esi_2006--Cecchelli_2

OVERVIEW OF THE BLOOD-BRAIN BARRIERAND ITS PROPERTIES

Laboratoire de Physiopathologie de laBarrire Hmato-Encphalique

EA 2465, IMPRT: IFR 114 Facult des Sciences Jean Perrin

Cellial technologies62307 Lens- FRANCE

Pr Romeo CECCHELLI

The Cerebral Vasculature and Neuroimaging in CNS Drug DicoveryAnd Development: Physiology,Pathology and Methodology

Astrazeneca june 2006

Goldmann 1913Ehrlich 1885

Peptide Drug Delivery to the Brain

William M. Pardridge 1991

The classical view of the BBBformulated by this pioneering work wasbased on evidence that blood-bornesubstances were excluded from thebrain

The BBB was located in brain capillariesand considered as a rigid structure

The Blood-Brain Barrier Cellular and Molecular Biology


Brain capillaries are a complex

structure

Where is located the barrier?



Brain capillary endothelial cells are the physical components ofthe BBB

Brain

7H6ZO-1AF6

/Cadherins / Cadherins

ZO-1

JAMs

Adherensjunction

Tightjunction

claudins

occludin

ZO-1ZO-2

ZO-1ZO-3

ZO-1ZO-3

ZO-2ZO-1

cingulincingulin

Actin

Apical plasma membrane

Blood BBB : a physical barrier

Endothelial Cell Biology

N. Simionescu and M. Simionescu 1988

Peripheral capillary

brain

blood

endothelial cell

BBB: PHYSICAL BARRIER

tightjunction

No paracellularpassage

Low transcellulartransport

Blood

Brain

Dopamine

L-DOPA

L-DOPA

MAODrug-metabolizing

enzymes

P-gp

BBB : a metabolic barrier

Degradation

CYP 450

MAO-B

Glutamyl aminopeptidase

Endothelial cells

BBB : A METABOLIC BARRIER

Pericyte

UGT-1A6

GST

CYP 1A1 (rat)CYP 1B1 (human)CYP 2B1 (rat)

CYP 2B6 (human)

Blood

Brain

Specific transport(receptor or transporter)

fluid-phasetranscytosis

Transport processes through a cerebralendothelium

ZO

ZA

Paracellularpathway

EXHAUSTIVE LIST OF BBB TRANSPORTERS ?

from Terasaki T 2003

Different techniques used to study the BBB

14C/3H sucrose/inulin(to measure cerebro-vascular volume

3H/14C-labelled drug(to measure BBB permeability)

endothelial cells

glial cells

brain

blood

BBB model

IN VITRO BBB MODEL

TRANSPORTERS AT THE BBB

OCT?OCTN2

ATP

P-gpBCRP

ORGANIC CATIONSNUTRIENTS

A, B0,+

ASC

Na+

GLUT 1

ORGANIC ANIONS

MCT 1OATP Aoatp 2

OAT4?

OAT3

DD

D

D

D

P-gpP-gp proteinprotein ((ConfocalConfocal) - BBCEC) - BBCEC

Luminal membrane

Abluminal membrane

P-gp OCTN2nuclei

24m0 5 10 15

0

10

20

30

Slides (1 = 0.16m)

Flu

ore

sce

nce

inte

nsity

Developing new technology is necessary for progression of the BBB research

There is no selective inhibitors for distinguishing ABCC subtypes and ABCG2.

Alternative strategy: siRNA

siRNA can selectively suppress target transporter(s).

*

0

50

100

150

200

250

300

AB

CG

2 m

RN

A (

% o

f c

on

tro

l)

siRNA

E2 (-)

-G

2-03 NC

E2 (+)

-G

2-03 NC

*

0

50

100

150

200

250

300

AB

CB

1 m

RN

A (

% o

f c

on

tro

l)

siRNA

E2 (-)

-G

2-03 NC

E2 (+)

-G

2-03 NC

0

50

100

150

200

250

300

AB

CC

1 m

RN

A (

% o

f c

on

tro

l)

siRNA

E2 (-)

-G

2-03 NC

E2 (+)

-G

2-03 NC

ABCG2 ABCB1/MDR1 ABCC1/MRP1

G2-03, siRNA for ABCG2; NC, negative control siRNAHori et al. J. Neurochem. 93:63-71 (2005)

G2-03 siRNA selectively suppressed the expression of rat ABCG2 in brain capillary

endothelial cells.

Regulation of A in the CNS

(1) Peripheral and central production

(2) Rapid receptor-mediated transport of soluble formsacross the BBB

from blood to brain via RAGE [30]

(3) Similar transport across the BBB from brain to blood[41,43] via

LRP [21,45]

(4) Binding to transport proteins such as apoE, apoJ and

a2-macroglobulin (a2M), which can influence: Absequestration in

plasma, brain ISF and CSF; the form of Ab accumulation inbrain

(i.e. soluble versus fibrillar) [6,46,47]; and/or transportacross the BBB

and bloodCSF barrier [6,65]

(5) Degradation, mediated by proteins such asenkephalinase [48],

insulinase, plasmin, tissue plasminogen activator or matrixmetalloproteinases

[49], or mediated by astrocytes [50,51] and microglia [66]

(6) Slow removal via ISFCSF bulk flow [57]

(7) Oligomerization and aggregation [5,6]

Brain Ab homeostasis is controlled bynumerous pathways (Berislav,2005):

Interaction between t-PA with the blood-brain barrier

tPA and the Fibrinolytic System

Benchenane et al., Trends Neurosci 2004

PAI-1

release

inhibition inhibition

FDPs

activationtPA

tPA

FibrinFibrin clotclot

2-APpln

plnplgtPA

Breakdown

Therapies

ThrombolysisThrombolysis

tPAActilyse

HoweverHowever, , increasingincreasing evidenceevidence supports supports thethe ideaidea thatthat t-PAt-PA couldcould

alsoalso potentiatepotentiate strokestroke damage damage

Does tPA cross the intact BBB in vivo?

Collagen IV GFAP

Intravenous Injection of Biotinylated Albumin

Collagen IV

Biot-albumin

Albumin remains intravascular

tPA

Albumin

Collagen IVBiotin Merged

Intravenous Injection of Biotinylated Albumin or Biotinylated tPA

tPA crosses the intact BBB in vivo

tPA does not affect BBB integrity or permeability

Luminal

Abluminal

tPA

?

tPA crosses the BBB in vitro

ZO-1ZO-1

ctrl tPA

Man

pe

10-3

cm/m

in (s

ucr

ose)

0

2

4

6

8

Ctrl tPA Mannitol

In vitro model of BBB

Tight junctionsHigh transendothelial resitivity

Cecchelli et al., 1999

Ab

lum

inaltP

A a

cti

vit

y(%

of lo

adedt

PA

)

0,0

1,0

2,0

3,0

4,0

5,0

0 20 40 60 80 100 120 140

time (min)

Time (min) 15 30 60 120 120

Abluminal tPA

Luminal-loadedtPA + + + + - rtPAA

blu

min

altP

A a

cti

vit

y(%

of lo

adedt

PA

)

0,0

1,0

2,0

3,0

4,0

5,0

0 20 40 60 80 100 120 140

time (min)

Time (min) 15 30 60 120 120

Abluminal tPA

Luminal-loadedtPA + + + + - rtPA

Receptor-mediated

How does tPA cross the BBB ?

Blood-brain barrier

Non specific

Transcellular Paracellular

Biot-tPA (green)

Ab

lum

inal

tPA

act

ivit

y (%

of c

ontr

ol)

0

20

40

60

80

100

120

140

37C 4C

**

tPA crosses the BBB by transendothelial pathway

tPA

Ctrl RAP Man

0

20

40

60

80

100

120

140

Ab

lum

inal

tPA

act

ivit

y(%

of c

ontr

ol)

Control RAP Mannose

*

Identification of the receptor involved in the passage of tPA

tPA crosses the intact BBB by LRP-mediated transcytosis

There are a lot a sophiticated receptor-mediated transportsat the BBB.

Tomorrow, I will presenetd you the cellular mechanisms ofthese transports in physiological conditions and the influence ofthe surrounding cells on these transports

TheThe simple observation simple observation thatthat epithelialepithelial but but notnot endothelialendothelialcelscels are able to are able to formform a a high-resistancehigh-resistance andand low-low-permeabilitypermeability barrierbarrier in vitro in vitro sheds light on sheds light on thethe importance importanceofof thethe microenvironnement microenvironnement in in thethe maintenance maintenance ofof thethebarrierbarrier propetiespropeties in vivo in vivo

Astrocytes

Endothelial

cells

Statistical study of 2D-PAGE area from BCECs

Co-culture

Solo-culture

Important variation : overexpressed in co-culture

No statistical significant variation

P-gp detection by Western Blot analysis

Isol

ated

capi

llarie

sBB

CE in

cocu

lture

BBCE

in s

olo

cultu

re

140

205

kDa

MRP1?

MRP4

MRP5

MRP1?

MRP4

MRP5MRP6

MRP1MRP4

MRP5MRP6

MRP6

P-gp

The definition of the BBB has shift today to amore integrated concept that takes into acount

not only the bidirectionality of the exchangeprocess, but also the discovery that besides theendothelium additional components (astrocytes,neurons) constitute integral parts of the barrierphysiology

Working forthe BBB

Rippe et al., 2002

Introduction

HOW TO CROSS A CONTINUOUS CAPILLARY?

HOW TO REACH THE BRAIN?

1. Interendothelial clefts

1

2. Transendothelial channel

2

3.Transcytosis

3

1. Not between cells because of tight junctions

2. Not by transendothelial channel because of its lack

3 & 4. By transcellular processes

4

4.Transporters

Histochemichal detection of -GT in braincapillaries endothelial cells

P-gpP-gp proteinprotein ((ConfocalConfocal) - BBCEC) - BBCEC

Luminal membrane

Abluminal membrane

P-gp OCTN2nuclei

24m0 5 10 15

0

10

20

30

Slides (1 = 0.16m)

Flu

ore

sce

nce

inte

nsity

CEREBRAL ENDOTHELIUM : METABOLIC AND TRANSPORT BARRIERS

Na+water

water

Na+

ATPaseK+

Na+

Na+

Glutamate

Glycine

Leucine

Glucose

P-glycoprotein

Alcaline PhosphataseGamma-GT

L-DOPAL-DOPA

DopamineDopamine

DCCA

MAO-B

DOPAC

L-DOPA

cerebral

capillary

Brainparenchyma

cells

endothelial cellsendothelial cells

BBB CEREBRAL VICINITYIntroduction

Astrocytes

Pericytes

010

20

30

40

0 15 30 60

Time (min)

Ctrl

tPA

Act

ivit

y in

the

ablu

min

al s

ide

(% o

f tP

A lo

aded

in t

he lu

min

al s

ide)

Control

OGDBiot-tPA (red)

Luminal

Abluminal

tPA

?

OGD 4h

15 30 6015 30 60

Ctrl OGD

Abluminal tPA

Time (min) 15 30 6015 30 660

Ctrl OGD

Abluminal tPA

Time (min)

OGD****

OGD potentiates the passage of tPA

esi_2006--Cecchelli_2

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