adenosine 50-triphosphate (atp) inhibits schwann cell demyelination during wallerian degeneration
TRANSCRIPT
ORIGINAL RESEARCH
Adenosine 50-Triphosphate (ATP) Inhibits Schwann CellDemyelination During Wallerian Degeneration
Youn Ho Shin • Hyung-Joo Chung •
Chan Park • Junyang Jung • Na Young Jeong
Received: 7 November 2013 / Accepted: 10 December 2013 / Published online: 23 December 2013
� Springer Science+Business Media New York 2013
Abstract Adenosine 50-triphosphate (ATP) is implicated
in intercellular communication as a neurotransmitter in the
peripheral nervous system. In addition, ATP is known as
lysosomal exocytosis activator. In this study, we investi-
gated the role of extracellular ATP on demyelination dur-
ing Wallerian degeneration (WD) using ex vivo and in vivo
nerve degeneration models. We found that extracellular
ATP inhibited myelin fragmentation and axonal degrada-
tion during WD. Furthermore, metformin and chlorprom-
azine, lysosomal exocytosis antagonists blocked the effect
of ATP on the inhibition of demyelination. Thus, these
findings indicate that ATP-induced-lysosomal exocytosis
may be involved in demyelination during WD.
Keywords ATP � Schwann cell � Lysosomal
exocytosis � Demyelination � Wallerian degeneration
Introduction
Wallerian degeneration (WD) following nerve injury
involves degradation of the myelin sheath. Demyelination
of Schwann cells during WD occurs through fragmentation
of the myelin sheath into ovoid-like structures near
Schmidt–Lanterman incisures (SLI) (Ghabriel and Allt
1979a, b; Webster 1965). Recent evidence indicates that
changes in actin polymerization through Rac1 activation in
an SLI are essential for the initiation of the myelin sheath
fragmentation near the SLI after nerve injury (Jung et al.
2011b). Previous studies reported that such extracellular
signals (e.g., neuregulin, a neuronal signal) are involved in
regulation of SLI actin dynamics and in the initiation of
WD (Jung et al. 2011b; Guertin et al. 2005). However, the
underlying initiation mechanism of the demyelinating
phenomenon in Schwann cells during WD through extra-
cellular signaling is unknown.
Adenosine triphosphate (ATP) is an essential neuro-
transmitter in the nervous system (Ralevic and Burnstock
1998). In the peripheral nervous system, nerve stimulation
increases the extracellular ATP level, and the secretion of
ATP from peripheral nerves involves the communication
between Schwann cells or neurons and Schwann cells
(Jung et al. 2013; Grafe et al. 2006; Holton 1959; Stevens
and Fields 2000). In addition, after nerve injury, ATP
released from premyelinated axons inhibits the prolifera-
tion and differentiation of developing Schwann cells in
in vitro (Stevens and Fields 2000), and extracellular ATP
inhibits dedifferentiation of Schwann cells during WD in
ex vivo models (Shin et al. 2013). Thus, ATP as an
extracellular signal seems to be involved in regulating
myelination after nerve injury.
In previous studies, ATP triggered the release of
vesicular contents through Ca2?-dependent exocytosis (Liu
et al. 2005; Jeftinija and Jeftinija 1998; Ansselin et al.
1997). Extracellular ATP is involved in the secretion of
ATP from Schwann cells through lysosomal exocytosis
during WD (Shin et al. 2012). Because Schwann cells are
Y. H. Shin � C. Park � J. Jung (&)
Department of Anatomy and Neurobiology, School of Medicine,
Biomedical Science Institute, Kyung Hee University, Heogi-
Dong 1, Dongdaemun-Gu, Seoul 130-701, Republic of Korea
e-mail: [email protected]
H.-J. Chung
Department of Anesthesiology and Pain Medicine, Kosin
University College of Medicine, 34 Amnam-dong, Seo-gu,
Busan 602-703, Republic of Korea
N. Y. Jeong (&)
Department of Anatomy and Cell Biology, College of Medicine,
Dong-A University, Busan 602-714, Republic of Korea
e-mail: [email protected]
123
Cell Mol Neurobiol (2014) 34:361–368
DOI 10.1007/s10571-013-0020-y
affected by lysosomal dynamics during WD (Jung et al.
2011a; Shin et al. 2012, 2013), ATP-induced lysosomal
exocytosis may be involved in ovoid formation and myelin
degradation in Schwann cells during WD. Thus, in this
study, we determined the effects of extracellular ATP on
Schwann cell demyelination and then assessed the rela-
tionship between lysosomal exocytosis and Schwann cell
demyelination in during WD.
Materials and Methods
Materials
The primary antibody used for immunostaining and wes-
tern blotting was raised against myelin protein zero (P0,
Santa Cruz Biotechnology, Santa Cruz, California, USA).
Neurofilament was obtained from Chemicon (Temecula,
California, USA). Alexa Fluor 594-conjugated secondary
antibody was purchased from Life Technologies (Grand
Island, New York, USA). Adenosine triphosphate, met-
formin (Met), vacuolin-1, bafilomycin A1, ammonium
chloride (NH4Cl), potassium cyanide (KCN), nocodazole,
and chlorpromazine (CP) were obtained from Sigma-
Aldrich Co. (St. Louis, Missouri, USA).
Animals and Surgical Procedures
All of the procedures were performed according to proto-
cols approved by the Kyung Hee University Committee on
Animal Research, which followed the guidelines of animal
experimentation established by The Korean Academy of
Medical Science. Adult male C57BL/6 mice (7 weeks old)
were anesthetized by an intraperitoneal injection of pen-
tobarbital sodium (50 mg/kg), and their sciatic nerves were
exposed mid-thigh. The sciatic nerves were cut 5 mm
proximal to the tibioperoneal bifurcation using fine iris
scissors (FST, Foster City, California, USA).
Explant Culture
Sciatic nerve explant cultures were performed according to
a previous study (Thomson et al. 1993). Briefly, the sciatic
nerves from adult C57BL/6 mice (7 weeks old, Samtako,
Osan, Korea) were removed, and the connective tissues
surrounding the nerves were detached under a stereomi-
croscope. The sciatic nerves were cut into three or four
explants of 3–4 mm in length. The explants were incubated
in Dulbecco’s modified Eagle’s medium containing
100 units/mL penicillin, 100 lg/mL streptomycin, 10 %
(vol/vol) heat-inactivated fetal bovine serum, and 2 mmol/
L L-glutamine. The cultures were maintained at 37 �C in a
humidified atmosphere containing 5 % CO2.
In Vivo Treatment
Adenosine triphosphate application in vivo was performed
as reported previously (Shin et al. 2013). Briefly, to iden-
tify the effect of ATP on demyelination in Schwann cells
during WD, the lesion site of the distal stump of the sciatic
nerve was inserted into a blind PVC tube (10 mm) that was
packed with gelfoam presoaked in 4 mM ATP (10 lL;
Fig. 4a). The tube was fixed by suturing it to nearby
muscles (Fig. 4a). Three days after sciatic nerve axotomy,
sciatic nerves with and without the ATP treatment were
removed and fixed with 4 % paraformaldehyde (PFA)
overnight.
Western Blot Analysis
For western blot analysis, cultured sciatic explants were
prepared with a modified sradioimmunoprecipitation assay
buffer [RIPA; 50 mm/L Tris–HCl (pH 7.4), 150 mmol/L
NaCl, 0.5 % deoxycholic acid, 0.5 % Triton X-100,
1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L
sodium o-vanadate, and 19 protease inhibitor mixture
(Roche Molecular Biochemicals, Nutley, New Jersey,
USA). Protein extracts were separated using 10 % sodium
dodecyl sulfate polyacrylamide gel electrophoresis and
transferred electrophoretically to a nitrocellulose mem-
brane (Amersham Bioscience, Piscataway, New Jersey,
USA). The blotted membranes were blocked with 5 % non-
fat milk in Tris-buffered saline containing 0.1 % Tween-20
(TBST) at room temperature (RT) for 1 h and then incu-
bated with primary antibodies diluted (1:1,000) in TBST
containing 3 % non-fat milk at 4 �C overnight. After three
washes in TBST, the blots were reacted with horseradish
peroxidase-conjugated secondary antibodies (1:3,000; Cell
signaling Technology, Beverly, Massachusetts, USA) for
1 h at RT and then washed again with TBST. Detection
was performed using an enhanced chemiluminescence-
Western blot system (Amersham Biosciences, Piscataway,
New Jersey, USA). For quantification, the X-ray films were
then scanned using a Samsung scanner and analyzed with
the LAS image analysis system (Fujifilm, Tokyo, Japan).
All of the experiments were repeated a minimum of three
times.
Myelin Ovoid Index
The myelin ovoid index calculation was performed as
reported previously by (Jung et al. 2011b). Briefly the
362 Cell Mol Neurobiol (2014) 34:361–368
123
myelin ovoid index is the number of myelin ovoids counted
from medium to large teased nerve fibers of 200 lm in
length under a Zeiss Axioimager upright microscope
equipped for differential interference contrast (DIC).
Sciatic Nerve Teasing and Immunofluorescent Labeling
Cultured sciatic explants or pieces of removed sciatic
nerve after axotomy were fixed with 4 % PFA for 6–12 h
and teased into single or several nerve fibers under a
stereomicroscope. The teased nerve fibers and longitudinal
section slides were blocked with phosphate-buffered sal-
ine (PBS) containing 0.3 % Triton X-100 and 10 %
bovine serum albumin for 1 h at RT. Tissue slides were
then incubated overnight with primary antibodies
(1:1,000) in PBS containing 0.3 % Triton X-100 at 4 �C
and washed three times with PBS. The tissue slides were
incubated with Alexa 594-conjugated anti-goat (1:1,000)
or anti-mouse IgG (1:1,000) for 2 h at RT. The slides
were washed three times with PBS; coverslips were
adhered to the slides with mounting medium (Gelmount,
Biomeda, Foster City, California, USA), and visualized
using a laser confocal microscope (LSM510, Carl Zeiss,
Oberkochen, Germany).
Statistical Analysis
The means of the collected data were determined for each
experimental group. The statistically significant differences
between the groups were tested by Student’s t-tests unless
otherwise noted in the text, p values\0.01 were considered
significant.
Results
As reported previously, ex vivo culture of sciatic nerve
explants displayed similar myelin sheath pattern degradation
in vivo (Lee et al. 2009). In ex vivo culture for 3 days
(3DIV), the transverse stripes of the intact sciatic nerves,
which were distinctive by stereomicroscopy, disappeared
during WD. These structures in sciatic nerves may be a
cluster of nodes of Ranvier and the clusters may create band-
like structures. We think that the gray tone of the transverse
stripes indicates nodes of Ranvier, and white regions indi-
cate myelin sheaths (internode) in Schwann cells (Fig. 1b).
Because the length of an internode (a portion between
nodes of Ranvier) is approximately 100–200 lm in Schw-
ann cells (Friede et al. 1981), the stripes could be observed
grossly by optical microscopy. To determine the relationship
between lysosomal exocytosis and myelination during
WD, we screened *10 drugs that possibly inhibited the
disappearance of the transverse stripes in ex vivo explant
cultures (Fig. 1a). In Fig. 1a, (???) indicates that the
number of the transverse stripes counted from a sciatic
explant of 2 lm in length is [10. Additionally, remaining
degrees, (??), (?), and (-), indicate 10 C (??) [ 5,
5 C (?) [ 0, and (-) = 0, respectively. We examined
whether lysosomal exocytosis agonists (i.e., bafilomycin A1,
zymosan, NH4Cl, KCN and ATP) (Tapper and Sundler
1995; Riches et al. 1983; Shin et al. 2012; Zhang et al. 2007)
could inhibit the disappearance of transverse stripes of the
intact sciatic nerves, indicating a possible marker of nerve
degeneration, ATP (2 mM) almost completely suppressed
the loss of transverse stripes and acquisition of the fatty
appearance of sciatic nerve explants at 3DIV (Fig. 1a, b).
Next, we examined lysosomal exocytosis antagonists (met-
formin; Met, chlorpromazine; CP, vaculonin-1, and noco-
dazole) (Elferink 1979; Labuzek et al. 2010; Huynh and
Andrews 2005; Rodrıguez et al. 1999). We found that ATP-
treated explants showed the maintenance of transverse
stripes (92.7 %; of 28 sciatic explants, 26 was given ???).
Percentage of ATP/vacuolin-1 and ATP/nocodazole-treated
explants which were given ??? were 90.3 % (28 of 31
sciatic explants) and 96.3 % (26 of 27 sciatic explants),
respectively. Thus, we found that Met (500 lM) and CP
(30 lM) combined with ATP treatment resulted in the dis-
appearance of transverse stripes of intact sciatic nerves
(Fig. 1a, b).
To determine whether extracellular ATP is involved in
myelin sheath fragmentation, we analyzed morphological
changes of sciatic nerve fibers using ex vivo explant cul-
ture. In the ex vivo culture, myelin ovoid formation was
accompanied by WD progression (Jung et al. 2011b; Lee
et al. 2009). At 3DIV, the mean number of myelin ovoids
in a 200 lm length of myelinated nerve fibers was
12.2 ± 3.2. However, ATP (2 mM) treatment resulted in
significant inhibition of myelin ovoid formation
(1.75 ± 1.4) (Fig. 2a, b), suggesting that extracellular ATP
is involved in myelin ovoid formation. Because ATP is a
lysosomal exocytosis agonist, to examine the involvement
of lysosomal exocytosis in myelin sheath fragmentation
directly, we tested myelin ovoid formation of sciatic nerve
fibers using two drugs that effectively inhibit the disap-
pearance of transverse stripes in the screening, Met, which
acidifies lysosomal compartments (Labuzek et al. 2010),
and CP, which competes with Ca2? for membrane binding
sites (Elferink 1979). The blockage of lysosomal exocy-
tosis with Met or CP in the presence of ATP restored the
appearance of myelin ovoids (10.8 ± 1.6 or 11.3 ± 4.1),
similar to the nerve fibers at 3DIV with no treatment
(Fig. 2a, b). Immunostaining against myelin basic protein
(MBP) showed that the control revealed intact myelin
sheaths, whereas the samples at 3DIV showed decreased
signals and ovoids or clustered structures (Fig. 2c). We
Cell Mol Neurobiol (2014) 34:361–368 363
123
also found that the intact myelin-like MBP staining was
significantly preserved in NEM-treated nerves, compared
with that of the explants at 3DIV (Fig. 2c). Taken together,
these findings indicate that extracellular ATP may protect
the myelin sheath from degradation.
Maintenance of nerve fiber myelin sheath by lysosomal
exocytosis led us to examine whether lysosomal exocy-
tosis is involved in axonal degeneration during WD. To
determine the involvement of extracellular ATP in axonal
degradation during WD, we used neurofilament (NF) as
an axonal marker. Interestingly, we found that ATP
treatment significantly delayed axonal degeneration in
sciatic nerves (Fig. 1a). We also found that Met blocked
the effect of ATP on the inhibition of axonal degradation,
suggesting the involvement of lysosomal exocytosis
through extracellular ATP in degeneration (Fig. 3a).
Quantitative results of axonal degradation (NF index) also
showed the effect of ATP on the inhibition of axonal
degradation and the effect of Met on the restoration of
axonal degradation (Fig. 3b). In addition, we confirmed
the effect of extracellular ATP on delaying myelin
degradation; western blotting against myelin protein zero
(P0), revealing that myelin degradation was inhibited in
the ATP-treated nerve fibers during ex vivo culture
(Fig. 3c, d). Thus, these findings suggest that extracellular
ATP induces the inhibition of myelin degradation and
axonal degeneration.
Lastly, to confirm the effect of extracellular ATP on
myelin fragmentation and degradation in vivo, we applied
ATP to the distal stump of axotomized nerves for 3 days
and performed immunofluorescent labeling of the teased
nerve fibers (Fig. 4a). We next confirmed the results by
means of differential interference microscopy imaging and
immunofluorescent staining for P0 (Fig. 4b). The applica-
tion of ATP to the axotomized sciatic nerves induced the
preservation of P0 immunostaining and the inhibition of
ovoid formation (Fig. 4b) in vivo. The numbers of myelin
ovoids were 1.7 ± 0.4, 13.4 ± 3.7, and 5.8 ± 2.5 for the
control, non-ATP-treated, and ATP-treated groups,
respectively (Fig. 4c). These results suggest that extracel-
lular ATP blocks myelin ovoid formation and myelin
degradation in vivo during WD.
Fig. 1 Involvement of
lysosomal exocytosis in the
disappearance of transverse
stripes. a Table shows the effect
of nine drugs on the
disappearance of intact sciatic
nerve transverse stripes in
ex vivo Wallerian degeneration.
??? : Strong inhibition.
- : No effect. b Sciatic nerve
explants were cultured for
3 days (3DIV) in the absence or
presence of ATP (2 mM) or
metformin (Met, 500 lM), and
then the explants were
photographed under a
stereomicroscope
364 Cell Mol Neurobiol (2014) 34:361–368
123
Discussion
ATP is critically involved in several fundamentally
important aspects of the peripheral nervous system (PNS)
as a neurotransmitter. In this study, we focused specifically
on the potential role of extracellular ATP in the regulation
of myelin fragmentation and degradation during WD. By
treating axotomized sciatic nerves with ATP, we were able
to show that an increase in extracellular ATP in environ-
ment around sciatic nerves delayed myelin fragmentation
and degradation in vivo (Fig. 4b, c). Furthermore, we
found that the effect of ATP on demyelination may be
involved in lysosomal exocytosis (Fig. 1). These findings
indicate that the regulation of extracellular ATP level in the
PNS may affect the process of Schwann cell demyelination
during WD.
Previous studies demonstrated that ATP is released from
peripheral nerves and Schwann cells after nerve injury and
in response to several stimuli (Jung et al. 2013; Grafe et al.
2006; Liu et al. 2005; Liu and Bennett 2003; Lazarowski
et al. 2003; Shin et al. 2012). This extracellular ATP is an
essential, activity-dependent axonal signal that inhibits the
proliferation and differentiation of Schwann cells during
Schwann cell development (Stevens and Fields 2000) and
dedifferentiation of Schwann cells after nerve injury (Shin
et al. 2013; Jessen and Mirsky 2008). Therefore, previous
studies showed that ATP as an extracellular signal is an
essential factor involved in Schwann cell dynamics.
Here, we propose that lysosomal exocytosis may be
involved in demyelination of Schwann cells during WD.
Extracellular ATP increases intracellular Ca2? in Schwann
cells and leads to increases in the lysosomal pH, resulting in
lysosomal exocytosis (Ansselin et al. 1997; Takenouchi et al.
2009). We found that the increased ATP in ex vivo and
in vivo models inhibited myelin degradation during WD. In
addition, in the presence of ATP in ex vivo culture, Met and
CP, as lysosomal exocytosis antagonists, reversed the effect
of ATP on the inhibition of myelin ovoid formation and
myelin degradation (Figs. 1 and 2). Thus, these findings
suggest that extracellular ATP-induced-lysosomal exocy-
tosis in Schwann cells may be involved in demyelination
during WD. During WD, LAMP1 is increased in injured
Fig. 2 Extracellular ATP
inhibits myelin sheath
fragmentation. a Differential
interference microscopy of
teased nerve fibers for 3DIV in
the absence or presence of ATP.
Scale bar 100 lm.
b Quantitative result of the
myelin ovoid index showing the
effect of ATP (2 mM), Met
(metformin, 500 lM), and CP
(chlorpromazine, 30 lM) on
myelin fragmentation. Three
mice were used for each ovoid
experiment. Each group
contained 18–24 sciatic
explants. c Teased nerve fibers
were immunostained with anti-
MBP antibody (green). Scale
bar 200 lm (Color figure
online)
Cell Mol Neurobiol (2014) 34:361–368 365
123
sciatic nerves; thus, lysosomal activation may be involved in
the increase in phagocytosis to remove myelin fragments in
Schwann cells (Lee et al. 2009; Jung et al. 2011a). Typically,
lysosomal acidification inhibits lysosomal exocytosis and
increases phagocytosis to remove intercellular debris or
foreign bodies, but the alkalization of lysosomal vesicles
induces lysosomal exocytosis (Blott and Griffiths 2002). In
the present study, we believe that the concept of ATP-
induced-lysosomal exocytosis during WD is different from
the previously reported lysosomal activation after nerve
injury. After nerve injury, Schwann cell lysosomes may be
acidified, which may inhibit lysosomal exocytosis and sub-
sequently increase the engulfment of myelin fragments.
When the extracellular ATP level is increased during WD,
the increased ATP may induce the alkalization of existing
acidified lysosomal vesicles through a Ca2?-dependent
manner (Dou et al. 2012, Ansselin et al. 1997), and then
lysosomal exocytosis may occur in Schwann cells. In addi-
tion, CP may regulate the Ca2? concentration to block the
effect of ATP (Labuzek et al. 2010), and Met may block
ATP-induced-lysosomal exocytosis in an AMPK-dependent
manner (Elferink 1979). Thus, it seems likely that some
secretory proteins induced by lysosomal exocytosis in
Schwann cells prevent myelin fragmentation and degrada-
tion. Further studies are needed to reveal the underlying
mechanisms of ATP-induced-lysosomal exocytosis in
demyelination during WD.
On the other hand, during WD, both Schwann cells and
macrophages are activated to engulf myelin sheaths (Rot-
shenker 2011; Dubovy 2011). Previous studies have
reported that the in vitro activated Schwann cells by P2X7
induce the release of IL-1b which is an inflammatory
molecule for the recruitment of macrophages (Colomar
et al. 2003; Martini et al. 2008). In this study, the increased
extracellular ATP may affect to increase the release the
inflammatory molecule and to accelerate the myelin deg-
radation. However, there are two possibilities to explain the
contradiction to the previous knowledge: (1) In ATP-trea-
ted ex vivo experiments, the effect of the recruitment of
macrophages on the myelin engulfment could be excluded
due to ex vivo sciatic nerve system; (2) Because Schwann
cells may have a much greater influence on the engulfment
of myelin sheaths than macrophages do in in vivo system,
the inhibited engulfment of myelin sheaths in ATP-treated
Schwann cells may be essential for the inhibition of myelin
degradation during Wallerian degeneration.
In conclusion, here, we found that extracellular ATP is
involved in demyelination during WD and that the role of
extracellular ATP in demyelination is dependent on lyso-
somal exocytosis. These results strongly suggest that the
Fig. 3 ATP delays axonal
degradation during WD. a The
sciatic nerve sections of
cultured explants were
immunolabeled with NF
antibodies. Scale bar 50 lm.
b Quantitative result showing
intact NF (longer than 40 lm)
from 300 nerve fibers in each
group under a microscopic field.
Three mice were used for the
immunohistochemistry.
c Sciatic nerve explants were
cultured for 3 days in the
absence or presence of ATP and
Met. Protein extracts from the
explants were analyzed by
western blotting. P0; myelin
protein zero. d Quantitative
analysis of western blotting data
illustrates the relative intensity
of the P0 band. Four
independent experiments were
performed for the western blot
analysis
366 Cell Mol Neurobiol (2014) 34:361–368
123
regulation of extracellular ATP might be a novel thera-
peutic target for myelination disorders.
Acknowledgments This work was supported by the Dong-A Uni-
versity research fund.
Conflict of interest The authors claim no conflict interests.
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a Schematic representation of
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b After sciatic nerve axotomy,
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