Tight junctions at the blood brain barrier: physiological architecture and disease-associated dysregulation
Fluids and Barriers of the CNS
Tight junctions at the blood brain barrier: physiological architecture and disease-associated dysregulation
The Blood-brain barrier (BBB), present at the level of the endothelium of cerebral blood vessels, selectively restricts the blood-to-brain paracellular diffusion of compounds; it is mandatory for cerebral homeostasis and proper neuronal function. The barrier properties of these specialized endothelial cells notably depend on tight junctions (TJs) between adjacent cells: TJs are dynamic structures consisting of a number of transmembrane and membrane-associated cytoplasmic proteins, which are assembled in a multimolecular complex and acting as a platform for intracellular signaling. Although the structural composition of these complexes has been well described in the recent years, our knowledge about their functional regulation still remains fragmentary. Importantly, pericytes, embedded in the vascular basement membrane, and perivascular microglial cells, astrocytes and neurons contribute to the regulation of endothelial TJs and BBB function, altogether constituting the so-called neurovascular unit. The present review summarizes our current understanding of the structure and functional regulation of endothelial TJs at the BBB. Accumulating evidence points to a correlation between BBB dysfunction, alteration of TJ complexes and progression of a variety of CNS diseases, such as stroke, multiple sclerosis and brain tumors, as well as neurodegenerative diseases like Parkinson's and Alzheimer's diseases. Understanding how TJ integrity is controlled may thus help improve drug delivery across the BBB and the design of therapeutic strategies for neurological disorders.
Blood-brain barrier; Tight junction; Neurovascular unit; Kinases; Signaling pathways
The BBB maintains the homeostasis of the central
nervous system (CNS) by (i) strictly limiting the passive
diffusion of polar substances from the blood to the brain,
(ii) mediating the transport of nutrients to the brain
parenchyma as well as the efflux from the brain of toxic
metabolites and xenobiotics, (iii) regulating the migration
of circulating immune cells [1-3]. Formed by specialized
vascular endothelial cells, the BBB is tightly controlled by
pericytes, embedded in the vascular basement membrane,
perivascular microglial cells, astrocytes and neurons which
1INSERM U1016, Institut Cochin, Paris, France
2CNRS, UMR 8104, Paris, France
Full list of author information is available at the end of the article
altogether constitute the neurovascular unit (NVU), a
concept highlighting the functional cell-cell interactions
supporting BBB function.
BBB endothelial cells display a unique phenotype
characterized by the presence of TJs and the expression of
specific polarized transport systems. TJs constitute the
most apical intercellular junctional complex in polarized
epithelium and endothelium, with three key biological
functions: a barrier to paracellular diffusion of
bloodborne polar substances , a fence preventing the lateral
diffusion of lipids and integral membrane proteins, thus
maintaining cell polarization [5-7] and an intracellular
signaling platform which will be described below.
Brain endothelial TJ strands, like epithelial TJs, are
composed of integral membrane proteins (occludin,
claudins and junctional adhesion molecules (JAMs))
involved in intercellular contacts and interactions with
cytoplasmic scaffolding proteins such as zonula occludens
(ZO) proteins, actin cytoskeleton and associated proteins,
such as protein kinases, small GTPases  and
heterotrimeric G-proteins .
Excellent reviews have recently been published on the
architecture of TJ complexes in epithelial and brain
endothelial cells [10,11]. Here we will briefly recall the
main features of the structural organization of TJs at the
BBB and will focus on transcriptional regulation,
posttranslational modifications and subcellular localization
of TJ proteins and their consequences for BBB integrity
with exposure to various environmental stimuli and
during CNS disorders.
Components of TJs in brain endothelial cells
As in polarized epithelial cells where TJs have been
mostly studied, the TJ backbone in brain endothelial
cells consists of transmembrane proteins (occludin,
claudins and JAMs) which recruit a number of
membrane-associated cytoplasmic proteins.
Transmembrane proteins as the BBB TJ backbone
Occludin (60kDa), a tetraspan integral membrane
protein, was the first TJ-specific protein identified [12,13] in
epithelial cells and shown to be functionally important
for barrier function . It is a member of the family
of TJ-associated marvel proteins (TAMP) with
tricellulin (marvelD2)  and marvelD3 [16,17]. Both the
MARVEL transmembrane domain of occludin,
encompassing the four transmembrane helices, and its coiled
coil cytosolic C-terminus were recently described to
mediate its lateral (i.e. cis-) oligomerization in epithelial
MDCK cells [18-20]. More precisely, cystein residues in
these domains are directly involved in oligomerization
through disulfide bridge formation. This process being
redox-sensitive, oligomerization of occludin likely
contributes to the redox-dependency of the TJ assembly
[20,21]: whereas normoxia conditions support occludin
oligomerization and contribute to TJ assembly, oxidative
stress associated with hypoxia-reoxygenation  or
inflammation [23,24] results in TJ disruption. This novel
concept that occludin plays a key role in the redox
regulation of TJs has been very recently reviewed .
In addition, the second extracellular domain of
occludin is required for its stable assembly in TJs . Indeed,
synthetic peptides corresponding to this domain were
shown to perturb TJ permeability barrier in epithelial
cells [27-29]. The important contribution of occludin to
TJ function is illustrated by the observations that ectopic
expression of chicken occludin induced the formation of
TJ-like structures in Sf9 insect cells , while increasing
electrical resistance in MDCK cells . Conversely,
occludin degradation induced by viruses or bacteria (like
HIV-1 Tat protein or Neisseria meningitidis), is associated
with increased permeability in primary or immortalized
human brain microvascular endothelial cells, respectively
[32,33]. However, well-developed TJ strands were reported
in cells lacking occludin (human or guinea pig testis) 
and between adjacent occludin-deficient epithelial cells
[34,35]; together with the report that occludin
deficientmice are viable, exhibiting normal TJs morphology as well
as intestinal epithelium barrier function, these observations
indicate that occludin is dispensable for TJ formation
Claudins constitute a large family of 20-27kDa
membrane proteins (with four transmembrane domains)
expressed in TJs in various cell types [4,38-40]
(endothelial and epithelial cells). Brain endothelial cells
predominantly express claudin-3 and claudin-5 [41,42], claudin-12
likely being also expressed [43,44]. A large corpus of data
clearly establishes the key contribution of claudin-3 and
claudin-5 to TJ formation and integrity at the BBB.
Indeed, exogenous expression of claudin-5 strengthens
barrier properties in cultured rat brain endothelial cells
, whereas depletion of claudin-5 induces the
disruption of the BBB in genetically-altered mice  and in
cultured human brain endothelial cells . Claudins
support TJ integrity via their capacity of cis- and
transhomodimerization as well as heterodimerization, notably
through their second extracellular loop, as recently
reported for claudin-5 [45-47]. Claudin-5 can interact
with claudin-3 [48,49] and the selective loss of the latter
during autoimmune encephalomyelitis or human
glioblastoma is associated with BBB breakdown .
Beside occludin and claudins, JAMs, although not
essential to TJ formation in epithelial and endothelial
cells, may be involved in the facilitation of assembly of
TJ components and in the establishment of cell polarity
by recruiting the polarity complex (Par-3/Par-6/aPKC:
see below) to TJs [50,51].
Membrane-associated cytoplasmic proteins in BBB TJs
A number of cytoplasmic proteins have been described
to associate with TJ transmembrane proteins and to
contribute somehow to TJ integrity in epithelial and
brain endothelial cells. Among them, the PDZ
domaincontaining, membrane-associated guanylate kinase
(MAGUK) family members have been largely documented:
zonula occludens-1 (ZO-1, 225kDa) , ZO-2 (160kDa)
, and ZO-3 (130kDa) . ZO-1 forms heterodimers
with ZO-2 and ZO-3 [54-56]. ZO proteins interact with the
C-terminal domain of claudins via their first PDZ domain
(PDZ1) , to JAMs by the third PDZ domain (PDZ3)
 and to occludin via their GUK domains [55,56,59]. It
is well established that ZO proteins are essential to the
assembly of claudins , occludin  and JAM-A 
at TJs, then anchoring this multimolecular complex to
the actin cytoskeleton . Par-3 (also known as ASIP)
 binds to JAM proteins [64-66] and recruits to TJs
atypical protein kinase C  and Par-6 , the three
proteins then forming a Planar Cell Polarity (PCP)
complex in polarized epithelial cells . Only very recently
was their expression confirmed also in brain endothelial
Among additional TJ-associated proteins, heterotrimeric
G-proteins (Gi) were first described, in association with
ZO-1, to contribute to TJ biogenesis and maintenance in
epithelial and brain endothelial cells [71-73]. Gi2 proteins
were reported to be involved in T-lymphocyte
extravasation, including in brain [74,75]. More recently, we reported
that Gi2 interacts with claudin-5 and that its depletion
increases brain endothelial cell permeability in vitro and
delays TJ reassembly after hyperosmotic shock (induced by
a high concentration mannitol treatment) . On the basis
of these observations, we proposed that claudin-5 and
Gi2, whether they interact directly or indirectly, might
control TJ integrity as components of a multiprotein
complex, including caveolin, ZO-1 linked to the actin
cytoskeleton and possibly also, occludin and MUPP-1.
Physiological regulation of TJ assembly by the
The NVU: regulation of TJ assembly by perivascular cells
Developmental role of astrocyte and pericyte secreted
Development and maintenance of the BBB requires
functional interactions between endothelial cells and
perivascular cells of the NVU: whereas astrocytes have
been well documented to regulate BBB formation and
integrity [76,77], only recently was the role of pericytes
unraveled (for reviews: [78-80]).
Indeed, early studies using co-culture of cerebral
endothelial cells and astrocytes (or culture in the presence of
astrocyte-conditioned medium) [81-87] highlighted the
role of astrocyte-derived soluble factors in maintaining
the specialized phenotype of brain endothelial cells
(Figure 1). In addition, more recent reports established
that pericytes also actively contribute to BBB formation
during development by the release of several growth
factors and morphogens [88-91].
Astrocyte- and pericyte-derived Wnt and hedgehog
morphogens were reported to control BBB formation
during development and TJ integrity. Indeed, the Wnt/
-catenin pathway has been recently discovered as a
major BBB-regulating pathway. Wnt ligation to its
membrane receptors, Frizzled4 (Fz4) and LRP5/6 expressed
by brain endothelial cells, inhibits the -catenin
repressor complex, allowing -catenin cytoplasmic
accumulation, nuclear translocation and transcription of various
genes, including claudin-3 in cultured murine brain
endothelial cells [96,99] (Figure 1). Moreover, in vivo
inactivation of Wnt factors (Wnt7a and Wnt7b) ,
Fz4 receptor  or injection of a soluble inhibitor of
the Wnt/ Frizzled receptor interaction  lead to
major vascular defects in the CNS (interestingly, not in
non-neuronal tissues) and to BBB breakdown, clearly
demonstrating a specific role for the Wnt/-catenin
pathway in BBB differentiation during development and
for BBB maintenance in adulthood. These exciting
observations (for review, see: ) open new research
avenues for controlling BBB permeability in pathological
situations as well as improving drug delivery to the CNS.
Sonic hedgehog (Shh), another well-known morphogen
protein, acting through its membrane receptors
Patched1 (Ptch1)/Smoothened (Smo), was also recently shown
to control BBB differentiation and to maintain the
immune privilege of the CNS by inhibiting the endothelial
production of chemokines and expression of adhesion
proteins supporting extravasation of leukocytes to the
In conclusion, these recent findings further document,
at the molecular and cellular levels, the functional
interactions between brain endothelial cells, pericytes and
astrocytes and emphasise the key importance of the
NVU in controlling BBB permeability and integrity. The
major cellular cross-talks at the NVU are illustrated in
Role of basement membrane-associated proteins
The vascular basement membrane (or basal lamina) is a
complex structure, composed of four glycoprotein
families: laminins, collagen type IV, nidogens and heparan
sulfate proteoglycans. Recent studies have unraveled the
contribution of the endothelial laminin isoform 5 to
the barrier property of the BBB by selectively inhibiting
lymphocyte infiltration; the basement membrane thus
contributes to maintain the well-known immune privilege
of the CNS .
The heparan sulfate proteoglycan agrin is found in the
basal lamina of brain microvessels . A strict positive
correlation has been reported between agrin deposition
and expression of occludin , whereas, conversely,
absence of agrin in glioblastoma vessels was shown to
correlate with the lack of TJ proteins (occludin, claudin-5):
these observations strongly suggest that agrin may
regulate TJ formation in brain endothelium. Recently, agrin
was described to be involved in the development of the
BBB by contributing to astrocyte polarity .
Moreover, 1-integrin-mediated attachment of brain
endothelial cells to the basement membrane has also been
reported to be critical for stabilizing claudin-5 localization
at TJs and maintaining BBB integrity in vitro and in vivo
. Genetic deletion of 1-integrin decreases the
expression of the polarity protein Par-3, leading to the
loss of endothelial cell polarity: these recent data
Figure 1 Schematic representation of TJ modulation by the NVU. (a) The basal lamina protein agrin increases claudin-5 (Cld5) and occludin
expression ; (b) Aquaporin-4 density, regulated by agrin, stabilizes TJ complexes through ZO-1 expression ; (c) 1-integrin engagement
stabilizes Cld5 localization at the TJ ; (d) astrocyte/pericyte-secreted TGF- induces Cld5 transcription through activation of Smad transcription
factor ; (e) Shh enhances expression of TJ proteins via its membrane receptor Ptch1/Smo and the transcription factor Gli-1 ; (f) Endothelial
PDGF- recruits pericytes which stabilize BBB phenotype ; (g) Wnt 7a/7b proteins, via their membrane receptors Frizzled-4 associated to
LRP5/6, induce Cld3 transcription through stabilization of -catenin ; (h,i,j) Angiopoietin-1 (Ang1), via its membrane receptor Tie2, enhances
VE-Cadherin clustering and Cld5 transcription through inhibition of FoxO1 activity by PI3K and -catenin sequestration [83,97] ; (k) VE-Cadherin
engagement recruits CCM1 and the polarity complex (PCP) leading to TJ stabilization .
suggest that 1-integrin-mediated brain endothelial cell
adhesion to the basement membrane may lead to the
development of cell polarity, TJ formation and BBB
VE-cadherin and -catenin as modulators of TJs
In addition to TJs, junctional complexes between
endothelial cells include adherens junctions (AJs), constituted
by transmembrane proteins VE-cadherin linked to the
actin cytoskeleton through catenins (eg: p120-catenin,
- and -catenin) [107-109]. Interestingly, AJ and TJ
complexes functionally interact in brain endothelial
cells: indeed, VE-cadherin engagement induces claudin-5
transcription through inhibition of FoxO1 activity (a
transcription repressor of claudin-5 gene) and -catenin
sequestration (a stabilizer of FoxO1 activity) in AJ
complexes , in line with the above-mentioned
capacity of -catenin, downstream of Wnt receptor
activation, to control claudin gene expression . These
findings clearly place VE-cadherin upstream of claudin-5
in the establishment, maturation and maintenance of
endothelial cell-cell junctions.
Contribution of shear stress to TJ modulation and
It is established that one important mechanical
stimulus contributing to BBB formation and maintenance is
shear stress , a tangential force generated by flow
across the apical surface of vascular endothelium
[111,112]. In line with the accepted concept that
cerebral microcirculation is highly heterogeneous, mean
shear stress levels in brain microvessels has been
estimated in a range as wide as 0.01 to 10 dynes/cm2
in capillaries and 10100 dynes/cm2 in arterioles [113-115].
Several dynamic in vitro models were developed in order
to mimic a physiological situation (using laminar, steady
flow) or a pathological condition (such as
atherosclerosis), using an irregular flow. Interestingly, culturing
human umbilical vein endothelial cells (HUVECs) in a
laminar flow chamber in the presence of meningococci
(N. meningitidis) was instrumental for unraveling some
key molecular mechanisms of CNS invasion by these
meningitis-causing human pathogens . Regarding
BBB differentiation, culture of brain endothelial cells
under flow has been reported to induce the expression of
the TJ proteins occludin and ZO-1, to promote actin
cytoskeleton reorganization and to reduce endothelial
permeability [113,115-117]. In addition, very recent
ings suggest that physiological shear stress (6 dynes/cm )
may increase the expression of a variety of
BBBassociated genes in human brain microvascular
endothelial cells, such as genes encoding for TJ proteins
(ZO1, claudin-3, claudin-5), several influx transporters
(Glut-1) and multidrug resistance efflux transporters
(ABCB1/P-gp, ABCC5/MRP5) . Nevertheless,
further investigation is still required to get a better
understanding of the contribution of shear stress to the
maintenance of BBB integrity.
Dysregulation of the BBB via phosphorylation and
relocalization of TJ proteins
Studies on CNS diseases associated with BBB dysfunctions
(e.g. stroke, multiple sclerosis, cerebral infection, brain
tumors, Parkinsons and Alzheimers diseases) have pointed
to various molecular mechanisms involved in disruption of
TJ integrity, notably including Serine/Threonine
(Ser/Thr)and Tyrosine (Tyr)-phosphorylation, down-regulation,
degradation or translocation of TJ proteins; a non
exhaustive list of related reports are presented in Table 1.
More than other TJ proteins (such as claudins or JAMs),
occludin has been the focus of numerous studies
investigating post-translational modifications and their
consequences on TJ integrity (see for review: [118,119]).
Table 1 Dysregulation of the BBB via phosphorylation or down-regulation of TJ proteins
Cld5 and Occludin
Cld5 (T207) Occludin (T382/S507)
Cld5, Occludin and ZO-1
Occludin and ZO-1 distribution
Alcohol / Reactive oxygen species
Hypoxia / Reactive oxygen species
Tyrosine phosphatase inhibition
Reactive oxygen species
Ser/Thr-phosphorylation of TJ proteins and regulation
of barrier permeability
Ser/Thr-phosphorylation forms of occludin are found
concentrated at TJs whereas dephosphorylated occludin
is rather detected on basolateral membranes and
associated with disrupted TJs in epithelial cells [142,143]
as well as in brain endothelial cells in experimental
autoimmune encephalomyelitis, a murine model of multiple
sclerosis characterized by brain inflammation .
Regarding claudin-5, phosphorylation of its C-terminal
domain on Thr207 residue in response to PKA or Rho
kinase activation [120,123,145] generally affected TJ
integrity in brain endothelial cells and increased permeability.
Differential regulation of TJs by Protein Kinases C (PKCs)
PKC-dependent pathways have been involved in
endothelial barrier disruption, as reported following treatment by
pertussis toxin, an inhibitor of Gi heterotrimeric G
proteins , or in response to the pro-inflammatory
cytokine interleukin-6 (IL-6) which plays a critical role
during hypoxia . However, early reports had clearly
established that PKC activity was crucial for BBB integrity
in epithelial cells, inasmuch as PKC inhibitors completely
blocked the formation of TJs [148,149]; in addition,
PKCmediated phosphorylation of occludin (on residue Ser338)
was involved in occludin targeting to TJs and TJ
stabilization in epithelial MDCK cells .
At least part of the interpretation of these apparently
conflicting data may be found in the heterogeneity of
the PKC family. The Ser/Thr-kinases PKCs are indeed
classified into conventional (cPKC: , I, II and ),
novel (nPKC: , , , , ) and atypical (aPKC: ,) PKC
isozymes  according to their modes of regulation.
Accumulating evidence has pointed to a differential
capacity of PKC isozymes to regulate BBB permeability.
Indeed, activation of nPKC- and aPKC- signaling by
hypoxia-mediated TJ proteins results in relocalization
(such as claudin-5, occludin, ZO-1) and increased BBB
permeability in rat brain microvascular endothelial cells
(in vitro and in vivo) [121,151]. In human brain
microvascular endothelial cells, cPKC, cPKC II and aPKC/
isoforms were activated by HIV-1 gp120 envelope
protein, leading to BBB disruption, intracellular calcium
increase and monocyte migration across cell monolayer
. Interestingly, when cPKC- was found to
contribute to TJ disassembly, nPKC- activation mediated
TJ formation in epithelial MDCK cells . In line
with this observation, over-expression of cPKC- in rat
epididymal microvascular endothelial cells was reported
to enhance thrombin-induced permeability, whereas
nPKC- expression promoted barrier function .
By contrast, IL-25, expressed by mouse brain capillary
endothelial cells, was shown to prevent
inflammationinduced BBB disruption and down-regulation of TJ
proteins (occludin, claudin-5, JAMs) through activation
of the nPKC- pathway . Altogether, these
observations strongly suggest that nPKC-selective activation
generally contributes to maintaining barrier integrity,
whereas cPKC activation has the opposite effect, both
in polarized epithelium and endothelium (Table 1).
Regarding aPKC isoforms ( and ), they have been
shown to contribute to the establishment of epithelial
cell polarity, via participation in the PCP complex
together with Par-3 and Par-6 [63,68,155]. As mentioned
above, the PCP complex is recruited to endothelial TJs by
Par-3 binding to JAM proteins [64-66]. Over-expression
of a dominant negative mutant of aPKC causes
mislocalization of Par-3 and affects the biogenesis of the TJs in
epithelial cells , suggesting that Par-3 is a substrate of
aPKC and that its localization in epithelial cells is
dependent upon its phosphorylation. In the same line,
the VE-cadherin/CCM1 (a protein encoded by the
CCM1 gene which is mutated in a large proportion
of patients affected by cerebral cavernous
malformation) complex controls aPKC- activation and Par-3
localization during early steps of brain endothelial cell
polarization . The participation of this PCP complex
to TJ integrity was further illustrated by the recent
observation that meningococcal adhesion to human cerebral
endothelial cells recruited Par-3, Par-6 and aPKC- under
bacterial colonies and induced disruption of cell-cell
junctions . Surprisingly, a distinct Par-3/Par-6
complex, directly associated with VE-cadherin and lacking
aPKC, has also been identified in endothelial cells .
Finally, although additional polarity complexes are
known in epithelial cells (the apical Crumbs complex and
the basolateral Scribble complex) where they also
contribute to TJ formation and regulation, no similar
observations have been reported, to our knowledge, in brain
BBB disruption mediated by Rho/ Rho kinase and MLCK
The RhoA GTPase signaling pathway, activated by several
membrane receptors, has been extensively documented in
various cell types to induce actin cytoskeleton
rearrangements involved in cell migration and proliferation. In
brain endothelial cells, RhoA activation increased
permeability, in response to inflammatory stimuli, through one
of its major effectors Rho kinase (ROCK) [158,159].
Among these inflammatory stimuli, chemokines like
MCP-1/CCL2, acting via their seven
transmembranedomain receptors, are known to activate the RhoA/ROCK
pathway in mouse brain endothelial cells, to induce
occludin, claudin-5 and ZO-1 Ser/Thr-phosphorylation,
followed by their delocalization from TJs, ultimately leading
to increased barrier permeability [124,160]. Similarly,
enhanced monocyte migration across human brain
endothelial cells was observed in an HIV-1 encephalitis
model [123,161]. Also, adhesion molecules like ICAM-1
and VCAM-1 were shown, in response to lymphocyte/
monocyte adhesion, to transduce signals in rat brain
endothelial cell lines including activation of the RhoA/
ROCK pathway [162,163]: activation of this pathway
ultimately leads to enhanced lymphocyte migration,
suggesting that this process may be involved in the massive
infiltration of immune cells into the CNS observed in
multiple sclerosis. It must be mentioned, however, that
lymphocyte migration across the BBB may also happen
via a transcellular pathway, leaving intact endothelial TJs
Rearrangements of the actin cytoskeleton have long
been recognized to be regulated, not only by the RhoA/
ROCK pathway, but also, often in a coordinated manner,
by the myosin light chain kinase (MLCK): MLCK
directly phosphorylates the myosin light chain, leading to
actomyosin contraction and endothelial barrier disruption
[165-167]. In the same line, inhibition of MLCK in bovine
brain endothelial cells was more recently reported to
prevent hypoxia-induced BBB disruption , whereas
alcohol increased human brain endothelial cell
permeability via activation of MLCK and phosphorylation of
occludin and claudin-5 [125,126]. Recently,
proinflammatory cytokines (IL1 and TNF), secreted by
lymphocytes chronically infected by the HTLV-1
retrovirus, were reported to induce barrier disruption in the
human brain endothelial cell line hCMEC/D3, associated
with loss of occludin and ZO-1 through activation of the
MLCK pathway .
In conclusion, as summarized in Table 1,
inflammationor infection-induced actin cytoskeleton rearrangements in
brain endothelial cells, mediated by the RhoA/ROCK
and/or MLCK pathways, are associated with the
phosphorylation, followed by delocalization or degradation of
TJ proteins, and BBB disruption.
BBB dysregulation by Tyr-phosphorylation of TJ proteins
Early studies with cultured bovine brain endothelial cells
and MDCK cells had pointed to Tyr-phosphorylation as a
mechanism for increasing TJ permeability .
Accumulating evidence demonstrated that Tyr-phosphorylation of
TJ proteins, as well as AJ proteins, was directly involved in
BBB disruption, as observed in various pathological
situations, although the identity of the Tyr-kinases involved
often remained unknown. Unlike occludin Ser/Thr
phosphorylation associated with barrier formation, as
mentioned above, occludin Tyr-phosphorylation was reported
to be associated with increased permeability of cultured
rat brain endothelial cells exposed to glutamate, as a way
to mimic cerebral ischemia  (Table 1). Like other
pro-inflammatory cytokines, transforming growth factor
(TGF)-1 is known to increase BBB permeability: as
recently reported in bovine retinal and human brain
endothelial cells, this effect was mediated by
Tyrphosphorylation of both claudin-5 and VE-cadherin
. Vascular endothelial growth factor (VEGF), a
major angiogenic factor, which is drastically enhanced in
response to hypoxia, promotes Tyr-phosphorylation of
TJ proteins (ZO-1, occludin) in mouse brain and retinal
endothelial cells [168,169] either directly via its
membrane receptor tyrosine kinase VEGFR2 or via the
activation of the cytosolic tyrosine kinase c-src [128,170].
VEGF-mediated Tyr-phosphorylation of TJ proteins in
brain endothelial cells was often followed by their
down-regulation and/or re-localization, leading to TJ
destabilization and permeability increase [136-138,171].
Alterations of expression and localization of TJ proteins
Caveolae are specialized plasma membrane
microdomains, abundantly found in endothelial cells where they
mediate various biological events such as transcytosis,
vascular permeability and angiogenesis [172,173]. They are
enriched in the small membrane protein caveolin-1 which
has been shown to recruit TJ proteins [9,174].
Caveolaemediated endocytosis induced by actin depolymerization
was reported to evoke occludin internalization in MDCK
cells . Interestingly, exposure of cultured rat brain
endothelial cells to the HIV-1 Tat protein was reported to
increase TJ permeability, through alterations in expression
and distribution of TJs proteins: occludin, claudin-5, ZO1,
ZO2 [134,176]. In the same line, the increase in TJ
permeability observed in mouse brain endothelial cell response
to the inflammatory cytokine CCL2 was recently shown to
be associated with claudin-5 and occludin internalization
in a caveolae-dependent manner . Altogether these
results strongly support the conclusion that alterations in
expression and localization of TJ proteins, associated or
not with their phosphorylation in response to various
pathological stimuli, directly contribute to TJ disruption
and BBB permeability increase (Table 1); in addition, they
suggest a role of caveolin-1/caveolae in such TJ remodeling.
The brain endothelial TJ complex, which constitutes a
key feature of the BBB, is now understood as a
scaffolding and signaling platform in close interaction
with the actin cytoskeleton and the AJ complex. It
also appears as a dynamic complex, submitted to
post-translational modifications in response to
physiological and pathological stimuli. Indeed, perivascular
cells of the NVU, notably astrocytes and pericytes,
secrete multiple growth factors and morphogens that
contribute to TJ formation and integrity. Conversely,
various pathological situations associated with the
presence of inflammatory cytokines, reactive oxygen
species or pathogens, lead to TJ disruption following
phosphorylation and/or internalization of TJ proteins.
Although our understanding of TJ architecture and
function has significantly increased over the last ten
years, a number of issues will have to be addressed in
the next future, in particular taking advantage of new
and/or global analysis technologies. For example,
superresolution light microscopy (time-lapse stimulated
emission depletion (STED) imaging) recently appeared as a
very powerful approach to unravel synapse assembly and
plasticity ; in the same line, super-resolution
microscopy of TJs (with a resolution down to 5080 nm)
of cerebral microvessels in brain slices should provide a
more accurate understanding of TJ organization and
dynamics. Also, thanks to the availability of validated
BBB in vitro models, identification by mass spectrometry
(MS/MS analysis) of the secreted proteins (so-called
secretome) from brain endothelial co-cultures with
astrocytes or pericytes may unravel new paracrine
signaling pathways in the NVU which contribute to the
stabilization of TJs at the BBB; in addition, similar
analyses in the presence of inflammatory agents or
pathogens  may highlight unsuspected mechanisms of TJ
disruption. This approach will complement quantitative
targeted absolute proteomics (also known as selected
reaction monitoring (SRM)), an emerging approach to
quantify membrane proteins . This technology
will also greatly benefit the field, allowing absolute
quantification of TJ proteins in physiological and
various pharmacological situations, as recently proposed
. The treatment of neurological diseases is
currently hampered by difficulties encountered in
delivering therapeutic compounds to the brain, across the
BBB. Because previous drug delivery strategies based
on transcellular transport machinery have shown
limited efficacy so far, it is tempting to propose that
transient modulation of TJs at the BBB, using in vitro
models of the BBB and in vivo models of human
pathologies, may constitute an alternative approach for
drug delivery to the brain. Clearly, this field will
benefit greatly from an in-depth understanding of TJ
architecture and functional regulatory mechanisms.
AJ: Adherens junction; BBB: Blood brain barrier; CNS: Central nervous system;
ECL: Extracellular loop; Fz: Frizzled; HUVEC: Human umbilical vein endothelial
cell; IL: Interleukin; JAM: Junctional adhesion molecule; MAGUK: Membrane
associated guanylate kinase; MLCK: Myosin light chain kinase;
NVU: Neurovascular unit; PCP: Planar cell polarity; PKC: Protein kinase C;
Ptch1: Patched-1; ROCK: Rho kinase; Shh: Sonic hedgehog;
Smo: Smoothened; TAMP: Tight junction-associated marvel proteins;
TGF: Transforming growth factor; TJ: Tight junction; VEGF: Vascular
endothelial growth factor; ZO: Zonula occludens.
The authors declare no conflict of interest.
ACL and CA: were responsible for the collection of data and references, and
for the drafting of the document. FG and KG were involved in the collection
of data and drafting of the document. POC was responsible for the drafting
and editing of the document, and for the discussion of the data. All authors
read and approved the final manuscript.
3Universit Paris Descartes, Sorbonne Paris Cit, Paris, France.
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