Cellular and Molecular Life Sciences
(2022) 79:90
https://doi.org/10.1007/s00018-022-04136-1
Cellular and Molecular Life Sciences
ORIGINAL ARTICLE
Aquaporin‑4 expression in the human choroid plexus
Felix Deffner1 · Corinna Gleiser1 · Ulrich Mattheus1 · Andreas Wagner1 · Peter H. Neckel1 · Petra Fallier‑Becker2 ·
Bernhard Hirt1 · Andreas F. Mack1
Received: 11 November 2021 / Revised: 2 January 2022 / Accepted: 5 January 2022
© The Author(s) 2022
Abstract
The choroid plexus (CP) consists of specialized ependymal cells and underlying blood vessels and stroma producing the
bulk of the cerebrospinal fluid (CSF). CP epithelial cells are considered the site of the internal blood-cerebrospinal fluid
barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In this
study, we analyzed the expression of aquaporins in the human CP using immunofluorescence and qPCR. As previously
reported, AQP1 was expressed apically in CP epithelial cells. Surprisingly, and previously unknown, many cells in the CP
epithelium were also positive for aquaporin-4 (AQP4), normally restricted to ventricle-lining ependymal cells and astrocytes
in the brain. Expression of AQP1 and AQP4 was found in the CP of all eight body donors investigated (3 males, 5 females;
age 74–91). These results were confirmed by qPCR, and by electron microscopy detecting orthogonal arrays of particles.
To find out whether AQP4 expression correlated with the expression pattern of relevant transport-related proteins we also
investigated expression of NKCC1, and Na/K-ATPase. Immunostaining with NKCC1 was similar to AQP1 and revealed
no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation
of their expression. We hypothesized that AQP4 expression in the CP was caused by age-related changes. To address this,
we investigated mouse brains from young (2 months), adult (12 months) and old (30 months) mice. We found a significant
increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Taken together, we provide evidence
for AQP4 expression in the CP of the aging brain which likely contributes to the water flow through the CP epithelium and
CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism
influencing the previously observed CSF changes during aging.
Keywords Ependyma · Cerebrospinal fluid · Aquaporin · Choroid plexus · Astroglia
Abbreviations
AQP1
Aquaporin-1
AQP4
Aquaporin-4
BCSFB Blood-cerebrospinal fluid barrier
CNS
Central nervous system
CP
Choroid plexus
CPCs
Choroid plexus epithelial cells
CSF
Cerebrospinal fluid
NKCC1 Na+–K+–2Cl−cotransporter1
OAPs
Orthogonal arrays of particles
* Andreas F. Mack
an.mack@uni-tuebingen.de
1
Institute of Clinical Anatomy and Cell Analysis, University
of Tübingen, Österbergstr. 3, 72074 Tübingen, Germany
2
Institute of Pathology and Neuropathology, University
of Tübingen, Tübingen, Germany
Introduction
The internal environment in the central nervous system
(CNS) is separated from blood and surrounding tissues by
several barrier-forming structures. Besides the blood–brain
barrier, there is an outer and inner blood-cerebrospinal fluid
barrier (BCSFB), the outer BCSFB formed by the arachnoid cells, the inner by the choroid plexus epithelial cells
(CPCs). The choroid plexus (CP) itself is a structure found
in all brain ventricles (I-IV) [1] and thought to be the main
production site of the cerebrospinal fluid (CSF). The CP
consists of blood vessels, the overlaying epithelial cells, and
varying amounts of stromal cells and matrix in between.
CPCs express ion channels, transport proteins, and tight
junction proteins which form the actual BCSBFB, reviewed
in [2, 3].
Besides its main role in CSF production, the CP has been
attributed with other functions such as water homeostasis,
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endocrine regulation, immune surveillance [4–6], and secretion of stimulating stem cells factors [7]. In addition, the CP
might be used as an entry site for parasites [8] or viruses
including SARS-CoV2 into the brain [9].
The CPCs can be considered as specialized ependymal
cells which otherwise cover the walls of the ventricles, since
both originate from neuroepithelium [10] and form a continuous sheet of cells [11]. However, 'regular' ependymal cells
differ from CPCs structurally and molecularly: ependymal
cells do not form tight junctions in mammals, are derived
from radial glia, and mostly bear kinocilia whereas CPCs
mostly have apically located microvilli and form a true epithelium attached to a basal lamina. Moreover, many transport and channel proteins on CPCs are involved in the CSF
production.
In this study, we first focused on the distribution of
water channel proteins in ependymal cells and CPCs of
the human choroid plexus. It has been well established that
CPCs express aquaporin-1 (AQP1) apically. The first identified water channel was AQP1, at the time called CHIP, and
was localized in the brain on choroid plexus cells [12]. Subsequently, another aquaporin abundantly expressed in the
brain was discovered, designated aquaporin-4 (AQP4) [13]
which was then localized to astrocytic endfeet and ependymal cells [14]. This localization coincided with the square
arrays or orthogonal arrays of particles (OAPs) [15] detected
by freeze-fracture electron microscopy prior to the discovery
of AQP4 (reviewed then in [16]). Since then, many aspects
on the functions of aquaporins in the brain have been studied
(see reviews by [17–20]) including implications in brain diseases [21]. More recently, the role of aquaporins in the brain
have been implicated with the homeostasis of cerebrospinal
fluid (CSF) [22], and the glymphatic waste removal system,
also with respect to aging [23].
Whereas ependymal cells show a basolateral localization
of AQP4, the expression of AQP1 on CPCs is mostly apical.
Given the continuity of the ependymal ventricular lining
and CPCs, the initial goal of the present study was to establish where in the human ependyma-CP transition the AQP
expression would switch from AQP4 to AQP1. Unexpectedly, we discovered that in the brains of body donors, there
was also expression of AQP4 in CPCs. We compared this
expression pattern to the aquaporin distribution and expression in the murine choroid plexus and ependyma of different
age groups.
Materials and methods
Post‑mortem specimens
Human CP were taken from eight individuals who voluntarily donated their bodies to the Institute of Clinical
13
Anatomical and Cell Analysis, Tübingen. They gave their
informed consent in agreement with the declaration of Helsinki to use the cadaver for research purposes. This procedure was approved by the Ethics Committee of the Medical
Department of the University of Tübingen under the project
number 237/2007BO1. Details on the female and male body
donors aged 74 and 94 years are provided in supplementary
table 1. CP samples were collected and processed within 8 to
19 h post-mortem. Parenchyma from the striatum was used
as reference tissue for histology and RNA isolation.
Animals
For this study we used the CP from C57BL/6 mice bred
in the facility of our institute. All procedures were performed according to University of Tübingen and governmental guidelines, and were approved by local authorities
(Regierungspräsidium Tübingen). For PCR analysis, we
used 15 mice from both sexes and three age cohorts: young
(2–6 months old), adult (12 months old) and old (more than
30 months old), five animals each. Immunohistochemistry
was performed on tissues from ten animals. For the removal
of the CP and parenchyma from the striatum, mice were
anaesthetized with CO2 and decapitated.
RNA isolation
For qPCR analysis, the CP was removed immediately and
placed in ice-cold, RNAse-free phosphate buffered saline
(PBS, Sigma Aldrich, St. Louis, MO, USA). Care was taken
to obtain solely tissue from the CP. Unfortunately, a pure CP
epithelial preparation cannot be performed in such a way that
no surrounding tissue is attached, especially for the mouse
CP. For this reason, parenchyma from the striatum was used
as control tissue for human and murine brains. For each prepared mouse, CPs from the left and right lateral ventricles
were combined. In addition, the CP from the fourth ventricle
was collected as a single sample, so that two CP samples
were obtained from each mouse. The reference striatal tissue
was always taken from the corresponding brain.
Immunohistochemistry
After removal, the excised CP was fixed in 4% paraformaldehyde overnight, afterwards rinsed with PBS, and placed
into 30% (w/v) sucrose for another 24 h for cryoprotection.
The fixed samples were frozen in isopentane-nitrogen cooled
TissueTek® (Sakura, Staufen, Germany), stored at -80 °C
before cryosectioned at 18 µm.
Sections were re-hydrated and washed in PBS for 10 min,
followed by incubation in blocking solution containing PBS,
4% (v/v) goat serum (Biochrom, Berlin, Germany), 0.1%
(v/v) bovine serum albumin (Roth, Karlsruhe, Germany),
Aquaporin-4 expression in the human choroid plexus
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90
and 0.1% (v/v) Triton® X-100 (Roth, Karlsruhe, Germany)
for 90 min. at room temperature. Next, the sections were
incubated with primary antibodies (Table 1) diluted in the
preincubation solution overnight at 4 °C in a humidified
chamber. After washing with PBS three times for 10 min,
the secondary antibodies (Table 1) were applied for 90 min
at room temperature. Afterwards, sections were stained
with the nuclear stains DRAQ5 (1:1000; Thermo Fisher,
Waltham, MA, USA) or DAPI (1:1000) and washed with
PBS three times for 10 min before mounting with Mowiol
4–88 (Roth).
freeze fracture apparatus (BAF400D; Balzers, Liechtenstein)
at 5 × 10–6 mbar and -150 °C. The fracture faces were contrasted with platinum/carbon (3 nm, 45°) and stabilized with
carbon (30 nm, 90°) for stabilization of the replica. Remaining cell material was removed with 12% sodium hypochlorite, and the rinsed replicas were collected on Pioloformcoated copper grids.
Ultrathin sections and freeze-fracture replicas were
analyzed, and images recorded on a Zeiss EM10 or a LEO
912AB transmission electron microscope (both Zeiss,
Oberkochen, Germany).
Light microscopy
RT and qPCR analysis
The cryostat sections were analyzed on a Zeiss LSM510
Meta confocal microscope (Zeiss, Oberkochen, Germany)
equipped with an argon laser excitation wavelength at
488 nm and two helium–neon lasers with wavelengths for
excitation at 543 nm and 633 nm, respectively and appropriated filter set. Alternatively, images were taken on an
Axio Imager Z1 fluorescence microscope (Zeiss) with an
Apotome module. The systems’ software Black and Blue
ZEN were used for image acquisition, and image plates were
assembled and processed with Adobe Photoshop CS2 (San
José, CA, USA).
Preparation material was placed in a Precellys® Lysing Kit
(VWR Life Science Competence Center, Erlangen, Germany) filled with 900 µl QIAzol® Lysis Reagent (Qiagen,
Hilden, Germany) immediately after collection and placed
on ice. After thawed on ice, the samples were homogenized
for 10 s at 3000 rpm in a Minilys system (Bertin Instruments, Montigny-le-Bretonneux, France). The homogenized
samples were stored at −80 °C until further processing.
The collected tissues were incubated for 5 min at room
temperature. After addition of 100 µl gDNA eliminator (Qiagen), the homogenate was transferred to a MaXtract High
Density Tube (Qiagen) and 200 µl chloroform was added.
Centrifugation was performed at 12,000 rpm and 4 °C for
5 min. The upper aqueous phase containing the nucleic acids
was pipetted into a new Eppendorf reaction tube (2 ml SafeLock). The RNA was automatically isolated in a QIAcube®
(Qiagen) using the RNeasy® Plus Universal Mini Kit (Qiagen) and the corresponding QIAcube® (Qiagen) protocol.
The QIAxcel Advanced System (Qiagen) was used to
determine both RNA integrity and RNA concentration. Only
RIS values (RNA integrity score) of at least 6.0 were used
for murine samples, and RIS values of at least 5.8 were used
for human samples.
Electron microscopy
CP tissue was fixed in 2.5% glutaraldehyde buffered in 0.1 M
cacodylate (pH 7.4) for 2 h. For ultrathin sections, samples
were post-fixed in 1% osmium tetroxide in PBS (pH 7.4) for
1 h and subsequently dehydrated in a graded ethanol series
and acetone, and embedded in epoxy resin (Sigma Aldrich,
Darmstadt, Germany).
For freeze-fracture sample preparation, fixed tissues were
cryoprotected in 30% glycerol and snap-frozen in nitrogen
slush (-210 °C). Subsequently, they were fractured in a
Table 1 Primary and secondary
antibodies used in this study
Antibody
Primary AB
AQP1
AQP1
AQP4
Na/K-ATPase
NKCC1
Laminin
Secondary AB
Anti-mouse Alexa 488
Anti-mouse Alexa 546
Anti-rabbit Alexa 488
Anti-rabbit Alexa 546
Host
Dilution
Source
Mouse
Rabbit
Rabbit
Mouse
Mouse
Rabbit
1:100
1:100
1:100
1:100
1:100
1:50
Santa Cruz Biotechnology, Dallas, USA
Thermo Fisher Scientific, Walmart, USA
Santa Cruz Biotechnology, Dallas, USA
Hybridoma Bank, Iowa, USA
Abcam, Cambridge, England
Abcam, Cambridge, England
Goat
Goat
Goat
Goat
1:400
1:400
1:400
1:400
Invitrogen, CA, USA
Invitrogen, CA, USA
Invitrogen, CA, USA
Invitrogen, CA, USA
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The Reverse Transcription was performed with the QuantiTect Reverse Transcriptase Kit (Qiagen) according to the
manufacturer's instructions. For negative controls, the Reverse
Transcriptase was replaced with nuclease-free water. The total
cDNA concentration of each sample was measured using
the Qubit™ ssDNA Assay Kit on a Qubit 2.0 Fluorometer
(Thermo Fisher Scientific, Waltham, MA, USA).
The primers/probes used to quantify mRNA expressions of
aquaporin genes were acquired from TaqMan®GenExpression
assays (Thermo Fisher) as summarized in supplementary
table 2.
cDNA with a concentration of 5 ng/µl was used for analysis.
Measurements were conducted in triplicates, and a no-template blank served as the negative control (duplicates). Parenchyma from the striatum was used as reference tissue. qPCR
was performed on Applied Biosystems Step One™ (Applied
Biosystems—Thermo Fisher) for 40 cycles: 15 s denaturation
at 95 °C, followed by 1 min annealing at 60 °C. The data were
collected with the StepOne™ Software v2.3 and Ct values
were exported to Microsoft Excel.
qPCR data evaluation and statistical analysis
Analysis of relative mRNA expression was performed using
qbase + software (Biogazelle, Zwijnaarde, Belgium), with
relative abundance (RQ values) calculated using a series of
normalization methods based on the classical delta-delta Ct
method and MIQE—compliant procedures [24]. The RTqPCR cycle threshold (Ct) values were the input data in the
qbase + program. Results were calculated for 100% PCR efficiency and ‘unpaired’ experimental design.
Statistical analysis for mouse AQP4 expression was performed with GraphPad Prism 6.07 (GraphPad Software, San
Diego, USA) by processing the qbase + RQ values using an
unpaired t test. P values < 0.05 were considered statistically
significant.
After the amplification, PCR products were analyzed
by high-resolution capillary electrophoresis with the Qiaxcel DNA High Resolution Kit, QX Alignment Marker
15 bp/600 bp and the QX DNA Size Marker 25–500 bp at a
concentration of 30 ng/µl was used (Qiagen). The separation
was performed using the OM800 method of the Qiaxcel System with the following parameters: 4 kV and 5 s for alignment
marker injection, 5 kV and 10 s for the sample injection and
3 kV for 800 s for separation. The results were displayed as gel
images as obtained from QIAxcel system software.
Results
Localization of aquaporins in the choroid plexus
In this study, we focused on the distribution pattern of
water channels and other transport proteins in the human
CP. First, to investigate the transition area between the
CP epithelium and ependyma, we stained for the water
channel aquaporin-1 (AQP1) and the extracellular protein laminin, an essential component of the basal lamina
(Fig. 1). As expected, AQP1 was strongly expressed by
plexus epithelium cells in the apical membrane domain,
in some cells there was also a weak expression basolaterally. Occasionally, a reduction or even complete absence
of AQP1 was observed. Laminin showed a continuous
expression delineating the basal lamina of the CP epithelium (Fig. 1a). The CP epithelium covers underlying blood
vessels embedded in connective tissue, collectively called
the Tela choroidea. The endothelial layer of capillaries
and the Tunica intima of larger blood vessels is delimited
by a basal lamina as well. Thus, in many CP villi, two
basal laminae (epithelial and endothelial) were observed
separated by a thick layer of connective tissue (Fig. 1b).
At the entrance of blood vessels into the choroid plexus,
the endothelial basal lamina was continuous whereas the
epithelial basal lamina appeared in the transition zone
where ependymal cells connect with the CP epithelium.
Along a similar gradient, the AQP1 immunoreactivity in
the transition zone covering epithelium became patchy and
was entirely lacking in the ependymal lining. Thus, we
confirmed previous findings of the absence of AQP1 and
a basal lamina in the ependyma, and their presence in CP
epithelium.
Since it is known that the ependyma expresses the water
channel aquaporin-4 (AQP4) in the basolateral membrane
domain, we stained the human CP and ependyma for both
aquaporins, AQP1 and AQP4. As expected, AQP4 was
clearly located on ependymal cells. Surprisingly however,
we found AQP4 positive cells in the CP of all human body
donors (Fig. 2). This AQP4 immunofluorescence was in
most cells located in the basolateral membrane domain.
Additionally, in few CP epithelial cells there was a remarkable immunoreactivity in the cytoplasm as well as in the
membrane (Fig. 2a).
Aquaporin mRNA detection in the choroid plexus
To gather further evidence for the expression of AQP4
in the human CP, we performed a gene expression analysis using TaqMan® assays on tissues from three human
body donors (Fig. 2b). In all three human CP samples,
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Fig. 1 Human choroid plexus (CP) and ependyma in the lateral ventricle immunostained for AQP1 (red) and laminin (green), cell nuclei
labeled with Draq5 (blue). a Overview of the transition zone between
the CP and the ependymal lining of brain parenchyma. AQP1 is
restricted to the CP epithelium and is not present in ependymal cells.
Laminin, as part of the basal lamina (BL), is found in the CP in the
BL of the epithelium, and in the BL of the blood vessels (BV) in the
stroma of the CP and brain parenchyma. Note that the ependymal lin-
ing does not have a basal lamina (except where BV are present). BV
supplying the CP are surrounded by astroglia and ependyma in the
transition zone (arrows). b Close-up view of a CP villus. AQP1 is
expressed continuously mostly on the apical side of the CP epithelial
cells. The laminin staining indicates a thick stromal layer of connective tissue between the two BLs highlighting a considerable diffusions
distance
we found AQP4 mRNA to be expressed even though in a
lower amount than AQP1 mRNA. In contrast, the striatal
control tissue showed high levels of AQP4 mRNA expression and very small relative amounts of AQP1 mRNA (SM
Fig. 1). We confirmed the specificity of amplified PCR
products by QIAxcel high-resolution capillary electrophoresis showing the expected product size for AQP1,
AQP4, NKCC1, and the three reference genes (Fig. 2c).
This clearly demonstrates the expression of AQP4 at the
mRNA level in the CP.
connective tissue in the stroma under the CP epithelial cells
but found no clear evidence for such a relationship.
Distribution of aquaporin‑4 expression
Immunostaining and evaluating sections of the CP from
eight body donors for AQP1 and AQP4, we could not detect
a particular pattern in any of the samples with the following observations: We found cells that expressed AQP4, and
these cells appeared as individual cells or in groups. These
AQP4 positive cells were also immunoreactive for AQP1,
i.e. double labeled, or they were lacking AQP1 immunoreactivity. Examples of this heterogenous distribution are
shown in Fig. 2d–i.
We attempted to correlate the expression of AQP4 with
the occurrence of psammoma bodies or amounts of increased
Ultrastructural detection of AQP4
We performed ultrastructural analysis since AQP4 is known
to form orthogonal arrays of particles (OAPs) in freezefracture electron microscopy. The analysis of ultrathin sections revealed an abundance of collagen fibers in the stroma
underneath the CP epithelial cells. Most of CP epithelial
cells displayed microvilli, basal membrane foldings, mitochondria, and many vesicular bodies (Fig. 3a, b). In freezefracture replicas, we detected OAPs in membranes of CP
epithelial cells (Fig. 3). This is additional evidence for the
presence of AQP4 in the human CP.
Co‑localization of aquaporins with NKCC1
and Na/K‑ATPase
To find out how this unusual AQP4 expression relates to
the expression of relevant transport proteins we expanded
our analysis with stainings for NKCC1 (Fig. 4a) and Na/KATPase (Fig. 4b) in our analysis. NKCC1 showed an
almost continuous homogeneous expression on the apical
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Fig. 2 Expression of AQP1 and
AQP4 in the human choroid
plexus. a A CP villus in the lateral ventricle stained for AQP1
(green) and AQP4 (red). The
arrows indicate CPCs expressing AQP4 mostly basolaterally
whereas AQP1 is expressed
mostly apically. b Gene expression analysis for AQP1, AQP4,
and NKCC1 using TaqMan®
assays of three human body
donors, HPRT, TBP and UBC
served as reference genes.
Besides the expected AQP1
mRNA and NKCC1 mRNA,
AQP4 mRNA was present in
the samples of all three body
donors. c Exemplary gel image
of RT-PCR products obtained
from the QIAxel system
software showing the expected
product size for AQP1 (96 bp),
AQP4 (92 bp), HPRT (82 bp),
TBP (91 bp), UBC (71 bp),
NKCC1 (97 bp). Lane 1-size
marker, 2-AQP1, 3-AQP4,
4-NKCC1, 5-HPRT, 6-TBP,
7-UBC. d–i Distribution pattern
of AQP1 and AQP4 on cryostat
sections of the human CP in the
lateral ventricle from different
body donors. There were areas
(d, e) with single AQP4-positive
cells, and areas with clusters of
cells expressing AQP4 (h, i),
over 50% of all epithelial cells
in this region). When investigating cellular localization,
we found cells expressing both
AQP1 and AQP4 (e, h; arrows),
as well as cells expressing only
one of the two water channels
(e, f, i; arrow heads)
side of the plexus epithelium, which was colocalized with
AQP1. Vice versa, most AQP4-positive cells also showed
expression of NKCC1. Like NKCC1, Na/K-ATPase was
expressed apically but not homogeneously: There was a
tendency that Na/K-ATPase and AQP4 were inversely
distributed, more specifically, there were areas (arrows in
Fig. 4b) where AQP4- positive cells did not reveal Na/KATPase immunoreactivity. AQP1 showed continuous apical expression. This result could be confirmed in the CP
tissue of all body donors investigated.
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Changes of AQP4 expression in aging
Since all our body donors were more than 70 years old, we
hypothesized that AQP4 expression in the CP was caused by
age-related changes. To address this, we used TaqMan® Gene
Expression assays for quantitative real-time PCR analysis
in mice of various ages. We investigated mouse brains from
young (2 months), adult (12 months), and aged (30 months)
mice. We analyzed five biological replicates for each age
cohort and found that the relative amount of AQP4 mRNA in
Aquaporin-4 expression in the human choroid plexus
Page 7 of 13
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Fig. 3 Electron micrographs of human CP epithelial cells in ultrathin
sections (a, b) and freeze fracture preparations (c). Note the extensive
stroma (STR) between blood vessels (BV) and epithelial cells. In c,
the high power views revealed orthogonal arrays of particles (circles)
which are known to be formed by AQP4. The inserts in the middle
indicate the location of the membrane faces on the CP epithelial cell
older mice was significantly increased (Fig. 5a). We confirmed
the specificity of amplified PCR products by QIAxcel high-resolution capillary electrophoresis showing the expected product
size for AQP1, AQP4, NKCC1, and the three reference genes
(Fig. 5b). We also stained tissue from the three age groups
for AQP1 and AQP4. AQP1 was located apically in CPCs
with no apparent differences between age groups (Fig. 5c, d).
We did not find AQP4 positive cells in the CP of old mice.
Thus, although AQP4-RNA was present and increased in older
mice, the AQP4 protein does not seem to be expressed at levels
detectable by immunofluorescence (Fig. 5e, f).
immunofluorescence in the epithelium of the CP. In addition,
we found OAPs formed by AQP4 on the ultrastructural level.
This was a surprising result since plexus epithelial cells so
far have been known to express AQP1 only [2, 12, 25],
whereas ventricle-lining ependymal express only AQP4 [26,
27]. Although there have been reports on the presence of
AQP4 in the CP of rats, these studies showed either a weak
in-situ hybridization signal [28], or a diffuse cytoplasmic
immunofluorescence reactivity [29] in the choroid plexus.
Our results, however, show a mostly basolateral expression of AQP4 in many CPCs. Furthermore, we showed the
expression of AQP4 at the mRNA level for the human choroid plexus. The post-mortem interval for the three body
donors used for quantitative RT-PCR of 11 h or less is well
within the range when suitable RNA is still preserved in the
brain [30, 31] and was confirmed by the RIS values.
As a possible cause for the appearance of AQP4 in the
plexus epithelium we suspected a relationship with the
Discussion
In this study we report the expression of the water channel AQP4 in the human choroid plexus. In each of the 8
body donors, AQP4-positive cells could be visualized by
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Fig. 4 Triple immunostains for transport proteins and aquaporins in
the human CP. Cell nuclei were counterstained with Draq5 (blue). a
Immunostains for AQP4, AQP1, and the Na+-K+-2Cl− cotransporter
NKCC1. NKCC1 showed an almost continuous homogeneous expression on the apical side of the plexus epithelium, which was colocalized with AQP1. Vice versa, most AQP4-positive cells also showed
expression of NKCC1. b Immunostains for the Na+/K+-ATPase,
AQP1, and AQP4. Na+/K+-ATPase was expressed apically yet not
as homogeneously as the apical AQP1 expression, nor the apical
NKCC1 expression shown in a. In areas (arrows) where AQP4-positive cells were present, there was weaker or lacking Na/K-ATPase
immunoreactivity. AQP1 showed continuous expression
process of aging. Indeed, we did observe presumably agerelated characteristics such as psammoma bodies [32]. In
addition and consistent with previous studies [33, 34], we
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found extensive connective tissue in the thickened CP stroma
which very likely results in an impaired diffusion since the
distance between plexus epithelium and blood vessels is
increased (see Figs. 1 and 3). Lower expression of AQP1
has been reported in aging rats [35], and a decrease in CSF
production was observed in old sheep (7–10 years) compared to young sheep (1–2 years), with an increase in protein CSF/plasma ratio while protein plasma levels remained
constant [36]. In addition, a decreased CSF production has
been suggested to play a role in Alzheimer’s disease [34, 37],
and a lower CSF flow in elderly patients has been linked to
cognitive impairment [38]. Thus, age-related changes in the
CP seem to be more pronounced in Alzheimer’s disease (for
a recent review see [21, 39].
In the context of these morphological and functional
changes associated with age and disease in the CP, the
detection of AQP4 in CPCs and can be interpreted in two
alternative scenarios: First, AQP4 expression could serve
as a compensatory mechanism in old age to maintain CSF
production known to be decreased. The compensation could
be implemented by constitutive expression of AQP4 or a
regulated mechanism similar to that described for antidiuretic hormone (ADH) and aquaporin-2 in the kidney. Here,
antidiuretic hormone (vasopressin) which is produced in
the hypothalamus, activates a signaling cascade resulting
in AQP2 membrane incorporation [40]. A similar process
could regulate AQP4 in the CP at low CSF production levels
since there was immunoreactivity for AQP4 in the cytoplasm
of some cells. Although such a mechanism has not been verified for AQP4 so far, vasopressin has been reported to modulate water flux in the cerebral cortex likely via AQP4 [41].
Recently, it was shown that AQP4 expression in the mouse
CP can be induced by experimental hypoxic conditions [42].
Moreover, cytoplasmic isoforms of human AQP4 generated
by alternative splicing have been shown to influence AQP4
membrane expression [43] and might affect the formation of
OAPs and therefore water homeostasis [44].
An alternative hypothesis can be inferred from AQP4
expression in the ependyma adjacent to the plexus epithelium. Here, AQP4 is expressed in the basolateral membrane
domain of ependymal cells [11, 14]. The basolateral expression corresponds to the histological localization we found
in CPCs. Therefore, CPCs might differentiate over time into
AQP4-positive cells taking on characteristics of ependymal
cells. This could include a partial basolateral instead of apical water outflow along an osmotic gradient [45] and would
be consistent with reduced CSF production in the aging
brain. The two hypotheses about the possible consequences
of AQP4 expression in the aging CP affecting water flow are
illustrated in Fig. 6.
Since CP tissue of young and healthy human adults is
difficult to obtain, we turned to a mouse model to compare
different age groups. Indeed, we could observe an increase
Aquaporin-4 expression in the human choroid plexus
Page 9 of 13
90
Fig. 5 AQP4 expression in
the mouse choroid plexus
from different age groups. a.
Quantitative RT-PCR analysis
using TaqMan® gene expression
assays for mice of three different ages (n = 3 for each group),
HPRT, TBP and UBC served as
reference genes and the striatum
as reference tissue. The relative
mRNA expression of AQP4
based on the qbase + exported
relative quantity (RQ) values,
calculated from Cq values.
Qbase + results are scaled to
the average across all unknown
samples per target showing
the relative quantity for AQP4
was significantly higher in the
30 month-old group compare to
the younger groups (* indicates
p < 0.05, ** p < 0.0005). b
Exemplary gel image of RTPCR products obtained from
QIAxel system software showing the expected product size for
AQP1 (94 bp), AQP4 (69 bp),
HPRT (65 bp), TBP (138 bp),
UBC (92 bp). Lane 1-size
marker, 2-AQP1 2-months,
3-AQP1 12-months, 4-AQP1
30-months, 5-AQP4 2-months,
6-AQP4 12-months, 7-AQP4
30-months, 8-HPRT 2-months,
9-TBP 2-months, 10-UBC
2-months. c–f Immunofluorescence staining for AQP1 (c, d)
and AQP4 (e, f) in CP tissue
from the lateral ventricle of
young, 2 months old mice (c, e),
and old, 30 months old mice (d,
f). AQP1 was apically expressed
in CPCs with no obvious difference between age groups.
AQP4 immunofluorescence was
not detected on CPCs in neither
young nor older mice, with
strong reactivity in adjacent
ependymal and subependymal
regions
in the AQP4/AQP1 ratio in correlation to the age but only
at the mRNA level. In contrast, antibody staining for AQP4
was negative in the CP epithelium of old mice. Thus, it is
possible that the translation of AQP4 protein is suppressed
by some regulatory mechanisms in the mouse. For example,
several miRNAs have been reported to downregulate the
expression of AQP4 (summarized in [46]). More recently,
the RNA binding protein DDX4 has been identified as a
negative regulator of AQP4 translation in mice [47]. Consistent with our results, Trillo-Contreras et al. [42] reported
expression of AQP4 in the CP especially of aged mice under
hypoxic condition on the mRNA level, and even moderately
on the protein level.
The reasons for the difference in human and mouse AQP
expression on the protein level observed in this study could
be due to a variety of reasons. Obviously, the maximum life
span of the mouse (3–4 years) differs greatly with that of
humans (≥ 100 years [48]), yet a recent MRI study showed
a clear reduction of water delivery through the CP to the
ventricles in aged mice [49]. However, the macroscopic
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Page 10 of 13
F. Deffner et al.
Fig. 6 A graphical illustration of the channels and transporters examined in this study, and their suggested impact on the waterflow and
CSF production in aging. a A choroid plexus epithelial cell in an
adult human with normal waterflow and CSF production. b An aged
choroid plexus epithelium cell with reduced waterflow and reduced
CSF production without basolateral AQP4 expression. Note increased
diffusion distance between the blood vessel and the epithelium. c
Explains our first hypothesis: AQP4 is expressed to compensate the
reduced CSF production in aged humans. The CP cells express AQP4
basolaterally to generate a higher transcellular waterflow. This leads
to a normal waterflow and CSF production. d Explains our alternative
hypothesis: The expression of AQP4 leads to an inverted transcellular
waterflow carrying a reduced CSF production with it
and microscopic structure of the CP of the mouse differs
from that of the human. While the CP of the lateral ventricle in mice consists of a thin vascular layer covered by CP
epithelium on both sides [50], the human CP is a highly
branched structure with villi and extensive connective tissue. In addition, we observed thickened subepithelial stroma,
and deposits such as psammoma bodies (see Fig. 1b) not
found in the CP of old mice. Thus, changes occurring with
age might be compensated for in other ways in the mouse,
and despite increased mRNA levels, the AQP4 channel is
not incorporated into the plexus epithelium. In humans, our
data show not only the occurrence of AQP4 at the mRNA
level but also at the protein level as basolateral expression
in the CP epithelium.
Either one of the proposed scenarios shown in Fig. 6
would also have an impact on the so-called 'glymphatic
pathways', which describes an exchange of cerebrospinal
fluid and interstitial fluid as well as metabolites in the brain
[51]. The transport is thought to take place peri-arterially
and peri-venously, and then via the AQP4 endfeet of astrocytes. CSF is taken up by the astrocytes via AQP4, and
dissolved substances are washed out paracellularly [52].
Such CSF uptake from the ventricles and delivery to the
CP stroma could be enabled by AQP4. An MRI study with
aquaporin-KO mice suggested a higher contribution to CSF
production by AQP4 than AQP1 [53].
The direction of water flow through aquaporins is determined by the osmotic and hydrostatic gradient [54]. By
decreasing Na/K-ATPase [35] (this study Fig. 5) thus lowering oncotic pressure, CSF could be absorbed from the
ventricles in the reversed direction. In a recent study [45],
explants from rat CP provide evidence for a substantial
13
Aquaporin-4 expression in the human choroid plexus
amount of apical outward water flow through the NKCC1
co-transporter, in addition to AQP1. Another report on
isolated CP cells, however, suggest an inward flow through
NKCC1 to contribute to cell water volume maintenance
needed for CSF secretion [55]. This debate is far from
settled (see recent discussions [56, 57]). Our data of the
expression of the fast water channel AQP4 in the CP suggests that the water balance across the epithelium can be
changed, and it remains to be shown whether this is a beneficial compensatory mechanism or not. To confirm one
of these alternatives, more investigations on isolated CP
tissue or cells, and further age comparisons of CP tissues
will be necessary.
Conclusions
In summary, we could demonstrate the expression of
AQP4 in the epithelium of the human CP and age-related
changes in the murine CP on the RNA level. The expression of AQP4 in the aging CP has consequences for the
models of water flow through the CP epithelium discussed
so far. Thus, an increased water inflow though AQP4 could
compensate for impaired perivascular diffusion; alternatively, AQP4 in the CP could be misexpressed and slow
down CSF production.
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00018-022-04136-1.
Acknowledgements We like to thank Ria Knittel for preparing the
freeze fracture replicas, Sarah Frosch and Lidia Garcia-Pradas for help
with the qPCR. The study was supported by the IZKF-Promotionskolleg of the medical faculty of the University of Tübingen.
Author contributions FD generated and analyzed most of the histological and molecular data and wrote large parts of the manuscript. CG
analyzed the molecular data and performed the statistics involved, and
wrote parts of the manuscript. UM performed some of the histological
processing and confocal analysis. AW performed some of the human
choroid plexus preparations and contributed to the manuscript writing. PHN performed some of the human choroid plexus preparations,
contributed to the data analysis, the concept of the study, and the writing of the manuscript. PF-B oversaw the freeze-fracture preparations
and performed electron microscopy examination. BH conceptualized,
oversaw, and interpreted the study. AFM designed the study, performed
preparations of the mouse choroid plexus, analyzed the histology, wrote
and finalized the manuscript. All authors read and approved the final
manuscript.
Funding Open Access funding enabled and organized by Projekt
DEAL. The study was supported by the IZKF-Promotionskolleg of
the medical faculty of the University of Tübingen.
Data availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable
request.
Page 11 of 13
90
Declarations
Ethics approval Body donors gave their informed consent in agreement with the declaration of Helsinki to use the cadaver for research
purposes. This was approved by the Ethics Committee of the Medical
Department of the University of Tübingen under the project number
237/2007BO1. Animals (mice) were used according to University of
Tübingen and governmental guidelines, and were approved by local
authorities (Regierungspräsidium Tübingen).
Consent for publication Not applicable.
Conflict of interest The authors declare that they have no competing
interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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