Cathepsin D deficiency delays central nervous system myelination by inhibiting proteolipid protein trafficking from late endosome/lysosome to plasma membrane
Experimental & Molecular Medicine
Cathepsin D deficiency delays central nervous system myelination by inhibiting proteolipid protein trafficking from late endosome/lysosome to plasma membrane
This study aimed to investigate the role of cathepsin D (CathD) in central nervous system (CNS) myelination and its possible mechanism. By using CathD knockout mice in conjunction with immunohistochemistry, immunocytochemistry and western blot assays, the myelination of the CNS and the development of oligodendrocyte lineage cells in vivo and in vitro were observed. Endocytosis assays, real-time-lapse experiments and total internal reflection fluorescence microscopy were used to demonstrate the location and movement of proteolipid protein in oligodendrocyte lineage cells. In addition, the relevant molecular mechanism was explored by immunoprecipitation. The increase in Fluoromyelin Green staining and proteolipid protein expression was not significant in the corpus callosum of CathD− / − mice at the age of P11, P14 and P24. Proteolipid protein expression was weak at each time point and was mostly accumulated around the nucleus. The number of oligodendrocyte lineage cells (olig2+) and mature oligodendrocytes (CC1+) significantly decreased between P14 and P24. In the oligodendrocyte precursor cell culture of CathD − / − mice, the morphology of myelin basic protein-positive mature oligodendrocytes was simple while oligodendrocyte precursor cells showed delayed differentiation into mature oligodendrocytes. Moreover, more proteolipid protein gathered in late endosomes/lysosomes (LEs/Ls) and fewer reached the plasma membrane. Immunohistochemistry and immunoelectron microscopy analysis showed that CathD, proteolipid protein and VAMP7 could bind with each other, whereas VAMP7 and proteolipid protein colocalized with CathD in late endosome/lysosome. The findings of this paper suggest that CathD may have an important role in the myelination of CNS, presumably by altering the trafficking of proteolipid protein. Experimental & Molecular Medicine (2018) 50, e457; doi:10.1038/emm.2017.291; published online 16 March 2018
CathD (Cathepsin D) is a lysosomal, aspartic endoproteinase
that requires an acidic pH to be proteolytically active. Although
the level of its expression in different cells varies considerably,
CathD is expressed in all tissues1 and has been involved in many
fundamental functions of cells, such as the degradation of
intracellular proteins in the lysosomal compartment, apoptosis,
inflammation and tumor progression.2–4 Mutations in the
CathD gene cause fatal neurological diseases, which are
characterized by an extensive loss of neurons and myelin,
pronounced gliosis and the accumulation of lipofuscin within
the remaining cells in human infants, as well as some lysosomal
storage disorders.5,6 CathD − / − mice, generated by gene
targeting, also develop neurological diseases resembling those in
human, characterized by signs including the accumulation of
storage materials in neurons as well as neurodegeneration,
which is particularly significant within the thalamus, the
hippocampus and the deep laminae of the cerebral cortex.7,8
Several reports also revealed the disruption of myelin sheaths
within the corpus callosum and the thalamus in CathD − / −
mice.9,10 Recent in vivo neurobiochemistry studies indicated
that myelin-related proteolipid protein (PLP) and myelin basic
protein (MBP) were both markedly reduced at P24, and myelin
sheaths became significantly atrophic in CathD − / − mice.11
Furthermore, there was a pronounced accumulation of
cholesteryl esters and abnormal levels of proteins related to cholesterol
transport.11 However, the mechanism underlying the defects in
myelin sheaths of CathD − / − mice remains unknown.
As the most abundant protein present in the central nervous
system (CNS) myelin of higher vertebrates, PLP (~80% of the
total myelin proteins) has a low molecular weight and is a
highly hydrophobic proteolipid protein containing four
transmembrane domains that interact with the cholesterol and
galactosylceramide-enriched membranes during its biosynthetic
transport in oligodendrocytes.12–14 Different from other CNS
myelin proteins such as MBP, myelin-associated glycoprotein
(MAG) and CNP, PLP is synthesized in membrane-bound
polysomes, followed by the incorporation in the endoplasmic
reticulum (ER) membrane, further processing through the
Golgi apparatus, and vesicular transport to the myelin
membrane.15 When initially expressed in cultured
oligodendrocytes, PLP resides in a compartment showing the
characteristics of late endosome/lysosome (LEs/L).16 Co-culture
with neurons leads to an increased level of PLP on the plasma
membrane and its detachment from the LEs/L, followed by the
maturation of oligodendrocytes. Such observation has
supported the idea that the amount of PLP in LEs/Ls and on the
plasma membrane of oligodendrocytes is regulated by neural
signal molecules.6 Recently, Anke Feldmann et al.17
demonstrated that PLP is transported to the cell surface from LEs/Ls
via distinct trafficking pathways. Hence, we postulate that
CathD deficiency may affect the metabolism or the transport
of PLP in oligodendrocyte lineage cells (OLs).
In this study, the development of OLs and their intracellular
PLP transportation were investigated using CathD knockout
mice. It was found that myelin maturation and OL
development were delayed by inhibiting the movement of PLP from
LEs/Ls to the plasma membrane of the OLs. The findings
shown in this paper may point to a new potential mechanism
underlying the myelin defects caused by CathD deficiency.
MATERIALS AND METHODS
Heterozygous mice (C57/BL CathD+/ − ) carrying a mutation in the
CathD protein, obtained from Christoph Peters (Institute of Molecular
Medicine and Cell Research, University of Freiburg, Freiburg,
Germany), were used to generate homozygous animals for the
experiment. The use and care of animals followed the guidelines of the
Zhejiang University Animal Research Advisory Committee. Using the
polymerase chain reaction and oligonucleotide primers as described
previously,8 the genotype of individual animals was determined from
the genomic DNA isolated from a small sample of the tail.
For immunoelectron microscopy, mice were deeply anesthetized and
perfused with 1% paraformaldehyde, 0.2% picric acid and 0.05%
glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4). Ultrathin sections
of 80 nm thickness processed from Lowicryl-embedded blocks of the
brain were placed on coated nickel grids and incubated for 45 min in a
blocking solution consisting of 10% human serum albumin in 0.05 M
Tris-buffered saline and 0.03% Triton X-100 (TBST). The grids were
incubated with PLP antibodies (1:1000#x0FF1B;polyclone; Abcam,
Shanghai, China) and CathD antibodies (1:200; N-19; polyclone;
SantaCruz, Shanghai, China) in TBST with 2% human serum albumin
(HSA) at 28 °C overnight. After rinsing, the grids were incubated for
3 h with donkey anti-rabbit IgG antibodies conjugated to colloidal
gold particles (5 nm, Sigma-Aldrich Corp., St Louis, MO, USA) and
donkey anti-goat IgG antibodies conjugated to colloidal gold particles
(15 nm, Sigma) diluted 1:30 in 2% HSA and 0.5% polyethylene glycol
in TBST. The grids were then soaked in Tris-buffered saline for
30 min and counterstained for electron microscopy, using saturated
aqueous uranyl acetate followed by lead citrate.
Western blot analysis
CathD+/+ and CathD − / − mice at the age of P11, P14 and P24 were
deeply anesthetized using 10% chloral hydrate and subsequently
perfused transcardially with ice-cold phosphate-buffered saline
(PBS). The brain was carefully isolated and subsequently homogenized
in a radioimmunoprecipitation assay buffer (Beyotime, Shanghai,
China), using a hand-held homogenizer. Protein concentration was
determined using a Bio-Rad protein assay. Thirty milligrams of each
sample were separated on a 10 or 12% Tris HCl gel (Bio-Rad
Laboratories, Hercules, CA, USA) and transferred to a polyvinylidene
fluoride (PVDF) membrane. Membranes were blocked in 0.1% Tween
20 TBS containing 5% nonfat dry milk for 1 h before incubation with
primary antibodies. Primary antibodies were diluted in the same
solution as follows: anti-PLP (1:1000; Abcam), anti-CNP (1:500;
Sigma), anti-Olig2 (1:500; Millipore, Burlington, MA, USA),
anti-βactin (1:15 000; Sigma), anti-CathD (1:800, N-19; SantaCruz) and
anti-VAMP11 (1:500; Abcam). Samples were incubated with
antimouse, anti-rabbit or anti-goat secondary antibodies conjugated to
horseradish peroxidase for 1 h before the proteins were detected using
an Immun-StarTM Western CTM detection system (Bio-Rad
Laboratories), the blots were exposed using ChemiDocTM XRS+ with Image
labTM (Bio-Rad Laboratories) and band intensities were measured
using Image-lab software (Bio-Rad Laboratories). Protein expression
was normalized to β-actin.
Immunohistochemistry and immunocytochemistry
For immunohistochemistry, the brain was isolated and fixed with
icecold 4% paraformaldehyde in PBS overnight at 4 °C and subsequently
cryoprotected using 30% sucrose in PBS. The brain was embedded in
optimal cutting temperature compound (OCT) (Tissue-Tek, Sakura,
Torrance, CA, USA) and sliced into 10 or 20 μm sections using a
freezing microtome. Tissue sections underwent immunostaining, first
by blocking and permeabilizing in 10% bovine serum albumin/0.3%
Triton X-100, followed by overnight incubation at
4 °C with corresponding primary antibodies. For
immunocytochemistry, cultured cells were fixed by 4% paraformaldehyde in PBS at 4 °C
for 10 min and were permeabilized with 0.2% Triton X-100 in PBS for
10 min, or with methanol for 10 min at − 20 °C. Dilutions for primary
antibodies were as follows: rabbit anti-PLP (1:1000; Abcam), rabbit
anti-NG2 (1:200; Chemicon, Rolling Meadows, IL, USA), mouse
antiAPC/CC1 (1:500; Abcam), rat anti-MBP (1:200; Chemicon), rabbit
anti-cleaved caspase-3 (1:200; Cell Signaling Technology, Danvers,
MA, USA), rabbit anti-Olig2 (1:200; Millipore), mouse anti-LAMP1
(1:200; Assay designs, Hines Drive, Ann Arbor, MI, USA), anti-NG2
(1:300; Millipore), rat anti-MBP (1:200; Chemicon), rabbit anti-PLP
(1:1000; Abcam), mouse anti-O10 (1:50; R&D, Minneapolis, MN,
USA), goat anti-CathD − / − (1:200; SantaCruz), mouse anti-LAMP1
(1:200; Assay designs) and mouse anti-VAMP11 (1:200; Abcam).
Samples were incubated with corresponding primary antibodies
overnight at 4 °C, followed by rinsing in PBS and staining with Cy3
(1:1000; Invitrogen, Carlsbad, CA, USA) or Alexa Flour
488conjugated secondary antibodies (1:1000; Invitrogen) for 1 h at room
temperature. After antibody incubation, some brain sections were
stained with FluoroMyelin Green Fluorescent Myelin Stain (1:300;
Invitrogen) for 20 min at room temperature. For samples with a
determined number of total cells, their nuclei were counterstained for
5 min with 100 nM 4, 6-diamidino-2-phenylindole dihydrochloride
Isolation and culture of mouse oligodendrocyte precursor cells
The following procedures were modified based on the Chen et al.18 In a
laminar flow hood, the embryos of CathD+/+ and CathD − / − mice were
removed on the embryonic day (E)14.5–17.5 and placed in clean Petri
dishes containing ice-cold HBSS. The embryos were decapitated and
their skins on the telencephalic bulb and the skull were removed gently
from the head by holding the neck region with a pair of forceps. The
telencephalic bulb was detached using a pair of 451 angled Dumont
forceps. The medial portion was removed while the lateral part of the
cerebral cortex was retained. The cortex was placed in a clean Petri dish
containing ice-cold HBSS and the meninges were removed using the
pair of forceps. Each cortex was cut into two to three pieces, which
were transferred into an ice-cold neurosphere growth medium (0.5 ml
per brain) containing 20 ng ml-1 epidermal growth factor (EGF) and
20 ng ml− 1 basic fibroblast growth factor (bFGF). The cortices were
dissociated by mechanical trituration using a fire-polished glass Pasteur
pipette (~35 strokes) until the cell suspension had no or very few small
clumps. The suspension was kept on ice for 2 min and subsequently
passed through a 50-mm nylon pouch placed on a 15-ml conical tube
to obtain a single cell suspension. The cells were counted with a
hemocytometer and added into a six-well plate (4 ml of neurosphere
growth medium per well) at a concentration of 5 × 104 cells per ml.
The plate was incubated in an incubator at 37 °C and 5% CO2. The
cells were fed once every 2 days by replacing half of the medium with a
fresh neurosphere growth medium. Near the fourth day after the
formation of neurospheres, the EGF/bFGF-containing neurosphere
growth medium was gradually changed to a B104
CM-containing oligosphere medium by replacing one-fourth of the
former medium with the latter medium every other day in a period of
2 weeks. The oligospheres should be either mechanically dissociated
using a fire-polished pipette as described above, followed by passage
through a 50-mm nylon mesh to obtain single cells, or by using an
enzymatic treatment of trypsin. The single cells were resuspended in
4 ml oligosphere medium, spun down at 120 g (1000 r.p.m.) for 5 min
at RT and passed through a 50-mm nylon mesh to obtain a cell
suspension. The cell suspension was cultured in the oligosphere
medium on an uncoated plate at a density of ~ 3 × 104 cells per ml.
Oligospheres would be formed again after 5–7 days. Alternatively, the
cell suspension could be plated on a poly-ornithine-coated plate in an
oligodendrocyte precursor cell (OPC) medium to achieve OPC
proliferation, or be cultured in an oligodendrocyte differentiation
medium to achieve differentiation.
Endocytosis was assessed by antibody internalization as described
previously.17 OLs from CathD+/+ and CathD − / − mice were incubated
on ice for 45 min with mouse anti-O10 monoclonal antibodies bond
to PLP extracellular epitopes in DMEM containing 10% horse serum,
followed by incubation with goat anti-mouse Cy3 antibodies (Red) for
30 min on ice. The cells were incubated at 37 °C to allow endocytosis
and were subsequently incubated with anti-goat Alexa Flour 488
(Green) to stain the proteins on the cell surface. Finally, the cells were
fixed in PBS containing 4% paraformaldehyde. In the assay, the signal
of endocytosed epitopes would appear solely in the red channel,
whereas the signal of surface-localizing epitopes would appear in both
red and green channels. The percentage of endocytosing cells was
determined from three independent experiments (100 cells were
counted in each experiment).
Fluorescence images were acquired on a Confocal microscope
(Fluoview 1000; Olympus; or Fluoview 500; Olympus, Tokyo, Japan)
equipped with a ULPLANAPO × 60/1.2 w immersion objective
(Olympus). The images were taken with a resolution of 1024 × 1024.
The gain of the photomultiplier was adjusted to maximize the signal/
noise ratio without causing image saturation.
For live cell imaging, coverslips containing the cells were mounted
in a live cell imaging chamber and observed in ECS (NaCl 145 mM,
KCl 3 mM, CaCl2 3 mM, MgCl2 6H2O 2 mM, HEPES 10 mM, Glucose
8 mM, pH 7.4) at room temperature. Time-lapse imaging was
performed on a confocal laser scanning microscope (IX71; Olympus).
Images were acquired at free intervals for the indicated time, using
sequential line excitations at 488 and 543 nm and appropriate band
pass emission filters. Image processing and analysis were performed
using Image-Pro Plus5.1 (Media Cybernetics, Shanghai, China).
Quantification of colocalization was performed using the
colocalization module of the software. Vesicle movement was analyzed by
subtracting from each image the information in its preceding image of
a time stack. The different image stack generated from the above
operation was used to identify vesicle motility events. TIRFM (total
internal reflection fluorescence microscopy) was performed on an
Olympus IX81 inverted microscope equipped with a × 60 1.45 NA oil
immersion objective (PlanApoN, Olympus). Evanescent field
excitation was obtained by focusing 488- and 568-nm laser lights that
resulted in a field depth of ~ 100 nm. Images were acquired with a
cooled CCD camera (iXon, ANDOR Technology, Beijing, China)
controlled by Andor iQ software (Version2.0, Media Cybernetics,
Shanghai, China). Each pixel corresponded to 133 nm in the
Biotinylation of cell surface proteins was carried out using a modified
approach of Duan et al.19 After 1 h of preincubation and 6 min of
HBSS wash, the cells were rinsed twice with HBSS/Ca2+–Mg2+
containing 138 NaCl, 2.7 KCl, 1.5 KH2PO4, 9.6 Na2HPO4, 1 MgCl2
and 0.1 CaCl2 (mM, pH 7.3). The cells were then incubated in
sulfoNHS-biotin solution (1 mg ml − 1 in HPBS/Ca2+–Mg2+) for 20 min at
4 °C. Biotinylation was terminated by washing twice in a quenching
solution of HBSS/Ca2+–Mg2+, in which the NaCl content was replaced
by an equimolar concentration of 100 mM glycine (to maintain the
300 mOsm). This was followed by an additional 45 min incubation in
the quenching solution at 4 °C. Subsequently, the quenching solution
was removed and the cells were lysed with 100 ml per well of a
radioimmunoprecipitation assay buffer containing protease inhibitors
(100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM iodoacetamide,
1 mg ml − 1 leupeptin, 5 mg ml − 1 aprotinin and 250 mM
phenylmethylsulfonyl fluoride). The lysis step was continued for 1 h at
4 °C with vigorous shaking. The lysates were centrifuged at 14 000 g
for 15 min at 4 °C. An aliquot of 300 ml of the supernatant was used
as the whole-cell fraction for western blot analysis. The remaining
supernatant (600 ml) was incubated with 300 ml of avidin bead
suspension for 1 h at room temperature with gentle shaking. The
avidin–lysate solution was then centrifuged for 15 min at 14 000 g, and
the supernatant was collected as the intracellular fraction for western
blot analysis. The pellet was washed four times with 1 ml
radioimmunoprecipitation assay buffer and resuspended in 300 ml
Laemmli buffer (62.4 mM Tris-HCl, pH 7, 2% SDS, 20% glycerol
and 5% 2-mercaptethanol) for 1 h with gentle shaking at room
temperature. After centrifugation for 15 min at 14 000 g, the
supernatant was collected as the biotinylated (plasma membrane) fraction
for western blot analysis.
Briefly, the tissue samples were harvested as described above and
homogenated in 1 ml of lysis buffer (1% Triton X-100, 50 mM Tris,
pH 7.4, 150 mM NaCl, 1 μg ml − 1 aprotinin, 5 μg ml − 1 leupeptin,
1 μg ml − 1 pepstatin, 1 mM phenylmethanesulfonyl fluoride (PMSF)
and 1 mM N-ethylmaleimide). After the nuclei were removed (5 min,
3200 r.p.m.) and nonspecific binding to goat IgG was eliminated, the
lysates were incubated with polyclonal goat anti-CathD and goat
antiPLP antibodies overnight at 4 °C, followed by protein A/G-Sepharose
for 1 h at 4 °C to precipitate the IgG. The pelleted beads were washed
five times in 50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton
X-100, 1% deoxycholate, 0.1% SDS, 1 mM 1,4-dithiothreitol, 1 mM
Nethylmaleimide, 1 μg ml − 1 aprotinin, 5 μg ml − 1 leupeptin, 1 μg ml − 1
pepstatin and 1 mM PMSF. Finally, the beads were resuspended in a
SDS-PAGE sample buffer and heated for 5 min to 95 °C, and the
supernatant was analyzed by SDS-PAGE and western blot. The
polyclonal CathD antibodies were used to verify CathD precipitation,
whereas polyclonal PLP antibodies were used to verify PLP
precipitation and to detect bond CathD or VAMP7.
Statistical analysis was performed using GraphPad Prism 6.00 software
(La Jolla, CA, USA). Data were expressed as mean ± s.e.m. All groups
were compared using two-tailed unpaired Student’s t-test unless
otherwise specified. P＜0.05 indicated significant difference.
CNS myelination is disrupted in CathD − / − mice
The myelin formation in the corpus callosum of CathD+/+ and
CathD − / − mice was assessed on P11, P14 and P24 using the
lipophilic dye Fluoromyelin Green and the immunostaining
against PLP (red; Figure 1a). The positive signals for
Fluoromyelin Green and PLP began to appear on P11 in the corpus
callosum of both CathD − / − and CathD+/+ mice. However,
such signals were much weaker in the CathD − / − mice than
those in the CathD+/+ mice, suggesting a delay in the onset of
myelination. On P14 and P24, a gradual increase in the density
of Fluoromyelin Green staining and PLP immunofluorescence
was observed in the corpus callosum of CathD+/+ mice.
However, the density of Fluoromyelin Green staining and
PLP immunofluorescence in the corpus callosum of CathD − / −
mice was lower at each progressing time point and the increase
in the density of Fluoromyelin Green staining and PLP
immunofluorescence in the corpus callosum of the
CathD − / − mice was much lower than that in the CathD+/+
mice. Interestingly, it was found that lots of PLP were located
around the cell nuclei, by forming large punctas in the corpus
callosum from P14 to P24 in CathD − / − mice, whereas finely
distributed punctas along axons could be seen as lines in CathD
+/+ mice at the same time point. To assess PLP expression
quantitatively during the corpus callosum development,
western blot analysis was performed on brain samples at P11,
P14 and P24 (Figure 1b). Consistent with the corpus callosum
staining, age-related increases in terms of PLP expression were
observed in CathD − / − and the control mice. At P11, PLP
expression was not readily detectable with western blot analysis.
At P14 and P24, PLP expression in CathD+/+ mice was twice as
high as that in the CathD − / − mice (Figure 1b and c).
Furthermore, the levels of CNP and Olig2, markers of OL
specification and CNS myelination in the corpus callosum,
were also examined. Compared with the CathD+/+ mice, the
expression level of CNP and Olig2 in P14 and P24 but not P11
in brain samples of the CathD − / − mice was lower (Figure 1b, d
and e). These results indicated that CNS myelination was
disrupted in the brain of CathD − / − mice.
OL maturation is inhibited in the CathD − / − mice
The dysmyelination in a CathD − / − mouse could be caused by
the defect of proliferation, migration or maturation in OLs.
The expression of Olig2 and CC1, a marker for post-mitotic
oligodendrocytes,20 was examined in the corpus callosum of
CathD+/+ and CathD − / − mice at P11, P14 and P24 (Figure 2a
and b). It was found that the density of Olig2﹢ cells was similar
in the CathD − / − and CathD+/+ mice at P11, but was reduced
at P14 and P24. At P24, the decrease of Olig2﹢cell density was
greater in the corpus callosum of the CathD − / − mice than that
in the CathD+/+ mice. On the other hand, as compared with
the CC1﹢cell density that continuously increased from P11 to
P24 in the corpus callosum of CathD+/+ mice, the CC1﹢cell
density in the CathD − / − mice was not significantly different
from that of the controls at P11 and only slightly
increased from P14 to P24. It is worth noting that there was
no difference in terms of Olig2﹢/CC1 − cells between CathD+/+
and CathD − / − mice at P11, P14 and P24, thus further
demonstrating that the reduction of Olig2﹢cells in the
CathD − / − mice was the reason for their different CC1﹢cell
counts. Therefore, it is postulated that CathD deficiency leads
to a selective loss of mature oligodendrocytes.
To identify apoptotic cells, the corpus callosum was
immunostained with an antibody against activated caspase-3
(Figure 3a). The numbers of activated caspase-3+ cells in the
corpus callosum of CathD − / − mice and controls were not
statistically different from P11 to P24 (Figure 3b), suggesting
that the rates of apoptotic cell death were similar at each time
point and apoptosis was not the main cause for the decreased
mature oligodendrocytes. The development of OLs was further
examined in vitro by culturing primary pure OPCs collected
from the CathD+/+ and CathD − / − mice. After culturing for 3,
5, 7 and 14 days in a differentiation medium, the mature
oligodendrocytes were immunostained with the antibody
against MBP, a marker for mature oligdendrocytes
(Figure 4a). The percentage of total MBP+ mature
oligodendrocytes in the CathD − / − mice continuously increased from
day 3 to day 14, but the increase was lower than that in the
OPCs of CathD+/+ mice. On day 14, the percentage of total
MBP+ mature oligodendrocytes was similar in both groups
(Figure 4d). Furthermore, compared with the CathD+/+ mice,
the MBP+ mature oligodendrocytes of CathD − / − mice
displayed simpler morphologies with fewer membranes at each
time point (Figure 4b, c and e), suggesting that CathD
deficiency inhibited oligodendrocyte maturation.
More PLP accumulates in LEs/Ls of OLs in CathD − / − mice
PLP is initially targeted to LEs/Ls by using a
cholesteroldependent and clathrin-independent endocytosis pathway, and
then transported from LEs/Ls to the plasma membrane and
myelin membrane upon activation by neuronal cells.21 To
analyze whether CathD deficiency had an impact on the
subcellular localization of PLP, the intracellular localization of
PLP and LAMP1, a lysosome marker, was observed in vitro
(Figure 5a). Using confocal microscopy in conjunction with
z-stack, it was discovered that the OPCs of CathD+/+ mice
started to express PLP during the first 2 day of culture in the
proliferation medium, and the colocalization of PLP and
LAMP1 was observed in ~ 90% of the cell bodies. However,
little colocalization of PLP and LAMP1 in the processes of OLs
was observed after 24 h in the differentiation medium. On the
other hand, in the OPCs of CathD − / − mice, PLP accumulated
in the LEs/Ls of both the cell bodies and processes of OLs,
where the colocalization of PLP and LAMP1 was nearly
~ 100%. Meanwhile, it was found that a fewer number of
processes were present in the OLs of CathD − / − mice than
those of the CathD+/+ mice after 24 h of culturing in the
differentiation medium, suggesting that the deficiency of CathD
not only disturbed PLP distribution, but also inhibited
Similar results were obtained in vivo. Significant
colocalization of PLP and LAMP1 was observed in the corpus
callosal cells of CathD+/+ mice at P11 and such colocalization
was increased by twofold in the P14 mice but decreased
at a level close to 100% was noted for the PLP and LAMP1
in the corpus callosum cells of CathD − / − mice, which
also formed larger punctas than those in the controls
Less PLP distribution on the plasma membrane of OLs in
CathD − / − mice
Next, experiments were carried out to examine whether the
developmental regulation of PLP distribution between LEs/Ls
and the plasma membrane of OLs was disturbed by the
deficiency of CathD. Surface staining showed that, in the
differentiation medium, fewer PLP was located at the plasma
membrane of cells in the CathD − / − mice than those in the
CathD+/+ mice (Figure 6a); quantitative analysis showed that
only 5% of O10 mAb colocalized with PLP in the OLs of
CathD − / − mice as compared with 49.7% in the CathD+/+ mice
(Figure 6b). Biotinylation labeling of cell surface proteins
showed that, compared with cells of the CathD − / − mice,
the cells of CathD+/+ mice were associated with a decreased
ratio of biotinylated (membrane-bound) to nonbiotinylated
(cytoplasmic) PLP (Figure 6c). There were no significant
alternations between the CathD+/+ and CathD − / − mice in
terms of total cell PLP expression. Densitometry measurements
of the PLP bands showed that the ratio of membrane bound to
cytoplasmic PLP was 1.69 ± 0.04 in the CathD+/+ mice, and
this ratio decreased to 0.18 ± 0.04 in the CathD − / − mice
Subsequently, TIRFM was used to analyze the behavior of in the immune complexes. In the brain of a P20 mouse, a
the vesicle pool containing peripheral PLP. It was observed that 30-kDa PLP-bound complex was associated with CathD
many PLP-EGFP containing LAMP1-positive vesicles localized and was found in the immunoprecipitate, whereas a 26-kDa
within the 100-nm vicinity of the plasma membrane in both DM20-bound complex was not associated with CathD and
the cell bodies and processes of living OLs of the CathD+/+ was not found in the immunoprecipitate. No PLP was
mice. In contrast, few or no PLP-EGFP that contained LAMP1- detected in a control immunoprecipitation experiment
positive vesicles was seen in the proximity of plasma membrane that used an irrelevant IgG (Figure 8d). In the reciprocal
of the living OLs from the CathD − / − mice (Figure 6e). These co-immunoprecipitation experiments using a goat anti-PLP
results suggested that fewer PLP appeared on the plasma antibody, interestingly, only the intermediate 48-kDa and the
membrane of OLs in the CathD − / − mice. mature light-chain 14 kDa of CathD were found to obviously
co-precipitate with the PLP in the immune complex, whereas a
52-kDa pro-CathD and 34-kDa mature heavy chains did not
co-precipitate with the PLP in the immune complex
(Figure 8e). Confocal imaging experiments revealed that PLP/
VAMP7 and CathD/VAMP7 colocalized in a high degree
(Figure 8f). In addition, in a co-immunoprecipitation
experiment using goat anti-PLP antibody, VAMP7 was found to
significantly co-precipitate with the PLP in the immune
complex (Figure 8g).
The transport of PLP from LEs/Ls to the plasma membrane
of OLs slowed down in the CathD − / − mice
Two possibilities may lead to the abnormal distribution of
PLP between the LEs/Ls and the plasma membrane of
OLs in the CathD − / − mice. One possibility is that fewer PLPs
were transported out of LEs/Ls to the plasma membrane
of OLs, whereas the other one is that more PLPs were
endocytosed and taken up in LEs/Ls. To analyze the putative
exocytic trafficking of PLP from LEs/Ls, live cell imaging
experiments were performed using LysoT in the
PLP-EGFPexpressing OLs from the CathD+/+ and CathD − / − mice.
A high degree of colocalization between PLP-EGFP and LysoT
was present in the perinuclear and peripheral pools of OLs
collected from both types of mice, similar to the
immunocytochemistry results related to PLP and LAMP1. The analysis
regarding the dynamics of PLP-EGFP/LysoT-positive vesicles
revealed that, in the OLs from CathD − / − mice, most
PLPcontaining LE/L vesicles moved more slowly toward the plasma
membrane at the distal end of the processes than those in the
CathD+/+ mice (Figure 7a). A quantitative analysis revealed
that, as compared with OLs from the CathD+/+ mice, fewer
PLP-EGFP/LysoT-positive vesicles were mobile in the OLs
from the CathD − / − mice (26.68 ± 1.98 versus 6.27 ± 0.35%;
To test the latter hypothesis described above, endocytosis
experiments were performed using the O10 mAb. There was
no significant difference in terms of the uptake of O10 in OLs
from the CathD − / − and the CathD+/+ mice (Figure 7c and d).
Thus, neither clatherin-dependent nor clatherin-independent
endocytosis of PLP seemed to be the underlying mechanism for
the accumulation of PLP in the LEs/Ls of OLs from the
CathD − / − mice.
VAMP7 may act as the mediator between CathD and PLP
Confocal imaging revealed that CathD was colocalized with
PLP in the perinuclear regions and processes of OLs
(Figure 8a). Immunoelectron microscopy showed that gold
particles marked CathD (larger, 15 nm particles) and PLP
(smaller, 5 nm particles) colocalized in large vesicles inside
LEs/Ls (Figure 8b). In vitro pull-down assay showed that
PLP-myc was recovered from immunoprecipitation of
CathD (Figure 8c). Subsequently, co-immunoprecipitation
experiments were carried out to determine whether
endogenous CathD also bond native PLP. Under non-denaturing
conditions, antibodies against CathD precipitated PLP isoforms
CathD and myelination
In peripheral nervous system, an important event before
myelination is the precise arrangement of Schwann cells along
the nerves. Initially, immature Schwann cells encircle a group
of nerves. Subsequently, individual cells migrate away from the
cohort and start to find the axons to the periphery of the nerve
bundle, where a 1:1 relationship with the nerve bundle was
adopted.21 Unlike myelination of peripheral nervous system by
Schwann cells, myelination of CNS is a more complex process
and begins with the specification of proliferating and migratory
OPCs, followed by the differentiation of these cells into
postmitotic oligodendrocytes that myelinate multiple axons. These
processes must be carried out for a large number of different
axons.22 Hence, a defect in any one of these steps could cause a
severe dysmyelination of CNS.
In human, CathD is a protein encoded by the CTSD gene.
As a member of the peptidase A1 family, CathD is an aspartic
endoprotease and is ubiquitously distributed in lysosomes.
CathD accounts for 11% of all proteolytic enzymes in
lysosomes. During its transportation to lysosomes, the
52-kDa pro-CathD is proteolytically processed to form a
48kDa single-chain intermediate that is an active enzyme located
in the endosomes. Further proteolytic processing yields a
mature and active lysosomal protease, which is composed of
both heavy (34 kDa) and light (14 kDa) chains.23 The main
functions of CathD include digestion of proteins and peptides
in acidic lysosomes, and participation in biological processes
such as cellular protein renewal and tissue homeostasis.24
CathD deficiencies are fatal neurological diseases that are
characterized by a significant loss of neurons and myelin in
human infants and sheep.25 CathD knockout mice also show
pronounced myelin changes in the brain, and myelin-related
PLP and MBP are both markedly reduced on postnatal day 24,
and the amount of lipids characteristically high in myelin are
significantly lowered as compared with the controls.11
However, few studies have investigated the myelination and the
mechanisms underlying the defects of myelin sheaths in
CathD − / − mice.
Our data demonstrated that the formation of myelin in the
CathD − / − mice was delayed as compared with the CathD+/ − +
mice. Although the myelin content (as was indicated by PLP
expression levels and Fluoromyelin Green staining) in the
corpus callosum of the CathD − / − mice increased from P11 to
P24, the extent of myelination in the CathD − / − mice was
lower than that in the CathD+/+ mice. However, CathD − / −
mice showed normal development during the first 2 weeks but
stopped in the third week. Eventually, they died on day 25 ± 1
in a state of anorexia due to widespread intestinal necrosis,
thromboemboli, fulminant loss of T and B cells, and massive
destruction of the thymus and spleen. Therefore, it is difficult
to investigate the changes of myelination causing by CathD
deficiency. Conditional CathD knockout mice may be useful to
achieve further elucidation.
CathD and OL development
In the CNS, OLs develop from OPCs. OPCs can differentiate
into oligodendrocytes, type II astrocytes and neurons in the
brain, and are now considered as adult stem cells.26 Many
known factors can regulate the differentiation of OPCs,
including SOX10 and Hes5.27,28 There are also many factors
regulating the maturation of OLs, including LINGO-1 and
PSA-NCAM.29,30 However, no report has shown that lysosomal
hydrolysis is related to OL development.
Several studies have shown that the activity and localization
of CathD can be changed because of aging or different
pathophysiological conditions. In patients with Alzheimer's
disease, the activity of CathD increases markedly and can
degrade beta amyloid, a soluble human brain extract under pH
4–6.31 At the time of cell death, mature CathD is present in the
nucleus and is involved in regulating the proteolytic activation
of endonuclease.32 In tumors, part of CathD fragments (amino
acids 27–44) can interact with an unknown receptor on the cell
surface, to promote the mitosis of cancer cells and stimulate
the growth of cancer cells.33–38 These studies demonstrate that
CathD has an important role in controlling cell transformation
The in vivo data of this study showed that CathD deficiency
leads to a selective loss of mature oligodendrocytes not related
to apoptosis. However, as CathD has both anti-apoptotic and
pro-apoptotic functions under different situations, the function
of this protease in apoptosis is still paradoxical up to now.30,39
In order to find a way to study OL development in CathD − / −
mice, primary OPC cultures were used in vitro to evacuate
other factors. It was found that CathD deficiency did not
inhibit oligodendrocyte maturation by promoting cell death.
Both CathD − / − mice and CathD+/+ mice exhibited the same
number of mature oligodendrocytes in vitro, suggesting that
CathD might have a regulating function in OL development.
CathD and PLP transportation
The biogenesis of the specialized myelin membrane requires
intricate machinery: the glial cells are associated with the axons
at the appropriate developmental time, whereas relevant
mechanisms must initiate to ensure the vectorial delivery
of newly synthesized myelin-membrane components to the
axons. That is, a specific set of lipids and proteins have to be
correctly assembled on the myelin membrane in time.40
In most cells, the majority molecules localizing to the internal
vesicles of the endosomal system are destined for lysosomal
degradation by lysosomal proteases. However, in OLs,
LE/L compartments may be particularly useful as storage
compartments, as they are able to harbor a large amount of
membrane materials for myelin biogenesis in a multilamellar
and multivesicularmanner.41 As a structural protein, PLP has a
major role in the correct apposition of the extracellular leaflets
of the membrane, thereby stabilizing the multilayered myelin
membrane structure upon compaction.42,43 Other functions of
PLP have been suggested as well, including a role in OL survival
and adhesion relevant to migration.44 It is initially targeted to
LEs/Ls and then redistributed from LEs/Ls to the plasma
membrane upon receiving mature signals from neurons.45
Therefore, LEs/Ls may be required for the trafficking of PLP
or other myelin contents from the membrane of distal
processes to myelinate axons. LEs/Ls may also be required
for OL maturation.
Our in vitro and in vivo experiments showed that more PLP
accumulated in LEs/Ls but fewer PLP was distributed on the
plasma membrane of OLs in the CathD − / − mice, suggesting
that CathD deficiency affects the location of PLP in OLs. PLP
contains a cholesterol recognition/interaction sequence and is a
major cholesterol-interacting protein in oligodendrocytes.
Pulse-chase experiments, together with the biochemical
isolation of CHAPS-resistant membrane fractions and the use of a
photoactive crosslinking cholesterol derivative, revealed that
PLP was associated with myelin lipids after leaving the
endoplasmic reticulum but before exiting the Golgi apparatus.
These assemblies will eventually pinch off as transport vesicles
from the trans-Golgi network and move to the plasma
membrane, by using unknown motor proteins during vesicular
transport.13 Therefore, some experiments were performed in
this study to observe whether the transportation of PLP in OLs
was obstructed in the CathD − / − mice. It was found that the
transport of PLP from the LEs/Ls to the plasma membrane of
OLs was slowed down in the CathD − / − mice, but neither the
clatherin-dependent nor the clatherin-independent endocytosis
of PLP was increased. However, it cannot be ruled out that
CathD deficiency interferes with the transport of PLP by
interfering with the transport of cholesteryl and esters.
Possible interactions between CathD and PLP
VAMP7 (also called tetanus neurotoxin-insensitive-VAMP,
TIVAMP) is involved in various important cellular functions,
including phagocytosis, mitosis, cell migration, autophagosome
biosynthesis as well as membrane repair and growth, and it acts
mainly through mediating the fusion of vesicles that are derived
from Golgi, late-endosomal and lysosomal compartments with
the plasma membrane.46–50 The specific role of VAMP7 in
regulating the exocytosis of LEs/Ls has been well established in
several cell types51–53 and appears to be primarily associated
with cellular processes requiring rapid expansion and
remodeling of the plasma membrane.54,55 The long N-terminal domain
(longin domain) of VAMP7 could be identified as a key factor
that acts as an ‘on/off’ switch for its activity. In PC12 cells,
overexpression of the VAMP7 longin domain inhibits neurite
outgrowth, whereas its deletion activates the SNARE-complex
assembly and stimulates neurite outgrowth.56 In mature
hippocampal neurons, removal of the longin domain increases
spontaneous Ca2+-independent fusion of VAMP7-containing
synaptic vesicles with the pre-synaptic plasma membrane.57
Moreover, the selective sorting of VAMP7 is mediated by direct
interactions between its longin domain and the coat
components of clathrin-coated vesicles.58 There are few studies on
how CathD regulates PLP transport from LEs/Ls to the cell
membrane in oligodendrocyte lineage cells. Recent data showed
that VAMP7 colocalized with PLP in the LEs/Ls of
oligodendrocyte lineage cells, and controlled the exocytosis of PLP from
LE/L organelles as a part of a transcytosis pathway, whereas the
missorting of VAMP7 caused a mild dysmyelination
characterized by reduced select myelin proteins, including PLP.17 In
addition, VAMP7 participated in the degradative function of
lysosomes by mediating proper post-Golgi maturation of
CahtD.59 Thus, we hypothesized that VAMP7 may act as the
mediator between CathD and PLP. Our results suggest that
CathD, PLP and VAMP7 may form a binding complex,
indicating that the loss of CathD may affect some interaction
between CathD and longin domain of VAMP7, and lead to
difficult exocytosis of PLP from LE/Ls to cell membrane of
oligodendrocyte lineage cells, thus delaying the morphological
maturation of oligodendrocytes. However, it remains unknown
whether CathD, PLP and VAMP7 could bind directly or there
are any other mechanisms. Further experiments are required to
demonstrate the interactions among CathD, PLP and VAMP7.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
This study was funded by the Natural Science Foundation of China
(81401063, 31471013), Beijng Nova Program (Z161100004916144),
The natural Science Foundation of Beijing (7153175) and the
Shanghai Pujiang Program (17PJ1410900).
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