Cystatin C as a potential therapeutic mediator against Parkinson’s disease via VEGF-induced angiogenesis and enhanced neuronal autophagy in neurovascular units
Citation: Cell Death and Disease
Cystatin C as a potential therapeutic mediator against Parkinson's disease via VEGF-induced angiogenesis and enhanced neuronal autophagy in neurovascular units
Jing Zou 0
Zhaoyu Chen 0
Xiaobo Wei 0
Zhigang Chen 0
Yongmei Fu 0
Xiaoyan Yang 0
Dan Chen 0
Rui Wang 0
Peter Jenner 0
Jia-Hong Lu 0
Min Li 0
Zhuohua Zhang 0
Beisha Tang 0
Kunlin Jin 0
Qing Wang 0
0 The role of cystatin C in the PD pathogenesis J Zou et al
Cystatin C (CYS C, Cst3) is an endogenous cysteine protease inhibitor that plays neuroprotective roles in neurodegenerative diseases. We aimed to explore the association of CYS C with Parkinson's disease (PD) models and investigate its involvement in the role of neurovascular units (NVUs) in PD neuro-pathogenesis. We used A53T α-synuclein (SNCA) transgenic mice and 6-hydroxydopamine-lesioned DAergic PC12 cells as experimental PD models to investigate the mechanisms behind this association. The injections of CYS C were administered to the right substantia nigra (SN) of A53T SNCA transgenic mice to measure the effects of CYS C in transgenic A53T SNCA mice. To explore the angiogenesis in vivo and in vitro, we used the chick embryo chorioallantoic membrane (CAM) assay and tube formation (TF) assay. We found that CYS C has a neuroprotective effect in this in vivo PD model. We observed increased VEGF, NURR1 and autophagy markers LC3B and decreased SNCA and apoptosis marker cleaved CASP3 in different brain regions of CYS C-treated A53T SNCA transgenic mice. In vitro, we observed that CYS C-induced VEGF, a secreted protein, attenuated 6-OHDA-lesioned DAergic PC12 cell degeneration by regulating p-PKC-α/pERK1/2-Nurr1 signaling and inducing autophagy. VEGF-mediated angiogenesis was markedly enhanced in the conditioned media of 6-OHDA-lesioned PC12 cells with CYS C-overexpression, whereas blockage of autophagy in CYS C-overexpressing PC12 cells significantly downregulated VEGF expression and the associated angiogenesis. Our data indicate that CYS C displays dual neuronal-vascular functions, promoting PC12 cell survival and angiogenesis via regulating the level of secreted VEGF in NVUs. Our study provides evidence that may aid in the development of an alternative approach for the treatment of PD through modulation of CYS C-mediated neuronal-vascular pathways. Cell Death and Disease (2017) 8, e2854; doi:10.1038/cddis.2017.240; published online 1 June 2017
Neural cells and vascular cells form a functionally integrated
network that is collectively termed the neurovascular units
(NVUs), which regulate important pathological functions in
neurodegenerative diseases such as Alzheimer’s disease
(AD) and Parkinson’s diseases (PD).1,2 Several lines of
evidence indicate that NVUs disruption, especially abnormal
neuronal-vascular relationships, play an important role in PD
pathogenesis.3,4 In the NVUs, some secreted molecules such
as vascular endothelial growth factors (VEGFs) are key
components that not only mediate neuronal survival but also
maintain vascular homeostasis and promote angiogenesis.5–7
In PD patients, increased VEGF in the CSF is associated
with blood–brain barrier (BBB) dysfunction and neural
degeneration.8 Several lines of evidence have also indicated
that the secreted molecule VEGF could regulate angiogenesis
and promote neuronal survival.9–11 On the other hand, recent
studies have shown that neuronal events such as autophagy
could regulate the cerebral microenvironment, leading to
disruption of the NVUs.12 Therefore, the neuronal–vascular
relationship is critical for cerebral functions in aging-related
diseases such as PD.
Cystatin C (CYS C), a secreted cysteine inhibitor encoded
by the CST3 (Cst3) gene, is a 13-kDa protein that consists of
120 amino acids encoded by a 7.3-kb gene located on
chromosome 20.13,14 It is commonly used as a biomarker of
renal function and is a strong predictor of cardiovascular
events and cerebral ischemia.15 Moreover, recent studies
have shown that CYS C has a neuroprotective role in
diseases, such as AD, amyotrophic lateral sclerosis (ALS)
and subarachnoid hemorrhage (SAH), by inducing cellular
autophagy.16,17 CYS C also exerts its function by regulating
vascular remodeling and integrity.18 Taken together, these
findings strongly suggest that CYS C could be a novel
secreted protein that induces cellular autophagy and
induces angiogenesis in the cerebral microenvironment.
Whether there is an association between CYS C and
VEGF and how this relationship influences
neurodegenerative diseases such as PD is an interesting topic to
In the present study, we therefore sought to determine: (
how VEGF/NURR1 levels and autophagy change in the brain
of CYS C-treated A53T α-synuclein (SNCA) mice, an in vivo
PD model; (
) in an in vitro study, whether CYS C exerts
neuronal–vascular dual functions in promoting DAergic PC12
cell survival and angiogenesis via regulating the secreted
protein VEGF in NVUs; (
) how CYS C-mediated enhanced
DAergic neuronal autophagy influences
VEGF-induced angiogenesis in NVUs.
In vivo study: increased VEGF, NURR1 and autophagy,
and decreased SNCA in CYS C-treated A53T SNCA
mice. We observed 5.8-fold and 5.5-fold increases in CYS
C expression, 3.75-fold and 3-fold increases in VEGF and
1.86-fold and 3.2-fold NURR1 among the striata and
substantia nigra (SN) of CYS C-treated A53T SNCA mice,
respectively, compared to A53T SNCA mice (***Po0.001,
CYS C-treated A53T versus A53T, n = 5; Figure 1A and B).
However, significant 3.1-fold (striatum) and 6.2-fold (SN)
decreases in Ser129-phosphorylated SNCA expression were
found in CYS C-treated A53T SNCA mice (***Po0.001, CYS
C-treated A53T versus A53T, n = 5; Figure 1A and B)
compared to A53T SNCA mice. The expression levels of
CYS C, VEGF, NURR1 and Ser129-phosphorylated
SNCA in the frontal cortex and hippocampus showed
similar trends to those in the striatum and SN (Figure 1A
LC3B and SQSTM1, robust markers of autophagosomes
and autophagy substrate, respectively, were used to measure
autophagic induction in A53T SNCA mice. Our data showed
significant 2.9-fold (frontal cortex), 3.53-fold (striatum), 1.5-fold
(hippocampus) and 3.2-fold (SN) decreases in the levels of
LC3B-II/LC3B-I expression in CYS C-treated A53T SNCA
mice compared to A53T SNCA mice (**Po0.01, ***Po0.001,
CYS C-treated A53T versus A53T, n = 5; Figure 1A and B). In
contrast, we observed that SQSTM1 decreased by 3.4-fold
(frontal cortex), 1.6-fold (striatum), 2.1-fold (hippocampus) and
4.3-fold (SN) in CYS C-treated A53T SNCA mice compared to
A53T SNCA mice (***Po0.001, CYS C-treated A53T versus
A53T, n = 5; Figure 1A and B).
Immunofluorescence with confocal microscopy in the frontal
cortex and SN indicated that CYS C co-localized with VEGF,
NURR1 and the autophagsome marker LC3B (Figure 1C–G).
Double labeling showed that the immunoreactivity of VEGF,
NURR1 and LC3B were stronger in CYS C-treated A53T
SNCA mice than in the A53T SNCA mice; while the
immunoreactivity of accumulated endogenous SNCA,
Ser129-phosphorylated SNCA, the apoptosis
markercleaved caspase-3 (cleaved CASP3) were lower in CYS
C-treated A53T SNCA mice than in the A53T SNCA mice
In vitro study: CYS C’s essential function in neurovascular interactions
CYS C knockdown enhanced apoptosis and inflammation in
6-OHDA-lesioned DAergic PC12 cells: CYS C levels were
increased in a time and dose-dependent manner in
6-OHDAlesioned PC12 cells, with a 24 h 6-OHDA (100 μM) incubation
producing the most obviously increase in CYS C release
compared to the other groups (Figure 2a). Therefore, we
chose to use this concentration in the subsequent
experiments. The total apoptotic values (Figures 2b and 4c,
Supplementary Figure 1) and the expression of TNF-α and
IL-1β were increased (Figure 2c), while the expression levels
of the DAergic PC12 cells markers DAT and TH were
significantly reduced (Figure 2d), in 6-OHDA-incubated
PC12 cells with Cst3 knockdown, compared to those cells
incubated with only 6-OHDA. These data suggest that CYS C
knockdown aggravates apoptosis and inflammation in
DAergic PC12 cells.
Overexpression of CYS C attenuated PC12 cell degeneration
by regulating VEGF-p-PKC-α/p-ERK1/2-NURR1 signaling:
NURR1 is an orphan nuclear receptor that has been
characterized as a transcription factor important for DAergic
neuron development.19 Recent studies have shown that
VEGF potently induces the expression of NURR1 in
endothelial cells.20 Interestingly, we found overexpression of
CYS C increases NURR1 and VEGF expression
(***Po0.001, n = 5; Figure 2d). In addition, 6-OHDA
incubation significantly decreased the density of cytosolic and
nuclear NURR1 protein, while VEGF overexpression
obviously restored the density of cytosolic and nuclear
NURR1 to normal control levels (*Po0.05, ***Po0.001,
n = 5; Figure 3b). These results indicate that VEGF may act
as a mediator between CYS C and downstream NURR1
expression. Previous reports also showed that Nurr1 can be
phosphorylated by the ERK1/2 and PKC signaling pathways
and translocate to the nucleus, where it is activated.21–23 Our
observations led us to explore whether VEGF exerts its
effects on NURR1 by regulating ERK and PKC signaling.
Following 6-OHDA incubation, knockdown of Vegf led to
significant (50% and 62%, respectively) decreases in the
expression of p-PKC-α and p-ERK1/2, as compared to
6-OHDA incubation alone (***Po0.001, n = 5; Figure 3a).
Following 6-OHDA incubation, U0126 (an inhibitor of
ERK1/2), GF109203 (an inhibitor of PKC), or SU5614 (an
inhibitor of VEGFR2/KDR) incubation significantly attenuated
this VEGF overexpression-induced restoration of nuclear
NURR1 protein expression, with SU5614 displaying the
most pronounced effect (*Po0.05, ***Po0.001, n = 5;
Figure 3b). These data suggested that VEGFR2/KDR
contributes the most to VEGF-regulated nuclear NURR1
expression, followed by the PKC pathway. When the
VEGFregulated nuclear NURR1 pathway was blocked, NURR1
translocated to the cytosol from the nucleus, leading to an
opposite trend in cytosolic NURR1 protein expression
compared with the nuclear NURR1 protein expression. These
results indicate that overexpression of CYS C in
6-OHDAlesioned DAergic PC12 cells profoundly attenuates PC12
cellular degeneration, probably through translocation of
NURR1 from the cytosol to the nucleus via VEGF-p-PKC-α/
Overexpression of CYS C in 6-OHDA-lesioned DAergic
PC12 cells attenuated PC12 cell degeneration by
upregulating VEGF-mediated autophagy: We observed that 6-OHDA
induced early autophagy (starting at 6 h) through
LC3B-II conversion and a significant increase in the LC3B-II
conversion rate was present at 12 h; however, 6-OHDA
then inhibited autophagy at 24 h of incubation (Figure 4A).
These results suggest that 6-OHDA treatment causes
a biphasic change in autophagy: it initially leads to
autophagic induction, followed by decreased autophagy
with 24 h of incubation (*Po0.05, **Po0.01, n = 5;
Figure 4A). This finding was consistent with the study by
In et al.24
To test whether CYS C regulated autophagy in
6-OHDAlesioned DAergic PC12 cells, we overexpressed CYS C in
6-OHDA-lesioned PC12 cells. CYS C overexpression
significantly unregulated the levels of LC3B-II/LC3B-I by
1.75-fold and downregulated the levels of SQSTM1 by
1.8-fold compared to cells without CYS C overexpression
(***Po0.001, n = 5; Figure 4B), while Cst3 knockdown yielded
the opposite trend in LC3B-II and SQSTM1 expression
(***Po0.001, n = 5, Figure 4B). Our immunofluorescence
findings showed results consistent with these findings for the
expression levels of LC3B and SQSTM1, as indicated in
Figure 4D and E. Furthermore, our transmission electron
microscopy (TEM) analysis demonstrated that overexpressing
CYS C in 6-OHDA-lesioned PC12 cells increased the number
of autophagic vesicles (Figure 4Fa and c). Taken together, our
data strongly indicates that overexpression of CYS C
upregulated autophagy in the 6-OHDA-lesioned PC12 model.
Recent studies have revealed an enrichment of
Ser129phosphorylated SNCA is found in Lewy bodies, suggesting
that Ser129 phosphorylation is involved in the pathogenesis of
PD.25 In addition, Ser129-phosphorylated SNCA/SNCA is
degraded mainly by the autophagy pathway.26,27 In
accordance with these findings, our western blot data showed that
CYS C overexpression significantly downregulated SNCA/
Ser129-phosphorylated SNCA levels (***Po0.001, n = 5;
Figures 2d and 5A); while 3-MA treatment (an autophagy
inhibitor) significantly reduced the number of autophagic
vesicles and attenuated the CYS C overexpression-induced
downregulation of SNCA/Ser129-phosphorylated SNCA
levels (***Po0.001, n = 5; Figures 4F and 5A). Strikingly, we
noted that the immunoreactivity of the autophagy marker
LC3B was discordant with that of
SNCA/Ser129phosphorylated SNCA levels under oxidative stress
conditions and the opposite pattern was observed with CYS C
overexpression (Figure 5B,C(n, o, v, w) and D). Therefore, we
reasonably concluded that CYS C promotes
6-OHDAlesioned DAergic PC12 cell survival by enhancing autophagic
clearance of SNCA aggregates.
The potential association among microvascular endothelial
cells, VEGF and autophagy has been previously
documented.28,29 To assess the effects of VEGF on CYS C
overexpression-mediated enhancement of autophagy, we
knocked down Vegf by lentivirus-mediated Vegf short hairpin
ribonucleic acid (shRNA) after overexpression of CYS C in
PC12 cells under oxidative stress conditions. Interestingly, we
observed that the levels of LC3B-II/LC3B-I in
6-OHDAincubated PC12 cells with Vegf knockdown after
overexpression of CYS C was significantly reduced by 33%, compared to
those with only CYS C overexpression (**Po0.01,
***Po0.001, n = 5; Figure 5E). The expressions of SQSTM1
and Ser129-phosphorylated SNCA were also significantly
increased by 1.2-fold and 1.16-fold, respectively (**Po0.01,
***Po0.001, n = 5; Figure 5e). Our data demonstrates that
VEGF mediates CYS C-induced autophagic clearance of
SNCA/Ser129-phosphorylated SNCA aggregates in
6-OHDAlesioned PC12 cells.
Conditioned media of 6-OHDA-lesioned, CYS
C-overexpressing DAergic PC12 cells induced VEGF-mediated
angiogenesis: In the current study, we found that VEGF may be a
downstream mediator of CYS C in 6-OHDA-lesioned PC12
cells. The results of an ELISA (Figure 6B) showed that VEGF
levels dramatically increased in the conditioned media of
6-OHDA-lesioned, CYS C-overexpressing PC12 cells. We
further identified the effect of this conditioned media on
angiogenesis in vitro (Figure 6A). When human umbilical vein
endothelial cells (HUVECs) were placed on Matrigel, robust
and elongated tube-like structures were formed after
incubation in the conditioned media from 6-OHDA-lesioned PC12
cells with CYS C-overexpression (Figure 6Af).
We next sought to examine whether CYS C could also
promote angiogenesis in vivo. As shown in Figure 6C,
angiogenesis was clearly observed in fertilized eggs after a
24 h of treatment with the conditioned media, and the group
treated with the conditioned media from 6-OHDA-lesioned
PC12 cells with CYS C-overexpression significantly promoted
the formation of branched blood vessels compared to the
untreated Chick Embryo Chorioallantoic Membrane (CAMs)
(Figure 6Cf). Based on these results, we conclude that, in both
in vitro and in vivo systems, VEGF expression and
VEGFmediated angiogenesis markedly increased upon exposure to
conditioned media of 6-OHDA-lesioned, CYS-C
overexpressing PC12 cells.
Blockage of autophagy in CYS C-overexpressing DAergic
PC12 cells treated with 6-OHDA reduced VEGF-induced
angiogenesis: To further confirm CYS C’s essential function
in neuro-vascular interactions in vitro, we blocked autophagy
in CYS C-overexpressing PC12 cells and examined whether
CYS C overexpression attenuated angiogenesis via
downregulating the levels of secreted VEGF. As shown in
Figure 6D, the tube formation rate in the group treated with
the conditioned media of CYS C-overexpressing PC12 cells
incubated with 3-MA and 6-OHDA was decreased to
56 ± 10.1% of the group without 3-MA treatment
(***Po0.001; Figure 6D(b and e)); our CAM assay findings
also showed that the conditioned media from CYS
C-overFigure 7 Schematic shows the signaling mechanisms underlying the –vascular dual functions of CYS C. CYS C exerts neuronal–vascular dual functions in promoting
neuronal survival and angiogenesis via regulating the secreted protein VEGF in NVUs. However, blockage of autophagy in CYS C-overexpressing DAergic PC12 cells
significantly aggravates neuronal degeneration by increasing SNCA aggregation and attenuates VEGF-mediated angiogenesis in NVUs
expressing PC12 cells incubated with 3-MA and 6-OHDA
significantly inhibited the formation of branched blood vessels
in vivo (***Po0.001; Figure 6D(d and f)). The ELISA results
(Figure 6G) demonstrated that VEGF levels dramatically
decreased in the conditioned media with 3-MA treatment.
These data suggested that blockage of autophagy in
6-OHDA-lesioned PC12 cells with CYS C-overexpression in
turn reduced the VEGF expression and subsequently
downregulated VEGF-induced angiogenesis both in vitro and
in vivo. Taken together, we confirmed that CYS C-induced
autophagy in DAergic PC12 cells had neuronal-vascular dual
functions, promoting PC12 cell survival and angiogenesis via
regulating the level of secreted VEGF protein.
CYS C has been previously documented to play important
roles in the pathogenesis of AD, vascular dementia (VaD) and
ALS.17,30,31 However, the exact mechanism still remains
unclear and needs extensive studies to explore. The current
study shows that CYS C is a potential mediator functioning to
induce angiogenesis and enhance cellular autophagy in the
NVUs of PD models. We obtained four principal findings in this
) we observed increased VEGF, NURR1 and
autophagy markers LC3B and decreased SNCA and
apoptosis marker cleaved CASP3 in different brain regions of CYS
C-treated A53T SNCA transgenic mice; (
) in an in vitro study,
we confirmed CYS C’s pivotal functions in the NVUs. In detail,
CYS C overexpression upregulated the levels of VEGF, while
CYS C-induced VEGF attenuated 6-OHDA-lesioned PC12
cell degeneration by regulating p-PKC-α/p-ERK1/2-Nurr1
signaling and inducing enhanced autophagy; (
) in the NVUs,
as a secreted protein, VEGF in the conditioned media of
6-OHDA-lesioned PC12 cells overexpressing CYS C
markedly increased angiogenesis. Interestingly, blockage of
autophagy by 3-MA in the CYS C-overexpressing PC12 cells
significantly decreased VEGF expression and
VEGFmediated angiogenesis. Taken together, we propose that
CYS C has neuronal–vascular dual functions, promoting
PC12 cell survival and angiogenesis, via regulating the levels
of secreted VEGF protein in the NVUs.
A53T SNCA transgenic mice were used here as an in vivo
PD model.32,33 Interestingly, the upregulated expression of
VEGF, NURR1 and autophagy markers LC3B, as well as
decreased Ser129-phosphorylated SNCA and apoptosis
marker cleaved CASP3 were observed in CYS C-treated
A53T SNCA transgenic mice. These findings strongly imply
that CYS C is involved in DA neuroprotection as indicated by
the upregulation of VEGF, NURR1 and downregulation of
Ser129-phosphorylated SNCA following CYS C injection into
the SN of A53T SNCA transgenic mice; while CYS
C-mediated neuroprotection is also associated with enhanced
autophagy, as shown by the upregulated LC3B and
downregulated SQSTM1 in the CYS C-treated A53T SNCA
transgenic mice. Moreover, we also observed that CYS C
colocalized with VEGF, NURR1 and autophagy markers in the
CYS C-treated A53T SNCA transgenic mouse brains,
including the frontal cortex and SN (Figure 1A–G). These findings
provide a hypothesis that CYS C may participate in NVU
activity via interacting with VEGF and autophagy pathways.
Based on our murine studies, we further investigated CYS
C’s functions in the NVUs in vitro. We found the
overexpression of CYS C increases VEGF expression and VEGF
overexpression significantly restored the 6-OHDA-mediated
downregulation of both nuclear and cytosolic Nurr1 proteins,
strongly indicating that VEGF may act as a mediator between
CYS C and downstream NURR1 expression and that the
upregulation of VEGF by CYS C overexpression might
promote DAergic neuronal survival. It has been well
documented that ERK and PKC signaling pathways are associated
with neuronal survival, and previous studies have suggested
that it is correlated with NURR1.34 Consistent with these
findings, our in vitro data verify (Figure 3a and b) that CYS
C-induced VEGF attenuated 6-OHDA-lesioned DAergic PC12
cells degeneration by regulating p-PKC-α/p-ERK1/2-Nurr1
signaling and inducing autophagy.
Our observation that 24 h of incubation with 6-OHDA inhibits
autophagy in PC12 cells is consistent with In et al.’s study.9
Recent studies have reported that SNCA is a crucial factor in
PD pathogenesis,33,35–37 and it is usually recognized as a
hallmark of PD. Its phosphorylation could accelerate PD
neurodegeneration,38 and the autophagy process could
prevent or reverse its phosphorylation.39,40 The current results
revealed that CYS C overexpression profoundly attenuated
the 6-OHDA-mediated increase in Ser129-phosphorylated
SNCA aggregation, and reversed these contra-directional
changes for LC3B-II/LC3B-I and SQSTM1, providing clear
evidence of the direct function of CYS C in the autophagic
clearance of SNCA aggregation in the in vitro PD model. This
enhanced autophagy and Ser129-phosphorylated SNCA
degradation induced by CYS C overexpression were
completely abolished by the autophagy inhibitor 3-MA; similar
findings were observed in our TEM data, further verifying the
enhancement in autophagy by CYS C overexpression. On the
other hand, Cst3 knockdown in 6-OHDA-lesioned PC12 cells
produced the opposite changes in LC3B-II/LC3B-I and
SQSTM1 levels compared to those with CYS C
overexpression, verifying this relationship. More importantly, we noted
that Vegf knockdown was able to partially attenuate the effects
of CYS C overexpression on enhanced autophagy and
Ser129-phosphorylated SNCA degradation under oxidative
stress, strongly implying that CYS C-induced VEGF
attenuates DAergic PC12 cell degeneration by enhancing
autophagic clearance of SNCA aggregates. These findings strongly
imply that CYS C promotes neuronal survival partially through
VEGF-mediated enhanced autophagy.
As a secreted protein, VEGF can exert its functions on both
neural cells and the surrounding cerebral microenvironment,
for example, by regulating vascular and neural differentiation,
proliferation and survival during development.9–11 To explore
the above hypothesis obtained from our in vivo studies, we
used the tube formation (TF) assay and CAM assay. The
conclusion that CYS C regulates angiogenesis is drawn from
the observations in the TF assays that the branch points of the
capillary-like structures markedly decreased with exposure to
PC12 cell-conditioned media incubated with 6-OHDA
compared to PC12 cell-conditioned media without 6-OHDA.
Furthermore, HUVECs developed more capillary-like
structures with exposure to conditioned media of PC12 cells
overexpressing CYS C, and it was noted that VEGF
expression was markedly increased in this conditioned media.
These results demonstrated that CYS C has positive effects
on VEGF-mediated angiogenesis in vitro. In addition, in the
CAM assay, usually recognized as an in vivo study, increased
branched vessel formation was observed with exposure to
conditioned media from 6-OHDA-lesioned PC12 cells
overexpressing CYS C compared to those without CYS C
overexpression. Consistent with previous studies showing that
angiogenesis was pivotal for neuron–vascular survival,11,41–44
our in vitro and in vivo findings indicate that CYS C exerts
angiogenesis functions via regulating the level of secreted
VEGF protein in the NVUs.
To further confirm CYS C’s function in neuro–vascular
interactions, we blocked autophagy in CYS C-overexpressed
DAergic PC12 cells and examined whether it attenuates
angiogenesis via regulating the level of secreted VEGF. It is
noteworthy that in the TF and CAM assay, the overexpression
of CYS C in 6-OHDA-lesioned PC12 cells upregulated the
level of VEGF and VEGF-induced angiogenesis; while
blockage of autophagy in 6-OHDA-lesioned PC12 cells
overexpressing CYS C downregulated the level of VEGF
and attenuated VEGF-mediated angiogenesis. Our findings
are consistent with the study by Poehler et al.,12 showing that
enhanced autophagy not only regulates secreted proteins but
also ameliorates the micro-environmental responses to
cellular damage. Taken together, we reasonably propose that
CYS C-induced autophagy in DAergic PC12 cells display
neuronal–vascular dual functions of promoting PC12 cell
survival and inducing angiogenesis via regulating the secreted
VEGF protein in the NVUs. As we know that NUVs consist of
multiple cell types such as neurons, endothelial cells,
astrocytes and microglia.1,2 In this study, we mainly focused
on neurons and endothelial cells. It is worthy of conducting
further in-depth studies in the future to explore the effects of
CYS C on other cell types in NUVs, that is, astrocytes and
In conclusion, our study demonstrates that CYS C displays
a neuroprotective effect in the A53T SNCA transgenic mice by
upregulating VEGF and autophagy and downregulating
a-synuclein and apoptosis. As shown in Figure 7, CYS
C-induced VEGF expression attenuated 6-OHDA-lesioned
DAergic PC12 cell degeneration by regulating
p-PKC-α/pERK1/2-NURR1 signaling and inducing autophagy (Figure 7).
VEGF-mediated angiogenesis was markedly enhanced upon
exposure to conditioned media from 6-OHDA-lesioned PC12
cells overexpressing CYS C. The blockage of autophagy in
CYS C-overexpressing DAergic PC12 cells significantly
downregulated secreted VEGF expression and subsequently
attenuated VEGF-mediated angiogenesis, strongly indicating
that CYS C-mediated enhanced neuronal autophagy plays an
important role in the NVUs. Importantly, we propose that CYS
C has the neuronal–vascular dual functions of promoting
PC12 cells survival and angiogenesis via regulating the level
of secreted VEGF protein in the NVUs. These in vitro and
in vivo findings suggest that CYS C could be used as a novel
angiogenesis target in clinical applications in PD. In addition,
our findings further confirm our hypothesis that CYS C
participates in NVU activity via interacting with the VEGF
and autophagy pathways. This study provides a clue for the
development of an alternative approach to the treatment of PD
through neuronal-vascular protection mediated by CYS C.
Materials and Methods
Investigation 1: how CYS C/VEGF levels and autophagy change
in A53T SNCA mouse brain tissues
Western blot analysis and immunofluorescence staining in A53T
SNCA mice and CYS C-treated A53T SNCA mice: Transgenic mice
expressing the mutant human A53T SNCA under the control of a prion promoter
(Prnp-SNCA*A53T),45 usually used as a transgenic PD mouse model, were
obtained from the State Key Laboratory of Medical Genetics of Central South
University (Changsha, China), and the wild-type littermates were used as the
controls. We certify that the mice in our study were carried out in accordance with
the National Institute of Health Guide for the Care and Use of Laboratory Animals
(NIH Publications No. 80–23) revised 1996 guidelines. The protocol was approved
by the Institutional Animal Care and Use Committee (Animal Ethic Approval No:
0014102402). We further attest that all efforts were made to minimize the number of
animals used and their suffering. The genotypes of all of the wild-type and A53T
SNCA transgenic mice were determined by polymerase chain reaction (PCR)
amplification analysis using tail DNA at three weeks of age and were verified at the
end of the experiment.46,47
All surgery was performed under Equithesin anesthesia (0.3 ml/100 g) and
adequate measures were taken to minimize pain or discomfort. The administration of
5 μg human cystatin C (Sigma, St. Louis, MO, USA) in 0.1% bovine serum albumin
(BSA) containing phosphate-buffered saline (PBS) or saline containing 0.1% BSA as
vehicle was performed. The animals was anesthetized and placed in a stereotaxic
frame (ASI Instrument, Warren, MI, USA) as described previously, and all injections
were using a 10-μl syringe at a rate of 0.4 μl/min. The injections of vehicle and
cystatin C were administered directly into the right SN using the following coordinates:
AP, +0.9 mm; L, ± 2.0 mm; and V, − 3.0 mm from skull.48–50 At the completion of
each injection, the needle was left in place for 5 min and then withdrawn at a rate of
1 mm/min. Four weeks after surgery, western blot and immunofluorescence staining
were performed according to the previously published protocols.51,52 Frontal cortex,
striatum, hippocampus and SN were selected. For additional details refer to the
Investigation 2: whether CYS C exerts neuronal–vascular dual
functions by influencing VEGF-mediated angiogenesis and
autophagy in NVUs in vitro
Cell culture and treatments: The 6-OHDA-lesioned DAergic PC12 cells have
been widely used as an in vitro PD model, since PC 12 cells could mimic the
pathological and biochemical characteristics of PD in vitro condition.53–56 They can
be used to define important cellular actors of cell death presumably critical for the
DAnergic degeneration. The PC12 cells were seeded in 96-well plates or 6-well
plates at a density of 1.0 × 105 cells/ml for 24 h. PC12 cells were subjected to
different concentrations of 6-OHDA (0, 10, 30, 50, 100 μM) for various time points
(0, 6, 12, 24 h). The released CYS C in the PC12 cells was analyzed by western
blot. For the measurement of the inflammatory mediators TNF-α and IL-1β, an
enzyme linked immunosorbent assay (ELISA, R&D Systems Inc., Minneapolis, MN,
USA) was performed at an absorbance of 570 nm with an ELISA plate reader. Each
treatment group was replicated in three wells. All of the results were normalized to
optical density (OD) values measured from an identically conditioned well without
cells. For enhancing or blocking Cst3 function, lentiviral vectors carrying Cst3 and
Cst3 siRNA oligonucleotides were added to 6-OHDA-incubated PC12 cells. In
contrast, for enhancing or blocking Vegf function, lentiviral vectors carrying Vegf and
lentivirus mediated Vegf shRNA (shVegf) were added to 6-OHDA-incubated PC12
Lentiviral vector construction and infection for the overexpression
of CYS C and VEGF and shRNA interference for the knockdown of
Vegf. Lentiviral vectors were used for the overexpression of CYS C and VEGF as
previously described with some modifications.57 Cst3, Vegf and green fluorescence
protein (GFP) cDNAs were cloned into the pRRL-cPPt-PGK-PreSIN vector. Lentivirus
mediated shVegf was cloned into p156RRL-sinPPT- CMV-GFP-PRE/NheI. The shRNA
design and sequences are available in the online data supplement. Viruses were
produced as described.58 PC12 cells were transduced for 24 h with recombinant
lentivirus at multiplicities of infection (MOIs) of 50 (overexpression of CYS C), 50
(overexpression of VEGF) and 100 (shVegf), in the presence of 10 μg/mL Polybrene.
After transduction, the cells were cultured in suspension for 72 h. Overexpression of
CYS C or VEGF was verified by flow cytometric analyses (GFP) and
immunofluorescence. Knockdown and transduction efficiency in the Vegf (shVegf)
constructs were verified by flow cytometric analysis (GFP) and were confirmed by
RT-PCR and immunofluorescence.
Construction of the Cst3 siRNA sequence and its transfection
into PC12 cells. Two Cst3 siRNA oligonucleotides were purchased and
identified using the primers S1 (5′-CCATACAGGTGGTGAGAGCTCdTdT-3′) and
S2 (5′-GTACCAAGTCCCAGACAAATTdTdT-3′). The negative control sequence
(Sn, UUCUCCGAAC GUGUCACGUUUGUGC) was formulated and synthesized.
Each sequence (100 nM) was transfected into the PC12 cell line (1 × 105 cells/ml)
using the oligofectamine liposome. The cells were divided into four groups: blank
control, negative control, S1 transfection (S1) and S2 transfection (S2). There were
no differences in the treatments of each group, with the exception that the blank
control and negative control were transfected with PBS and empty vector,
respectively, at the same working concentrations and volumes. Only the most
effective siRNA was used in the subsequent studies.
Protein extraction, subcellular fractionation and western blot
analysis of 6-OHDA-lesioned PC12 cells. The cells were harvested
using cell scrapers, washed in ice-cold PBS, and lysed with two different ice-cold
lysis buffers. The supernatants were collected for protein determination by BCA
assay (Pierce, Inc., Rockford, IL, USA), and the proteins were run on NuPage
Bis-Tris 10% gels (Invitrogen, Waltham, MA, USA) and transferred to PVDF
membranes (Amersham Bioscience, Ltd., Buckinghamshire, UK). The membranes
were blocked in 5% skim milk, 0.05% Tween 20, and Tris-buffered saline (TBS) for
1 h. The PVDF membranes were incubated in the primary antibodies overnight at
4 °C. For detailed antibody information, please refer to the Supplementary
Information. The next day, horseradish peroxidase-conjugated secondary antibodies
(Cell Signaling, Danvers, MA, USA) were applied. Peroxidase-conjugated
streptavidin and substrate were used for detection. Negative controls were
prepared by omitting the primary antibodies. For the protein extractions prepared
from the cytosolic and nuclear fractions, the method described by Garcia-Yagüe
et al.59 was used. The images were analyzed using NIH Image J software
(Bethesda, MD, USA). For additional details, please refer to the Supplementary
Immunofluorescence in 6-OHDA-lesioned PC12 cells. For
immunofluorescence analysis, previously describe methods were employed,60–62 with some
modifications. Briefly, 1 × 105 cells/ml from the different experimental groups were
plated on confocal Petri dishes in serum-containing media for 24 h. The cells were
then incubated in conditioned media alone, 6-OHDA (100 μM) alone, 6-OHDA
(100 μM)+Cst3 knockdown or overexpression of CYS C and 6-OHDA (100 μM)
+Vegf knockdown or overexpression of VEGF, before staining for
immunofluorescence. For additional details, please refer to the Supplementary Information.
In vitro TF assay. The procedure for the in vitro capillary-like TF assay was
performed as presented in the study by Fang et al.,63 with some modifications.
Briefly, Matrigel (356231, BD Bioscience, San Jose, CA, USA) was used to coat
culture plates according to the manufacturer’s instructions. Thawed Matrigel at a
volume of 150 ml was applied to each well of 24-multiwell plates and was
polymerized at 37 °C for 1 h. HUVECs were cultured in the presence of 90% PC12
cell-conditioned media from (
) PC12 cells without transfection; (
) PC12 cells
transfected with NC; (
) PC12 cells overexpressing CYS C; (
PC12 cells without transfection; (
) 6-OHDA-lesioned PC12 cells transfected with
) 6-OHDA-lesioned PC12 cells overexpressing CYS C; and (
C-overexpressing PC12 cells incubated with 3-MA and 6-OHDA. Phase contrast
images were taken after 12 h. The tube branching was photographed with
invertedphase contrast microscopy, and the number of tube branching points per field was
quantified using Image J software. Six fields under × 200 magnification were
randomly selected for each well. The results are expressed as the mean ± standard
deviation of the mean from five independent experiments.
CAM assay. The procedure for the CAM assay was performed as described in
the study by Wang et al.,64 with modifications. The fertilized chicken eggs were
placed in an incubator upon embryogenesis and maintained under constant
humidity at 37 °C. On day 8, a square window was opened in the shell after
removing 2–3 ml of albumen to detach the CAM from the shell. Substances treated
with the compounds being tested were added to the detached CAM that contained
PC12 cell-conditioned media from the groups indicated in the experiment for the
capillary-like tubular formation assay. The window was sealed with parafilm and
incubated for an additional 24 h. After the second incubation, the CAM arteriosus
branches in each treatment group were photographed. The angiogenic effect of
CYS C overexpression was indicated by the relative numbers of arteriosus
branches. The assay was performed five times to ensure reproducibility.
TEM. TEM was performed for the visualization and quantitation of autophagic
vacuoles.65,66 Cells were fixed in 2.5% glutaraldehyde in 0.1 M PBS buffer at 4 °C
overnight, and then post-fixed with 1% osmium tetroxide at room temperature for
2 h. After dehydration in a graded series of acetone, the cells were embedded in
Epon 812 resin. Ultrathin (60 nm) sections were collected on 200 mesh copper
grids, stained with 2% uranyl acetate in 50% methanol for 10 min, followed by 1%
lead citrate for 7 min. Subsequently, the sections were stained with uranyl acetate
and lead citrate and examined with a transmission electron microscope (Hitachi
H-7650, Chiyoda, Tokyo, Japan).
Statistical analysis. For the in vitro and in vivo studies, the data are
expressed as the mean ± S.E.M. The data related to the human continuous
variables, ELISA and flow cytometry analyses, and the different protein
quantifications by western blot were analyzed using one-way analysis of variance
(ANOVA) followed by Bonferroni’s comparison post hoc analysis (SPSS 15.0
program, Chicago, IL, USA). Differences with P values of less than 0.05 are
regarded as statistically significant.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. We thank Professor Raymond A. Swanson (Department
of Neurology, UCSF) and Professor Mark Hallett (Human Motor Control Section,
NINDS, NIH) for their comments on the discussion and critical review of the
manuscript. Neither contributor received any compensation for their contributions.
This work was supported by the National Natural Science Foundation of China (Grant
No: 81271427, 81471291), 973 Project (2011CB510000), Natural Science
Foundations of Guangdong of China (2014A020212068), Science and Technology
Program of Guangdong of China (2016A050502019), Fundamental Research Funds
for the Central Universities (Grant No.: 16ykjc22), and Scientific Research Foundation
of Guangzhou (Grant No.: 2014J4100210) to QW. ZZ and BT were supported by 973
JZ, ZC, DC, XW, ZGC and QW conceived and designed the experiments. JZ, ZC, RW,
YF, XY, BH, RY, BH and QW performed the experiments. JZ, XW, ZC, DC, RW, YF and
QW analyzed the data. BT and ZHZ contributed reagents/materials/analysis tools. JP,
ML, JHL, JK, ZHZ and BT revised the paper for intellectual content. JZ, ZC, JK and
QW wrote the paper. All authors read and approved the final manuscript.
The animal study protocol was approved by the Institutional Animal Care and Use
Committee (Animal Ethic Approval No: 0014102402).
Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis)
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