheimer’s disease

• Traumatic brain injury• Hypertension• Diabetes• Ischaemia, hypoxia• Inflammation• Migraine• Pain (inflammatory, surgical)• Multiple sclerosis• HIV-encephalitis • Cerebral malaria• Bacterial meningitis• Brain tumours• Epilepsy• Depression• Schizophrenia• Age-related dysfunctions• Alzheimer’s disease• Parkinson’s disease

Consequences of BBB opening

If mild or transient → BBB resealing → protection against further stress If severe or chronic → BBB dysfunction → brain oedema and persistent CNS pathology

• Removes protective/homeostatic actions of barrier• Allows CNS access of peripheral transmitters/

neuroactive agents and vice versa

• Plasma albumin toxic → glial proliferation and scar

formation• Leukocyte infiltration → CNS inflammation

The Blood-brain barrier (BBB) in disease:

REGULATION OF TIGHT JUNCTIONAL PERMEABILITY

BBB breakdown in the spinal cord of HTLV-1-infected HAM/TSP patients

NON-HAM/TSP

HAM/TSP

-ZO1

-OCLN

NON-HAM/TSP HAM/TSP

-fibrinogen Afonso et al. (2007) J Immunol 179, 2576

MOG and spinal cord immunization induces BBB breakdown in mice

Fabis et al. (2007)

PNAS 104, 5656- 5661

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Control Acute G1 Acute G4 Recovery Remission Chronic

Com

para

tive

expr

essi

on (

2-??

CT)

occludin

claudin-5

0

1

2

3

4

5

6

7

8

sAβ 1-40 Aβ 1-40 sAβ 1-42 Aβ 1-42

Pe (c

m/m

in x

10-5

)

Extracellular pathogenic stimuli increases hCMEC/D3 paracellular permeability

D3CEMHUT102C81-66

0 5 10 15 200.0

2.5

5.0

7.5

t (h)Afonso et al. (2007) J Immunol

179, 2576

HTLV-1-infected lymphocytes*

0

2

4

6

8

10

12

14

Cont

rol

TNF2

hTN

AFan

dIFN2

h

TNF6

hTN

AFan

dIFN6

h

TNF2

4hTN

AFan

dIFN2

4h

Pe

(1x1

0-5

cm/m

in)

TNF-αIFN-γ

- +- -

++

+-++ +- ++

2 h 6 h 24 h

Cytokines

Amyloid-

Tai et al. (2010) J Cell Mol Med 14(5):1101

TNFα and IL-1

are necessary for HTLV-1-

induced BBB breakdown

- IL1 TNF IL1+TNF0.0

2.5

5.0

7.5

10.0

0.0

2.5

5.0

7.5

Afonso et al. (2007) J Immunol 179, 2576

Mechanisms involved in disruption of tight junctions

• Redistribution of transmembrane and adaptor proteins from cell-cell junctions: transient BBB opening

– Alterations in actin cytoskeleton– Posttranslational modifications of AJ & TJ proteins– Proteolytic

degradation of AJ & TJ proteins

• Changes in expression of AJ & TJ proteins: chronic BBB opening


Actin: FITC-phalloidin

Control HTLV-infected

Pathogenic stimuli induce reorganization of

actin cytoskeleton and TJ proteins

hCMEC/D3+PBL sAβ 1-40 Aβ

1-40

50 μm

B

F

A

C

Claudin-5

ZO-1

Occludin

50 μm

Amyloid-

Afonso et al. (2007) J Immunol

179, 2576

Tai et al. (2010) J Cell Mol Med 14(5):1101

00.20.40.60.8

11.21.41.61.8

mR

NA

rela

tive

leve

ls

0

0.2

0.4

0.6

0.8

1

1.2

Expression of occludin and claudinExpression of occludin and claudin--5 protein and mRNA is 5 protein and mRNA is decreased by combination of cytokines in hCMEC/D3 cellsdecreased by combination of cytokines in hCMEC/D3 cells

2

Occludin

Time hTNFα+IFNγ

6 24 2 6 24

Control

Actin

2 6 24Time h

% o

f con

trol

0

0.2

0.4

0.6

0.8

1

1.2

2

TNFα+IFNγ

6 24 2 6 24

Control

2 6 24Time h

% o

f con

trol

Claudin-5

Actin

1 2 3 4 5 6 12 24

Time (h)

occludinclaudin-5

TNF-α+IFN-γ

Signalling at the TJ

• INTERACTIONS WITH G PROTEINS: heterotrimeric

G proteins (e.g. Gi2) and small G proteins (e.g. Rho)

• INTERACTIONS WITH PROTEIN KINASES: PKC, PKA

• PHOSPHORYLATION OF TJ COMPONENTS: ZAK1, Casein kinase II, MLCK

Time (h) 0 1 2 3 4

5 6


Identification of signalling pathways mediating BBB breakdown by pathogenic stimuli: HTLV-1-infected

T lymphocytes

JNK inhibition suppresses Aβ1-40 - and cytokine-

induced increased permeability of hCMEC/D3 cells

*

Aβ1-40

0

1

2

3

4

5

6

Control SP600125 TAT-TI-JIP153-163

SB203580

Pe(X

10

-5cm

/min

)

sA 1-40

A 1-40

TNF +INF

Pe( f

old

over

con

trol

)

PBS+ BSA

PBS+ BSA +

SP6001

25

PBS+ BSA +

TAT-TI-JIP

SP6001

25

TAT-TI-JIP

SP6001

25

TAT-TI-JIP

TNF-α TNF-α +

IFN-γ

0

2

4

6

8

10

12

14

16

18

**

**

JNK inhibitors

p38MAPK inhibitor

Regulation of claudin-5 and occludin levels at the BBB by cytokines and amyloid-

TNF

Claudin-5 Occludin

amyloid

JNK

IFN

p38PKC Rho

ROCK

Regulation of TJ protein expression

Actin-myosin contraction

TJ protein redistribution

Apoptosis

MLCK

• The hCMEC/D3 cell line is a suitable in vitro human

model to study BBB function in health and CNS pathologies

• Understanding the molecular basis of BBB function may

provide a rationale for targeted delivery to the CNS

• Understanding common molecular mechanisms mediating

BBB dysfunction in CNS pathologies may provide tools for targeted prophylaxis and therapies to BBB

Summary

Acknowledgments:

• Institut

Cochin

Pierre-Olivier Couraud

Paris, France

Babette Weksler

• Institut

Pasteur

Simona Ozden

Paris, France

Pierre-Emmanuel Ceccaldi

• National Research Council

Arsalan

Haqqani

Ottawa, Canada

Danica Stanimirovic

• QMUL, London, UK

David Baker

Greg Michael

• University of Sheffield, UK

Basil Sharrack

• The Open University

David Male

Milton Keynes, UK

Jane Loughlin Mark Hirst

Karen Logan Evanne

Subileau

Hadassah Sade Leon

Tai

Alejandro Lopez-Ramirez Camilla Cerutti

http://www.mrc.ac.uk/

THE BLOOD-BRAIN BARRIER

3-year Post-Doctoral Research Associate:

microRNAs in the cerebral vasculature and multiple sclerosis

[email protected]

Deadline: 30th November

mailto:[email protected]

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