The p75 neurotrophin receptor localization in blood-CSF barrier: expression in choroid plexus epithelium.

Abstract

The presence of neurotrophins and their receptors Trk family has been reported in the choroid plexus. High levels of Nerve Growth Factor (NGF), Neurotrophin-4 (NT-4) and TrkB receptor were detected, while nothing was know about p75 neurotrophin receptor (p75NTR) in the choroid plexus epithelial cells. In neurons, p75NTR receptor has a dual function: promoting survival together with TrkA in response to NGF, and inducing apoptotic signaling through p75NTR. We postulated that p75NTR may also affect the survival pathways in the choroid plexus and also undergoes regulated proteolysis with metalloproteases.
Here, we demonstrated the presence of p75NTR receptor in the choroid plexus epithelial cells. The p75NTR receptor would be involved in cell death mechanisms and in the damaged induced by amyloid beta (Aβ) in the choroid plexus and finally, we propose an essential role of p75NTR in the Aβ transcytosis through out choroid plexus barrier.
The presence analysis reveals the new localization of p75NTR in the choroid plexus and, the distribution mainly in the cytoplasm and cerebrospinal fluid (CSF) side of the epithelial cells. We propose that p75NTR receptor plays a role in the survival pathways and Aβ-induced cell death. These data suggest that p75NTR dysfunction play an important role in the pathogenesis of brain diseases. The importance and novelty of this expression expands a new role of p75NTR.

Full text

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RESEARCH ARTICLEOpen Access
The p75 neurotrophin receptor localization in
blood-CSF barrier: expression in choroid plexus
epithelium
Carlos Spuch1,2*and Eva Carro1,3*
Abstract
Background: The presence of neurotrophins and their receptors Trk family has been reported in the choroid
plexus. High levels of Nerve Growth Factor (NGF), Neurotrophin-4 (NT-4) and TrkB receptor were detected, while
nothing was know about p75 neurotrophin receptor (p75NTR) in the choroid plexus epithelial cells. In neurons,
p75NTR receptor has a dual function: promoting survival together with TrkA in response to NGF, and inducing
apoptotic signaling through p75NTR. We postulated that p75NTR may also affect the survival pathways in the
choroid plexus and also undergoes regulated proteolysis with metalloproteases.
Results: Here, we demonstrated the presence of p75NTR receptor in the choroid plexus epithelial cells. The
p75NTR receptor would be involved in cell death mechanisms and in the damaged induced by amyloid beta (Ab)
in the choroid plexus and finally, we propose an essential role of p75NTR in the Ab transcytosis through out
choroid plexus barrier.
Conclusions: The presence analysis reveals the new localization of p75NTR in the choroid plexus and, the
distribution mainly in the cytoplasm and cerebrospinal fluid (CSF) side of the epithelial cells. We propose that
p75NTR receptor plays a role in the survival pathways and Ab-induced cell death. These data suggest that p75NTR
dysfunction play an important role in the pathogenesis of brain diseases. The importance and novelty of this
expression expands a new role of p75NTR.
Background
The choroid plexus, located in the cerebral ventricles, is
a highly vascularised tissue, in which blood microvessels
are enclosed by a single layer of cubical epithelial cells
[1]. Choroid plexus epithelial cells are closely connected
to each other by tight junctions and constitute the
structural basis of the blood-CSF barrier [2]. The barrier
function can be subjected to modulation and thereby
regulates the entry of physiologically important sub-
stances. However, choroid plexus epithelial cells are also
an important target organ for polypeptides, with capa-
city to produce and secrete numerous biologically active
neurotrophic factors into the CSF. In mammalian, brain
CSF is produced by the choroid plexus, which not only
regulates homeostasis in central nervous system (CNS),
but also participates in neurohumoral brain modulation
as well as in neuroimmune interaction. Several peptides
are shown to be actively transported by the choroidal
epithelial cells to the CSF and most of the transported
hormones evidently have, at least, a hypothalamic desti-
nation. These peptides circulate throughout the brain
and spinal cord, maintaining neuronal networks and
associated cells [3].
Studies in the past few years have promoted insight
into the molecular structure and function of the choroid
plexus. Even modest changes in the choroid plexus can
have far reaching effects and changes in the anatomy
and physiology of the choroid plexus, and have been
linked to several neurological disorders such as Alzhei-
mer’s disease or multiple sclerosis. Given a widely pos-
tulated role in neuronal cell survival, the p75NTR is
also though to be associated with many neurodegenera-
tive diseases.
* Correspondence: carlos.spuch@gmail.com; carroeva@12o.es
1Neuroscience Group, Research Institute Hospital 12 de Octubre, Madrid,
Spain
Full list of author information is available at the end of the article
Spuch and Carro BMC Neuroscience 2011, 12:39
http://www.biomedcentral.com/1471-2202/12/39
© 2011 Spuch and Carro; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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The choroid plexus is involved in a variety of neurolo-
gical disorders, including neurodegenerative, inflamma-
tory, infectious, traumatic, neoplastic, and systemic
diseases. It is well known that Ab is accumulated in the
choroid plexus of Alzheimer’s disease patients [4]; there
is also evidence that circulating Insulin-Like Growth
Factor-I (IGF-I) participates in brain Ab clearance by
modulating choroid plexus function [5]. It has been
published that substances, such as transthyrretin (TTR)
or apolipoprotein J (ApoJ), are produced by the choroid
plexus and secreted into the CSF [6]. In multiple sclero-
sis and in animal models of multiple sclerosis, the chor-
oid plexus is the main route of leukocyte entry into the
brain [7].
Many studies have demonstrated the presence of pro-
teins and mRNA for a large number of cytokines,
growth factors and hormones in the choroid plexus, for
example: interleukin-1b [8], interleukin-6 [9], Tumor
Necrosis Factor (TNF)-a [10], IGF-I [11], NGF [12],
IGF-II [13], Transforming Growth Factor (TGF)-a [14],
TGF-b [15], Vascular Endothelial Growth Factor
(VEGF) [16], transferrin [17], TTR [6,18], gelsolin [19]
and vasopressin [20]. Most of these substances have
their own receptors in the choroid plexus [21]. The pro-
duction and secretion of all these substances and their
receptors are strongly associated with the health of cen-
tral nervous system (CNS).
The mammalian neurotrophins comprise a family of
related secreted factors required for differentiation, sur-
vival, development, and death of specific populations of
neurons and non-neuronal cells. The effects of the neu-
rotrophins (NGF, Brain-Derived Neurotrophin Factor
(BDNF), NT-3, NT-4) are mediated by binding to TrkA,
TrkB and TrkC receptor tyrosine kinases and to the
p75NTR [22,23]. The Trk receptors play critical roles in
mediating neuronal survival, growth and synaptic func-
tion [24]. The p75NTR serves as a receptor for the four
mentioned neurotrophins and it is an important compo-
nent of distinct cell surface signaling platforms, which
induce apoptosis and neuronal growth inhibition [25].
The presence of the neurotrophins and their receptors
has been investigated in the choroid plexus of rats and
humans. The choroid plexus contains high levels of
NGF, NT-4, and TrkB, and low levels of NT-3 and
BDNF, while TrkA and TrkC levels remain undetectable
[12].
Regarding the neurotrophins receptors in the choroid
plexus, the expression of p75NTR has not been fully
investigated. The present research shows the distribu-
tion of p75NTR in the epithelial cells of the choroid
plexus and its possible role in the normal transportation
of molecules of the blood-CSF-barrier. The importance
and novelty of this expression expands a new role of
p75NTR.
Results
1. The p75 neurotrophin receptor is expressed in the
epithelial cells from choroid plexus
Figure 1 shows p75NTR immunofluorescence, in three
different species, with a well-established antibody against
p75NTR receptor, donated by M. Chao (9651). p75NTR
receptor signal was detected in a monolayer of epithelial
cells from a choroid plexus primary culture (Figure 1A).
p75NTR immunofluorescence staining was also shown
in human samples from biopsied choroid plexus (Figure
1B, upper), and in samples of mouse choroid plexus,
using 3,3’-Diaminobenzidine (Figure 1B, bottom). The
p75NTR expression was specifically detected in the
cytoplasm of choroid plexus epithelial cells and mainly
located in CSF side of the blood-CSF barrier (Figure
1B). In order to test p75NTR expression by Western
blot analysis we lysed different samples of choroid
plexus. p75NTR expression was detected in primary cul-
tures of choroid plexus and in tissue of mouse choroid
plexus (Figure 1C).
2. Proteolytic processing of p75NTR by amyloid-b (Ab) in
choroid plexus
Ab1-40 aggregates of undetermined structure have been
reported to be death-inducing ligands of p75NTR [26].
Although Ab is also known to bind many other targets,
such as megalin, the most important multicargo receptor
in the choroid plexus [27]. It has been known for many
years that p75NTR undergoes ectodomain shedding.
Ectodomain cleavage of full length p75NTR releases the
extracellular domain from the membrane bound C-term-
inal fragment (CTF) ≈24 KDa. The CTF is subsequently
cleaved by g-secretase to give rise to the soluble p75NTR
intracellular domain (ICD) ≈19 KDa [28].
The cleavage of p75NTR has been followed essentially
in transfected cells [29] and in primary Schwann cells
[28]. Previous observations with cell lines have suggested
that amyloid could regulate the cleavage of p75NTR. To
examine whether p75NTR is cleaved endogenously in
epithelial cells from choroid plexus, we performed tests
in primary culture cells from the choroid plexus epithe-
lium previously applied with proteasome inhibitor (Cal-
biochem) and then treated with Ab1-40 (2.5 μg/mL)
during 15, 30 and 60 minutes. Western blotting with an
antibody directed against the cytoplasmic domain of
p75NTR (ICD), donated by B. Carter and M. Chao
(9992), revealed an immature underglycosylated form of
45 KDa and fragments at 24KDa consistent with the
p75NTR-CTF. A band at 19KDa consistent with
p75NTR-ICD fragment was also observed weakly. The
treatment with Ab1-40 induced a release of ICD frag-
ment at 15 and 30 minutes (Figure 2A). The cellular
distribution of p75NTR-ICD was determined in these
cells by immunofluorescence and confocal microscopy.
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A)
B)
C)
p75NTR
? ?-actin
95
49
75
p75NTR
? ?-actin
95
49
75
10 ? ?m 5 ? ?m
40 ? ?m
20 ? ?m
40 ? ?m
20 ? ?m
Figure 1 p75NTR receptor is expressed in the epithelial cells from choroid plexus. A) Figure 1A shows p75NTR staining of epithelial cells
from choroid plexus in vitro with p75NTR-ECD antibody (9651) donated by M. Chao and B Carter. The expression is mainly in the cytoplasm of
the cells an immunofluorescence (red) and the nucleus is staining with DAPI. B) Figure 1B shows p75NTR staining of human choroid plexus with
p75NTR-ECD antibody (9651) (upper panel). The bottom panel shows p75NTR staining developed with DAB and shows the p75NTR expression in
the cytoplasm and in the apical membrane of mouse choroid plexus corresponding with CSF side of epithelial cells. C) Western blot analysis of
homogenates from primary culture cells from epithelial cells of choroid plexus (left) and mouse choroid plexus tissue (right). Representative blots
are shown.
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Control
A? ?1-4060 minutes
ICD-p75
A)
B)
C)
10 ? ?m 10 ? ?m
75
50
20
37
Control
A? ?1-40
p75NTR
CTF
ICD
? ?-actin
15’ 30’60’
25
100
A? ?1-40
Cont15’30’ 60’
Cytoplasm
p75NTR
ICD
? ?-actin
Nucleus
75
50
37
20
25
75
50
75
50
CREB
50
Figure 2 Proteolytic processing by Amyloid-b (Ab) in choroid plexus cells. Western blot analysis of homogenates from primary culture cells
from choroid plexus treated with Ab1-40 2.5 μg/mL during 15, 30 and 60 minutes. The samples were prior applied with proteosome inhibitor
during 1 hour. The membranes were developed with an antibody directed against the cytoplasmic domain of p75NTR (ICD) donated by B.
Carter and M. Chao (9992). The blot revealed fragments at 24 KDa consistent with the p75NTR-CTF, and a fragment at 19 KDa consistent with
p75NTR-ICD, also was observed weakly. The treatment with Ab1-40 induced a release of ICD fragment at 15 and 30 minutes. a-actin was used
as a loading control. B) Similar analysis as in A) but in immunofluorescence treated with Ab1-40 2.5 μg/mL, 60 minutes. C) Similar analysis as in
A) but we separated cytoplasm and nuclear fractions. We observed that p75NTR-ICD was accumulated in the nucleus of the cells after Ab
treatment. a-actin was used as a loading control of cytoplasm fraction and CREB was used as a loading control of nuclear fraction.
Representative blots are shown.
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To avoid instability of the ICD fragment, primary cul-
ture of choroid plexus epithelial cells were treated with
proteasome inhibitor (Calbiochem) 1.5 hours before
treated with Ab1-40 during 60 minutes. In control cells
we observed the intensity of p75NTR-ICD labelling in
the most part of the cytoplasm and in some of the
nuclei (Figure 2B, left image). In the presence of Ab1-
40, a considerable accumulation of p75NTR-ICD
occurred in the majority of nuclei shifted from cyto-
plasm to nuclei (Figure 2B, right image).
To confirm that the p75NTR-ICD does indeed accu-
mulate in the nucleus in response to Ab1-40, nuclear
and cytoplasmic extracts were subjected to Western
blotting with a p75NTR-ICD specific antibody (9992). A
19 KDa band, characterized as p75NTR-ICD fragment
[28], was enriched in nuclear extracts from primary cul-
ture cells from choroid plexus exposed to Ab1-40 for
15, 30 and 60 minutes (Figure 2C).
3. p75NTR receptor involved in Ab transport in the
choroid plexus
In order to investigate the influence of p75NTR receptor
on choroid plexus function, we used two different meth-
ods. Firstly, with in vitro double-chamber well used to
mimic the blood (lower chamber)-CSF (upper chamber)
interface (Figure 3A). Choroid plexus epithelial cells
were grown in the floor of the upper compartment on
the top of a porous membrane. In other systems the
incubation with antibodies against extracellular domains
of membrane receptors are able to inhibit the activity of
some ligands, due to antibody and ligand compete for
the same localization in the receptor. With previous
experiments we checked that incubations with high con-
centrations of p75NTR antibody block the activity of
p75NTR in our cell system. Based on this results, prior
Ab1-40 addition in the upper chamber, we had inhibited
the p75NTR activity by incubation during 24 hours the
upper chamber with high concentrations (1:50) of
p75NTR antibody directed against extracellular domain
(9651). Indeed, as determined by Western blotting ana-
lysis in control situation, Ab is transported from inner
chamber (CSF) to outer chamber (blood) (Figure 3B).
Ab translocation is enhanced through out megalin, as
shown by immunoprecipitation analysis (Figure 3B), and
was already suggested by Zlokovic’s group [27]. When
p75NTR biological activity was inhibited in this in vitro
model with p75NTR antibodies, Ab CSF-to-blood trans-
port is interrupted (Figure 3B). We then analyzed
expression and release of TTR, a protein specifically
synthesized in the brain by the choroid plexus [30], and
associated with Ab transport via megalin in the choroid
plexus [31]. We observed, in our in vitro blood-CSF-bar-
rier model, that the inhibition of p75NTR biological
activity induced TTR expression in the choroid plexus
A)
B)
TTR
Control
Anti-p75
A? ?
TTR
? ?-actin
C)
CP cells
CSF
Blood
A? ?1-40
5? ?g/mL
Anti-p75NTR(9651)
1:50 (24h)
Figure 3 p75NTR is involved in Ab transport through choroid
plexus. A double-chamber choroid plexus epithelial cell-culture
system mimicking the blood-CSF interface was used for in vitro
studies A) Figure 3A shows a scheme of one of these chambers.
Choroid plexus epithelial cells were grown in the floor of the upper
compartment on the top of a porous membrane. Ab1-40 5 μg/μL
was added to the upper chamber. For inhibition of p75NTR
biological activity experiments, prior Ab1-40 were added in the
upper chamber, where we had inhibited the p75NTR activity
incubating 24 hours with high concentrations (1:50) of p75NTR
antibody directed against extracellular domain (9651). B) In control
cells Ab is normally translocated to the lower chamber, however
the cells with p75NTR biological activity inhibiting Ab translocation
was interrupted. TTR is a protein specifically synthesized in the brain
by the choroid plexus and associated with Ab transport via megalin
in the choroid plexus. Interestingly, we evidenced that the inhibition
of p75NTR biological activity induced a strong secretion of TTR to
the upper chamber. C) Inhibition of p75NTR biological activity
increased TTR expression in the choroid plexus cells. Representative
blots are shown.
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(Figure 3C) and strong TTR release in the culture med-
ium of the upper chamber (Figure 3B).
4. The p75NTR receptor is implicated in cell death
induced by Ab
In control situation we corroborated that Ab induced an
increase of death cell of 29% upon control cells. When
we blocked the biological activity of p75NTR, Ab-
induced cell death was inhibited (Figure 4A). After that,
we transfected with p75NTR plasmid primary culture
cells of choroid plexus epithelial cells and we corrobo-
rated that over-expression of p75NTR receptor induced
an increase of caspase-3 and caspase-9 expression (Fig-
ure 4B). When we added Ab we observed the increase
of cleaved-caspase-3 in both situations (control and
p75NTR over-expression). However, when p75NTR was
over-expressed, the cleaved-caspase-3 was strongest
compared with control (Figure 4C).
Discussion
The p75NTR receptor is a transmembrane protein that
binds all neurotrophins and has multiple functions in
the nervous system, where it is widely expressed during
developmental stages of life in neurons and also in a
variety of glial populations. Expression of p75NTR
receptor can increase in pathological states related to
neural cell death. In choroid plexus only transcripts cod-
ing for the TrkB molecule have been described [23].
Our data clearly demonstrate that p75NTR receptor is
also expressed in the choroid plexus and is mainly
located towards the apical membrane in the epithelial
cells. In Madine-Darby canine kidney (MDCK) cells
transfected with the plasmid p75NTR receptor, the
expression is restricted to the apical domain [32]. Sev-
eral studies demonstrated that p75NTR participates in
more diverse biological events including neuronal cell
death, migration and axonal elongation. In the present
study we have suggested the role of p75NTR receptor in
the epithelial cells from choroid plexus and it seems to
be involved in the damage and death of cells induced by
Ab.
It is well described in neurons and different cells lines
that p75NTR receptor undergoes sequential proteolytic
cleavage by a-secretase and g-secretase activities, releas-
ing its ICD into cytoplasm, which is in a manner analo-
gous to the cleavage-dependent signalling pathway of
Notch and APP [29]. Although neurotrophins did not
regulate p75NTR processing, the a-secretase and g-
secretase mediated cleavage of p75NTR is modulated by
TrkA and TrkB receptors [33] and the choroid plexus
epithelial cells were able to express TrkB receptors [23].
In this context, we have presented evidence that the
predicted ICD of p75NTR was detectable by Western
blot analysis and immunostaining after treatment with
Ab, and this fragment was translocated to the nucleus.
The ICD of p75NTR was unstable in the choroid plexus;
we therefore attempted to detect it by inhibiting a pro-
teasomal pathway that had been previously shown to
mediate degradation of Notch-ICD or p75-ICD in neu-
rons [34]. We have not evidence about the function of
ICD nuclear accumulation in choroid plexus, however,
there were many evidences that p75NTR-ICD have
nuclear functions [35], such as apoptosis [36], transcrip-
tional and cell cycle regulations [37]. Numerous proteins
interact with p75NTR-ICD to activate different path-
ways (NF-kappaB, Akt or JNK) and some of them have
been associated with the translocation to the nucleus
and the induction of apoptosis [38]. However we have
not evidence that some of these adaptors, such as NRIF,
TRAFs, SC1, MAGE, RIP2, are expressed in choroid
plexus. It remains to be determined whether adaptor
proteins of p75NTR are also expressed in choroid
plexus and can interact with p75NTR. The identification
of p75NTR interactors and signaling pathways will spark
new directions in blood-CSF-barrier research and will
provide better understanding of this enigmatic receptor.
A)
0
anti-p75
20
40
60
80
100
120
140
*
*
A? ?1-40
-
-
-
+
+
-
+
+
Arbitrary units
p75NTR
Caspase-9
Caspase-3
? ?-actin
Control
p75
B)
p75NTR
Cleaved
Caspase-3
? ?-actin
A? ?1-40
p75NTR
+
+
-
+
+
-
-
-
C)
Figure 4 p75NTR is involved in the cell death induced by Ab.
A) Cell death quantification with ELISA PLUS Kit. In control cells Ab
induced a 29% of cell death, when we blocked the p75NTR
biological activity with prior p75NTR antibody incubation (1:50)
during 24 hours, the Ab did not produce any cell death in choroid
plexus. B) Increased levels of caspase-3 and caspase-9 after p75NTR
transfection in primary cell cultures. C) Increased of cleaved-caspase-
3 in primary culture cells transfected with p75NTR. The choroid
plexus culture cells that over-express p75NTR were more sensitive
to damage induced by Ab increasing the cleaved-caspase-3 band in
the blot.
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The choroid plexus play pivotal roles in basic aspects
of neural function including maintaining the extracellu-
lar milieu of the brain by actively modulating chemical
exchange between the CSF and brain parenchyma. In
this context, it is well known the role of megalin, a mul-
tifunctional endocytic receptor. Megalin is a multicargo
transmembrane protein with a large extracellular
domain containing multiple binding sites for its numer-
ous ligands. Megalin is the major receptor for the
uptake of Ab complexed with different proteins, such as
TTR, clusterin, ApoE, albumin [39,40]. Based on pre-
vious observations, megalin mediated clearance mechan-
isms of Ab have been proposed to play a crucial role in
the elimination of Ab from the brain through the blood-
CSF-barrier into the periphery [27,41]. To investigate
the possible role of p75NTR receptor in an artificial
model of blood-CSF-barrier, we corroborated the nor-
mal Ab transport from CSF to blood; however, when we
inactivated the biological activity of p75NTR we demon-
strated that Ab transcytosis was completely blocked.
These results suggest that p75NTR receptor could be
modulating megalin activity in the blood-CSF-barrier,
and modifying the Ab clearance. The precise mechanism
as to how the p75NTR modified megalin activity
remains elusive.
Our findings have led to the suggestion that the
p75NTR signaling in choroid plexus might be deter-
mined by the type of co-receptor involved in the com-
plex. Our studies revealed that Ab could be able to
engage a functional interaction between p75NTR and
megalin. Further, TrkB expression was detected in chor-
oid plexus [23], and in neurons it was well know to
engage functional interaction between p75NTR and
TrkB [42]. The mechanism of TrkB and p75NTR in
choroid plexus remains elusive and needs more experi-
ments; however we suggest that the possible interaction
between p75NTR, TrkB and megalin will provide new
directions in the p75NTR signaling and better under-
standing in the blood-CSF-barrier research.
The mechanism underlying this phenomenon and its
relevance to neurodegenerative diseases is unclear.
Therefore, it is reliable to speculate that p75NTR recep-
tor could modify TTR expression and release it to CSF.
Previous studies confirmed by data from our laboratory
suggested that TTR is reduced in CSF samples from
subjects with Alzheimer’s disease and frontotemporal
dementia [43]. Experiments investigating the response of
choroid plexus TTR synthesis after inhibition of
p75NTR biological activity produced an increase of TTR
synthesis; alternatively when we investigated the inhibi-
tion of p75NTR biological activity in an in vitro model
of blood-CSF-barrier, we confirmed a strong release of
TTR (upper chamber). It is well established in our
laboratory that Ab induced cell death in choroid plexus
epithelial cells [4]. We confirmed the implication of
p75NTR receptor in Ab-induced cell death by blocking
the p75NTR biological activity. These experiments were
corroborated when we over-expressed p75NTR receptor
in primary culture of choroid plexus epithelial cells
increasing the expression of caspase-3 and caspase-9,
and cleaved of caspase-3. Dietrich’s study shows Ab
accumulation in choroid plexus in different stages of
Alzheimer’s disease [44] and induced oxidative stress
and mitochondrial alterations [45]. These results showed
the impact of p75NTR receptor in the survival and pro-
tection of epithelial cells from choroid plexus and con-
firmed the inhibition of p75NTR biological activity such
as a protective mechanism in neurodegenerative dis-
eases, but further studies using animal models will be
required to confirm this hypothesis.
Conclusions
In summary, our results have been revealed p75NTR
expression in epithelial choroid plexus, mainly localized
in the apical membrane in contact with CSF side.
Indeed, p75NTR plays an important role in cell surface
platforms to induce neuronal apoptosis [25]. In our con-
text we suggest that p75NTR is also regulating the sur-
vival/apoptosis pathways and the Ab-induced damage.
The mechanism of TrkB and p75NTR in choroid plexus
remains elusive and needs more experiments, although
we suggest that p75NTR and TrkB could interact with
megalin providing new directions in the p75NTR signal-
ing and better understanding in the blood-CSF-barrier
research.
Methods
Cell Cultures
Primary cultures from choroid plexus epithelial cells
from P3-P5 Wistar rats were prepared as described pre-
viously [11]. Cells were grown to confluence for 5-7
days and serum starved for 2 hours. Cultures were
maintained at 37°C in a humidified atmosphere contain-
ing 5% CO2, and cultivated for 7 days prior to experi-
mentation. Human analogue peptides corresponding to
Ab1-40 and scrambled Ab1-40 (AnaSpec, Inc.) were
added. 24 hours after stimulation, cells were either fixed
for immunocytochemical analysis or homogenized for
immunoblot determination. For ICD p75NTR detection
primary cultures cells proteasome inhibitor epoxomycin
(1 μM) (Sigma) was applied 1.5 hours prior to Ab
treatment.
A double-chamber choroid plexus epithelial cell-cul-
ture system mimicking the blood-CSF interface was
used for in vitro studies, as described previously [11].
Thereafter, cells were incubated another 24 hours with
excess of p75NTR (9651) antibody (1:50) (gifted from
Dr. M. Chao and Dr. B. Carter). The antibody was
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added to the upper and lower chamber and 24 hours
later the upper chambers were treated with Ab1-40 (5
μg/mL). Twenty-four hours later, lower chamber med-
ium was collected and content of Ab and TTR was
determined by immunoblotting (see below). Treatments
were done in triplicate wells per experiment.
All animals were handled and cared for in accordance
with European Community Council Directive (86/609/
EEC). Animals were perfused transcardially with saline
buffer and 4% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.4, for immunohistochemical analysis.
Immunoassays
For Western blot analysis cultures cells were washed
once with ice-cold PBS and lysed in PIK buffer (150 mM
NaCl, 20 mM TrisHCl pH 7.4, 1% NP40 and protease
inhibitors: 1 μg/mL aprotinin, 1 μg/mL leupeptin and 1
μg/mL phenylmethylsulfonyl fluoride, PMSF). For cell
fractionation cells were lysed with C buffer (10 mM
HEPES, 60 mM KCl, 1 mM EDTA, 0.075% Triton X100,
1 mM DTT with protease inhibitors. The pellet nuclei
was obtained by centrifugation 325 g, 4 minutes and
resuspended in NB buffer (20 mM TrisHCl pH 8, 420
mM NaCl, 15 mM MgCl2, 0.2 mM EDTA, 25% Glicerol
and protease inhibitors). Thereafter, the nuclei superna-
tant were obtained after centrifugation 9000 g, 10 min-
utes. After running the samples in acrylamide gels,
proteins were transferred (immobilon, Bio-Rad) and
membranes were incubated with the corresponding pri-
mary at 4°C overnight. Afterwards, membranes were
washed and incubated with secondary antibodies. Mem-
branes were washed several times with Tween-TBS and
developed with ECL plus (Amersham). Western blot
membranes were re-blotted with unrelated proteins (a-
actin) as an internal standard and normalized for protein
load. Densitometric analysis was performed using ImageJ
software (NIH Image). A representative blot is shown
from a total of at lest three independent experiments.
For immunoprecipitation, cells were lysed with PIK
buffer and centrifuged at 11000 g for 15 minutes. Super-
natants were incubated with primary antibody overnight.
Protein A-agarose (Amersham) was added to the anti-
gen-antibody mixture and incubated with gentle agita-
tion 4 hours. The immunoprecipitate was washed
several times with the same PIK buffer, resuspended in
SDS loading buffer and analyzed by western blot.
For cell death quantification, after incubation for 24 h
with Ab40 (5 mg/ml), choroid plexus epithelial cells in
vitro were measured with Cell Death Detection ELISA-
PLUSkit (Roche Diagnostics) as described previously [4].
Immunofluorescence
Immunocytochemistry was performed with primary cul-
tures from choroid plexus plated on 20 mm coverslips
and fixed. Rat and human choroid plexus were also fixed
with 4% paraformaldehyde and processed for histochem-
ical analysis. Coverslips and choroid plexus sections were
blocked with 5% BSA and incubated overnight at 4°C
with the respective antibody in PB containing 0.5% BSA
and 0.1 Triton X100. After several washes in PB, sections
were incubated with Alexa-coupled secondary antibody
in the same PB buffer. Omission of primary antibody was
used as control. Confocal analysis was performed in a
Leica confocal microscope. Human brain tissue was
obtained from the Neuropathology Institute, anatomy
pathology service, IDIBELL. Hospital “Universitario de
Bellvitge” (Barcelona, Spain).
Antibodies
The following antibodies were used: rabbit polyclonal
anti-p75NTR against extracellular domain (9651) and
intracellular region (9992) gifted from Dr. M. Chao and
Dr. B. Carter. Mouse monoclonal anti-a-actin (Sigma),
mouse monoclonal anti-b-amyloid (MBL), rabbit poly-
clonal anti-transtyrretin (Santa Cruz), mouse monoclo-
nal anti-caspase 3 and 9 (Cell Signalling), goat
polyclonal anti megalin (Santa Cruz). All alexa fluor
antibodies were purchased from Invitrogen.
Acknowledgements
We thank Bruce Carter (Vanderbilt University Medical Center, Nashville, TN)
and Moses Chao (Skirball Institute, New York, NY) for p75-ICD and p75-ECD
antibodies. We also thank Mark Bothwell (University of Washington, Seattle)
for expert comments and revision of the manuscript. Also, we would like to
thank to Tania Vazquez for editorial assistance. This work was supported by
Grants from Fondo de Investigacion Sanitaria (FIS) (CP04/00179, PI060155),
Fundación Investigación Médica Mutua Madrileña (2006.125), (CP04/00011,
PI050379) and Xunta de Galicia (INCITE2009, 09CSA051905PR) and “Isidro
Parga Pondal” programme. The authors would like to thank to Brain Bank for
Neurological Research (Universidad Complutense, Madrid, Spain) and the
“Banco de Tejidos Biológicos de Vigo, BTN-Vigo” (Neurological Tissues
Biobank of Vigo), which is part of the CHUVI-Biobank, recently integrated
into the Spanish Cooperative Health Research Thematic Network on
Biobanks (RETICS-Biobancos, Code RD09/0076/00011) for the human choroid
plexus samples.
Author details
1Neuroscience Group, Research Institute Hospital 12 de Octubre, Madrid,
Spain.2Department of Pathology and Neuropathology, University Hospital of
Vigo (CHUVI), Vigo, Spain.3Biomedical Research Networking Center in
Neurodegenerative Diseases (CIBERNED), Madrid, Spain.
Authors’ contributions
Conceived and designed the experiments: CS and EC. Performed the
experiments: CS. Analyzed the data: CS and EC. Wrote the paper: CS. CS and
EC read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 10 February 2011 Accepted: 11 May 2011
Published: 11 May 2011
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doi:10.1186/1471-2202-12-39
Cite this article as: Spuch and Carro: The p75 neurotrophin receptor
localization in blood-CSF barrier: expression in choroid plexus
epithelium. BMC Neuroscience 2011 12:39.
Spuch and Carro BMC Neuroscience 2011, 12:39
http://www.biomedcentral.com/1471-2202/12/39
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