Neuroscience Letters 760 (2021) 136094

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Research article

microRNA-106b-containing extracellular vesicles affect autophagy of

neurons by regulating CDKN2B in Parkinson’s disease
Xue Bai *, Qiaoyun Dong , Li Zhao , Yan Yao , Bo Wang
Department Five of Neurology, Cangzhou Central Hospital, Cangzhou 061000, Hebei, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder, and autophagy dysfunction is
Parkinson’s disease involved in the pathogenesis of PD. Mesenchymal stem cells (MSC)-derived extracellular vesicles (EVs) have been
Mesenchymal stem cell-derived extracellular established as an attractive therapeutic tool, since they can serve as biological nanoparticles with beneficial
vesicles
effects in PD. Herein, the study aimed to investigate the effects of EVs derived microRNA (miR)-106b on
microRNA-106b
autophagy of neurons in PD. Following the development of a mouse model of PD, we conducted behavior test,
CDKN2B
Autophagy TUNEL assay and HE staining to verify the success of modeling. Afterward, MSC-derived EVs were extracted and
Neuron identified. In hippocampal tissues and neurons of PD mice, miR-106b was poorly expressed, while CDKN2B was
highly expressed. miR-106b shuttled by MSC-derived EVs increased neuronal survival, autophagy, LC3II/LC3I
ratio and Bcl-2 protein expression, while inhibited neuronal apoptosis and Bax expression in PD mice. It was also
confirmed that CDKN2B is a downstream target of miR-106b. Overexpression of CDKN2B reversed the protective
effects of miR-106b-containing EVs on neurons in mice with PD. Collectively, miR-106b-containing EVs alleviate
neuronal apoptosis and enhance neuronal autophagy in PD by downregulating CDKN2B.

1. Introduction physiology of neuronal circuits by delivering proteins, RNA, lipids, and

Parkinson’s disease (PD), a neurological disorder, is characterized by
the motor features of parkinsonism related to Lewy bodies and loss of
dopaminergic neurons in the substantia nigra [1]. PD is the second most
prevalent neurodegenerative disease globally, disturbing more than 10
million people, about 1% of the global population over 60 years old [2].
The exposure to pesticides, consumption of dairy products, history of
melanoma, as well as traumatic brain injury pose the risk factors of PD
[3]. Despite decades of research dedicated to developing therapy op­
tions, multiple agents have failed in clinic, and the task of improving PD
seems increasingly daunting as the intricacies of the genotype, pheno­
type, and pathogenesis of PD continue to emerge [4]. Autophagy, a
multifaceted intracellular process, exists to identify and capture a large
repertoire of intracellular components and bring them to the lysosomal
compartment [5]. Autophagy, which is suppressed in aging cells, could
protect the cells against aging and disease [6]. Therefore, detailed illu­
mination of the mechanism underlying autophagy has great signifi­
cances for exploring effective therapies against PD.
Extracellular vesicles (EVs), having the competence to modify the
metabolites, are involved in the development of neurodegenerative
diseases, including PD [7]. EVs, from specific cell types, can alleviate
some diseases by regulating autophagy [8]. Moreover, EVs can mediate
the pathogenesis of PD, such as the dysfunction of autophagy and
lysosomal degradation [9]. Mesenchymal stem cell (MSC)-derived EVs
may be applied as a possible therapeutic approach or as an adjuvant
therapy to halt the disease progression and improve PD symptoms [10].
In addition, the activation of autophagy stimulated by human umbilical
cord MSC (hUCMSC)-derived EVs can efficiently reduce the nephro­
toxicity of cisplatin [11]. Therefore, we assumed that hUCMSC-derived
EVs could be related to autophagy of neurons in PD as well. Interest­
ingly, homo sapiens-microRNA (miR)-106b-3p was found as one of the
five miRNAs that were downregulated in plasma EVs from PD patients
than that in controls, indicating that it might be a possible biomarker for
the diagnosis of PD [12]. The close interaction between miR-106b and
autophagy has been appreciated in inflammatory bowel disease [13].
Hence, we hypothesized that EVs released from hUCMSC, as carriers,
could effectively deliver miR-106b into the neurons to regulate auto­
phagy in PD. All efforts made in the present study were to offer a new

* Corresponding author at: Department Five of Neurology, Cangzhou Central Hospital, No. 50, Xinhua West Road, Yunhe District, Cangzhou 061000, Hebei, PR
China.
E-mail address: baixue5191@126.com (X. Bai).

https://doi.org/10.1016/j.neulet.2021.136094
Received 12 May 2021; Received in revised form 24 June 2021; Accepted 26 June 2021
Available online 30 June 2021
0304-3940/© 2021 Elsevier B.V. All rights reserved.
X. Bai et al.
idea for the discovery of new strategies for the treatment of PD.
2. Materials and methods
2.1. Ethics statement
The experiments regarding animals were performed as per the pro­
tocols and guidelines and were approved by the Ethics Committee of
Cangzhou Central Hospital. Best efforts were made to minimize the
suffering of the included animals.
2.2. Establishment and treatment of the PD mouse model
Forty-five male healthy C57BL mice (8 weeks old, 18–22 g) were
from the Laboratory Animal Center of Sun Yat-sen University
(Guangzhou, Guangdong, China). The animals were kept on in a
specific-pathogen-free environment at 24–26 ◦ C with 45–55% humidity.
The feed and drinking water were sterilized at high temperature before
use.
Ten mice were randomly selected as the control group and 35 mice
were used for PD model generation using an intraperitoneal injection of
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) [14]. MPTP
(Sigma-Aldrich Chemical Company, St Louis, MO, USA) was dissolved in
sterile saline and injected intraperitoneally into the mice at a dose of 25
mg/kg once daily for 7 d. Control mice were injected with equal
amounts of saline.
After 25 d of modeling, the mice were anesthetized by intraperito­
neal injection of 2% sodium pentobarbital (60 mg/kg), and the skull was
exposed by a longitudinal median incision after alcohol disinfection of
the skin on the top of the head. A small hole was drilled with a 5-gauge
needle (1 mm from bregma and 2 mm next to the midline). A micro­
injector was inserted vertically to a depth of 2.15 mm, and 10 μL len­
tiviruses (titer 2 × 108 U/mL) or 5 μL phosphate-buffered saline (PBS)
containing 50 μg human umbilical cord MSC (hUCMSC)-EVs was slowly
injected. The needle was left in place for 5 min after injection. Ten PD
mice were left untreated, and the remaining 25 PD mice were randomly
categorized into five groups (n = 5): PD + EVs group (PD mice injected
with 50 μg hUCMSC-EVs suspended in 5 μL PBS), PD + EVs-NC group
(PD mice injected with 50 μg hUCMSC-EVs-mimic NC suspended in 5 μL
PBS), PD + EVs-miR-106b group (PD mice injected with 50 μg hUCMSC-
EVs-miR-106b mimic suspended in 5 μL PBS), PD + EVs-miR-106b + oe-
NC group (PD mice injected with 50 μg hUCMSC-EVs-miR-106b sus­
pended in 5 μL PBS and control lentivirus), and PD + EVs-miR-106b +
oe-CDKN2B group (PD mice injected with 50 μg hUCMSC-EVs-miR-106b
suspended in 5 μL PBS and CDKN2B overexpressing lentivirus). After 2
d, the pole test and swimming test were performed (as described in
Supplementary File). After the above experiments were completed, the
mice were euthanized by intraperitoneal injection of excessive sodium
pentobarbital, and the brain tissues were harvested for subsequent ex­
periments. Pathologically damaged structures in mouse hippocampal
tissues and the proportion of apoptosis were evaluated using
hematoxylin-eosin (HE) staining and Terminal deoxynucleotidyl trans­
ferase (TdT)-mediated 2′ -Deoxyuridine 5′ -Triphosphate (dUTP) nick
end labeling (TUNEL) (as described in the Supplementary File).
2.3. Treatment of primary mouse hippocampal neurons
Mouse primary hippocampal neurons were isolated as described in
the Supplementary File. The neurons were treated with 500 μg hUCMSC-
derived EVs suspended in 500 μL PBS or 500 μg EVs derived from
hUCMSC transfected with miR-106b mimic in 500 μL PBS. The neurons
were infected with GV287 (Genechem, Shanghai, China) overexpression
lentiviral vectors. Normal neurons were also infected with lentiviruses
harboring mimic NC (5′ -UUCUCCGAACGUGUCACGUTT-3′ ) or miR-
106b (5′ -TAAAGTGCTGACAGTGCAGAT-3′ ). An appropriate amount of
lentivirus was supplemented to the cell culture plate according to the
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Neuroscience Letters 760 (2021) 136094
multiplicity of infection = 5, and the 6-well plate was incubated in an
incubator at 37 ◦ C with 5% CO2. After 24 h, the cells were then collected,
and the cell proliferation and apoptosis and the expression level of
autophagy-related protein LC3 were detected by cell counting kit-8
(CCK-8), flow cytometry, and immunofluorescence, respectively
(described in detail in the Supplementary File).
2.4. Isolation and identification of hUCMSC-derived EVs
hUCMSCs (HUXUC-01001, Cyagen, Suzhou, Jiangsu, China) were
cultured with DMEM containing 10% FBS, 100 U/mL penicillin and 100
μg/mL streptomycin for 48 h in six-well plates at 37 ◦ C in 5% CO2. All
experiments were performed using MSC at passage 3–8.
MSCs were cultured in FBS-free medium and the supernatant was
centrifuged sequentially (CP100WX/CR22N Hitachi Tokyo Japan) at
300 g for 10 min at 2000 g for 20 min and at 10,000 g for 30 min. After
each centrifugation step the precipitate was removed. The EVs were then
precipitated by ultracentrifugation of the supernatant (70 min at
100,000 g). The precipitate was resuspended in PBS. The EVs were
passed through a 0.22 μm filter and stored at − 20 ◦ C until use. The
identification method of the extracted EVs is shown in the Supplemen­
tary File
2.5. Reverse transcription-quantitative polymerase chain reaction (RT-
qPCR) and western blot
We used RT-qPCR and western blot to detect the expression of the
relevant genes in cells and tissues (shown in the Supplementary File).
2.6. Dual-luciferase reporter assay
Starbase (http://starbase.sysu.edu.cn/) was utilized to predict miR-
106b binding sites to CDKN2B, and dual-luciferase assays were per­
formed to verify whether CDKN2B is a target of miR-106b. The CDKN2B
3′ UTR with binding sites were artificially synthesized and cloned into
pmirGLO vector (Promega, Madison, WI, USA) using endonuclease sites.
The complementary sequence mutation (MUT) of the seed sequence was
designed on the CDKN2B wild type (WT), and the target fragment was
subcloned into the pGL3-control vector by restriction endonuclease
digestion using T4 DNA ligase. The 293 T cells were co-transfected with
correctly sequenced luciferase reporter plasmids WT or MUT and miR-
106b mimic, respectively. The cells were lysed 48 h after transfection,
and luciferase activity was measured on a Luminometer TD-20/20
analyzer (model: E5311, Promega) using the Dual-Luciferase Reporter
Assay System kit (Promega).
2.7. Data analysis
SPSS 21.0 software (IBM Corp. Armonk, N.Y., USA) was applied for
the analysis of the data. The results are expressed as the means ± SD. All
the experiments were repeated at least three times. Comparisons be­
tween every two groups were analyzed using an unpaired t-test, while
comparisons among three groups were analyzed by one-way ANOVA.
The post hoc test was conducted using the Tukey’s in multiple com­
parisons. Statistical significance was defined as p < 0.05.
3. Results
3.1. miR-106b expresses poorly in PD mice
PD miRNA dataset GSE16658 from the GEO database was inquired
for analysis, and we found 49 significantly upregulated miRNAs and 19
significantly downregulated miRNAs (Fig. 1A). The heat map shows the
top 15 differentially expressed miRNAs (Fig. 1B), of which miR-106 is
the most pronounced one. For validation, we induced a PD mouse model
using MPTP and conducted RT-qPCR to detect the miR-106b expression
X. Bai et al. Neuroscience Letters 760 (2021) 136094

Fig. 1. miR-106b is poorly expressed in PD mice. A, 68 differentially expressed miRNAs in GSE16658 dataset in the GEO database; B, the first 15 differentially
expressed miRNAs in GSE16658 dataset where horizontal coordinates indicate sample numbers, vertical coordinates indicate gene names, each small square in the
figure indicates the expression of a gene in a sample, and the histogram on the top right is the color scale; C, miR-106b expression in hippocampal tissues of control
and PD mice by RT-qPCR. *p < 0.05.

in the hippocampal tissues of PD mice. The results showed that miR-
106b expression in the hippocampal tissues of PD mice was signifi­
cantly lower compared with the control mice (Fig. 1C).
3.2. Identification of EVs-miR-106b
We isolated MSC-derived EVs for identification, and TEM observa­
tion showed that the EVs released by MSC showed a cup-shaped or
spherical morphology (Fig. 2A). NTA analysis exhibited that EVs were
40–120 nm in diameter (Fig. 2B), and Western blot demonstrated that all
EVs from MSC expressed CD63, CD81 and TSG101 and did not express
Calnexin (Fig. 2C). After transfection of miR-106b mimic in MSC
(Fig. 2D), EVs released from MSC also showed high miR-106b expression
(Fig. 2E).
3.3. EVs-miR-106b promotes autophagy and inhibits neuronal apoptosis
in PD
Neurons from mice in the control and PD groups were extracted, and
the expression of MAP-2 protein, a marker of primary neurons, was
detected by immunofluorescence. The results showed that the positive
expression rate of MAP-2 in the primary neurons extracted from the
control and PD mice reached more than 95%, indicating that the
extracted neurons were of high purity and could be used for subsequent
experiments (Fig. 3A). Neurons in PD mice were treated with MSC-
derived EVs or EVs-miR-106b. CCK8 assay and flow cytometry exhibi­
ted that (Fig. 3B, C): compared with normal neurons, PD neuronal
viability was decreased and apoptosis was significantly increased;
relative to the PD + EVs group, neuronal survival was increased and
apoptosis was significantly decreased in the PD + EVs-miR-106b group.
Immunofluorescence detection showed that neuronal autophagy was
reduced in the PD group compared with the normal group, and over­
expression of miR-106b in EVs facilitated the neuronal autophagy

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Fig. 2. Identification of hUCMSC-EVs-miR-106b. A, observation of EVs morphology by TEM (bar = 100 nm); B, the size distribution of EVs determined by NTA; C,
detection of EVs-specific surface marker proteins determined by Western blot; D, miR-106b expression in MSCs after transfection with miR-106b mimic by RT-qPCR;
E, expression of miR-106b in MSC-EVs detected by RT-qPCR after transfection of MSCs with miR-106b mimic. All the experiments were repeated at least three times.
*#p < 0.05.

(Fig. 3D). Western blot results in Fig. 3E revealed that compared with
the normal neurons, the LC3II/LC3I ratio and Bcl-2 expression were
reduced, while Bax protein expression was enhanced in PD neurons. By
contrast, compared with the EVs treatment, LC3II/LC3I ratio and Bcl-2
expression were increased, whereas Bax protein expression was
decreased in PD neurons treated with EVs overexpressing miR-106b.
3.4. EVs-miR-106b promotes neuronal autophagy and alleviates neuronal
damage in PD mice
The mice were injected with EVs or EVs overexpressing miR-106b
after successful PD modeling. As shown in Fig. 4A, B, mice in the PD
group spent significantly more time in the pole test and had significantly

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X. Bai et al. Neuroscience Letters 760 (2021) 136094

Fig. 3. EVs-miR-106b promotes autophagy and inhibits apoptosis in PD neurons. A, immunofluorescence detection of MAP-2 protein expression in cells; B, neuronal
activity determined by CCK8 assay; C, detection of apoptosis rate of neurons tested by flow cytometry; D, LC3 protein expression in neurons examined by immu­
nofluorescence detection; E, the expression of autophagy-related proteins LC3II and LC3I, apoptosis-related proteins Bcl-2 and Bax in each group of neurons
examined by western blot. *#p < 0.05.

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X. Bai et al. Neuroscience Letters 760 (2021) 136094

A Control B Control
PD PD
PD + EVs PD + EVs
30 PD + EVs-NC 4 PD + EVs-NC
PD + EVs-miR-106b PD + EVs-miR-106b
The total time (s)

20
* 3

Score
# #
2

10
1
*

0 0
C

50 μm 50 μm 50 μm

Control PD PD + EVs

50 μm 50 μm

PD + EVs-NC PD + EVs-miR-106b
D Control
PD
PD + EVs
50 PD + EVs-NC
PD + EVs-miR-106b
Apoptosis rate (%)

40

30
*
20 #

10

0
Fig. 4. EVs-miR-106b affects behavioral changes and alleviates neuronal damage in PD mice. A, the time spent on pole climbing by each group of mice; B, the score
of each group of mice examined by swimming test; C, histopathological changes of hippocampal neurons in the brain of mice were observed by HE staining; D,
apoptosis of hippocampal neurons in mice examined by TUNEL assay. *#p < 0.05.

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X. Bai et al. Neuroscience Letters 760 (2021) 136094

lower scores in the swimming test versus control mice. Compared with
the PD + EVs group, mice in the PD + EVs-miR-106b group spent
significantly less time in the pole test and had higher scores in the
swimming test. The results of HE staining (Fig. 4C) showed that the
hippocampal neurons in control mice were evenly distributed with clear
nuclei structure; some neurons in PD and PD + EVs groups were swollen
with interstitial edema and shriveled nucleus. However, the hippo­
campal tissues in the PD + EVs-miR-106b group had a more intact
structure and clearer nuclei results. The TUNEL assay in Fig. 4D showed
that few tan positive granular cells were seen in the hippocampal tissues
of control mice, while there was an increase in hippocampal tan positive
cells in PD group, PD + EVs group, and PD + EVs-miR-106b group.
3.5. CDKN2B is a target gene of miR-106b
To investigate the mechanism of miR-106b affecting PD, Starbase
website demonstrated that CDKN2B shares a binding relation with miR-
106b (Fig. 5A). Therefore, we speculated that miR-106b may influence
PD progression by regulating the CDKN2B expression.
Dual-luciferase assay revealed that the fluorescence intensity of cells

Fig. 5. CDKN2B is a target gene of miR-106b. A, CDKN2B shares a specific binding relation to miR-106b predicted by starbase website; B, the target binding
relationship between miR-106b and CDKN2B verified by dual-luciferase assay; C, miR-106b and CDKN2B mRNA expression in each group of neurons by RT-qPCR; D,
CDKN2B protein expression in neurons in response to miR-106b mimic or mimic NC examined by western blot; E, CDKN2B protein expression in PD neurons co-
cultured with EVs-miR-106b mimic by western blot. *#p < 0.05.

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X. Bai et al. Neuroscience Letters 760 (2021) 136094

transfected with miR-106b mimic + CDKN2B-WT was significantly
decreased compared with those transfected with mimic-NC + CDKN2B-
WT (Fig. 5B). In addition, miR-106b mimic was transfected into
extracted normal neurons, and miR-106b and CDKN2B mRNA expres­
sion in cells were detected using RT-qPCR and CDKN2B levels were
detected by Western blot, and the results showed (Fig. 5C, D): compared
with the mimic NC treatment, miR-106b expression was upregulated
and CDKN2B mRNA and protein levels were significantly
downregulated after miR-106b mimic treatment. Western blot was
implemented to detect CDKN2B expression in neurons after PD
modeling, and the results showed that CDKN2B expression was highly
expressed in neurons upon PD modeling, and was significantly reduced
after EVs-miR-106b treatment (Fig. 5E).

Fig. 6. EVs-miR-106b inhibits CDKN2B to promote neuronal autophagy and suppress neuronal apoptosis in PD mice. Neurons extracted from PD mice treated with
EVs-miR-106b + oe-NC or EVs-miR-106b + oe-CDKN2B. A, miR-106b and CDKN2B mRNA expression in neurons by RT-qPCR; B, CDKN2B protein expression in PD
neurons by western blot; C, neuronal activity determined by CCK8 assay; D, detection of apoptosis rate of neurons tested by flow cytometry; E, LC3 protein expression
in neurons examined by immunofluorescence detection; F, the expression of autophagy-related proteins LC3II and LC3I, apoptosis-related proteins Bcl-2 and Bax in
each group of neurons examined by western blot. *p < 0.05.

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3.6. EVs-miR-106b inhibits CDKN2B to promote neuronal autophagy and
suppress neuronal apoptosis in PD mice
CDKN2B was then overexpressed in PD neurons treated with EVs-
miR-106b. RT-qPCR and western were carried out to detect expression
pattern changes, and the results showed that (Fig. 6A, B): compared with
the PD + EVs-miR-106b + oe-NC group, there was no significant dif­
ference in miR-106b expression in neurons of PD + EVs-miR-106b + oe-
CDKN2B group, while CDKN2B mRNA and protein expression were
significantly increased.
CCK8, immunofluorescence and flow cytometry were performed to
detect neuronal survival, autophagy, and apoptosis, respectively.
Compared with the PD + EVs-miR-106b + oe-NC group, the neuronal
survival rate of PD + EVs-miR-106b + oe-CDKN2B group decreased,
autophagy was alleviated, and the proportion of apoptotic cells was
significantly increased (Fig. 6C-E). Next, western blot was performed to
measure the expression of autophagy-related proteins LC3II and LC3I,
apoptosis-related proteins Bcl-2 and Bax. Fig. 6F exhibited that
compared with the PD + EVs-miR-106b + oe-NC group, the LC3II/LC3I
ratio and Bcl-2 protein were decreased and Bax protein expression was
increased in the PD + EVs-miR-106b + oe-CDKN2B group. In summary,
the EVs-miR-106b promotes autophagy and inhibits neuronal apoptosis
in PD neurons by targeting and negatively regulating CDKN2B.
3.7. EVs-miR-106b inhibits CDKN2B to alleviate neuronal damage in PD
mice
CDKN2B overexpression treatment also was performed on EVs-miR-
106b-treated mice after successful PD modeling to observe the behav­
ioral changes in mice. As shown in Fig. 7A, B, compared with the PD +
EVs-miR-106b + oe-NC group, mice in the PD + EVs-miR-106b + oe-
CDKN2B group spent significantly more time in the pole test, and had
significantly lower scores in the swimming test. The results of HE
staining (Fig. 7C) indicated that the neurons in the hippocampus of mice
with PD + EVs-miR-106b + oe-NC were more uniformly distributed, and
the nuclei were more clearly structured. However, some neurons in the
hippocampus of mice overexpressing CDKN2B were swollen, with
interstitial edema and shriveled nucleus. TUNEL assay results showed

A PD + EVs-miR-106b + oe-NC
B PD + EVs-miR-106b + oe-NC
PD + EVs-miR-106b + oe-CDKN2B PD + EVs-miR-106b + oe-CDKN2B

*
The total time (s)

25 2.5

20 2.0
Score

15 1.5
*
10 1.0

5 0.5

0 0.0
C

50 μm 50 μm

PD + EVs-miR-106b + oe-NC PD + EVs-miR-106b + oe-CDKN2B

D PD + EVs-miR-106b + oe-NC
PD + EVs-miR-106b + oe-CDKN2B
50
Apoptosis rate (%)

40 *
30

20

10

0
Fig. 7. EVs-miR-106b inhibits CDKN2B to alleviate neuronal damage in PD mice. PD mice were treated with EVs-miR-106b + oe-NC or EVs-miR-106b + oe-CDKN2B.
A, the time spent on pole climbing by each group of mice; B, the score of each group of mice examined by swimming test; C, histopathological changes of hip­
pocampal neurons in the brain of mice were observed by HE staining; D, apoptosis of hippocampal neurons in mice examined by TUNEL assay. *p < 0.05.

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X. Bai et al.
(Fig. 7D) that the level of apoptosis was significantly enhanced in the
hippocampal tissues of mice in the PD + EVs-miR-106b + oe-CDKN2B
group relative to the PD + EVs-miR-106b + oe-NC group. The above
finding indicates that the EVs-miR-106b ameliorates behavioral deficits
and pathological changes in PD mice by targeting and negatively regu­
lating the CDKN2B.
4. Discussion
In recent years, EVs derived from all cell-types have been under­
scored as mediators of PD which remains the second most frequent
neurodegenerative diseases worldwide [15]. Moreover, the regulation
of dopaminergic neuronal loss and autophagy is of great importance to
reveal the pathogenesis of PD [16,17]. Moreover, therapeutic strategies
that boost autophagy may be theoretically beneficial for PD [18]. The
current study aimed to investigate the effect of MSC-secreted EVs con­
taining miR-106b on autophagy and apoptosis of neurons in PD. MPTP, a
neurotoxin which recapitulates the neuropathology of PD, could be
specifically uptaken by dopaminergic neurons and targets the mito­
chondria of neurons, thus triggering loss of dopaminergic neurons in
animals [19]. Taken together, our findings revealed that MSC-derived
EV-miR-106b can promote autophagy and alleviate apoptosis of neu­
rons from MPTP-induced mice by downregulating CDKN2B to relieve
PD.
The capability of EVs to deliver proteins and genetic material be­
tween cells, including miRNAs, provides vital insights as to how EVs
may involve in pathological progression in PD [20–22]. More relevantly,
MSCs-released EVs have been proposed as a capable therapeutic tool
because it has been established that they can serve as biological nano­
particles with advantageous effects on various pathological conditions,
including PD [23]. Firstly, we discovered via a miRNA dataset
GSE16658 that miR-106b was downregulated in PD, which was further
proved in the hippocampal tissues of PD mice. In line with our study, the
expression of miR-106b was decreased in the temporal cortex of patients
with Alzheimer’s disease [24]. MAP-2 has been indicated to be specif­
ically expressed in neuronally differentiated cells, and has been formerly
applied as a sensitive and specific marker for neurons [25]. Therefore,
we characterized the extracted neurons from PD mice with MAP-2
expression using immunofluorescence staining. Subsequently, EVs
derived from MSC transfected with miR-106b were used for neuron co-
culture or mouse treatment. It was found that MSC-EVs rescued the
neuronal loss induced by MPTP and enhanced autophagy, while more
pronounced trends were observed in neurons treated with EV-miR-106b.
Pretreatment with EVs augmented 6-hydroxydopamine-stimulated SH-
SY5Y cells to proliferate and inhibit apoptosis by inducing autophagy
[26], which were largely in agreement with our finding. Next, we
revealed the neuroprotective role of EV-miR-106b in vivo, as evidenced
by increased LC3II/LC3I ratio and Bcl-2 expression, as well as decreased
Bax protein expression.
Furthermore, the current study also indicated that EV-miR-106b
promoted autophagy and inhibited apoptosis of neurons via down­
regulation of CDKN2B. CDKN2B was first predicted by Starbase to be a
putative target of miR-106b and validated through dual-luciferase re­
porter assay. CDKN2B was then identified to be overexpressed in neu­
rons extracted from PD mice, which was downregulated by miR-106b
overexpression. CDKN2B (also known as INK4B, p15), lies in band 9p21
and encodes a protein that encourages a G1-phase cell cycle arrest via
the suppression of cyclin-dependent kinase 4/6 [27]. CDKN2B was
identified by Chung et al. to share an association with the risk of PD or
Alzheimer’s disease and the severity of cognitive impairment in both
Caucasian and Asian populations [28]. Folch et al. also found that acute
exposure of rat cerebellar granule neurons to micromolar concentrations
of pentachlorophenol enhanced the transcriptional activity of genes
related to mitogenic response, including CDKN2B [29]. CDKN2B
knockdown has been reported to noticeably increase the axon outgrowth
in dorsal root ganglion neurons, indicating it might be a novel potential
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Neuroscience Letters 760 (2021) 136094
target during nerve repair in post-peripheral nerve injury [30]. Over­
expression of CDKN2B resulted in a reduction in retinal ganglion cell
viability in N-methyl-d-aspartate-injected retinas, and adeno-associated
virus type 2-CRISPR-Cas9-mediated knockout of CDKN2B attenuated N-
methyl-d-aspartate-induced retinal ganglion cell death [31]. However,
whether CDKN2B is also involved in the modulation of neuronal auto­
phagy remains largely unknown. The rescue experiments in vitro and in
vivo exhibited that CDKN2B repressed the neuronal autophagy and
augmented apoptosis.
5. Conclusion
In conclusion, our findings reveal that EV-miR-106b released from
MSC may relieve PD symptoms via inhibiting neuronal apoptosis and
enhancing autophagy. The downregulation of CDKN2B may be a key
downstream mechanism to mediate the role of EV-miR-106b in PD
development. Our study provided a possible novel strategy for the
treatment of PD. Further studies are still essential to verify our findings.
Funding
None.
Competing interests
The authors declare that there are no competing interests associated
with the manuscript.
CRediT authorship contribution statement
Xue Bai: Data curation, Conceptualization, Methodology, Visuali­
zation, Writing - original draft, Validation. Qiaoyun Dong: Writing -
original draft, Writing - review & editing, Formal analysis, Data cura­
tion, Validation. Li Zhao: Writing - original draft, Writing - review &
editing, Formal analysis, Data curation, Validation. Yan Yao: Data
curation, Supervision, Resources. Bo Wang: Visualization, Investigat,
ion, Resources, Formal analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.neulet.2021.136094.
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