Autophagy is required for human umbilical cord mesenchymal stem cells to improve spatial working memory in APP/PS1 transgenic mouse model
Li et al. Stem Cell Research & Therapy
Autophagy is required for human umbilical cord mesenchymal stem cells to improve spatial working memory in APP/PS1 transgenic mouse model
Wen Li 0 1
Kai Li 0
Jing Gao 0
Zhuo Yang 0
0 School of Medicine, State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive for Materials Ministry of Education, Nankai University , 94 Weijin Road, Tianjin 300071 , China
1 Tianjin Third Central Hospital , Tianjin 300170 , China
Background: Recent studies have shown that autophagy plays a central role in mesenchymal stem cells (MSCs), and many studies have shown that human umbilical cord MSCs (huMSCs) can treat Alzheimer's disease (AD) through a variety of mechanisms. However, no studies have looked at the effects of autophagy on neuroprotective function of huMSCs in the AD mouse model. Thus, in this study we investigated whether inhibition of autophagy could weaken or block the function of huMSCs through in vitro and in vivo experiments. Methods: In vitro we examined huMSC migration and neuronal differentiation by inhibiting or activating autophagy; in vivo autophagy of huMSCs was inhibited by knocking down Beclin 1, and these huMSCs were transplanted into the APP/PS1 transgenic mouse. A series of related indicators were detected by T-maze task, electrophysiological experiments, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA), and Western blotting. Results: We demonstrated that regulation of autophagy can affect huMSC migration and their neuronal differentiation. Moreover, inhibition of autophagy in huMSCs could not realize neuroprotective effects via anti-apoptosis or promoting neurogenesis and synapse formation compared with those of control huMSCs. Conclusions: These findings indicate that autophagy is required for huMSCs to maintain their function and improve cognition impairment in APP/PS1 transgenic mice.
Autophagy; Human umbilical cord mesenchymal stem cells; Long-term potentiation; Neurogenesis; Synaptic formation
Autophagy plays a key role in normal physiology and
pathology; it degrades cell organelles and misfolded
proteins by fusing autophagosomes with lysosomes to prevent
waste accumulation and achieves intracellular homeostasis
and cell organelle self-renewal. Autophagy is administered
by a series of well-characterized proteins: for example,
Beclin 1 is responsible for the initiation of the
autophagosome; ATG and LC3 members are responsible for the
elongation and formation of the autophagosome; and P62
is a polyubiquitin-binding protein that is incorporated into
the autophagosome and undergoes degradation in
autolysosomes, and is thus inversely related to the autophagy
]. Abnormal expression of these proteins will lead
to failure of the autophagy process and some diseases such
as cancer, neurodegenerative disease, and immune disease.
Human umbilical cord mesenchymal stem cells
(huMSCs) are stromal cells isolated from the fetal
umbilical cord with Wharton’s jelly. huMSCs are positive
for CD29, CD44, and CD90, and negative for CD31,
CD34, and CD45 [
], and in line with the characteristics
of stem cells. Mesenchymal stem cells (MSCs) are
multipotent and can be differentiated into various cells of
mesodermal lineage in vitro [
]. In vivo, MSCs have
the ability to migrate to sites of injury and differentiate
as well as releasing trophic and growth factors to protect
tissue from damage [
]. Recent studies have shown
that autophagy could regulate the differentiation of
MSC-derived cell lineages, stemness maintenance, and
cell senescence [
]. For instance, autophagy
promoted the differentiation of MSCs into neurons [
and osteoblasts [
]. Rapamycin-induced autophagy
contributed to maintaining MSC stemness, while using
3-methyladenine (3-MA) to inhibit autophagy led to loss
of stemness. Similarly, inhibition of autophagy with 3-MA
leads to a reduced expression of senescence-related
proteins in the process of MSC senescence [
Moreover, there is a close relationship between autophagy and
]. The autophagic process serves a
physiologic function to maintain cellular viability and
delays cell apoptosis during periods of starvation. A
previous study has shown that autophagy prevents MSC
apoptosis under hypoxia/serum deprivation, and inhibition of
autophagy allows MSCs to exhibit higher rate of apoptosis
]. In summary, autophagy is crucial for the fate and
function of MSCs.
Alzheimer’s disease (AD) is the most common
neurodegenerative disorder. It is caused by: 1) synapse loss,
such as PSD95 expression decrease; 2) neuronal
apoptosis, which leads to abnormal brain function; and
3) the presence of amyloid-β (Aβ) plaques and tau
tangles, which result in learning and memory impairment
]. Aβ is regulated by amyloid precursor protein
(APP), and failure to clear Aβ will lead to a series of
downstream events in AD. Recent research has proposed
that MSC transplantation provides neuroprotective
effects for neurodegenerative disorders [
Furthermore, in vivo MSCs can affect the recovery of cognitive
function through complex mechanisms such as secreted
neurotrophic factors [
], increased neurogenesis [
and reduced neuronal apoptosis. However, no studies
have investigated what effect these functions of MSCs
have in AD. In this study, we investigated whether
inhibition of autophagy could weaken or arrest the function
of huMSCs, and whether inhibition of autophagy in
huMSCs transplanted into an AD mouse model can
exert neuroprotective effects or generate worse effects
through in vitro and in vivo experiments.
huMSC cultures, drug treatments, and lentiviral
Human umbilical cords were obtained from full-term
births after either cesarean section or normal vaginal
delivery with the consent of parents in Tianjin First Center
Hospital, Tianjin, China. The procedure of primary
huMSC separation was according to Yang et al. [
huMSCs were cultured in Dulbecco’s modified Eagle’s
medium (DMEM-F12; HyClone) containing 10% fetal
bovine serum (FBS; Sigma-Aldrich, F2442). huMSCs
underwent three passages for this study. huMSCs were
co-treated with 20 mg/ml
tricyclodecane-9-yl-xanthogenate (D609; Sigma-Aldrich, T8543) [
] and 10 ng/ml
] (Rap; Sigma-Aldrich, 37094) or 5 mmol
] (Sigma-Aldrich, M9281) in a humidified
incubator at 37 °C and 5% CO2 for 2 h and 4 h. huMSCs
were then collected for assay. All experiments were
replicated three times.
To investigate the migration ability of huMSCs, the
cells were cultured in six-well plates and a wound was
created by scratching with a sterile plastic pipette tip.
huMSCs were then treated with 10 ng/ml Rap or 5 mmol
3MA in DMEM-F12 without FBS for 24 h. After
incubation, the cells were washed with phosphate-buffered
saline (PBS) and the migrated cells of the wound area
were observed by microscope (Olympus). The area of
cell migration was counted by ImageJ software.
Beclin-1 levels of huMSCs were knocked down by a
lentivirus containing small-hairpin (sh)RNA and green
fluorescent protein (GFP) reporter gene. The lentivirus
was purchased from GeneChem (Shanghai, China). The
following nucleotide sequences were used for the
cloning of shRNA encoding sequences into a lentiviral
vector: Beclin-1 (Becn 1): 5′-ccggga CAGTTTGGCAC
CTTTTTg-3′; and negative controls (NC): 5′-CCGG
CACGTTC GGAGAATTTTTG-3′. huMSCs were stably
infected with negative control lentivirus
(huMSCsshNC) or lentivirus expressing shRNA inhibiting the
gene Beclin-1 (huMSCs-shBecn 1).
Heterozygous APPswe/PS1dE9 double-transgenic male
mice (6 months old) were bred with
backgroundmatched C57BL/6 mice; this type of transgenic mouse
has been widely used [
] and exhibits early Aβ
accumulation which is a typical characteristic in AD. All
mice were purchased from Beijing HFK Bio-Technology
Co. Ltd. (Beijing, China).
huMSC transplantation in APP/PS1 double-transgenic
huMSCs-shNC and huMSCs-shBecn 1 were suspended
in saline at a density of 2 × 105 cells/μl. Mice were
anesthetized with an intraperitoneal injection of chloral
hydrate (0.4 g/kg; Sigma-Aldrich), and 5 μl of saline or
huMSCs-shNC or huMSCs-shBecn 1 suspension was
then injected into the left lateral ventricles (0.1 mm
caudal, 0.9 mm bilateral to bregma, and 2.0 mm ventral
from the dura mater) of the brain at a delivery rate of
1 μl/min using a 10-μl Hamilton microsyringe fixed on a
stereotaxic apparatus (Narishige, Japan). After the
injection, the needle was kept in place for 5 min before
it was slowly retracted. The animals were divided into
three groups (n = 6): 1) the AD-Veh group—APP/PS1
mice were subjected to saline; 2) huMSCs-shNC
group—APP/PS1 mice were subjected to huMSCs-shNC
suspension; and 3) huMSCs-shBecn 1 group—APP/PS1
mice were subjected to huMSCs-shBecn 1 suspension.
All mice were sacrificed on post-transplantation day 14.
Spatial working memory on the elevated T-maze
To assay working memory performance, a T-maze task
was employed. The protocol followed that of Deacon
and Rawlins [
]. The T-maze consisted of a start arm
(30 × 10 cm) and two goal arms (30 × 10 cm),
surrounded by a 20-cm high wall. In brief, prior to the
start of the formal experiment, the body weight of the
mice was reduced to 90% of their original weight by
restricting food intake. Then followed the habituation
phase, when mice became adapted to the T-maze, and
condensed milk as a reward (0.07 ml/reward; Nestle)
was given in the food well at the end of the arm. During
the trial phase, each trial consisted of a forced choice
and a free choice. For the forced choice, one of two goal
arms was blocked by a wall and the mouse was directed
towards the open arm with a condensed milk reward,
and then the mouse was returned to the start box. For
the choice phase, the wall was removed and the mouse
had to select the formerly closed arm to receive a second
] (this was the rewarded alternation and
recorded as correct, if not it was recorded as wrong)
(Fig. 3a). In this study, the time interval between the
forced choice and the free choice was approximately
1 min. Mice were subjected to 10 trials per day for 4
consecutive days. A percentage of correct choices per
animal was calculated.
Long-term potentiation (LTP) and depotentiation (DEP) recordings
Following the T-maze test, LTP and DEP were assessed
by an in vivo electrophysiological test, based on previous
]. The mouse was anesthetized with an
intraperitoneal injection of urethane (1.2 mg/kg;
SigmaAldrich) and then positioned on the stereotaxic
apparatus prepared for surgery. First, the skull was exposed
and a hole was drilled for inserting electrodes. Then the
bipolar stimulating electrode was positioned in the
performant pathway (PP; 2.1 mm lateral and 3.8 mm
posterior to the bregma, 1.8 mm from the brain surface)
and the monopolar stainless steel recording electrode
was positioned in the dentate gyrus (DG; 1 mm lateral
and 1.7 mm posterior to the bregma, 1.8 mm from the
brain surface) of the hippocampus. The stimulation
intensity (range 0.3–0.5 mA, stimulus pulse 0.2 ms at
0.03 Hz) was used to stimulate a response at 70% of its
maximum to deliver baseline, LTP, and DEP
recordings (Scope software, PowerLab; AD Instruments,
New South Wales, Australia). The baseline was
recorded every 30 s for 20 min. After the baseline,
theta burst stimulation (TBS) was delivered to induce
LTP, and then the same single plus stimulating
intensity was recorded every 60 s for 1 h as LTP.
Following LTP, low-frequency stimulation (LFS; 900 pulses,
1 Hz for 15 min) was delivered to induce DEP, and
the same method as used for recording LTP was used
to record DEP. The field excitatory postsynaptic
potential (fEPSP) slope was measured by Clampfit 10.0
(Molecular Devices, Sunnyvale, CA, USA).
Mouse brain tissue was embedded in OCT
compound (Tissue-Tek, Miles) and sectioned at 10-μm
intervals (Leica CM 1850). The sections were stained
with hematoxylin and eosin (H&E). Survival of
neurons in the hippocampus and cortex was
calculated from six sections of each sample, and the
average was taken. Microscopic images were
analyzed by ImageJ software.
Brain sections and cultured huMSCs were fixed in 4%
paraformaldehyde for 10 min and then incubated with
0.5% Triton-X100 for 10 min. After each step, the
sections were washed three times with PBS. The sections
were blocked with 10% normal goat serum for 1 h at
room temperature, followed by incubation with the
primary antibodies rabbit anti-NSE (1:100, Abcam,
ab53025), rabbit anti-MAP2 (1:500, Abcam, ab32454),
mouse anti-Human Nuclear Antigen (hNu; 1:200,
Abcam, ab191181), mouse anti-LC3 (1:1000, MBL,
M186-3), rabbit anti-Cleaved Caspase-3 (CCaspase-3;
1:500, Cell Signaling, 9661), rabbit anti-Sox2 (1:1000,
Abcam, ab97959), mouse anti-SQSTM1/P62 (P62;
1:1000, Abcam, ab56416), Rabbit anti-Aβ 1-42 (1:100,
Bioss, China, bs-0107R), rabbit anti-DCX (1:500, Abcam,
ab77450), rabbit anti-Ki67 (1:250, Abcam, ab16667), and
rabbit anti-Postsynaptic density protein 95 (PSD95;
1:250, Abcam, ab16667) for 24 h at 4 °C. After washing
three times with PBS, the sections were stained with the
secondary antibodies Alexa 488-conjugated goat
antimouse IgG (1:1000, CA11008S; Invitrogen) and Alexa
594-conjugated goat anti-rabbit IgG (1:1000, A21235;
Life Technologies) for 1 h at room temperature. They
were then washed three times with PBS. Subsequently,
the nucleus was stained with DAPI (1:1000, Solarbio,
China) for 5 min. Images were taken on a confocal laser
scanning microscope (Olympus). The fluorescent value
was quantified by ImageJ software, with six sections of
each sample being calculated and the average taken.
Western blot assay and enzyme-linked immunosorbent assay (ELISA)
Collected cell pellets and mouse brain lysates were
prepared, and the procedure for Western blotting was
performed as previously described [
]. The primary
antibodies for the Western blot analysis were as follows:
rabbit anti-Beclin 1 (1:1000, Cell Signaling, 3495), rabbit
anti-ATG 7 (1:500, Cell Signaling, 2631), rabbit
anti-SDF1 (1:1000, Abcam, ab18919), rabbit anti-PARP (1:1000,
Cell Signaling, 9542), rabbit anti-APP (1:5000, Abcam,
ab180140), rabbit anti-Presenilin1 (PS1; 1:5000, Abcam,
ab76083), rabbit anti-Bcl-xl (1:2000, Cell Signaling, 2764),
rabbit anti-Bax (1:2000, Abcam, ab32503), rabbit
antiCaMKII (1:2000, Abcam, ab52476), rabbit anti-CaMKII
(phospho) (p-CaMKII; 1:2000, Abcam, ab32503), rabbit
anti-NMDAR2B (1:1000, Cell Signaling, 4212), rabbit
antiACTB (β-actin; 1:5000, Sangon, China), and anti-LC3
antibody (1:1000), anti-MAP2 antibody (1:1000),
antiSQSTM1/P62 antibody (1:2000), anti-Sox2 antibody
(1:1000), anti-CCaspase-3 antibody (1:1000), and
antiPSD95 antibody (1:2000) as previously described. The
secondary antibodies were anti-rabbit IgG (H + L), HRP
conjugate (1:5000, Promega) or anti-mouse IgG (H + L)
HRP conjugate (1:5000, Promega). Immunoreactivity was
obtained by a chemiluminescence imaging system (GE
Healthcare, RPN2108), and ImageJ software was used to
evaluate the differences between the samples.
Brain-derived neurotrophic factor (BDNF) and nerve
growth factor (NGF) in the hippocampus and cortex
levels were measured by an ELISA method using the
mouse BDNF (SEA011Mu, Cloud-Clone Corp., China)
or NGF (SEA105Mu, Cloud-Clone Corp., China) assay
kits in accordance with the manufacturer’s instructions.
For all animal experiments, rats were selected by a
completely randomized design to each group. A
doubleblinding method was used for group assignment and
outcome assessment. The method of assessing the sample
size was according to our previous studies [
]. All results
are expressed as mean ± standard error of the mean
(SEM). Data were generated from three independent
experiments. Statistical analysis was performed by SPSS 22.0
(SPSS Inc.) and GraphPad Prism 6 (GraphPad Software).
One-way analysis of variance (ANOVA) followed by
further Dunnet’s multiple comparison was used to analyze
the statistical differences between three or more groups,
and P < 0.05 was considered statistically significant.
Autophagy promotes migration and neuronal differentiation of huMSCs in vitro
In order to investigate whether autophagy was involved
in migration and neuronal differentiation of huMSCs,
we first conducted a scratch test and treated huMSCs
with Rap, an autophagy inducer, or 3MA, an autophagy
inhibitor. The data showed that huMSC migration was
significantly boosted by Rap and significantly inhibited
by 3MA compared with the control group (Fig. 1a).
Previous studies have reported that D609 can induce
MSC differentiation into neuron-like cells [
Therefore, in this study D609 was employed to treat huMSCs.
It was found that the morphology of huMSCs became
neuron-like (huMSCs-NCs) after treatment for 4 h
(Fig. 1b); simultaneously, the expression of LC3 II (an
autophagy marker) was significantly increased from 2 h
to 4 h of treatment (Fig. 1c). To further understand the
relationship between autophagy and neuronal
differentiation of huMSCs, neuron-specific enolase (NSE) and
microtubule-associated protein 2 (MAP2) (neuronal
markers) and LC3 II were stained with their specific
antibodies and observed using confocal microscopy. The
increased expression of NSE and MAP2 indicated that
huMSCs-NCs have the potential to function as neurons
(Fig. 1d). Then MAP2 and LC3 II were co-localized by
double immunofluorescence staining. The data showed
that the expressions of MAP2 and LC3 in huMSCs-NCs
were simultaneously increased (Fig. 1e). In addition,
MAP2 and Beclin 1 (autophagy-related protein) were
examined with Western blot assay. At 2 h and 4 h of
huMSC differentiation, activation of autophagy with Rap
can promote the expression of MAP2; in turn, inhibition
of autophagy with 3MA prevented neuronal
differentiation (Fig. 1f and g). Beclin 1 expression was consistent
with MAP2 expression (Fig. 1f and h).
Taken together, these data imply that in vitro
migration and neuronal differentiation of huMSCs is tightly
regulated by the autophagy pathway.
The effect of inhibition of autophagy on huMSC function in vitro
To explore the mechanisms of autophagy-regulated
huMSC function, Beclin 1 was knocked down, and the
proteins associated with autophagy (Beclin 1, ATG7,
LC3, P62), migration and differentiation (stromal
cellderived factor-1 (SDF-1)), stemness (Sox2), and
apoptosis (caspase-3 and poly-ADP-ribose polymerase
(PARP)) were examined with Western blot assay. The
results showed that the expression of the autophagy
pathway proteins Beclin 1, ATG7, LC3, and P62 were
significantly decreased in the huMSCs-shBecn 1 group
compared with those in the huMSCs-shNC group
(Fig. 2a and b). This suggested that autophagy was
precisely suppressed in huMSCs. SDF-1 plays a key role
in the migration and differentiation of MSCs [
Sox2 as a transcriptional factor is essential for
maintaining self-renewal/proliferation/pluripotency of
undifferentiated stem cells [
]. Our data demonstrated that the
inhibition of autophagy markedly reduced the
expressions of SDF-1 and Sox2 in the huMSCs-shBecn
1 group (Fig. 2a and c). Furthermore, inhibition of
autophagy can promote the expression of the
apoptotic proteins caspase-3 and PARP in huMSCs
(Fig. 2a and d).
To investigate the relationship between autophagy and
CCaspase-3 and Sox2, LC3 and CCaspase-3, and P62
and Sox2 were co-localized by double
immunofluorescence staining, respectively. We observed that impaired
autophagy led to increased apoptosis (Fig. 2e) and
reduced pluripotency in huMSCs (Fig. 2f ).
These data indicated that autophagy is essential for
maintaining huMSC function, including migration,
differentiation, stemness, and survival.
The effect of huMSC transplantation on spatial working memory, LTP, and DEP
It is generally believed that AD is commonly
characterized by a progressive learning and memory impairment.
To assay the working memory performance of huMSC
transplantation in an AD model mouse, a T-maze task
was employed. In this task, the AD-Veh and
huMSCsshBecn 1 groups showed profound spatial working
memory impairment. In the free choice phase, they
failed or delayed the alternate response on two-choice
mazes. Even at the end of the testing, they were still at
accidental levels (percent correct: AD-Veh, 64%;
huMSCs-shBecn 1, 63%). In contrast, in the
huMSCsshNC group the spatial response was improved from
trial to trial and they obtained a gradual choice accuracy
level of 80% (Fig. 3b).
LTP assays are a key indicator for estimating learning
and memory. fEPSPs were evoked in the PP-DG region of
the hippocampus pathway. After TBS stimulation, the
fEPSP slopes increased abruptly from baseline in the
huMSCs-shNC group, and the mean fEPSP slopes reached
132% of baseline. In contrast, the fEPSP slopes were not
obviously increased with TBS stimulation in the AD-Veh
and huMSCs-shBecn 1 groups (mean fEPSP slopes:
ADVeh, 110%; huMSCs-shBecn 1, 108%) (Fig. 3c and d). DEP
assays are an index of reversal learning behavior, and are
the opposite of LTP. DEP was stimulated with LFS and
the mean slope of LTP was normalized and used as the
baseline of DEP in each group. The data showed that DEP
was significantly suppressed in the AD-Veh and
huMSCsshBecn 1 group (mean fEPSP slopes: AD-Veh, 87%;
huMSCs-shBecn 1, 85%), but the DEP of the
huMSCsshNC group displayed good flexibility and the fEPSP slope
dropped to 62% (Fig. 3c and e).
These results indicated that inhibition of autophagy
in huMSCs to transplant could not ameliorate the
impaired learning and memory in APP/PS1 transgenic
The effect of huMSC transplantation on Aβ clearance in
the cortex and hippocampus
To examine whether huMSC transplantation exhibited
the ability to clear Aβ, we immunofluorescence stained
Aβ with specific antibodies in the cortex and
hippocampus region. The data analysis showed that the Aβ plaque
significantly degraded in the huMSCs-shNC group
compared with the AD-Veh and huMSCs-shBecn 1
groups (Fig. 4a). APP and PS1 protein levels, which
lead to Aβ production, were then examined by
Western blot assay. The levels of APP and PS1 were
substantially reduced in the huMSCs-shNC group
compared with the AD-Veh group. However,
inhibition of autophagy in huMSCs failed to reduce the
expression of APP and PS1 (Fig. 4b).
The effect of huMSC transplantation on neuronal apoptosis of the cortex and hippocampus
For the analysis of neuronal apoptosis in the cortex and
DG of the hippocampus, H&E staining was employed. As
shown in Fig. 5a, neurons in the AD-Veh and
huMSCsshBecn 1 groups were arranged loosely, with obvious
nucleus shrinkage and neuron loss in the cortex and DG
region. However, neurons in the huMSCs-shNC group
were arranged in an orderly and dense manner. The
number of surviving neurons was counted by ImageJ in each
group, and the results showed that there were significantly
more of these in the huMSCs-shNC group compared with
the AD-Veh and huMSCs-shBecn 1 groups (Fig. 5b, c). To
further understand the mechanism of apoptosis,
proteins associated with apoptosis (Bcl-xl, Bax,
CCaspase-3, and PARP) in the cortex and
hippocampus lysates were examined by Western blot. The
results showed that the ratio of Bcl-xl/Bax in the
huMSCs-shNC group was higher than that in the
AD-Veh and huMSCs-shBecn 1 groups, and that
huMSCs-shNC significantly inhibited the expression
of the apoptotic CCaspase-3 and PARP compared
with the huMSCs-shBecn 1 group (Fig. 5d).
Thus, inhibition of autophagy in huMSCs blocked its
anti-apoptosis function in the APP/PS1 transgenic
Migration, apoptosis, and differentiation of huMSCs following transplantation in the APP/PS1 transgenic mouse
As shown in Fig. 6, labeled GFP huMSCs were observed
at 14 days post-transplantation. huMSCs-shNC were
widely distributed in the whole brain, but the majority of
huMSCs-shBecn 1 were located in the left side of the
brain and gathered into groups (Fig. 6a). The gathered
blocks were labeled with CCaspase-3 antibody, and the
expression of CCaspase-3 was obviously increased in the
huMSCs-shBecn 1 group compared with the
huMSCsshNC group (Fig. 6b).
To investigate the differentiation of huMSCs in vivo,
the newly born neuron marker doublecortin (DCX) and
the mature neuron marker MAP2 were detected. The
results indicated that huMSCs-shNC expressed DCX and
(See figure on previous page.)
Fig. 5 The effect of human umbilical cord mesenchymal stem cell (huMSC) transplantation on neuronal apoptosis of the cortex and hippocampus.
a Cell apoptosis was evaluated by H&E staining. The boxes indicate neurons arranged loosely and neuron loss. The arrows indicate nucleus shrinkage.
Relative number of surviving neurons in b the cortex and c the hippocampus. d Representative cropped Western blots and statistical analysis of the
apoptosis-related proteins Bcl-xl, Bax, cleaved caspase-3 (CCaspase-3), and cleaved poly-ADP-ribose polymerase (CPARP) in the cortex and hippocampus. All
data are expressed as mean ± SEM, n = 3. Data were generated from three independent experiments. *P < 0.05, **P < 0.01, vs. AD-Veh; #P < 0.05, ##P < 0.01,
MAP2 at high levels and, in turn, huMSCs-shBecn 1 did
not (Fig. 6c and d).
These data showed that the ability of huMSCs, which
migrated to the injury site and differentiated neurons,
was blocked by inhibiting autophagy, consistent with the
in vitro data.
The effect of huMSC transplantation on neurogenesis of the subgranular zone (SGZ) and the subventricular zone (SVZ)
In the adult brain, neurogenesis allows for continuous
development under physiological and pathological
stimuli. Neural stem cells (NSCs) are mostly located in the
SGZ of the DG and the SVZ [
]. In this study, the
proliferation marker Ki67 in the SGZ and SVZ was analyzed
in APP/PS1 transgenic mice. The number of
Ki67positive cells in the huMSCs-shNC group was
significantly increased compared with the AD-Veh group.
However, in the huMSCs-shBecn 1 group, inhibition of
huMSC autophagy failed to promote neurogenesis, and
the number of Ki67-positive cells was equally matched
with those of the AD-Veh group (Fig. 7a–d).
Furthermore, we measured the levels of the
neurotrophic factors BDNF and NGF, which contribute to
neurogenesis, by ELISA. The data suggested that the
levels of BDNF and NGF significantly increased in the
huMSCs-shNC group compared with the AD-Veh
group, but the inhibition of autophagy in huMSCs could
not promote the secretion of BDNF and NGF in the
brains of APP/PS1 transgenic mice compared with that
in the huMSCs-shNC group (Fig. 7e–h).
The effect of huMSC transplantation on synaptic transmission
PSD-95 is an important factor that contributes to synaptic
]. We next performed immunoreactivity
studies to explain the functional recovery in the APP/PS1
transgenic mouse after huMSC transplantation, and
focused on the possible links between neurogenesis and
synapse formation. Immunoreactivity images showed a higher
intensity of PSD-95 in the DG of the hippocampus in the
huMSCs-shNC group compared with the AD-Veh and
huMSCs-shBecn 1 groups (Fig. 8a). In addition, we
checked the synaptic transmission-related proteins
CaMKII, p-CaMKII, NMDAR 2B, and PSD95, which
contributed to LTP generation in the hippocampus as seen by
Western blot. The results indicated that the ratio values of
p-CaMKII/CaMKII and NMDAR 2B were significantly
decreased in the huMSCs-shNC group compared with the
AD-Veh group. huMSCs-shBecn 1 transplantation did not
produce a meaningful improvement. The level of PSD95
was significantly increased in the huMSCs-shNC group
compared with the AD-Veh and huMSCs-shBecn 1 groups,
consistent with the immunoreactivity data (Fig. 8b).
These data indicated that inhibition of autophagy in
huMSCs failed to restore synaptic transmission injury in
APP/PS1 transgenic mice.
Autophagy is crucial for regulating the stemness
maintenance, expansion, and differentiation of stem cells [
Previous studies suggested that cultured MSCs continuously
kept a high level of autophagy to maintain stemness [
], and activating autophagy can promote MSC
differentiation by various signaling pathways . Moreover,
activating autophagy can also block MSC apoptosis [
Based on the above, in this study we aimed to investigate
the effect of autophagy in huMSCs. In vitro we found that
migration of huMSCs was regulated by autophagy, and
that activation of autophagy with Rap or inhibition with
3MA promoted or prevented the migration of huMSCs to
the wound (Fig. 1a). We next examined the effect of
autophagy on huMSC differentiation. huMSC-derived
neurons were induced with D609 (Fig. 1b), and they showed a
high level of the neuronal markers NSE and MAP2
(Fig. 1d). Moreover, autophagy was activated in the
process of differentiation as indicated by the levels of LC3
at 2 h and 4 h (Fig. 1c). LC3 and MAP2 were then
colocalized by immunofluorescence labeling (Fig. 1e) and
further cell lysates, collected from Rap- or 3MA-treated
huMSCs-NCs, were analyzed (Fig. 1f–h). These results
were consistent and suggested that the level of autophagy
was increased during huMSC differentiation, and that
activation or inhibition of autophagy promoted or
suppressed their differentiation.
To ascertain how autophagy controls huMSCs, Beclin
1 plays a central role in mediating the localization of
other autophagy-related proteins to the phagophore
membrane in autophagy [
]. Beclin 1 was
downregulated in huMSCs by lentiviral transfection. We
performed a huMSCs-shBecn 1 lysate analysis and found
that, under conditions where autophagy was precisely
suppressed as indicated by downregulation of Beclin 1,
ATG7, LC3, and P62 expression (Fig. 2a and b), SDF-1
and Sox2 were reduced and represented the level of
huMSC migration and stemness maintenance,
respectively (Fig. 2c). In addition, downregulation of
Beclin 1 led to increased apoptosis in huMSCs, which
correlated with increased CCaspase-3 and CPARP
(Fig. 2d). These results were consistent with those from
(See figure on previous page.)
Fig. 7 The effect of human umbilical cord mesenchymal stem cell (huMSC) transplantation on neurogenesis of the subgranular zone (SGZ) and the
subventricular zone (SVZ). a–d Ki67 was detected by immunofluorescence staining and quantitative analysis by image J software in the SGZ and SVZ,
respectively. e–h The effect of huMSC transplantation on brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) secretion in the
cortex and hippocampus by ELISA. All data are expressed as mean ± SEM, n = 3. Data were generated from three independent experiments. *P < 0.05,
**P < 0.01, vs. AD-Veh; #P < 0.05, ##P < 0.01, vs. huMSCs-shNC. DG dentate gyrus, LV lateral ventricle
immunostaining (Fig. 2e, f ), and showed that autophagy
influenced huMSC function via a variety of specific
Based on the above in vitro experiments, we further
studied the effects of autophagy on huMSC
transplantation in APP/PS1 transgenic mice used as an AD model.
AD is characterized by massive neuronal death caused
by Aβ plaques and tangle formation, and cognitive
impairment caused by synaptic loss in several brain regions
]. Previous research has suggested that stem cell
transplantation could have a therapeutic potential for
AD via various mechanisms. This is consistent with our
results, as we found that huMSCs-shNC transplantation
could improve the learning and memory ability of the
mouse AD model by inhibition of neuronal apoptosis,
promoting neurogenesis and synapse formation. However,
and more importantly, inhibition of huMSC autophagy by
knocking down Beclin 1 expression did not allow these
neuroprotective effects in the mouse AD model.
The mouse AD model was associated with impairment of
spatial working memory, which was managed by the
hippocampus LTP [
]. Moreover, LTP is responsible for synaptic
transmission. In this study, we found that huMSCs-shNC
transplantation could ameliorate the spatial working
memory (Fig. 3b), and the mean fEPSP of LTP was obviously
enhanced in the mouse AD model (Fig. 3c, d). DEP is the
opposite to LTP, responsible for balance between synaptic
attenuation and enhancement, and involved in the
forgetting and storage of information [
]. The mean fEPSP of
DEP was reduced after huMSCs-shNC transplantation in
the mouse AD model (Fig. 3c, e). In contrast,
huMSCsshBecn 1 transplantation could not restore the impaired
working memory, and remained at the same level as the
AD-Veh group. These findings suggested that autophagy is
essential for huMSC neuroprotection.
We also demonstrated that huMSC-shNC
transplantation could reduce Aβ production (Fig. 4a) and cell
death (Fig. 5a–c) in the transgenic mouse brain. We
identified the molecular mechanisms involving APP and
PS1 (Fig. 4b) which related to Aβ peptide synthesis [
and cell apoptosis-related proteins Bcl-xl, Bax,
CCaspase-3, and PARP were decreased (Fig. 5d).
However, inhibition of huMSC autophagy could not play a
useful role in the mouse AD model.
To explore why transplanted huMSCs-shBecn 1 could
not play a neuroprotective effect, we observed that most
of the transplanted huMSCs-shBecn 1 were located in
the left brain and clustered into groups. Transplanted
huMSCs-shNC could extensively migrate, however, and
aggregation rarely occurred (Fig. 6a). Furthermore, the
gathered cells had undergone significant apoptosis as
indicated by CCaspase-3 expression (Fig. 6b). Using the
specific antibodies anti-hNu with DCX or MAP2
colocalization, we found that huMSCs-shNC migrated to
the DG region of the hippocampus and differentiated
into neurons. However, we found hardly any
huMSCsshBecn 1 in the DG region (Fig. 6c, d). We summarized
that inhibition of autophagy in huMSCs caused them to
fail to migrate to the damaged hippocampus, which was
then related to cognitive dysfunction and further to
neuronal differentiation. Furthermore, extensively
distributed huMSCs-shNC promoted neurogenesis (Ki67)
in SGZ and SVZ by increasing BDNF and NGF secretion
in the transgenic mouse brain (Fig. 7), and due to
inhibition of huMSC autophagy causing migration defects
and increased apoptosis, huMSCs-shBecn 1
transplantation could not promote the neurogenesis.
In conjunction with the cognitive impairment and reduced
LTP, a significant loss of synapses was found in the AD
mouse model. In the AD mouse model, Aβ accumulation
induced increased NMDAR2B expression [
huMSCs-shNC transplantation blocked CaMKII
phosphorylation and inhibited the expression of NMDAR2B. PSD-95 is
a molecular partner with NMDAR, and forms a molecular
complex to contribute to synaptic formation. We confirmed
that huMSCs-shNC transplantation promoted the synapse
formation by enhancing PSD-95 expression in the AD
mouse model; when huMSC autophagy was inhibited, all of
the above functions ceased (Fig. 8).
Our results clearly show that autophagy dominates
huMSC function and that inhibition of autophagy in
huMSCs leads to the disappearance of functions
including migration, differentiation, and anti-apoptosis, and
the promotion of neurogenesis and synapse formation in
the AD mouse model (Fig. 9). In conclusion, autophagy
is required for huMSCs to maintain their function and
improve cognition impairment in APP/PS1 transgenic
mice. Our findings suggest that the therapeutic effect of
huMSCs can be improved by increasing the level of
autophagy, such as with traditional Chinese medicine or
small molecule drug intervention. In addition, MSC
senescence is also an important factor affecting its function,
and autophagy is impaired in MSC senescence [
Therefore, a clear relationship between autophagy and
senescence can also help researchers to improve the
therapeutic effect of MSCs.
3MA: 3-Methyladenine; AD: Alzheimer’s disease; APP: Amyloid precursor protein;
Aβ: Amyloid-β; BDNF: Brain-derived neurotrophic factor; D609:
Tricyclodecane-9-ylxanthogenate; DCX: Doublecortin; DEP: Depotentiation; DG: Dentate gyrus;
DMEM: Dulbecco’s modified Eagle’s medium; ELISA: Enzyme-linked immunosorbent
assay; FBS: Fetal bovine serum; fEPSP: Field excitatory postsynaptic potential;
GFP: Green fluorescent protein; H&E: Hematoxylin and eosin; huMSC: Human
umbilical cord mesenchymal stem cell; LFS: Low-frequency stimulation; LTP:
Longterm potentiation; MAP2: Microtubule-associated protein 2; MSC: Mesenchymal
stem cell; NC: Negative control; NGF: Nerve growth factor; NSE: Neuron-specific
enolase; PARP: Poly-ADP-ribose polymerase; PBS: Phosphate-buffered saline;
PP: Performant pathway; PS1: Presenilin 1; Rap: Rapamycin; SDF-1: Stem cell-derived
factor-1; SGZ: Subgranular zone; shRNA: Small-hairpin RNA; SVZ: Subventricular zone;
TBS: Theta burst stimulation
This work was supported by grants from the National Natural Science
Foundation of China (81571804, 81771979).
Availability of data and materials
ZY and WL conceived and designed all the experiments. WL, KL, and JG
performed the experiments and analyzed the data. WL and ZY drafted and
revised the article. All authors approved the final version.
Ethics approval and consent to participate
All experiments involving animals were performed in accordance with guidelines
approved by the Committee for Animal Care at Nankai University and coincided
with the National Institutes of Health’s Guide for the Care and Use of Laboratory
Animals. Human umbilical cords were obtained from full-term births after either
cesarean section or normal vaginal delivery with the consent of parents in Tianjin
First Center Hospital, Tianjin, China. This study was performed in accordance with
the principles of human subject protection in the Declaration of Helsinki and with
Nankai University’s Institutional Review Board approval.
Consent for publication
All authors consent for the publication of this study.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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