STATE-OF-THE-ART REVIEW

The dynamic blood–brain barrier

James Keaney and Matthew Campbell
Smurfit Institute of Genetics, Trinity College Dublin, Ireland

Keywords
Alzheimer’s disease; astrocytes; blood–brain
barrier; GLUT-1; gut microbiota; Mfsd2a;
neurovascular unit; pericytes; tight
junctions; transcytosis
Correspondence
M. Campbell, Smurfit Institute of Genetics,
Trinity College Dublin, Dublin 2, Ireland
Fax: +353 189 638 48
Tel: +353 189 624 84
E-mail: Matthew.Campbell@tcd.ie
(Received 29 May 2015, revised 20 July
2015, accepted 12 August 2015)
doi:10.1111/febs.13412
With the endothelium as its central unit, the blood–brain barrier (BBB) is
a complex multicellular structure separating the central nervous system
(CNS) from the systemic circulation. Disruption of the BBB has now been
implicated in a multitude of acute and chronic CNS disorders indicating
the potentially devastating effects of BBB breakdown on brain function.
However, the healthy BBB is not an impermeable wall, but rather a com-
munication ‘centre’, responding to and passing signals between the CNS
and blood. New studies are identifying BBB-specific transport pathways
that tightly regulate the entry and exit of molecules to and from the brain.
They are revealing a highly plastic barrier in which dynamic changes in
BBB components like paracellular tight junction complexes can contribute
to BBB maintenance. Here, we provide a succinct overview of the current
state-of-play in BBB research and summarize novel findings into BBB
regulation in homeostatic regulation of the brain.

Introduction
In the mammalian body, numerous biological barriers
separate the circulating blood from the interstitial fluid
that surrounds tissues. Positioned along the blood ves-
sels of the central nervous system (CNS), the blood–
brain barrier (BBB) is perhaps the most selective and
tightly controlled of these barriers, reflecting the
brain’s critical roles in cognition, regulating metabo-
lism and co-ordinating the functions of peripheral
organs. Because communicating this information
depends on fine control of electrical and chemical sig-
nals between neurons, the brain requires a precise and
balanced microenvironment. The BBB is therefore
essential in regulating the influx and efflux of ions,
oxygen and nutrients between the blood and brain
compartments and protecting the delicate neural tissue
by restricting the invasion of toxins and pathogens. To
sustain this robust barrier, CNS endothelial cells have
properties distinct from endothelial cells in other tis-
sues: the presence of BBB-specific transporter and
receptor proteins to control entry and exit of metabo-
lites across cells (transcellular transport) and high
electrical resistance tight junctions (TJs) to limit move-
ment between adjacent cells (paracellular transport);
and low levels of transcytotic vesicles compared to
peripheral endothelia and an absence of fenestrae
(small pores that allow rapid passage of molecules in
peripheral endothelial cells) [1,2].
Although the endothelial cells of the CNS vascula-
ture act as the principal barrier unit, it is now recog-
nized that an intricate network of molecular crosstalk
among a variety of cell types is required to form the
BBB during embryonic development and to maintain
its integrity in adulthood [3]. Cell–cell interactions
within the ‘neurovascular unit’ (NVU) – the milieu of

Abbreviations
Ab, amyloid-b; AD, Alzheimer’s disease; AJ, adherens junctions; BBB, blood–brain barrier; CNS, central nervous system; CSF, cerebrospinal
fluid; LRP1, lipoprotein receptor-related protein 1; NVU, neurovascular unit; RAGE, receptor for advanced glycation end products; TBI,
traumatic brain injury; TEM, transendothelial leukocyte migration; TJ, tight junction; VE, vascular endothelial; WNV, West Nile virus; ZO-1,
zonula occludens-1.

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The dynamic blood–brain barrier
neurons, astrocytes, pericytes, microglia and other
components of the brain parenchyma that communi-
cate with endothelial cells – are helping to explain how
the unique characteristics of the BBB are induced and
how disease-associated dysfunction of any one NVU
component can impact BBB permeability [4]. Recent
work has also identified how deficiencies in specific
macromolecule transporters (e.g. GLUT-1, Mfsd2a)
[5–7] and paracellular TJ complexes [8] at the BBB can
affect overall cerebrovascular integrity. This has impli-
cations in the study of CNS disorders like Alzheimer’s
disease (AD) and in developing novel drug-delivery
systems based on BBB biology. Insights from studies
of infection and innate immunity in the CNS are also
challenging the oft-used analogy of the BBB as a
‘brick wall’ guarding the precious neuronal tissue [9].
There is an increased awareness that dynamic changes
in the levels and cellular distribution of BBB compo-
nents such as TJ proteins help to maintain a healthy
and robust BBB under physiological conditions.
Indeed, we discuss the emerging influences of diverse
biological processes like sleep–wake behaviour and gut
microbiota on BBB development and integrity, high-
lighting a complex interplay between CNS- and
peripheral-derived signals converging at the BBB on a
daily basis.

lange of
The ‘neurovascular unit’: a me
cell types
A dominant question in early BBB research concerned
whether the unique properties of cerebral endothelial
cells were intrinsic to this cell type or whether signals
from the surrounding brain microenvironment induced
BBB formation. In early transplantation experiments by
Stewart and Wiley [10] using quail chick chimeras, it
was found that following grafting of immature avascu-
lar brain tissue from embryonic quails into the coelom
of chick embryos, the abdominal vessels vascularizing
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J. Keaney and M. Campbell
the grafted tissue developed endothelial cells with
characteristics of the nonleaky BBB such as TJs and
minimal transcytotic vesicles. When dorsal mesoderm
from embryonic quails was transplanted into the embry-
onic chick brain, the vessels developing in the grafted
tissue lacked barrier properties [10]. This suggested that
interactions between the nascent vascular tissue and the
surrounding neural microenvironment are crucial to
BBB development. Much attention has since focused on
the roles of two cell types, astrocytes and pericytes,
which exist in close anatomical association with brain
endothelial cells. Along with neurons, microglia (the
brain’s resident macrophages), peripheral immune cells,
the noncellular basement membrane and other compo-
nents, it has become clear that no cell type in the brain
functions in isolation, but instead is in constant commu-
nication with other NVU members. See Fig. 1 for sche-
matic representation of the BBB.
Astrocytes
Astrocytes are glial cells that help support and protect
neurons by controlling neurotransmitter and ion con-
centrations to maintain the homeostatic balance of the
neural microenvironment, by modulating synaptic
transmission and by regulating immune reactions [11].
Astrocytes are also known to interact with endothelial
cells through their end-feet projections that encircle the
abluminal side of cerebral capillaries [12]. In the adult
brain, such interactions are important in synchronizing
metabolite levels with cerebral blood flow and vasodi-
lation, and regulating brain water content [13]. For
example, the most abundant water channel protein,
aquaporin 4, is predominantly expressed in astrocytic
end-feet surrounding CNS vessels [14]. Astrocytes are
also a key cellular support of BBB integrity. Recent
in vitro and in vivo molecular studies have revealed
several effector molecules released by astrocytes that
function to enhance and maintain barrier tightness,
Fig. 1. The neurovascular unit comprises
endothelial cells, pericytes and astrocytes
that together confer unique properties on
the blood–brain barrier. The endothelial
cells that line the cerebral vasculature
have numerous transcellular transporters
in addition to well-evolved tight junctions
that limit the passive paracellular diffusion
of all but the smallest of solutes and ions.
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J. Keaney and M. Campbell
primarily members of the Hedgehog family [15], the
renin–angiotensin hormone system [16], and the
cholesterol and phospholipid transporter molecule
apolipoprotein E [17]. Release of apolipoprotein E
from astrocytes, for example, regulates endothelial TJs
by signalling through the low-density lipoprotein recep-
tor-related protein 1 (LRP1) on both pericytes and
endothelial cells of CNS microvessels [17,18].
The role of astrocytes in BBB development in the
immature brain is more controversial. When Janzer
and Raff [19] injected neonatal rat astrocytes into the
anterior chamber of the eye, only vessels formed in the
presence of astrocytes excluded Evans blue dye (in-
jected systemically). Based on these results, it was pro-
posed that astrocytes were necessary for the formation
of impermeable TJs, but in a follow-up study by a sep-
arate group, no ultrastructural changes in the develop-
ing vessels of the astrocyte grafts was observed [20].
In vitro co-cultures of endothelial cells with astrocytes
or astrocyte-conditioned media have been shown to
induce more complex TJs, elevated expression of trans-
porters and increased transendothelial electrical resis-
tance although these studies are limited by the use of
adult endothelial cells [21,22]. Furthermore, the finding
that astrocytes are not present in the developing brain
during the time of initial vascularization lends support
to a role for astrocytes in TJ maintenance and not for-
mation [23]. More recently, however, production and
release of retinoic acid by astrocyte precursors (radial
glial cells) has been shown to act through the retinoic
acid receptor-b on developing brain endothelial cells to
induce BBB properties [24].
Pericytes
Although astrocyte–endothelial interactions along
mature vessels are essential for BBB integrity and may
also help seal the barrier in the final stages of BBB
formation, pericytes have emerged as a central NVU
component in the first stages of ‘barriergenesis’. Peri-
cytes are perivascular cells of mesodermal origin whose
elongated processes ensheath CNS vessel walls. The
CNS vasculature has significantly higher pericyte cov-
erage compared with peripheral vessels and pericytes
have an important role in regulating capillary diame-
ter, cerebral blood flow and extracellular matrix pro-
tein secretion and levels [25]. Indeed, a recent study
has highlighted an essential role of capillary pericytes
in regulating cerebral blood flow – the neurotransmit-
ter glutamate promotes the release of messengers
including prostaglandin E2 and nitric oxide that help
dilate capillaries by actively relaxing pericytes [26].
During early angiogenesis and barrier formation along
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The dynamic blood–brain barrier
nascent vessels of the embryonic brain (driven by
vascular endothelial growth factor and Wnt/b–catenin
modelling among other pathways) [3,27–32], pericyte
recruitment is crucial to establishing BBB characteris-
tics. Complete loss of pericytes in platelet-derived
growth factor (Pdgfb) or Pdgfrb knockout mice has
been shown to result in CNS microhaemorrhages, dys-
functional TJs, increased vascular permeability and
embryonic lethality [23,33]. It is thought that PDGFb
release by endothelial cells of sprouting vessels pro-
motes the recruitment and proliferation of PDGFRb-
positive pericytes, which in turn decrease transendothe-
lial trafficking and inhibit the expression of molecules
in endothelial cells that promote immune cell infiltra-
tion [4,23]. Following pericyte recruitment to develop-
ing vessels, further communication between pericytes
and neighbouring endothelial cells is established via
transforming growth factor b and its receptor
(TGFRb) [34]. Importantly, these pericyte effects on
BBB formation precede astrocyte differentiation by
one week [23].
Pericytes are also vital to BBB integrity during
adulthood because Pdgfrb knockout mice exhibit age-
dependent BBB dysfunction as a result of reduced TJ
protein expression [35], whereas young adult mice with
hypomorphic alleles of Pdgfb display defects in BBB
integrity as a result of increased rates of endothelial
transcytosis [36]. Intriguingly, Armulik and colleagues
[36] also found that pericytes not only regulate mature
BBB integrity, but also function to guide astrocytic
foot processes to cerebral vessel walls and mediate the
polarization of astrocytic end-feet, again emphasizing
the complex interdependency among components of
the NVU. Another recent study has highlighted the
involvement of pericytes in disease phenotypes such as
neurodegeneration. Progressive loss of pericytes in a
mouse model of AD lacking a single Pdgfrb allele
(APPSw/Pdgfrb+/ ) accelerated the hallmarks of AD
pathology including elevated brain amyloid b (Ab)
levels, tau pathology and early neuronal loss [37].
These combined findings underscore the importance of
pericytes not only in barrier differentiation during
embryonic development, but also in maintaining a
healthy and robust BBB and proper neural function in
adulthood.
Microglia
Derived from haematopoietic precursors that migrate
from the yolk sac into the CNS parenchyma, microglia
act as the brain’s main line of defence past the BBB
and play a vital role in innate immune responses in the
CNS [38]. Although located in close proximity to
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The dynamic blood–brain barrier
endothelial cells of brain microvessels, little is known
about potential microglial–endothelial communication
in forming and regulating the homeostatic BBB. One
study has demonstrated that microglia associate with
endothelial tip cells along nascent vessels in the devel-
oping brain and promote the fusion of tip cells in the
stages following vascular endothelial growth factor-
mediated tip cell induction [39]. At present, more is
known about the activation of microglia in CNS disor-
ders like AD and multiple sclerosis, which are associ-
ated with BBB breakdown and neuroinflammation. In
these conditions, microglial activation may be both a
cause and consequence of BBB dysfunction [40].
Microglia can exist in one of two active states: in the
classically activated M1 pathway, microglia primarily
release proinflammatory cytokines like interleukin-1b
and tumour necrosis factor-a, whereas in the alterna-
tive M2 pathway microglia are involved in tissue
repair, phagocytosing damaged neurons and foreign
material, releasing chemokines and vascular endothe-
lial growth factor, and activating neurotrophic path-
ways [41]. In multiple sclerosis, the release of
proinflammatory cytokines and reactive oxygen species
by activated microglia can exacerbate myelin damage
and increase BBB permeability potentially via down-
regulation of adherens junction [AJ, such as vascular
endothelial (VE)-cadherin] and TJ (occludin and clau-
din-5) proteins [42,43]. In AD, T-cell transmigration
across the BBB can also be driven by microglia-
derived signals such as tumour necrosis factor-a [44].
Tumour necrosis factor-a secretion by microglia in
response to Ab leads to upregulation of MHC class I
in brain endothelial cells, which in turn provides a
mechanism for T-cell migration into the brain [44].
Recruitment of these peripheral lymphocytes may help
or hinder resident microglia in resolving brain injury
depending on the CNS disease and the disease stage
[9]. Brain endothelial cells can also secrete molecules
that cause microglial activation. Microvessels from
AD patients have been found to release high levels of
the neurotoxic protease thrombin [45]. Previous work
has highlighted how thrombin can increase nitric oxide
levels, activate microglia and contribute to the loss of
dopaminergic neurons [46]. Along with bone-marrow-
derived perivascular macrophages that can act as anti-
gen-presenting cells [47], a complex interplay between
systemic- and CNS-derived immune cells exists at the
BBB.
Basement membrane
An often-overlooked component of the NVU is the
basement membrane – the extracellular matrix of
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J. Keaney and M. Campbell
structural proteins secreted by astrocytes, pericytes and
endothelial cells. The basement membrane helps to
regulate crosstalk between NVU components and to
this end, Yao and colleagues [48] have recently shown
that a brain-specific basement membrane component,
astrocytic-derived laminin, helps to regulate pericyte
differentiation via binding to the integrin a2 receptor
on pericytes. Disruption of this basement membrane
protein in the brain converts pericytes from a resting
to a contractile phenotype, which in turn decreases
aquaporin 4 levels in astrocytic end-feet and decreases
endothelial TJ expression [48].
It is now clear that molecular communication
between cellular and noncellular members of the
NVU, first established during early CNS angiogenesis
and barriergenesis, must remain in place to ensure
BBB tightness in adulthood. Moreover, it suggests that
if this fine balance is disrupted, it may directly or indi-
rectly contribute to CNS disease pathogenesis. As we
see in the next sections, these NVU components help
to establish and maintain transport pathways located
across brain endothelial cells, the breakdown of which
can lead to uncontrolled BBB permeability.
BBB transport routes in development
and disease
Passive diffusion across brain endothelia is limited to
lipid-soluble molecules of approximately < 180 Da in
size and with fewer than 10 hydrogen bonds [49].
Instead, distinct BBB transcellular transport systems
exist to tightly regulate the CNS entry and exit of
hydrophilic molecules. Carrier-mediated transport is
the major route for essential nutrients like glucose,
fatty acids and amino acids to travel across the BBB
[1]. New studies are revealing BBB-enriched compo-
nents that lead ‘double lives’ – not only as trans-
porters of essential molecules, but also as important
regulators of BBB permeability. In the context of
CNS disease, we also consider the BBB transcytosis
of Ab – the major pathogenic component in Alzhei-
mer’s disease.
GLUT-1
The glucose transporter GLUT-1 (encoded by the
Slc2a1 gene) is enriched in CNS endothelial cells com-
pared with peripheral endothelia [50]. The importance
of GLUT-1 in brain glucose metabolism is reflected in
human GLUT-1 deficiency syndromes in which
affected individuals suffer from infantile seizures, cog-
nitive impairments and developmental delay [51]. Fur-
thermore, GLUT-1 deficiency has profound impacts
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J. Keaney and M. Campbell
on BBB physiology and neurovascular function
because Slc2a1+/ mice have been found to have
decreased brain glucose uptake, reduced cerebral blood
flow and increased BBB permeability due to lower
TJ protein levels [5]. This suggests that loss of BBB-
specific transporters can ultimately impact general
BBB integrity and also has implications in neurodegen-
erative disorders. GLUT-1 levels are known to be
decreased in brain microvessels of AD patients [52]
and correlate with reduced brain sugar uptake and
subsequent cognitive decline [53]. It is still unclear
whether reduced GLUT-1 levels in AD represent lower
energy demands in a degenerating brain or whether
the primary insult occurs at the BBB and precedes
brain atrophy. However, PET imaging studies have
suggested that altered glucose transport occurs early in
AD [54,55]. Analysis of phenotypes in APPSw/
Slc2a1+/ mice, an AD mouse model with deficits in
GLUT-1, indicates that BBB breakdown following
reduced GLUT-1 precedes and exacerbates features of
AD pathology including reduced Ab clearance across
the BBB, elevated brain Ab levels, impaired neural
activity and neuronal loss [5]. In light of these findings,
studying perturbations of BBB transporters in the
early stages of other CNS disorders may yield novel
insights into disease mechanisms.
Mfsd2a
Mfsd2a has been recently identified as a critical com-
ponent of BBB formation and integrity. Mfsd2a is a
brain endothelium-enriched receptor for the omega 3
fatty acid, docosahexaenoic acid (transported in the
form of lysophosphatidylcholine), which is essential
for normal brain growth and function [6]. Two human
genetic analyses have recently identified missense
mutations in the Mfsd2a gene that lead to autosomal
recessively inherited microcephaly syndromes [56,57].
Interestingly, all three Mfsd2a mutations identified in
these studies did not affect Mfsd2a protein expression
or cell surface localization. Instead, the Mfsd2a variant
affecting individuals in one pedigree (encoding
p.Ser339Leu) leads to partial inactivation of Mfsd2a’s
lysophosphatidylcholine transport activity and non-
lethal microcephaly and intellectual disability pheno-
types [56]. Two other Mfsd2a variants discovered in
separate families (p.Thr159Met and p.Ser166Leu)
cause complete Mfsd2a inactivation and these individ-
uals present with severe microcephaly, ventricu-
lomegaly (enlarged lateral ventricles), and seizures that
proves fatal in the early years of life [57]. In rodents,
Mfsd2a knockout mice have reduced brain levels of
docosahexaenoic acid [6] but also display a leaky BBB
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The dynamic blood–brain barrier
in embryonic and adult stages as a result of increased
endothelial vesicular transcytosis [7]. Similar to
GLUT-1, this suggests a double function for Mfsd2a
in mediating nutrient supply from the blood into the
brain while also regulating BBB integrity. Further-
more, because pericyte-deficient mice display reduced
endothelial Mfsd2a levels and increased vesicular traf-
ficking is seen in both pericyte-deficient and Mfsd2a
knockout mice, this suggests that one way in which
pericytes support BBB transport pathways and BBB
integrity is via the control of Mfsd2a expression [7].
Determining whether individuals with mutant Mfsd2a-
associated microcephaly syndromes display BBB
breakdown in addition to deficient lysophosphatidyl-
choline transport will be of great interest.
Ab transcytosis
Both brain-derived and peripheral Ab is transported
across the BBB via receptor-mediated transcytosis.
Based on recent measurements of venous to arterial
Ab concentration ratios in humans, ~ 25% of Ab
clearance from the brain occurs at the BBB [58].
Highly expressed in brain endothelial cells of the BBB,
the ATP-binding cassette efflux transporter P-glyco-
protein (Pgp) has been implicated in Ab transport out
of the brain as APP/Pgp-null transgenic mice show
increased brain Ab levels [59,60]. However much atten-
tion has focused on the receptor for advanced glyca-
tion end products (RAGE) and low-density LRP1 as
the central Ab transporters at the BBB. Elucidating
these transcellular pathways of Ab movement has
revealed how disrupting their normal activity can
accelerate Ab build up and AD pathogenesis. RAGE
is an Ab receptor found on the surface of neurons and
microglia and is also the major endothelial receptor
for Ab influx from the blood to the brain [61]. RAGE
expression at the BBB is increased in the brain
endothelium of many AD patients as well as in AD
transgenic mouse models, likely due to accumulation
of its ligand Ab [61]. LRP1 receptor mediates cellular
uptake and internalization of Ab for lysosomal degra-
dation in neurons, astrocytes, microglia and vascular
smooth muscle cells [62,63]. Working in parallel with
RAGE, LRP1 is a major Ab efflux transporter and
LRP1/Ab binding at the abluminal side of the BBB
mediates Ab clearance from brain to blood [64].
Decreased LRP1 levels have been detected in brain
microvessels in aged rodents, nonhuman primates and
humans, as well as in AD mouse models and AD
patients [64,65]. More recently, the Zlokovic group
have identified PICALM as being involved in the
internalization and trafficking of LRP1/Ab complexes
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The dynamic blood–brain barrier
across the BBB [66]. PICALM was previously identi-
fied from genome-wide association studies as a genetic
risk factor in AD [67].
Paracellular multiprotein complexes: adherens
junctions and tight junctions
Located between neighbouring brain endothelial cells,
adherens junctions (AJs) and TJs are distinct multipro-
tein complexes that interconnect at a structural and
functional level to seal the paracellular gap. Cadherins
are the major protein family of AJs and include VE-
cadherin, which forms homophilic interactions across
adjacent endothelial cells [68]. Although the roles of
AJs specifically at the BBB are not fully resolved, AJs
are believed to initiate cell–cell contacts, promote
endothelial cell survival, establish cell polarity and
respond to stimuli via interactions with the catenin
complex and actin cytoskeleton [69,70]. By contrast to
AJs that are present in all vascular beds of the mam-
malian body, high electrical resistance continuous TJs
are specific to the brain vasculature. TJ protein com-
plexes play a critical role in neuronal homeostasis by
restricting the paracellular diffusion of ions and
macromolecules across the BBB and by linking extra-
cellular stimuli with intracellular signalling in the brain
endothelium. Major TJ proteins at the BBB include a
number of interacting transmembrane components:
occludin, claudins (in particular, claudin-5 and clau-
din-12) and members of the junctional adhesion mole-
cule family (e.g. JAM-4) [71,72]. Anchored to these
transmembrane TJ proteins, intracellular components
such as zonula occludens-1 (ZO-1) act as a scaffold
connecting TJ strands with the actin cytoskeleton of
brain endothelial cells [73]. Where three brain endothe-
lial cells meet, tricellulin and lipolysis-stimulated
lipoprotein receptor have been identified as potential
regulators of paracellular permeability at tricellular
contacts [74,75].
Increasing evidence of crosstalk between compo-
nents of both AJs and TJs highlights a sophisticated
system in establishing and regulating the paracellular
barrier. ZO-1, for example, controls VE-cadherin-
mediated cell–cell tension and cytoskeletal organization
[76]. Conversely, VE-cadherin can upregulate claudin-5
and promote its junctional localization [77]. In
response to shear stress, VE-cadherin can also transmit
signals via Tiam1/Rac1, which reduces levels of tyro-
sine-phosphorylated occludin and increases barrier
strength [78]. In addition to transcriptional up- and
downregulation and changes in cellular localization,
post-translational phosphorylation of junctional pro-
teins at serine, threonine and tyrosine residues can reg-
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J. Keaney and M. Campbell
ulate paracellular permeability [8]. Loss of AJ proteins
like VE-cadherin has been reported in brain microves-
sels of multiple sclerosis patients [79], although more
research is needed to understand the involvement of
AJs in CNS disease pathology. TJ breakdown has
been linked to increased BBB permeability and neu-
ronal dysfunction in chronic neurological disorders
like amyotrophic lateral sclerosis and AD [80–82].
However, detailed analyses of events at the BBB fol-
lowing ischaemic stroke and traumatic brain injury
(TBI) reveal highly dynamic and kinetic rearrange-
ments of TJ complexes and other pathways in the min-
utes, hours and days after injury. Along with recent
studies of infection and innate immunity in the CNS,
this is helping to consider TJ complexes and the BBB
as adaptive structures that can quickly respond to sig-
nals emanating from either the brain or the blood.
Dynamic BBB remodelling: the flexible
TJ
Stroke and TBI
In rodent studies, both ischaemic stroke and TBI are
characterized by a ‘biphasic’ opening of the BBB fol-
lowing the initial insult. Although reperfusion of the
infarct site after an ischaemic stroke is necessary to
support damaged neuronal tissue, return of the blood
supply can also lead to further neuronal damage and
cerebrovascular injury [83]. In the transient middle
cerebral artery occlusion model of ischaemic stroke, a
biphasic increase in BBB permeability during reperfu-
sion (at ~ 6 h and 48–72 h) corresponds to increased
vasogenic oedema formation [84]. In an elegant study
using fluorescent tracers and two-photon time-lapse
imaging in the transient middle cerebral artery occlu-
sion model, Knowland and colleagues [85] have
recently mapped the dynamic events at the BBB that
lead to barrier deficits in the hours after stroke. Using
Claudin-5:eGFP transgenic mice to visualize endothe-
lial TJs, ultrastructural changes in TJ assemblies such
as gaps (discontinuous TJs) and protrusions (exten-
sions from linear TJ strands), and decreased occludin
and ZO-1 staining were found 48–58 h after stroke
and correlated with delayed leakage of biocytin-TMR
and IgG [85]. At early time-points, however, there
were minimal deficits in TJ integrity, instead increased
rates of endocytosis and transcytosis (measured by
uptake and transport of fluorescently labelled albumin)
were detected as early as 6 h after transient middle
cerebral artery occlusion [85]. This distinct sequence of
events at the BBB – increased transcytosis followed by
TJ remodelling – highlights the dynamic involvement
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J. Keaney and M. Campbell
of both transcellular and paracellular pathways in
modulating BBB permeability in the hours and days
following a cerebral infarction. TJ plasticity and
biphasic opening of the BBB are also observed in the
penumbral region or neural tissue surrounding the site
of a TBI. In a cortical cold-induced injury model of
TBI, TJ protein levels are altered post injury [86] and
BBB permeability is evident in the penumbra at 24
and 72 h, but not 48 h after injury [87]. Interestingly,
we have found that short interfering RNA-mediated
suppression of claudin-5 in this model enhances move-
ment of water from brain to blood, reducing cerebral
oedema and improving cognitive function in rodent
models [87]. Although BBB breakdown in the TBI
region likely exacerbates neuronal dysfunction and
inflammation, TJ complexes in the penumbra may
adapt to this fluid accumulation to alleviate swelling
and protect the surrounding neuronal tissue.
CNS innate immunity and infection
Because of its low rates of neurogenesis, the CNS was
long considered an ‘immune-privileged’ organ that
required a BBB to prevent potentially damaging
immune cell entry and inflammation. However, it is
increasingly being recognized that normal CNS physi-
ology involves continuous immune surveillance of
pathogen invasion and injury [9]. The BBB is integral
to this process as the mechanisms of peripheral leuko-
cyte contact with brain endothelial cells and migration
across the BBB (also known as transendothelial leuko-
cyte migration or TEM) become elucidated. In
response to danger signals (such as tumour necrosis
factor-a and interleukin-1b cytokines) on the ablumi-
nal or luminal side, brain endothelial cells become acti-
vated to release proinflammatory molecules and
upregulate cell adhesion molecules like ICAM-1,
VCAM-1 and E-selectin [88]. Contact between periph-
eral leukocytes and brain endothelial cells is then
mediated by interactions between cell adhesion mole-
cules and integrins like lymphocyte function-associated
antigen 1 on leukocytes [88]. Subsequent TEM is
believed to occur paracellularly via reorganization of
the actin cytoskeleton and TJ remodelling, although
the precise mechanism is still unclear [89]. Rapid
remodelling of TJ complexes during TEM has recently
been reported using 3D confocal imaging of mono-
cytes during the TEM process [90]. In the space of
10 min, the addition of monocytes is followed by dis-
continuous claudin-5 staining, specifically in areas of
monocyte TEM. These gaps are then rapidly resealed
following leukocyte migration across the TJ [90].
Intriguingly, barrier function was found to be pre-
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17424658, 2015, 21, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.13412 by CAPES, Wiley Online Library on [06/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
The dynamic blood–brain barrier
served during this process. This may be due to
PECAM-1-mediated recruitment of the lateral border
recycling compartment, a vesicular-membrane com-
partment, to the TJ that can laterally displace TJ com-
ponents while also sealing the intercellular gap [90].
Although chronic BBB breakdown and leukocyte infil-
tration in conditions such as multiple sclerosis is likely
detrimental [91], bone marrow-derived microglia are
known to be crucial for restricting senile plaque for-
mation in AD [92]. Recruitment of T cells to the CNS
can also contribute to hippocampal neurogenesis and
spatial learning in adulthood [93], thus the BBB likely
plays an active role in mediating contact between the
CNS and peripheral immune system.
The paracellular pathway of the BBB can also be
hijacked by pathogens like bacteria and viruses to
invade the CNS. Bacterial meningitis in newborns is
commonly caused by group B Streptococcus, a Gram-
positive bacteria, and is a potentially fatal CNS infec-
tion [94]. Kim et al. [95] have recently found that
group B Streptococcus infection of brain endothelial
cells induces upregulation of Snail1, a global repressor
of TJ gene expression. Snail1-mediated decreases in
levels of claudin-5, occludin and ZO-1 in turn allow
for bacterial passage through the dysregulated TJ com-
plexes [95]. Viral neuro-invasion has also been linked
to BBB disruption via TJ modulation. The West Nile
virus (WNV) is a mosquito-borne flavivirus that can
cause encephalitis (neuroinflammation), neuronal dys-
function and death. Although the mechanisms of
WNV entry into the CNS are unclear, WNV-induced
loss of major TJ proteins and increased matrix metal-
loproteinase production at the BBB have been
reported in WNV-infected mice [96]. A role for para-
cellular TJ disruption in WNV infiltration is also sup-
ported by the recent finding that interferon k
treatment of brain endothelial cells stabilizes claudin-5
and ZO-1 localization after WNV infection [97].
Administration of interferon k in WNV-infected mice
also reduced viral movement across the BBB [97].
Novel insights into BBB physiology
Pathological changes in BBB function have now been
implicated in a wide range of CNS disorders [4], how-
ever less is known about the influence of common bio-
logical processes on BBB development and integrity.
Some intriguing work is pointing to the role of diverse
phenomena such as sleep–wake behaviour and gut
microbiota in establishing and maintaining a homeo-
static BBB. We are also learning how the normal ageing
process can impact BBB permeability and potentially
trigger disease-associated changes in cognitive function.
4073

The dynamic blood–brain barrier
Sleep–wake cycle
Sleep is a universal and essential state among animals
that is characterized by decreased sensory responsive-
ness and reduced muscle tone [98]. The functions of
sleep are still unclear although sleep likely assists in
tissue repair, consolidation of memories and immune
system function [98]. Sleep deprivation can lead to
impairments in neurocognitive function and increase
the risk of developing conditions like Type 2 diabetes
[99]. Two recent studies have also found that sleep
deprivation, in the form of chronic sleep restriction
[100] or selective rapid eye movement sleep deprivation
[101], can increase BBB permeability. Chronic sleep
restriction, for example, was associated with reduced
GLUT-1 and TJ protein expression at the BBB,
decreased brain glucose uptake and increased paracel-
lular permeability to sodium fluorescein and biotin tra-
cer molecules [100]. In both studies, sleep recovery
restored BBB structure and function including a return
to baseline TJ protein levels [100,101]. Related work
has also elucidated the role of sleep in regulating the
exchange of solutes like Ab between the cerebrospinal
fluid (CSF) and interstitial fluid along paravenous
drainage pathways (the so-called ‘glymphatic’ system)
[102]. These combined findings suggest that sleep regu-
lation of cerebrovascular clearance pathways is vital to
maintaining brain homeostasis.
Gut microbiota
The collection of microorganisms in the human gas-
trointestinal tract is known as the gut microbiota.
There is accumulating evidence that gut microbiota
can communicate with the CNS to alter neural func-
tion and behaviour via changes in the autonomic ner-
vous system, microbial metabolites and/or systemic
inflammatory cytokines levels [103]. Braniste and col-
leagues [104] have recently shown that a lack of gut
microbiota is associated with reduced levels of claudin-
5 and occludin and increased BBB permeability.
Interestingly, this phenotype was sustained from devel-
opment into adulthood indicating that BBB function
may depend on constant communication with the gut
microbiota throughout life. The authors also found
that exposure of germ-free mice to Clostridium tyrobu-
tyricum, a bacterial strain that produces butyrate, or
administration of sodium butyrate alone upregulates
TJ protein expression and restores BBB tightness
[104]. Although the mechanism is still largely unclear,
isolating the factor(s) responsible for gut microbiota
regulation of BBB integrity may provide novel means
of restoring a leaky BBB in disease.
4074
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J. Keaney and M. Campbell
Ageing
One of the strongest environmental risk factors for a
host of neurodegenerative conditions is age. BBB dys-
function and vascular impairments have also been
identified in the pathophysiology of many of these dis-
eases including AD and amyotrophic lateral sclerosis,
but whether these vascular changes are a cause or con-
sequence of neurodegeneration remains a question of
active debate [105,106]. Some post-mortem histopatho-
logical studies have shown that in the absence of
comorbidities, BBB integrity in humans declines with
age as evidenced by IgG extravasation, TJ protein
changes and cerebral microbleeds [107,108]. Further-
more, a meta-analysis of 31 BBB permeability studies
(8 used post-mortem imaging, 21 of 23 others used
CSF/plasma albumin ratio) of normal ageing found
that BBB permeability increased with age [109],
although these imaging and biochemical approaches
have limitations including tissue processing and CSF
turnover rates [110]. New work by Montagne and col-
leagues [111] has used a high-resolution method of
dynamic contrast-enhanced MRI to assess BBB perme-
ability in the living human brain. In the CA1 and den-
tate gyrus regions of the hippocampus, they found an
age-dependent BBB breakdown in cognitively normal
individuals (from 23 to 91 years of age) based on the
blood-to-brain transfer constant (Ktrans) of gadolinium
contrast agent [111]. No obvious age-related changes
in BBB permeability were seen in cortical and subcor-
tical regions or in white matter. BBB permeability in
the hippocampus was further increased in individuals
with mild cognitive impairment (a disorder that can
precede AD) leading the authors to suggest that age-
ing-related BBB breakdown may contribute to demen-
tia onset [111]. CSF analyses also showed that CSF
levels of sPDGFRb, a potential biomarker of pericyte
injury, increased with age [111]. Because pericyte loss
and dysfunction in murine models have been associ-
ated with BBB breakdown [23,25,35], increased BBB
permeability in the ageing human hippocampus may
be due to pericyte damage. For future research, this
novel dynamic contrast-enhanced MRI method pro-
vides an exciting platform to investigate in ‘real time’
how genetics, diet and medicines can affect BBB integ-
rity in living humans.
Conclusions
Whereas transplantation and histological studies domi-
nated early BBB research, our knowledge of how
NVU components interact at the molecular level to
signal BBB development and maintenance has
FEBS Journal 282 (2015) 4067–4079 ª 2015 FEBS

J. Keaney and M. Campbell
advanced over the last decade. It is clear that early
and sustained communication in the NVU via a range
of secreted morphogens and cell receptors gives rise to
and maintains the unique barrier properties of the
BBB. Furthermore, dysfunction of any one NVU
member can ultimately lead to dysregulation of tran-
scellular and paracellular pathways across brain
endothelial cells and contribute to pathological BBB
breakdown. Much work to date has focused on the
role of astrocytes and pericytes in BBB regulation.
Elucidating how other cell types, in particular neurons
and microglia, interact with brain endothelial cells
may offer further clues to the complete recipe for a
fully functional BBB and to repairing the damaged
barrier. Conversely, new methods of circumventing the
BBB to deliver small or large molecule drugs into the
brain could take advantage of the dynamic nature of
TJ complexes. To conclude, as we increase our under-
standing of BBB involvement in brain homeostasis
and disease, it highlights the importance of considering
BBB biology in all basic and translational neuroscience
research.
Author Contributions
James Keaney and Matthew Campbell wrote the
review.
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