overview of the blood-brain barrier and its properties … · overview of the blood-brain barrier...
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OVERVIEW OF THE BLOOD-BRAIN BARRIERAND ITS PROPERTIES
Laboratoire de Physiopathologie de laBarrière Hémato-Encéphalique
EA 2465, IMPRT: IFR 114 Faculté 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
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Goldmann 1913Ehrlich 1885
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Peptide Drug Delivery to the Brain
William M. Pardridge 1991
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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
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The Blood-Brain Barrier Cellular and Molecular Biology
William M. Pardridge 1993
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Peptide Drug Delivery to the Brain
William M. Pardridge 1991
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• Brain capillaries are a complex
structure
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The Blood-Brain Barrier Cellular and Molecular Biology
William M. Pardridge 1993
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Where is located the barrier?
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Peptide Drug Delivery to the Brain
William M. Pardridge 1991
Brain capillary endothelial cells are the physical components ofthe BBB
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The Blood-Brain Barrier Cellular and Molecular Biology
William M. Pardridge 1993
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The Blood-Brain Barrier Cellular and Molecular Biology
William M. Pardridge 1993
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The Blood-Brain Barrier Cellular and Molecular Biology
William M. Pardridge 1993
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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
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Endothelial Cell Biology
N. Simionescu and M. Simionescu 1988
Peripheral capillary
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Endothelial Cell Biology
N. Simionescu and M. Simionescu 1988
Peripheral capillary
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brain
blood
endothelial cell
BBB: PHYSICAL BARRIER
tightjunction
No paracellularpassage
Low transcellulartransport
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Blood
Brain
Dopamine
L-DOPA
L-DOPA
MAODrug-metabolizing
enzymes
P-gp
BBB : a metabolic barrier
Degradation
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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)
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Blood
Brain
Specific transport(receptor or transporter)
« fluid-phase »transcytosis
Transport processes through a cerebralendothelium
ZO
ZA
Paracellularpathway
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EXHAUSTIVE LIST OF BBB TRANSPORTERS ?
from Terasaki T 2003
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Different techniques used to study the BBB
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14C/3H –sucrose/inulin(to measure cerebro-vascular volume
3H/14C-labelled drug(to measure BBB permeability)
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endothelial cells
glial cells
brain
blood
BBB model
IN VITRO BBB MODEL
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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
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D
D
D
D
D
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P-gpP-gp proteinprotein ((ConfocalConfocal) - BBCEC) - BBCEC
Luminal membrane
Abluminal membrane
P-gp OCTN2nuclei
24μm
0 5 10 150
10
20
30
Slides (1 = 0.16μm)
Flu
ore
sce
nce
inte
nsity
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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.
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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 blood–CSF 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 ISF–CSF bulk flow [57]
(7) Oligomerization and aggregation [5,6]
Brain Ab homeostasis is controlled bynumerous pathways (Berislav,2005):
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Interaction between t-PA with the blood-brain barrier
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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
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Therapies
ThrombolysisThrombolysis
tPAActilyse®
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HoweverHowever, , increasingincreasing evidenceevidence supports supports thethe ideaidea thatthat t-PAt-PA couldcould
alsoalso potentiatepotentiate strokestroke damage damage
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Does tPA cross the intact BBB in vivo?
Collagen IV GFAP
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Intravenous Injection of Biotinylated Albumin
Collagen IV
Biot-albumin
Albumin remains intravascular
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tPA
Albumin
Collagen IVBiotin Merged
Intravenous Injection of Biotinylated Albumin or Biotinylated tPA
tPA crosses the intact BBB in vivo
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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
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Receptor-mediated
How does tPA cross the BBB ?
Blood-brain barrier
Non specific
Transcellular Paracellular
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Biot-tPA (green)
Ab
lum
inal
tPA
act
ivit
y (%
of c
ontr
ol)
0
20
40
60
80
100
120
140
37°C 4°C
**
tPA crosses the BBB by transendothelial pathway
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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
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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
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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
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Peptide Drug Delivery to the Brain
William M. Pardridge 1991
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Astrocytes
Endothelial
cells
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Statistical study of 2D-PAGE area from BCECs
Co-culture
Solo-culture
Important variation : overexpressed in co-culture
No statistical significant variation
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P-gp detection by Western Blot analysis
Isol
ated
capi
llarie
sBBCE
inco
cultu
reBBCE in
sol
o
cultu
re
140
205
kDa
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MRP1?
MRP4
MRP5
MRP1?
MRP4
MRP5MRP6
MRP1MRP4
MRP5MRP6
MRP6
P-gp
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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
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Working forthe BBB…
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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
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Histochemichal detection of -GT in braincapillaries endothelial cells
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P-gpP-gp proteinprotein ((ConfocalConfocal) - BBCEC) - BBCEC
Luminal membrane
Abluminal membrane
P-gp OCTN2nuclei
24μm
0 5 10 150
10
20
30
Slides (1 = 0.16μm)
Flu
ore
sce
nce
inte
nsity
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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
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cerebral
capillary
Brainparenchyma
cells
endothelial cellsendothelial cells
BBB CEREBRAL VICINITYIntroduction
Astrocytes
Pericytes
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0
10
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