Scarpetta et al. Fluids and Barriers of the CNS
https://doi.org/10.1186/s12987-023-00420-9(2023) 20:19

Fluids and Barriers of the CNS

Open Access

RESEARCH

Morphological and mitochondrial changes
in murine choroid plexus epithelial cells
during healthy aging
Valentina Scarpetta1,6, Felipe Bodaleo2, Chiara Salio3, Amit Agarwal2,4, Marco Sassoè‑Pognetto1 andAnnarita Patrizi4,5,6*

Abstract
Background Choroid plexuses (ChPs) are intraventricular structures mainly composed by specialized epithelial cells
interconnected by tight junctions that establish the blood-cerebrospinal fluid (CSF) barrier. ChPs are essential to pro‑
duce CSF and transport solutes from and into the brain. Deterioration of ChP function and morphology has been cor‑
related to worsening of neurodegenerative disorders. We here map morpho-functional changes in the ChP epithelial
cells during healthy aging, starting from young adult to 2-years old mice.
Methods We used a multi-tiered approach, including transmission electron microscopy (TEM), immunohistochem‑
istry, RT-qPCR, Western Blot and 2-photon microscopy (2-PM) at multiple timepoints ranging from young adult to
2-years old mice.
Results We identified distinct morpho-functional modifications in epithelial cells of ChP starting from 8 to 12 monthsof age, which mostly remained stable up to 2 years. These changes include flattening of the epithelium, reductionof microvilli length and an augmentation of interrupted tight junctions. We also found a decrease in mitochondria
density together with elongation of mitochondria in older mice. Morphological mitochondrial rearrangements wereaccompanied by increased superoxide levels, decreased membrane potential and decreased mitochondrial motility
in aged mice. Interestingly, most of the age-related changes were not accompanied by modification of protein and/or
gene expression levels and aged mitochondria effectively responded to acute pharmacological stressful stimuli.
Conclusions Our study suggests a long-term progression of multiple morpho-functional features of the mousechoroid plexus epithelium during adulthood followed by structural remodeling during the aging process. These find‑ings can lead to a better understanding on how functional and morphological rearrangements of ChP are correlated
during aging.
Keywords Mitochondria motility, Mitochondria speed, Tight junctions, Microvilli*Correspondence:
Annarita Patrizi
a.patrizi@dkfz.de
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Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

Introduction
The Choroid Plexus (ChP) is a highly vascularized
structure located in the ventricular system of the brain,
where it forms a selective interface between blood andcerebrospinal fluid (CSF). The ChP has a distinct tissueorganization, which comprises: an external monolayerof specialized epithelial cells, a connective stroma and ahighly permeable fenestrated capillary network. Epithelial cells are interconnected by tight junctions and form
a permissive barrier, called blood-CSF fluid barrier. Different types of immune cells are also present in the ChP
and play a major role in the immune surveillance of thecentral nervous system (CNS) [1, 2].
CSF is a “nourishing liquor” containing essential
micro- and macro-nutrients, ions, and bioactive molecules that are synthesized and released by the epithelial cells of the ChP. In addition, epithelial cells
transport selective peptides and proteins (e.g., leptin) from blood into CSF [3]. ChP secretome and CSF
composition change throughout life, and it has beensuggested that such changes interfere with the physiological regulation of brain parenchyma [4, 5]. Recently,
it was demonstrated that aged brain can transfer signals via CSF that interfere with ChP production ofneurotrophic molecules, giving rise to decline in cognitive function and hippocampal neurogenesis [6].
Thus, the bidirectional flow of ‘molecular information’between ChP–CSF and the brain is essential for maintaining the brain homeostasis and is compromised inaged mice [7–9].
Early studies, in humans and rats, described agerelated morphological modifications of the ChP duringphysiological aging, such as reduction of epithelial cellheight, emergence of cytoplasmic inclusions and thickening of the epithelial basement membrane [10–12].
Surprisingly, we are only aware of one previous study
that analyzed morphological alterations of mouse ChP
during physiological aging. Given the importance ofmurine models for basic and translational research, we
decided to investigate age-related morphological modifications of the ChP epithelium in C57BL/6 mice. Using
transmission electron microscopy (TEM) and 2-photon
microscopy (2-PM), we mapped the architectural transition of the epithelial cells starting from 2-months old(m.o.) mice until up to 2-years old mice (24 m.o.). Overall, ChP epithelium showed multiple cellular changes
during adulthood and aging. Mitochondria were among
the most impacted organelles, displaying noticeable
shape shifting, increased superoxide levels, decreased
membrane potential and decreased motility. Interestingly, most of the observed alterations were detected in
8 m.o. animals and remained stable at later stages.

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Materials and methods
Experimental animals

Inbred C57BL/6J and C57BL/6N mice were purchasedfrom Janvier labs and housed in both the Interdisciplinary Neurobehavioral Core (INBC) at Heidelberg University and the German Cancer Research Center (DKFZ)
animal facility. Both male and female mice were includedin the study. Animals were housed in static cages. No
environmental enrichment toys were added to the cages,
only extra Kimwipes tissue. After weaning, animals weresocially housed in groups of two to five animals per cage.
Animals were housed in a temperature and humiditycontrolled room on a 12 h light/ 12 h dark cycle andhad food and water ad libitum. All animal experiments
were approved by the local governing regional council(Regierungspräsidium Karlsruhe, Germany). All methods
were carried out following the German Animal Welfare
Act regulations. Animal studies are reported in compliance with the ARRIVE guidelines.
Transmission electron microscopy (TEM)

Two m.o. (n = 3), 8 m.o. (n = 3) and 20 m.o. (n = 3) animals were perfused with PBS 0.1 M (Gibco) followed by
2.5% glutaraldehyde in cacodylate buffer (CaCodylade
buffer 50 mM, KCl 1 M, ­CaCl2 0.1 M, ­MgCl2 0.1 M, Glutaraldehyde 2.5% and sucrose 2%). The ChP was dissected
and post-fixed for 1–2 h in the same fixative at 4 °C. Afterwashing in PB 0.2 M, the ChP was post-fixed in osmium
ferrocyanide (1 volume of 2% aqueous osmium tetroxide:
1 volume of 3% potassium ferrocyanide) for 1 h at 4 °C,
dehydrated for 15 min in increasing concentrations ofacetone (30%, 60%, 90%, 100%), incubated in acetone/
Spurr resin (1:1, 30 min, 1:2, 30 min) and in Spurr resin(Sigma-Aldrich) overnight at room temperature (RT).
Finally, the ChP was embedded in Spurr resin in capped
00 BEEM capsules (Electron Microscopy Sciences) for
24 h at 70 °C. Ultrathin sections were cut with an ultramicrotome (EM UC6, Leica Microsystems), collected on
uncoated nickel grids (100 mesh) and counterstained for
30 s with UranyLess EM Stain and 30 s with lead citrate(Electron Microscopy Sciences). Sections were observedwith a JEM-1400 Flash transmission electron microscope(JEOL, Tokyo, Japan), and images acquired with a highsensitivity sCMOS camera. Electron micrographs in
Fig. 1 were obtained with the automated montage systemof the Jeol Matataki Flash camera. Shading correction
and contrast adjustment of the titled images was donewith Adobe Photoshop.
TEM image analysis

Epithelial choroid plexus cells (30 epithelial cells per
mouse; 90 cells per age) were selected such that both

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

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length of tight junctions (µm); tight junction contact area(%); average number of tight junction dilatations; mitochondria distribution; mitochondria localization, calculated based on their distance from the apical cell surface(the shortest distance was extracted to calculate the apical-basal proximity ratio, such that 1 corresponded to thebasal surface and 0 to the apical surface); mitochondria
density (mito/µm2); mitochondria surface area (µm2),
average of the surface occupied by all mitochondria profiles per cell; mitochondria perimeter (µm), average of theperimeter of all mitochondria profiles per cell; circularity,
calculated as 4π x Area/Perimeter2 (round-shaped mitochondria profile have a value close to 1, while elongatedmitochondria have a score closer to 0); Feret’s diameter(µm), longest distance between any two points within theselected mitochondrion; aspect ratio, representing major
to minor axis ratio of each mitochondrion.
Tissue processing for light microscopy

Two–three m.o., 8–12 m.o. and 20–24 m.o. mice were
either transcardially perfused with 4% paraformaldehyde(PFA) in sodium phosphate buffer (PBS 0.1 M, pH 7.4) oreuthanized by cervical dislocation. Perfused brains were
post-fixed in 4% PFA for 1 h at 4 °C, cryoprotected in
10, 20 and 30% sucrose in PBS at 4 °C and then embedded in cryostat medium (Tissue-Tek® O.C.T. Compound,
Sakura). Freshly dislocated brains were rinsed with HBSS,
embedded in O.C.T. and then frozen in dry ice.
Immunostaining

Fig. 1 Ultrastructure of the choroid plexus epithelial cells during
aging. Representative TEM images of choroid plexus epithelial cells at
2, 8 and 20 months old (m.o.). Scale bar: 5 µm

apical and basal borders of the cells were visible andnucleus was at its best fit. The analysis was performed
with a semiautomatic custom macro developed by Dr.
Damir Krunic (Light Microscopy Facility, DKFZ). The
following parameters were considered: surface cellular area (µm2); cell axis ratio, obtained by dividing thehorizontal and vertical cell axes; surface occupied by
microvilli (µm2); microvilli length (µm), average of threesegments traced from the apical border of the epithelial
cell until the end of the microvilli (the segments were
distributed at the two lateral extremes and in the centerof the structure); the area (µm2) occupied by basolateral
membrane invaginations; the percentage of invaginations distributed by size and animal age; tight junction
integrity, percentage of interrupted tight junctions; total

All brains were sliced in 20 µm-thick coronal or sagittal
sections using a cryostat (Leica CM1950). Before immunostaining, frozen sections were fixed with cold 4%-PFA
for 10 min, on ice, followed by rinses with PBS 0.01 M.
Sections were blocked and permeabilized (0.5% Triton
X-100 and 10% normal donkey serum in PBS) for 1 h at
RT, incubated in primary antibodies overnight followed
by secondary antibodies for 2 h (0.5% Triton X-100 and
3% normal donkey serum in PBS). Sections were counterstained with Hoechst (D9542-1MG, Sigma Aldrich;
1:4000) and mounted using Dako Fluorescent Mounting
Medium (GM304, Agilent Technologies, Inc). The following primary antibodies were used: mouse anti Ezrin(sc-58758, Santa Cruz Biotechnology; 1:500); mouseanti ATP5A (ab14748, Abcam; 1:1000); rabbit anti Claudin-1 (71-7800, Thermo Fisher Scientific, 1:1000); rat
anti Zonula occludens-1 (14-9776-82, Invitrogen, 1:500);
rabbit anti GLUT1 (ab116730, Abcam, 1:1000); mouseanti ­Na+/K+-ATPase (sc-48345, Santa Cruz Biotechnology, 1:800). Secondary antibodies were selected from
Alexa series: Alexa Fluor™ 555 (A-31570, Thermo Fisher
Scientific; 1:500); Alexa Fluor™ 488 (712-545-150, Jackson ImmunoResearch, 1:1000) and Alexa Fluor™ 555

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19(A-31572, Thermo Fisher Scientific; 1:500). For mitochondria labeling an additional antigen retrieval stepwith L.A.B Solution treatment (Polyscience) for 10 min
at RT was applied. Images were acquired using a Zeiss
LSM780 confocal microscope with × 20, × 40 or × 60
objective (oil immersion). Images were acquired with atleast 1024 × 1024-pixel resolution. ZEN Black software
was used for image acquisition and ImageJ (Fiji, NIH) forprocessing and analysis [13].
Confocal mean intensity analysis

For GLUT1 and ­Na+/K+-ATPase expression analysis, 1024 × 1024 fields of ChP were imaged using a × 20
objective. The mean pixel intensity of GLUT1 and ­Na+/
K+-ATPase signals was measured by manually drawing
regions of interest at basal and apical cellular segments,
respectively, using Fiji software (n = 4–8 images per animal, 3 animals per age).
Tight junction analysis

Sholl analysis was used to quantify tight junction (TJ)
organization (3 to 6 images per animal, 3 animals perage). We used a dedicated macro developed by Dr.
Anthony D. Hill with the Fiji software. TJs were analyzed
from images characterized by homogeneous cell distribution and staining covering at least 80% of the image
surface. Signal background was removed by appropriate
threshold, an initial circle was positioned in the center ofthe image followed by successive larger circumferences
increasing of a 10 µm step. The number of intersections
between the concentric circumferences and stained network was calculated.
Immunoblotting

Two–three m.o. (n = 3), 8–12 m.o. (n = 3) and 20–24 m.o.
(n = 3) mice were sacrificed by cervical dislocation. The
ChP was pulled from both lateral and fourth ventricles under a dissection microscope (stereomicroscope
Stemi DV4, Zeiss), frozen in liquid nitrogen and stored
at – 80 °C. Tissues were homogenized in 1X RIPA Lysis
reagent (9806S, Cell Signaling) supplemented with Pierce
Protease and Phosphatases inhibitor mini tablets (A32955
and A32957, Life Technologies) on dry ice. Tissues weremechanically dissolved and centrifuged at 12,000 rpm for
15 min at 4 °C. Protein concentration was measured with
BCA assay (23225, Life Technologies) and the supernatant was mixed with Laemmli buffer (1610747, Bio-Rad).
Approximately 35 µg of proteins were loaded onto gels,electrophoresed by SDS-PAGE and then transferred on
0.45 µm nitrocellulose or PVDF membranes. Membranes
were blocked for 1 h at RT with 5% non-fat dried milk
dissolved in TBST buffer (Tris-buffered saline containing
0.1% Tween 20) and then incubated on a shaker overnight

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at 4 °C in blocking buffer containing rabbit anti GLUT1(ab115730, Abcam, 1:1000), mouse anti ­Na+/K+-ATPase(sc-48345, Santa Cruz Biotechnology, 1:500), mouse anti
AQP1 (sc-55466, Santa Cruz Biotechnology, 1:500), total
OXPHOS antibody cocktail (ab110413, Abcam, 1:1500)
that included a positive control (rat heart mitochondria).
The membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for
1 h at RT. For allowing quantitative comparison and normalization, the membranes were incubated with a rabbit
anti-Calnexin antibody (ab22595, Abcam; 1:10,000). The
membranes were then developed using the Pierce ECL
chemiluminescence substrate (32106, Life Technologies),
visualized with ChemiDoc™ XRS (Bio-rad), and band
density was measured by ImageJ (Fiji, NIH).
RNA isolation

For evaluating mRNA levels, ChPs were processed as previously described [14]. For mitochondria related genes, 6
mice per age were used, while for TJs related genes 4–5
mice per age were. Briefly, mice were sacrificed by cervical dislocation and the ChP was pulled from both lateral and fourth ventricles under a dissection microscope(stereomicroscope Stemi DV4, Zeiss) and frozen in liquid
nitrogen. Total RNA was extracted using RNeasy Mini
Kit (Qiagen) following manufacturer’s protocol. Tissue
was disrupted and homogenized using Lysis buffer RTL
and QIAshredder spin columns (Qiagen). Genomic DNA
was removed using RNase-free DNase I (Qiagen) for
15 min at RT. RNA was eluted with 30 µl of RNase freewater. RNA quality and concentration were measured by
UV spectrophotometry on NanoDrop One (Thermo Scientific). RNA samples with absorbance ratios below 1.8
were precipitated using 100% ethanol, ammonium acetate and glycogen (R0551, Thermo Scientific) following
manufacturer’s protocol. RNA was reverse-transcribed
into cDNA using the High-Capacity RNA-to-cDNA kit(4387406, Thermo Scientific) and cDNA samples.
Reverse transcription quantitative real‑time PCR (RT‑qPCR)

RT-qPCR was performed using PowerUp SYBR Green
Master Mix (A25742, Thermo Scientific) following
manufacturer’s protocol, with 50 ng cDNA, 20 µM eachprimer in a 10 µl reaction volume. All RT-qPCR experiments were performed on LightCycler 480 (Roche) andgene expression levels were normalized to the expressionof the two stable housekeeping genes Tbp and Rlp13a.
The following primer sequences were used:
Cldn1 (FW: AGC​ACC​GGG​CAG​ATA​CAG​T; REV:
AGC​ACC​GGG​CAG​ATA​CAG​T);
Tjp1 (FW: GCA​CCA​TGC​CTA​AAG​CTG​TC REV: ACT​CAA​CAC​ACC​ACC​ATT​GC);

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

Ndufb8 (FW: GGC​CGC​CAA​GAA​GTA​TAA​CA; REV:TGA​TAC​CAC​GGA​TCC​CTC​TC);
Sdhd (FW: TGG​TCA​GAC​CCG​CTT​ATG​TG; REV:GGT​CCA​GTG​GAG​AGA​TGC​AG);
Uqcrc2 (FW: AAA​GTT​GCC​CCG​AAG​GTT​AAA; REVGAG​CAT​AGT​TTT​CCA​GAG​AAGCA);
Atp5a (FW: GGA​GCC​CAG​CAA​GAT​CAC​AAA​GTT;
REV: ATC​TGG​TGA​CAG​TGA​CAG​GGC​TTT​);
Rlp13a (FW: ACA​GCC​AAG​ATT​CAC​GGT​AGA REV:
GCT​TCT​TCT​TCC​GAT​AGT​GCATC);
Tbp (FW: CCT​TGT​ACC​CTT​CAC​CAA​TGAC; REV:ACA​GCC​AAG​ATT​CAC​GGT​AGA).
Raw data processing

Non-baseline-corrected RT-qPCR raw data wereextracted from the machine to provide input for LinRegPCR (version 2020.0) and analyzed accordingly to theprotocol published by Hoa and Patrizi [14]. In brief, thesoftware performed baseline correction for each sample
individually and calculated amplification efficiency (E),quantification cycle (Cq) and coefficient of determination ­(R2) by fitting a linear regression model to log-linear
phase. Technical replicates were examined, and arithmetic mean was taken as the Cq value representing biological samples.
Ex vivo two‑photon microscopy

ChP explants were dissected from 3–4 m.o. and
22–24 m.o. animals (n = 3–4 mice per age). Explants
were allowed to recover in artificial cerebrospinal fluid(ACSF) containing (in mM): 119 NaCl, 2.5 KCl, 2.5­CaCl2, 1.3 ­MgCl2, 1 ­NaH2PO4, 26.2 ­NaHCO3 and Dextrose (292–298 mOsm/L), bubbled with carbogen (95%
­O2/5% ­CO2) for 10 min at 37 °C, then maintained at RT
for the entire duration of the experiment. Live-cell imaging was performed using a custom built 2-PM (Bergamo
II, Thorlabs, USA) with a Nikon NIR, × 60 1.0 NA objective. 2-PM excitation was achieved using a mode-locked
Ti: sapphire laser (Coherent Ultra II).
MitoTracker loading, imaging and analysis

Explants were incubated with 100 nM MitoTracker Red
CMXRos (M7512, ThermoFisher) for 30 min at RT, andrinsed 3 times with carbogen-bubbled ACSF. Ti: sapphire laser (Coherent Ultra II) was tuned at 940 nm,
and each image frame was acquired at a rate of 0.52 s/
frame (about 1.9 Hz) for 400 s and was 90.31 µm2 at
512 × 512-pixel resolution. Time series were acquired
as z-stacks with a step size of 0.3 µm for 10.2 µm
z-depth. For mitochondrial dynamics analysis, images
were registered using ImageJ plugin Correct 3D drift,
and mitochondrial tracking was performed manually using the ImageJ plugin MTrackJ [15]. Analyzed

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parameters were total distance, displacement between
two consequential spatial points, and instantaneousand average speeds (n = 50 mitochondria per animal, 3animals per age).
TMRM and MitoSOX loading, imaging and analysis

For mitochondrial membrane potential experiments,
explants were either kept in ACSF or incubated with
10 µM Rotenone (HB5398, Hellobio) or with10 µM Oligomycin A (HB4488, Hellobio) for 15 min, followed byco-incubation with 25 nM TMRM (ab228569, Abcam)
and 100 nM MitoTracker Green (M7514, Thermofisher)for 45 min. For mitochondrial reactive oxygen species (ROS) measurements, explants were coincubatedfor 45 min with 200 nM MitoSOX Red (M36008, Thermofisher) and 200 nM MitoTracker Green. For all treatments, tissues were rinsed for 10 min with bubbled ACSF
before imaging. Ti: sapphire laser (Coherent Ultra II) was
tuned to 860 nm, and each image frame was 111.15 µm2
at 1024 × 1024-pixel resolution. Z-stacks were acquiredwith a step size of 0.5 µm for 50.5 µm z-depth. Acquisition parameters were maintained the same within
experimental groups. Fluorescence intensity was calculated by cell. Cells were randomly selected such as thenucleus was at its best fit. The analysis was manually
performed. Pixel mean fluorescent intensity values wereobtained using Measure tool in Fiji software (TMRM:
2–3 m.o., n = 180–250 cells, 22–24 m.o., n = 150–240
cells, 4 animals per age; MitoSOX: 2–3 m.o., n = 200 cells,
22–24 m.o., n = 170 cells, 3 animals per age).
Statistical analysis

Statistical analysis was performed using GraphPad Prism(version 9.4.1). Appropriate statistical tests were selectedbased on the distribution of data and sample size. Parametric tests (One-Way ANOVA) were used only ifdata were normally distributed. Otherwise, nonparametric alternatives were chosen (Mann–Whitney andKruskal–Wallis). For the ultrastructural cellular analysis (Figs. 2, 3, 4 and 5), a nonparametric test (Kruskal–
Wallis test) was chosen. For gene and protein levels
evaluation (Figs. 3 and 7), One-Way ANOVA analysis
was performed, followed by Tukey’s multiple comparison test. For the mitochondria motility analysis (Fig. 6),
Mann–Whitney nonparametric test was selected. For
the mitochondria functional analysis (Fig. 8), unpaired
t-test was selected. Data are either presented as medianvalues or as mean ± standard error of the mean (s.e.m.).
p values ≤ 0.05 were considered significant (*p ≤ 0.05,**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). The statistical test
used is indicated in each figure legend.

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

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Fig. 2 Remodeling of the gross morphology of choroid plexus epithelial cells during aging. A Representative TEM images of choroid plexus
epithelial cells at 2, 8 and 20 months old (m.o.). Yellow lines indicate horizontal and vertical cellular axes. Quantification of cell axis ratio (B) and cell
profile area (C) averaged per cell. n = 30 cells per animal, 3 animals per age; black dashed line: median; Kruskal–Wallis; *p < 0.05, **p < 0.01. Scale bar:
2 µm

Results
Remodeling of choroid plexus epithelial cells during aging

To address age-dependent morphological changes ofmurine ChP epithelial cells, we analyzed their ultrastructure at three time points: 2–4 m.o. (from now on referred
as young adult), 8–12 m.o. (adult) and 20–24 m.o. (aged)(Fig. 1). The most obvious change observed in adultanimals was a progressive flattening of epithelial cells(Figs. 1, 2A), as demonstrated by a significant decrease incell height (Fig. 2B) and cell profile area (Fig. 2C) in theadult and aged groups. The ChP of aged mice was alsocharacterized by an enrichment of intracytoplasmic electrodense structures (Fig. 1). These intracellular inclusions
increased in density (2 m.o., 0.8 ± 0.2; 8 m.o., 1.8 ± 0.3*;
20 m.o., 2.8 ± 0.4***; Kruskal–Wallis. n = 50 cells, 3 animals per age) and complexity from adult to the aged
stage.
The apical portion of choroid epithelial cells is covered by microvilli that project into the ventricular lumen(Figs. 1, 3A). In parallel with the remodeling of cell shape,
there was a significant reduction of microvilli length(Fig. 3B) and of the area occupied by microvilli (Fig. 3C)
in adult and aged mice compared with the young adultstage. When we assessed the localization of Ezrin, astructural protein component of microvilli, with confocal microscopy, we found that Ezrin remained concentrated on the apical surface of the epithelial cells [16] atboth ages. However, there was a clear reduction of Ezrinimmunopositive structures in epithelial cells of aged micecompared to young adult stage (Fig. 3D), confirming theultrastructural observations.
The basal portion of choroid epithelial cells was characterized by extensive invaginations of the basolateral
membrane in aged mice (Figs. 1, 3E). The area occupied
by basolateral membrane invaginations was significantly
increased in adult and aged ChP compared to young
adult stage. The median surface of these structures also
increased with aging (Fig. 3F, G).
To start understanding whether the observed morphological alterations may correlate with dynamic and/or functional consequences, we mapped the distributionof key transporters and channels known to be selectively
located at apical and basal cellular compartments. Dueto the limited choice of reliable antibodies, we used glucose transporter member 1 (GLUT1) and ion pump ­Na+/
K+-ATPase as basal and apical markers [17]. Low magnification confocal images did not reveal major differencesin the distribution of these proteins in both young adult(See figure on next page.)
Fig. 3 Ultrastructural and molecular remodeling of both apical and basal cellular compartments during aging. A High magnification TEM
images showing representative microvilli at 2 and 20 months old (m.o.). Yellow dashed lines indicate the apical cell surface from which microvilli
originate. Quantification of microvilli length (B) and surface occupied by microvilli (C) averaged per cell. D Representative images of Ezrin
immunofluorescence (red) at 2–3 and 20–24 m.o. E High magnification TEM images showing representative basolateral invaginations at 2 and20 m.o. F Quantification of the surface of basolateral invaginations. G Distribution in the three age groups of the surface occupied by basolateralinvaginations organized by median size. H Representative low-magnification immunofluorescence images of N
­ a+/K+-ATPase (magenta) and GLUT1(green) immunoreactivities at 2–3 and 20–24 m.o. I Quantification of fluorescence mean intensity at 2–3, 8–12 and 20–24 m.o. A.U., arbitrary unit. J
Western blot of GLUT1, ­Na+/K+-ATPase and AQP1. K Western blot quantification. L Representative high magnification immunofluorescence imagesof ­Na+/K+-ATPase (magenta) and GLUT1 (green) at 2–3 and 20–24 m.o. B, C, F n = 30 cells per animal, 3 animals per age; black dashed line: median;
Kruskal–Wallis; I, K n = 3 animals per age; Data are mean ± s.e.m.. *p < 0.05, **p < 0.01, ****p < 0.0001. Scale bars: 1 µm (A, E), 6 µm (H), 10 µm (L)Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

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Fig. 3 (See legend on previous page.)

and aged ChP (Fig. 3H). Neither mean intensity analysis nor western blot quantification detected differencesin the protein expression levels (Fig. 3I–K). Because
it was previously reported that aquaporin 1 (AQP1) is

downregulated in aged ChP [1], we measured its protein
expression using western blot. Surprisingly, AQP1 wassignificantly upregulated in adult ChP (Fig. 3J, K). We,
then, mapped the cellular localization of GLUT1 andScarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

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Fig. 4 Increased frequency of interrupted tight junctions in aged choroid plexus epithelium. A Representative TEM images of tight junctions in 2, 8
and 20 months old (m.o.) choroid plexus epithelial cells. Yellow asterisks represent junctional contact areas. B Representative immunofluorescence
images of Claudin-1 (red) at 2–3 and 20–24 m.o. Zoomed inserts show high-magnification Claudin-1-immunopositive tight junctions. C
Quantification of the number of Claudin-1-immunopositive intersections at 2–3, 8–12 and 20–24 m.o. n = 3–6 images per animal, 3 animals per age;
One-Way ANOVA. D Quantification of junctional contact areas (%) at 2, 8 and 20 m.o. n = 30 cells per animal, 3 animals per age. Black dashed line:
median; Kruskal–Wallis. **p < 0.01, ****p < 0.0001. Scale bars: 1 µm (A), 4 µm (B)­ a+/K+-ATPase in high-magnification confocal images
Nnoticing some differences between young adult and aged
epithelial cells (Fig. 3L). GLUT1 staining was more visible in the apical compartment of aged epithelial cells and­Na+/K+-ATPase staining was also observable descending
into the lateral compartments. These observations suggest that, despite the lack of a clear change in the molecular composition, the localization of key markers could
be altered in aged epithelial cells (Fig. 3L). All together
our results support the possibility that morphological cellular remodeling may be associated to molecularreorganization.
Finally, we evaluated whether aging alters the organization of tight junctions (TJs) (Figs. 1, 4A). At all analyzed ages, the junctional complex was located close to
the apical side of the epithelial cells, most likely due tothe presence of multiple invaginations of the basolateral membrane (Fig. 1). In young adult mice, TJs werestrongly interconnected with each other, although up to
17% of the junctional profiles appeared disrupted, revealing an interrupted structure. The percentage of interrupted junctions increased considerably in epithelial
cells analyzed in older animals (adult: 43%; aged: 40%)
(Fig. 4A, Table 1). In parallel, we performed immunofluorescence against selective TJ components, such as Claudin-1 (Cla-1) and the TJ intracytoplasmic marker Zonula
occludens-1 (ZO-1) [18]. Immunolabeling for Cla-1
and ZO-1 revealed a clear difference in the distributionof these proteins in the different mouse groups. In fact,in aged mice TJs lacked linear organization, but instead
appeared irregular and pseudo-spiked (Fig. 4B). Specifically, by using Sholl analysis we quantitatively characterized the spatial organization of TJs by counting thenumber of intersections of the TJ network (see details
in Materials and Methods). We observed a significant
reduction in the total number of Cla-1-positive intersections in the adult and aged groups (Fig. 4C). Similar
results were also obtained for ZO-1 (data not shown). In
agreement with this, ultrastructural analysis confirmed
a reduction of the total junctional contact areas starting from the adult stage (Fig. 4D). Moreover, there wasa reduction of the total TJ length and an increase of thenumber of TJ dilatations (Table 1). To further investigate
whether structural alterations could be a consequence oftranscriptional changes, we tested mRNA expression levels of Tjp1 and Cldn1 in ChP homogenates of the threegroups of mice. No age-dependent modification in these
key structural components was observed (Tjp1: 2–3 m.o.,
2.2 ± 0.9; 8–12 m.o., 1.3 ± 0.1; 20–24 m.o., 3.8 ± 1.7;
Cldn1: 2–3 m.o., 0.9 ± 0; 8–12 m.o., 0.9 ± 0.1; 20–24 m.o.,
1.2 ± 0.3; One-Way ANOVA, p > 0.05). Taken together,
these data indicate the occurrence of multiple age-relatedchanges in the cellular ultrastructure of the ChP epithelium, with prominent alterations of cell size, microvilli
length and TJ organization.

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

Page 9 of 16

Fig. 5 Increased mitochondrial elongation in aged choroid plexus epithelial cells. A Representative TEM images of choroid plexus epithelial
cells at 2 and 20 months ols (m.o.). Quantification of intracellular distribution (B), density (C) and sectional surface of mitochondria (D). E Highmagnification TEM images showing representative mitochondria at 2 and 20 m.o. Quantification of mitochondria circularity score (F) and Feret’s
diameter (G). n = 30 cells per animal, 3 animals per age; black dashed line: median. Kruskal–Wallis; *p < 0.05, ***p < 0.001, ****p < 0.0001. Scale bars:
2 µm (A), 500 nm (E)Elongated and stationary mitochondria in aged choroid
plexus epithelial cells

Abundant mitochondria are located in the cytoplasm ofepithelial cells to sustain the intense metabolic demandof the ChP. Right after birth, mitochondrial distribution shifts from the basal to the apical compartmentof the cell, likely in correlation with developmental
processes of the ChP epithelium [19]. To determine
whether aging also affects mitochondrial polarization
we semi-automatically mapped all the mitochondria
located in individual epithelial cell profiles (see Materials and Methods for details) (Fig. 5A). The subcellular distribution of mitochondria did not differ between
the apical and basal compartments of epithelial cells(Fig. 5B) at the three analyzed ages, suggesting that theenergy demand remains rather constant in the apicaland basal domains of epithelial cells throughout animal
life. On the contrary, we observed that mitochondrial
density decreased significantly in adult and aged micecompared with young adult (Fig. 5C), while their surface area increased with age (Fig. 5D). Moreover, wefound remarkable changes in the shape of mitochondria

across all age groups (Table 2), whereas the structureof cristae remained intact (Fig. 5E). On average, mitochondria of young adult mice were round-shaped,with a circularity close to 1 (Fig. 5E, F) and an average sectional profile of 0.53 ± 0.01 µm (Table 2). Starting from the adult stage, mitochondria appeared moreelongated, as determined by reduced circularity (closer
to 0) (Fig. 5E, F), increased Feret’s diameter (Fig. 5G),
increased aspect ratio and an average sectional profile
of 0.61 ± 0.01 µm (Table 2). It is of interest to note thatmost of the mitochondria phenotypes appeared in adultmice and then remained stable thereafter, suggesting a
possible persistent maturation of these organelles until
adulthood. Taken together, these results demonstratethat age impacts mitochondrial morphology in ChPepithelial cells, with mitochondria becoming moreelongated and acquiring a more complex shape in older
animals.
To study how re-shaping of mitochondria could affecttheir dynamics, we analyzed mitochondrial motility using 2-PM in whole ChP explants. The use of the
2-PM allowed us to analyze ChP samples without

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

Page 10 of 16

Fig. 6 Mitochondrial movement is reduced in aged choroid plexus epithelial cells. A Schematic of MitoTracker incubation and 2-PM imaging (ACSF:
artificial cerebrospinal fluid). B Representative 2-PM images of 3–4 and 22–24 months old (m.o.) epithelial cells. Scale bar = 5 µm. C Manually traced
mitochondria in 3–4 and 22–24 m.o. cells. Inserts show a single traced mitochondrion. Quantification of total length traveled by mitochondria (D),mitochondrial displacement at 400 s (E), mitochondrial instantaneous (Inst.) speed (F) and mitochondrial average (Avg) speed at 400 s (G) in 3–4
and 22–24 m.o. n = 50 mitochondria per animal, 4 animals per age; black line: median. E, G: Mann–Whitney; ****p < 0.0001

inflicting damage to the specimen and reducing overall cellular stress. Whole ChP explants were incubatedwith MitoTracker probe, and then mitochondria wererecorded for 8 min at 1.9 Hz (Fig. 6A, B). In young adultmice, motility of mitochondria was remarkably low,
mostly exhibiting a Brownian-like movement (wiggling)(Additional file 1: Video S1) characterized by short-range
motions as apparent from the schematic illustration ofmanually traced mitochondria (Fig. 6C). Interestingly,
mitochondria were even less motile in older mice (Additional file 2: Video S2). Manual tracing also revealed adecrease of the total distance traveled by each mitochondrion over time between two consequent temporal points
in the aged ChP compared to young adult ChP (Fig. 6B,
D). Moreover, after 400 s recording, the total mitochondrial displacement from its starting point was also

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

Page 11 of 16

Table 1 Measurements of gross morphological parameters

Cell axis ratio
Cell area (µm2)

2 m.o.

8 m.o.

20 m.o.

0.88 ± 0.04

0.72 ± 0.03*

0.78 ± 0.04*

20.35 ± 0.6

16.22 ± 0.49****15.97 ± 0.49****86.5 ± 1.99

Microvilli surface (µm2)
Microvilli length (µm)
2

Invagination area (µm )78.84 ± 1.92*

1.26 ± 0.02

1.05 ± 0.02****0.95 ± 0.02****; ##0.64 ± 0.13

2.05 ± 0.42**

2.2 ± 0.28****8.28 ± 0.54

Inclusion density (inclusion/µm2)

77.6 ± 2.11**

19.66 ± 0.76****22.54 ± 0.84****

Interrupted tight junctions (%)

17

42

41

Total length tight junctions (µm)

4.6 ± 0.14

4.14 ± 0.14

3.63 ± 0.16****

Total junctional contact area (µm)

4.32 ± 0.14

Number of tight junction dilatations

0.42 ± 0.06

2.7 ± 0.1****

1.98 ± 0.12****2.09 ± 0.1****; ##2.14 ± 0.13****

Mean ± s.e.m. n = 30 cells per animal, 3 animals in each age group. *Statistical differences compared to 2 months old (m.o.), # to 8 m.o., Kruskal–Wallis test, *p < 0.05;**p < 0.01, ***p < 0.001, ****p < 0.0001

reduced in aged samples (Fig. 6B, E). Finally, mitochondria from aged ChP exhibited a slower dynamic compared to young adult samples, as revealed by a decreasein both the instantaneous speed at different time points(Fig. 6F) and in the average speed after 400 s recording(Fig. 6G). Overall, these results demonstrate that mitochondria are lifelong stable organelles in the ChP, displaying limited mitochondria motility in aged epithelium.
To understand whether the morphological and dynamic
differences were correlated to changes in the mitochondrial molecular apparatus, we first stained against
ATP5A, mitochondrial membrane ATP synthase belonging to the electron transport chain, showing no majorchanges in mitochondrial cellular distribution between
the three animal groups. However, immunofluorescent
mitochondria appeared to be larger in size in older mice,
supporting electron microscopic observations (Fig. 7A).
To investigate whether these morphological alterations
were accompanied by changes of proteins implicated in
mitochondrial oxidative phosphorylation (OXPHOS),

Table 2 Measurement
parameters

Area (µm2)
Perimeter (µm)
Density (mito/µm2)
Apical:Basal ratio
Circularity
Feret’s diameter (µm)
Aspect ratio

ofmorphometricmitochondrial

2 m.o.

8 m.o.

20 m.o.

0.2 ± 0.00

0.25 ± 0.01****0.22 ± 0.00*; #7.63 ± 0.2

6.56 ± 0.2****

6.88 ± 0.2*

0.86 ± 0.00

0.85 ± 0.02***0.83 ± 0.00****0.53 ± 0.01
0.71 ± 0.05
0.2 ± 0.01

1.64 ± 0.02

0.61 ± 0.01****0.72 ± 0.05

0.23 ± 0.01****1.71 ± 0.02****0.58 ± 0.01****0.82 ± 0.11

0.23 ± 0.01****1.79 ± 0.02**

Mean ± s.e.m. n = 30 cells per animal, 3 animals in each age group. *Statisticaldifferences compared to 2 months old (m.o.), # to 8 m.o., Kruskal–Wallis test,*p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001

we measured OXPHOS levels by western blot in ChP
homogenates (Fig. 7B). No changes were detected in therelative protein levels of the OXPHOS complex between
the three groups (Fig. 7C). To further investigate possible
transcriptomic changes, we quantified mRNA expression
level of key OXPHOS genes using RT-qPCR. No significant differences were observed for selected representative genes for complex I (CI, NDUFB8), II (SDHD), III(UQCRC2) and V (ATP5A) (Fig. 7D), suggesting that neither transcriptomic nor protein composition is affectedduring aging.
Finally, we investigated key mitochondrial functional
properties, such as membrane potential and levels of
ROS, combining the use of gold standard probes and2-PM in whole ChP explants. MitoTracker Green wasused to determine the localization of mitochondria inepithelial cells (see material and methods for details)(Fig. 8A). First, we measured the mitochondrial superoxide levels by MitoSOX application. As expected,
aged ChP showed a significant increased level of mitochondrial ROS when compared to young adult explants(Fig. 8A–C). Secondly, we measured the ability of active
mitochondria to accumulate TMRM at both ages. AgedChP showed a decreased mitochondrial membranepotential as revealed by a significantly lower TMRM
accumulation compared to young adult explants (Fig. 8A,
D, E), suggesting a lower capability of mitochondrial ATP
synthesis. The application of rotenone and oligomycin A,
selective inhibitors of complex I and ATP synthase activity, reduced the accumulation of TMRM at both ages
with a similar kinetic (Fig. 8F–H), indicating that young
adult and aged ChP mitochondria can respond to thesame acute stressful event analogously. All together, ourdata demonstrate that aged mitochondria are stable organelles that, despite showing some hallmarks of aging suchScarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

Page 12 of 16

Fig. 7 Mitochondrial biochemical composition is preserved during aging. A Representative immunofluorescence images of ATP5A (red) at 2–3 and
20–24 months old (m.o.). Scale bar: 4 µm. B Western blot of OXPHOS complex subunits from 2–3, 8–12, 20–24 m.o. choroid plexuses and positive
control (+, heart). C OXPHOS protein expression levels at all ages. A.U., arbitrary unit. n = 3 animals per age. D Quantification of relative mRNA
expression levels of OXPHOS complex subunits. n = 6 animals per age. Data are mean ± s.e.m

as high ROS levels, can effectively buffer acute stressful
events.

Discussion
In this study, we have investigated structural changes
occurring in the ChP during the healthy aging process,from young adult mice to 2-years old mice. One of the
major findings is that the majority of identified morphological changes occur already at the adult stage(8–12 months) and remain rather stable thereafter. Interestingly, previous studies on human samples have demonstrated that many of the morphological aged-relatedchanges become apparent in samples from middle-aged
individuals (45 years old) [20]. In addition, a recent
human study confirmed that ChP transcriptomic differences become apparent at 50 years and stay stable until
80 years [21]. Together, these observations indicate thatboth in humans and in mice morpho-functional changes
in the ChP occur relatively early at the adult stage,

preceding the period more commonly associated withsenescent brain [22]. In the future, it will be importantto understand why age-related changes in the ChP starts
early during lifetime and how this event impacts overall
ChP function.
As previously reported for human and rat, we showedthat mouse ChP epithelial cells exhibit a progressive cellular flattening. Atrophy is one of the most common aging
phenotypes of epithelial organs, consisting in a shrinkof epithelial cells and an increase in connective tissue[12, 20, 21, 23–27]. In the ChP, such structural changes
have been correlated with functional variations, such as
decreased enzymatic activity, both during healthy aging[28] and in degenerative disorders of the nervous system[7, 29, 30]. Dynamics studies of the CSF revealed diminished CSF production and turnover in aged brains [31,32], decreased protein concentration starting from young
adult age, and changes of the ChP secretome over time
[33, 34]. We also demonstrated a significant reduction of

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

Page 13 of 16

Fig. 8 Aged choroid plexus epithelial cells display reduced mitochondrial membrane potential and increased mitochondrial ROS production. A
Colocalization of MitoTracker Green and TMRM in choroid plexus explants (left). Schematic of MitoSOX or TMRM incubation and 2-PM imaging(ACSF: artificial cerebrospinal fluid). B MitoSOX representative 2-PM images of 3–4 and 22–24 months old (m.o.) choroid plexus epithelial cells.
C Quantification of MitoSOX fluorescence mean intensity at both ages. n = 3 animals per age. D TMRM representative 2-PM images of 3–4 and
22–24 m.o. choroid plexus epithelial cells. E Quantification of TMRM fluorescence mean intensity at both ages. F Schematic of co-incubation of
TMRM and rotenone or oligomycin A (OLIG. A). G TMRM representative 2-PM images of 3–4 and 22–24 m.o. treated choroid plexus epithelial cells.
H Quantification of TMRM fluorescence mean intensity after treatment with rotenone (left) or OLIG. A (right). n = 3–4 animals per age. Data aremean ± s.e.m. Un-paired t-test, *p < 0.05. A.U., arbitrary unit. Scale bars: 5 µm (A), 10 µm (B, D, G)

microvilli length, essential to support flux of solutes andwater from plasma to CSF [3], suggesting a decrease of
the contact interface between the CSF and the epithelium [12]. On the contrary, we found that the basolateral

membrane invaginations increased in size and covered abigger cellular section in aged ChP. These structures are
known to play a crucial role in blood filtration in the kidney [35], it is therefore suitable to assume that they may

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

play a similar role in the ChP. It was previously shownthat immunofluorescence staining of some choroidal
proteins and transporters involved in CSF production(e.g. carbonic anhydrase II, AQP1 and N­ a+-K+-ATPase)is reduced in aged rats [16]. Interestingly, our analysis
did not detect changes in the protein expression level of
either apical or basal markers of aged epithelial cells, but
it distinctly showed a possible rearrangement of their cellular distribution. In conclusion, we demonstrated that
two cellular compartments dedicated to CSF production are structurally remodeled in different directions
throughout the mouse life. In the future, it will be important to understand the relationship between CSF milieu
and ChP cellular remodeling and how this relationship ismaintained in different species.
The epithelium flattening was also accompanied by theappearance of cytoplasmic electron-dense structures,
which progressively increased in density and size starting from the adult stage. These inclusions are presumablyof lipid nature, like shown in studies on human samples[11]. Healthy human samples and Alzheimer’s disease
patients are also characterized by the presence of Biondi
ring tangles. Interestingly, the onset of these type of accumulation starts around 45 years of age in healthy samples and peaks at 70 years of age in Alzheimer’s disease
patients [11, 36]. Despite the early age onset in humansamples, we did not observe Biondi ring tangles in mice
ChP.
The blood-CSF barrier ensures a well-controlled environment of the brain, which is necessary for proper functioning of the CNS. TJs that are present between the ChP
epithelial cells control paracellular transport between
blood and CSF [37, 38]. Therefore, dysregulation of TJs
could result in CSF compositional changes. Interestingly, we found discontinuous TJ strands and paracellular dilatations in adult and aged ChP compared to theyoung adult stage. In addition, the ultrastructural analysis showed that the surface of the contact area between
adjacent cells was also reduced with aging. Interestingly,
the aged-related morphological phenotypes were notaccompanied by changes in neither protein nor mRNA
expression levels of major TJ molecular components. In
agreement, a study in sheep showed a small increase inprotein permeability for the blood-CSF barrier but not
a complete disruption of the barrier in aged animals [39,40]. In humans, starting from the age of 45 years old, anincreased variability in the CSF/serum albumin ratio has
been observed [41–43]. To our knowledge, our study isthe first one demonstrating morphological reorganization of TJs in the ChP during aging. We, here, suggestthat minor but critical TJ morphological rearrangements
can be responsible for functional barrier permeability,
as already been shown for the blood–brain barrier andPage 14 of 16

also for other organs, such as the intestine [44, 45]. In the
future it will be important to understand which changes
are responsible for triggering such events and how themorphological TJ rearrangements influence the CSF
composition.
Because of their secretory function, ChP epithelial cells
contain a substantial number of mitochondria, that are
essential to maintain the cellular high demand of energy[2]. Here we showed that ChP mitochondria in adult epithelial cells are characterized by a small but significant
decrease in total density and by an increase in size. Similar age-related changes in mitochondria structure havebeen described in other organs (muscle, liver, heart andkidney) [46]. Notably, ChP mitochondria shifted fromround-shaped to elongate-shaped in adult mice compared to young adult animals. These differences weremaintained stable in aged ChP. In vitro models, elongated mitochondria most commonly display improved
mitochondrial function [47, 48], which has been interpreted as an adaptive mechanism to sustain cell survival[48]. The structural re-organization of the mitochondria
was accompanied by a significant increase in the level ofmitochondrial superoxide, suggesting possible mitochondria oxidative damage, a hallmark of aging [49]. The significant reduction of TMRM accumulation in aged ChP
mitochondria indicates a disruption of the mitochondrial
membrane potential [50], indirectly suggesting that theability to synthetize ATP through oxidative phosphorylation may be impaired at baseline. Interestingly, when apharmacological acute stressful stimulus was introduced,
young adult and aged ChP explants buffered it in a similar manner. It is, therefore, tempting to speculate that themorphological remodeling observed in mitochondria of
the ChP may represent an adaptive mechanism engaged
by ChP epithelial cells to endure stressful events.
The implementation of intact ChP explants and 2-PM
microscopy allowed us to demonstrate, for the first time,that ChP mitochondria show slow and short-range movements, generally defined as wiggling movements [51],that significantly decrease with age. A similar trend has
already been described in other cell types [52, 53], e.g.
vascular smooth muscle cells and cardiac myoblasts, inwhich the reduced mobility has been partially correlatedwith an increase of mitochondrial size [53]. Interestingly,
both mitochondrial motility and size can be regulated bycytoplasmic ­Ca2+ and can rearrange the spatial patternof ATP production influencing the cell functionality [54].
Interestingly, ChP epithelial cells do exhibit spontaneous ­Ca2+ transients [55] and our ongoing studies indicatethat the ­Ca2+ transients can change throughout mouse
lifetime (data not shown), raising the possibility that
intracellular ­Ca2+ dynamics could influence mitochondrial shape and motility in aged ChP epithelial cells.

Scarpetta et al. Fluids and Barriers of the CNS(2023) 20:19

In conclusion, this study provides an extensive description of morphological changes of ChP epithelial cells during aging, highlighting that most of them occur as early as
8 months and stay stable thereafter. We found that aging
phenotypes of ChP epithelial cells comprise not only cellular flattening but also an enlargement of basolateralinvaginations and the emergence of interrupted TJs. In
addition, we demonstrated, for the first time, that themorphology of mitochondria changes during adulthood,
becoming stable at 8 m.o. and stationary in aged ChPepithelium. Finally, we revealed that aged ChP exhibit alower mitochondrial membrane potential compared toyounger ages, although mitochondria responses to anacute challenging stimulus did not differ. In the future it
will be important to understand how these morpho-functional phenotypes can adjust to chronic stressful eventsand other functional reorganizations.
Abbreviations
ACSF	Artificial cerebrospinal fluidATP	Adenosine triphosphate
ChP	Choroid plexus
Cla-1	Claudin-1
CNS	Central nervous system
CSF	Cerebrospinal fluid
OXPHOS	Mitochondria oxidative phosphorylation
ROS	Reactive oxygen species
TEM	Transmission electron microscopy
TJ	Tight junction
ZO-1	Zonula occludens 1
2-PM	2-Photon microscopy

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12987-​023-​00420-9.
Additional file 1: Video S1. Mitochondria motility in young adult ChP
explant.
Additional file 2: Video S2. Mitochondria motility in aged ChP explant.
Acknowledgements
We would like to thank Interdisciplinary Neurobehavioral Core (INBC) at
Heidelberg University and the German Cancer Research Center (DKFZ) animal
facility for animal housing and caring, Drs. Duncan Odom’s and Michael
Milson’s labs to share aged tissues, the Genomics and Proteomics Core Facility(DKFZ) for providing the RT-qPCR machine Roche LightCycler480, Dr. Damir
Krunic (Light Microscopy Facility, DKFZ) and Anthony Hill, PhD for developing
the semiautomatic macros used for analysis, Marleen Trapp for technical sup‑port, Dorothea Mitschang and Jola Hoffert for helping with qRT-PCR, Martin
Wolfring for help in data analysis, Dr. Anthony Hill for reviewing/editing themanuscript and the all Patrizi group for helpful discussion.
Author contributions
VS and AP conceived the study; VS, CS, MSP, AA, and AP designed the study;
VS, CS, FBT and AP performed experiments; VS and FBT analyzed data; VS and
AP wrote the manuscript. All authors read and approved the final manuscript.
Funding
This project was supported by Chica and Heinz Schaller Foundation (Hei‑delberg, Germany) (AA and AP), Department of Neuroscience of Universityof Turin (MUR project "Dipartimento di Eccellenza 2018-2022") (MSP). VS isPage 15 of 16

recipient of a doctoral fellowship awarded by the Department of Neurosci‑
ence of Turin University (MUR project "Dipartimento di eccellenza 2018–2022"). Open Access funding enabled and organized by Projekt DEAL.
Availability of data and materials
All data generated during this study are included in this published article. The
developed analysis tools are available from the corresponding author uponrequest.

Declarations
Ethics approval and consent to participate
No applicable.
Consent for publication
No applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Neurosciences Rita Levi Montalcini, University of Turin,
10126 Turin, Italy. 2 Chica and Heinz Schaller Research Group, Institutefor Anatomy and Cell Biology, 69120 Heidelberg, Germany. 3 Department
of Veterinary Sciences, University of Turin, 10095 Turin, Italy. 4 Interdisciplinary
Center for Neuroscience, Heidelberg University, 69120 Heidelberg, Germany.
5
Zentrum Für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH
Alliance, Heidelberg, Germany. 6 Schaller Research Group, German Cancer
Research Center (DKFZ), 69120 Heidelberg, Germany.
Received: 25 August 2022 Accepted: 27 February 2023

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