Download Connexin-based channels contribute to metabolic pathways in the

© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
RESEARCH ARTICLE
Connexin-based channels contribute to metabolic pathways in the
oligodendroglial lineage
ABSTRACT
Oligodendrocyte precursor cells (OPCs) undergo a series of energyconsuming developmental events; however, the uptake and
trafficking pathways for their energy metabolites remain unknown.
In the present study, we found that 2-NBDG, a fluorescent glucose
analog, can be delivered between astrocytes and oligodendrocytes
through connexin-based gap junction channels but cannot be
transferred between astrocytes and OPCs. Instead, connexin
hemichannel-mediated glucose uptake supports OPC proliferation,
and ethidium bromide uptake or increase of 2-NBDG uptake rate
is correlated with intracellular Ca2+ elevation in OPCs, indicating a
Ca2+-dependent activation of connexin hemichannels. Interestingly,
deletion of connexin 43 (Cx43, also known as GJA1) in astrocytes
inhibits OPC proliferation by decreasing matrix glucose levels without
impacting on OPC hemichannel properties, a process that also
occurs in corpus callosum from acute brain slices. Thus, dual
functions of connexin-based channels contribute to glucose supply in
oligodendroglial lineage, which might pave a new way for energymetabolism-directed oligodendroglial-targeted therapies.
KEY WORDS: Oligodendroglia, Connexin hemichannel, Glucose
uptake, Intracellular Ca2+, Glial metabolism
INTRODUCTION
Oligodendrocyte precursor cells (OPCs) undergo proliferation,
migration and dynamic interactions with axons before myelination
(Kang et al., 2010; Richardson et al., 2011; Rivers et al., 2008). In
addition to myelination, oligodendrocytes also act as an energy
source for axons by actively participating in monocarboxylate
transporter 1 (MCT1, also known as SLC16A1)-mediated lactate
delivery (Funfschilling et al., 2012; Lee et al., 2012). Recently, it
has been found that OPCs directly promote angiogenesis to support
the highly energy-consuming myelination process (Yuen et al.,
2014), and oligodendroglial cells take up more energy substrates
than neurons do at least in culture (Sanchez-Abarca et al., 2001).
Thus, oligodendroglial cells require an extraordinary metabolic
demand to support their development and function (Harris and
Attwell, 2012; Nave, 2010). However, the detailed energy metabolism
pathways of the oligodendroglial lineage are currently unclear.
1
Department of Histology and Embryology, Faculty of Basic Medicine, Chongqing
Key Laboratory of Neurobiology, Third Military Medical University, Chongqing
2
400038, China. Collège de France, Center for Interdisciplinary Research in
Biology (CIRB)/Institut National de la Santé et de la Recherche Mé dicale U1050,
3
Paris 75231, Cedex 05, France. Southwest Eye Hospital, Southwest Hospital, Third
Military Medical University, Chongqing 400038, China.
*These authors contributed equally to this work
‡
Authors for correspondence ([email protected];
[email protected])
Received 17 August 2015; Accepted 14 March 2016
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In the present study, we considered the first step of energy
consumption: energy substrate uptake through selective
transporters (Hirrlinger and Nave, 2014). It is well known that
astrocytes are the major energy source for neurons because they
take up glucose through Gluts and provide the metabolite
lactate for neurons through the ‘MCT-mediated astrocyte neuron
lactate shuttle’ (Pellerin and Magistretti, 1994, 2012). Similarly,
oligodendrocytes can absorb extracellular glucose and/or lactate
through Glut1 (also known as SLC2A1) and MCT1, respectively
(Hirrlinger and Nave, 2014; Morrison et al., 2013; Rinholm et al.,
2011; Saab et al., 2013). By contrast, OPCs do not express MCT1
(Lee et al., 2012), and there is a lack of evidence showing the
expression of other Gluts in OPCs. Therefore, our main question is
whether OPCs can obtain energy supply through other pathways
such as non-selective energy uptake channels.
A typical feature of glial cells is their high expression of
connexins, which can form gap junctions and/or hemichannels in
different glial cell types. For instance, connexin 43 (Cx43, also
known as GJA1) and connexin 30 (Cx30, also known as GJB6)
are mainly expressed in astrocytes (Ransom and Giaume, 2013),
whereas Cx47, Cx32 and Cx29 (also known as GJC2, GJB1 and
GJC3, respectively) are present in oligodendroglial cells (Parenti
et al., 2010; Theis et al., 2005). Recently, pathological changes of
oligodendroglia or demyelination found in transgenic mice with
different subsets of connexins (i.e. a Cx43 and Cx30 double
knockout) suggest that glial connexins might participate in the
regulation of the myelination or remyelination processes (Li et al.,
2014; Markoullis et al., 2012a,b, 2014). In astrocytes, Cx43 gap
junction channels and hemichannels are permeable to glucose,
lactate and other metabolic substrates (Giaume et al., 2013;
Ransom and Giaume, 2013). At a more integrated level, the
astroglial networking mediated by gap junctions sustains neuronal
activity through the intercellular trafficking of metabolites
(Rouach et al., 2008). Based on these findings, it has been
hypothesized that gap junction communication between astrocytes
and oligodendrocytes might allow oligodendrocytes to obtain
energy supplies from astrocytes (Hirrlinger and Nave, 2014;
Morrison et al., 2013). However, it is not clear whether this gapjunction-mediated energy substrate pathway also exists between
OPCs and astrocytes. Given that connexin-based channel
functions are related to the development of astrocytes and
oligodendrocytes (Tress et al., 2012; Venance et al., 1995; Von
Blankenfeld et al., 1993), it is worthwhile exploring whether
connexin-based channels also contribute to energy uptake in
OPCs.
To address these questions, we took advantage of the ability of
the fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3diazol-4-yl)amino)-deoxyglucose (2-NBDG), which can permeate
connexin-mediated gap junction channels and hemichannels
(Retamal et al., 2007b; Rouach et al., 2008), to help analyze the
Journal of Cell Science
Jianqin Niu1, *, Tao Li1,*, Chenju Yi2, Nanxin Huang1, Annette Koulakoff2, Chuanhuang Weng3, Chengren Li1,
Cong-Jian Zhao3, Christian Giaume2,‡ and Lan Xiao1,‡
glucose trafficking in oligodendroglial lineage cells. Here, we
demonstrate that 2-NBDG can be taken up by OPCs through
connexin hemichannels in a Ca2+-dependent manner, whereas it is
transferred between astrocytes and oligodendrocytes through gap
junction channels. We also show that connexin-hemichannel-mediated
glucose uptake supports OPC proliferation. Taken together, our
findings indicate that connexin-based channels contribute to energy
uptake pathway in oligodendroglial lineage cells.
RESULTS
Oligodendroglial cells express connexins during
development
Oligodendroglial cells pass through distinguished development
stages with specific biomarkers expression. Briefly, platelet-derived
growth factor a (PDGFRa) is used as an early developmental marker
to identify OPCs. Immature oligodendrocytes are identified
by O4 (marker specific for the oligodendroglial lineage; Sommer
and Schachner, 1981; Bansal et al., 1989) or CNPase (also known as
CNP, 2′,3′-cyclic nucleotide 3′-phosphodiesterase), and MBP and
CC1 (also known as APC, adenomatous polyposis coli) are both
mature oligondendrocyte markers (Emery, 2010). In our study, most
OPCs (PDGFRa positive) differentiate into immature
oligodendrocytes (O4 positive) on the third day, and into mature
oligodendrocytes (MBP positive) on the sixth day after being induced
to differentiate in vitro (Fig. 1). Double immunostaining results
showed that only Cx29 and Cx47, but not Glut1, were detected in
OPCs and immature oligodendrocytes (Fig. 1A,B). At more advanced
stages of differentiation, namely after 6 days differentiation in culture,
all three connexins (i.e. Cx29, Cx32 and Cx47) and Glut1 expression
were observed in mature oligodendrocytes (Fig. 1C). The percentages
of oligodendroglial cells positive for connexins at different time in
culture showed that PDGFRa+ OPCs dominantly express Cx47 and
Cx29 (Fig. 1D), and the same expression pattern was also observed in
the corpus callosum of postnatal developing mice (Fig. S1). The
expression levels of connexins and Glut1 were further determined by
western blot analysis as well as quantitative PCR (qPCR) upon
differentiation of OPCs in cultures (Fig. 1E,F). Moreover, Glut2 and
Glut3 (neuronal specific) were not found in OPCs (Fig. S1D). Based
on the different expression patterns of connexins and Gluts between
OPCs and oligodendrocytes, we hypothesized that oligodendroglial
connexins might differently contribute to metabolic pathways (i.e.
glucose uptake) in oligodendroglial lineage cells.
Glucose analog can be exchanged between
oligodendrocytes and astrocytes, but not between OPCs and
astrocytes
To examine the possibility of metabolic coupling between
astrocytes and oligodendroglial lineage cells, we studied glucose
trafficking in an astrocyte-oligodendroglia co-culture system by dye
coupling (Giaume et al., 2012). Briefly, the cells were patched
with a whole-cell recording pattern, and the intercellular diffusion
of the dye was monitored after 20 min of recording. At the
beginning of the whole-cell configuration (1 min), 2-NBDG or
sulforhodamine B (SRB) filled up CC1+ mature oligodendrocytes.
After 20 min, these probes diffused from oligodendrocytes to the
astrocyte layer underneath (Fig. 2A). Specifically, 2-NBDG was
time dependently transferred to astrocytes (Fig. 2B), and this
intercellular diffusion was blocked by carbenoxolone (CBX)
(Fig. 2A). However, when the same experiment was performed on
OPCs (PDGFRa+), 2-NBDG and SRB only filled up the recorded
OPCs but were not detected in the underlying astrocytes after
20 min recording (Fig. 2C), indicating that glucose can be
Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
transferred between oligodendrocytes and astrocytes but not
between OPCs and astrocytes. This result raised the question of
how glucose enters OPCs.
Functional hemichannels contribute to glucose analog
uptake in oligodendroglial cells
As functional gap junctions between astrocytes and OPCs were not
detected in the early developmental stages of oligodendroglial cells
(Fig. 2) whereas Cx29 and Cx47 are already expressed, we
examined their hemichannel function by performing an ethidium
bromide (EtBr) uptake assay in cultured OPCs (Giaume et al.,
2012). As illustrated in Fig. 3, OPCs (PDGFRa+) exhibited
hemichannel activity in normal culture conditions (solution with
1 mM Ca2+), which could be blocked by CBX and La3+ but not by
the Glut1 inhibitor STF31. This hemichannel activity was increased
in Ca2+-free condition known to trigger hemichannel opening
(Fig. 3A). The uptake assay was also performed on mature
oligodendrocytes (CC1+) under the same condition, which
showed that EtBr uptake activity in oligodendrocytes was less
than that in OPCs (Fig. 3A,a1,B,b1). To test whether hemichannels
in OPCs and oligodendrocytes were permeable to glucose, we
performed a dye uptake assay with 2-NBDG (Retamal et al., 2007a).
The uptake ratio for this fluorescent glucose analog was similar to
that monitored by the uptake of EtBr, indicating that both OPCs and
oligodendrocytes uptake glucose from the extracellular medium
through connexin hemichannels; however, hemichannel-dependent
glucose analog uptake was more pronounced in OPCs than that in
oligodendrocytes (Fig. 3A,a2,B,b2). In addition, 2-NBDG uptake in
OPCs could be blocked by hemichannel inhibitors but not by the
Glut1 inhibitor STF31 or Cytochalasin B, which has also been
shown to inhibit Gluts (Griffin et al., 1982) (Fig. 3A,B; Fig. S2).
Thus, hemichannels might be the main contributor for glucose
uptake in oligodendroglial cells, specifically at the OPC stage.
Intracellular Ca2+ signaling actives hemichannels in OPCs
Because it has been reported that intracellular Ca2+ ([Ca2+]i)
elevation triggers the opening of Cx43 and Cx32 hemichannels in
other cell types (De Vuyst et al., 2006; Wang et al., 2013), we
wondered whether hemichannel activity in the oligodendroglial
lineage was also dependent on Ca2+. Thus, we monitored
cytoplasmic [Ca2+]i in OPCs and oligodendrocytes in normal
culture conditions following Rhod-2 loading. Real-time recordings
showed that most OPCs exhibited spontaneous ‘oscillatory’-like
Ca2+ signaling with peak and plateau transients, whereas
oligodendrocytes showed ‘flat’ Ca2+ signaling (Fig. 4A,B). As
OPCs were characterized by higher [Ca2+]i signal and hemichannel
activity compared to oligodendrocytes, we focused our
investigation on OPCs. The cytoplasmic [Ca2+]i and the
hemichannel activity were monitored under different conditions,
including chelating cytoplasmic Ca2+ with BAPTA-AM or
increasing [Ca2+]i with ionomycin. We found that treatment of
BAPTA-AM inhibited the spontaneous oscillatory-like Ca2+
signaling and reduced the 2-NBDG or EtBr uptake in OPCs
(Fig. 4C–E). However, the relative 2-NBDG uptake rate was
significantly increased in the ionomycin-treated group, and this
effect was inhibited by CBX treatment (Fig. 4D). Taken together,
these results indicate that hemichannel activity depends on
intracellular Ca2+ elevation in OPCs.
Inhibition of hemichannel activity impacts OPC proliferation
Glucose is considered as the most important energy source in the
brain and OPC development might rely on it. To determine the role
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RESEARCH ARTICLE
Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
Fig. 1. Oligodendroglial cells express connexins during development in vitro. (A) Double immunostaining of Cx29, Cx47, Cx32 and Glut1 (red) with the
OPC-specific bio-marker PDGFRa (PDGRaR, green). (B) Double immunostaining of connexins and Glut1 (red) with the immature oligodendrocyte-specific biomarker O4 (green). (C) Double immunostaining of connexins and Glut1 (red) with the oligodendrocyte-specific bio-marker MBP (green). Note: only Cx29 and
Cx47 can be detected in OPCs (PDGFRa+) and immature oligodendrocytes (O4+), and all three connexins and Glut1 can be observed in mature oligodendrocytes
(MBP+). Arrows highlight representative positive cells. (D) Quantification of connexin- or Glut1-positive oligodendroglial cells at different time in culture.
Development of oligodendroglia is identified by PDGFRa, O4 and MBP, respectively at the indicated time points. (E) Western blot showing the expression patterns
of connexins and Glut1 during oligodendroglial differentiation in vitro. (F) qPCR showing the connexins and Glut1 expression levels at indicated time points in
culture. Values are mean±s.e.m., three independent experiments were performed in triplicate. **P<0.01 compared to day 0 (unpaired t-test).
of connexin-hemichannel-mediated glucose uptake in supporting
OPC development, we firstly tested the effect of glucose on OPC
proliferation. Purely cultured OPCs were fed with OPCproliferation media containing three different concentrations of
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glucose (0, 0.75 and 1.5 mg/ml). Usually 1.5 mg/ml is considered
as the normal extracellular glucose concentration. Olig2 is an
oligodendroglial lineage marker which is expressed through the
whole development process, and we use Ki67 and Olig2 double-
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proliferating OPCs were significantly decreased in a
concentration-dependent manner (Fig. 5A,B). Moreover, in the
presence of a normal glucose concentration, the blockade of
connexin hemichannels in OPCs with CBX or La3+ resulted in a
significant decrease of OPC proliferation, indicated by decreased
viable cell numbers or Ki67 and Olig2 double-positive OPC
numbers (Fig. 5C–E). These results indicate that glucose uptake
through hemichannels contributes to OPC proliferation.
Fig. 2. Glucose analog is transferred between astrocytes and
oligodendrocytes, but not between astrocytes and OPCs. In a co-culture
system, astrocytes show flat cell bodies, thick processes, and form a confluent
monolayer; and OPCs show typical bipolar processes, round cell bodies, while
oligodendrocytes (OL) show multipolar processes above the astrocytes
monolayer. (A) Intercellular diffusion test in oligodendrocyte and astrocyte
co-cultures. At the beginning (1 min) of the whole-cell patch, 2-NBDG
(342.3 g/mol) and sulforhodamine B (SRB, 558.7 g/mol,) fill up the multi-polar
processes of oligodendrocytes and diffuse from oligodendrocytes to
neighboring astrocytes after 20 min. 2-NBDG and SRB diffusion can be
blocked by CBX (50 µM). Oligodendrocytes are positive for CC1 (red, white
arrowheads) immunostaining, and the underlying astrocytes are shown by
phase contrast imaging (black arrows). (B) Timecourse of 2-NBDG diffusion
from one oligodendrocyte to neighboring oligodendrocytes and underlying
astrocytes (green arrows show the cells containing newly diffused 2-NBDG).
(C) Intercellular diffusion test in OPCs co-cultured with astrocytes. 2-NBDG
and SRB only fill up the typical bipolar processes of OPCs but cannot be
detected in neighboring astrocytes after 20 min. OPCs are immunostained by
PDGFRa (PDGRaR, red, white arrowheads), and the underlying astrocytes
are shown by phase contrast imaging (black arrows). More than six injections
were performed for each group.
positive cells to mark the proliferating OPCs as described
previously (Niu et al., 2012b). When exposed to media with
reduced concentrations of glucose (0 mg/ml and 0.75 mg/ml), the
number of viable cells and Ki67 and Olig2 double-positive
Cx43 is highly expressed by astrocytes, and works as hemichannels
and/or homomeric gap junction channels (Cx43–Cx43) between
astrocytes (Evans et al., 2013; Mitterauer, 2015; Ransom and
Giaume, 2013; Ye et al., 2009), and also forms heteromeric gap
junctions (Cx43–Cx47) between astrocytes and oligodendrocytes
(Orthmann-Murphy et al., 2007; Theis et al., 2005). It has been
reported that in astrocytes, Cx43 deletion increases glucose uptake
by a compensatory upregulation of Glut transporters and/or glucose
metabolism enzymes (Gangoso et al., 2012). To determine the
influence of astroglial glucose over-consumption on the
oligodendroglial lineage, we used the astrocyte Cx43 conditional
knockout mouse (hGFAPCre/+:Cx43fl/fl, Cx43-KO). Interestingly, it
was found that the glucose concentration in the medium collected
from Cx43-KO astrocytes was significantly lower than that from the
wild-type astrocytes (Fig. 6A). Similar results were found in wildtype astrocytes treated with the connexin-based channel blocker
CBX (Fig. 6B).
In addition, we performed an EtBr uptake assay in corpus
callosum of acute brain slices from postnatal day (P)14 mice or
astrocyte–OPC co-cultures to detect hemichannel function in OPCs
under different conditions. We found that the hemichannel function
in OPCs was blocked by CBX and increased in Ca2+-free solution,
but was not affected by Cx43 deletion in astrocytes (Fig. 6D,E).
However, decreased OPC proliferation was found in the corpus
callosum of postnatal Cx43-KO mice as well as OPCs co-cultured
with astrocytes from Cx43-KO mice (Fig. 6F,G; Fig. S3), whereas
no significant difference in PDGF, bFGF, CNTF and lactate levels
were found after Cx43 deletion in astrocytes (Fig. S4). Moreover,
the expression of oligodendroglial connexins was not significantly
altered by Cx43 knockout in astrocytes (Fig. 6C).
Finally, we performed rescue experiments and found that the
decrease in OPC proliferation observed after Cx43 deletion in
astrocytes could be compensated by an external glucose supply in
astrocyte–OPC co-cultures (Fig. 7A). To further confirm that
astrocytes affect OPC proliferation in a non-cell-autonomous
manner, we designed sandwich co-cultures in which OPCs were
seeded on coverslips in contact with astrocyte medium but not
directly on astrocyte monolayers, as shown in the diagram (Fig. 7B).
In these sandwich co-cultures, similar results were obtained as that
in the astrocyte–OPC co-cultures (Fig. 7C,D). Taken together, these
results indicate that Cx43 deletion in astrocytes affects glucose level
in the extracellular medium but does not impact on the connexin
hemichannel function of OPCs.
DISCUSSION
OPCs and oligodendrocytes need to go through a series of energyconsuming developmental events, including proliferating to enrich
the population in central nervous system (CNS) tissues, migrating
and/or distributing into their destinations and wrapping axons to
form myelin (Kang et al., 2010; Richardson et al., 2011; Rivers
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Journal of Cell Science
Astrocytic Cx43 depletion decreases glucose concentration
in the extracellular medium and inhibits OPC proliferation,
which can be compensated by glucose supply
RESEARCH ARTICLE
Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
et al., 2008). However, the mechanisms underlying their energy
metabolism, in particular the pathways involved in energy
metabolite uptake and trafficking remain unidentified. Functional
tests have shown that astrocytes and oligodendrocytes are connected
by gap junctions to form panglial networks (Griemsmann et al.,
2014; Ransom and Giaume, 2013; Tress et al., 2012). Moreover,
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astroglial connexin channels (gap junction channels and
hemichannels) are permeable to glucose derivatives (2-NBDG) in
both in vitro and ex vivo models (Blomstrand and Giaume, 2006;
Retamal et al., 2007a), thus they provide the basis to form metabolic
intercellular networks (Rouach et al., 2008). Interestingly, gap
junction channels are ∼65% less permeable to the phosphorylated
Journal of Cell Science
Fig. 3. Functional hemichannels
contribute to glucose analog uptake in
oligodendroglial cells. (A) EtBr (394.3 g/
mol, red) uptake (a1) or 2-NBDG (green)
uptake (a2) in PDGFRa+ OPCs (PDGRaR,
green) or oligodendrocytes (CC1+, green)
under different conditions, including
normal culture medium and culture
medium pre-incubated with CBX, La3+
ions, Glut1 inhibitor (STF31) or Ca2+-free
solution. OL, oligodendrocyte.
(B) Quantification of the EtBr uptake ratio
(b1) or 2-NBDG uptake ratio (b2). Both
EtBr and 2-NBDG uptake can be blocked
by CBX (50 µM) and La3+ (200 µM) but not
by STF31 (5 µM), whereas the uptake ratio
is significantly increased in Ca2+-free
solution. Note: both OPCs and
oligodendrocytes take up EtBr or 2-NBDG
from the extracellular medium through
connexin hemichannels in normal
conditions, and OPCs can take up more
EtBr or 2-NBDG than oligodendrocytes do.
Values are mean±s.e.m., three
independent experiments were performed
in triplicate. *P<0.05, **P<0.01 compared
to normal conditions (unpaired t-test).
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Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
2-NBDG-6P than to 2-NBDG (Rouach et al., 2008), indicating that
the phosphorylated form of the fluorescent glucose analog is
restricted in the glial cells. As glial hemichannels exhibit similar
permeability properties with gap junction channels (Giaume et al.,
2013), it is expected that once absorbed in OPCs by hemichannels,
2-NBDG is phosphorylated and then stays trapped within the cells
for further metabolic requirement, such as glycolysis. Based on
these findings, and to further understand the metabolic role of glial
connexins in metabolic supply, we investigated whether connexinbased channels contribute to glucose trafficking pathways in
oligodendroglial cells. Here, we provide evidence demonstrating
that a glucose analog cannot exchange between OPCs and
astrocytes due to the absence of gap junction, whereas
hemichannel activity supports glucose uptake in OPCs, which is
essential for their proliferation. In addition, for the first time, we
show that a glucose analog can be transferred through
oligodendrocyte–astrocyte gap junctions, which provides the basis
for a panglial metabolic route that previously had only been
suggested but not demonstrated (Morrison et al., 2013).
During brain development, oligodendroglial-specific Cx47 and
Cx32 are detected at embryonic stages, whereas Cx29 appears at P0.
Cx47 expression increases successively in regions populated with
developing oligodendroglia and declines from early postnatal stage
to adulthood (Parenti et al., 2010). Here, we used OPC cultures to
further demonstrate the dynamic expression pattern of connexins
during oligodendroglial development in vitro, suggesting a role for
connexin-based channels in this process (Li et al., 2014). Although
the combination of different connexins between oligodendrocytes
and astrocytes (Cx47–Cx43, Cx47–Cx30, Cx32–Cx30 and Cx32–
Cx26) has been identified, and a heterotypic gap junction activity
has been confirmed by dye coupling and/or electrical measurements
(Magnotti et al., 2011), the types of metabolic substrates that are
exchanged between these two glial cell types have not been
investigated yet. Current data have shown that this ‘panglial’
networking of oligodendrocytes and astrocytes serves to spatially
buffer K+, allow water transport and serve as bi-directional channels
for the spreading of Ca2+ waves (Kamasawa et al., 2005; Menichella
et al., 2006; Parys et al., 2010; Wallraff et al., 2006). Here, we
provide direct evidence that oligodendrocyte–astrocyte gap junctions
are also permeable to the fluorescent glucose analog 2-NBDG.
Considering the capacity of astrocytes to uptake, store and supply
energy substrates (Rouach et al., 2008), it is likely that glucose
and/or its metabolites might be transferred from astrocytes to
oligodendrocytes through oligodendrocyte–astrocyte gap junctions
as recently hypothesized (Morrison et al., 2013). In this regard, our
findings might provide a new understanding of a connexin-channel1907
Journal of Cell Science
Fig. 4. Intracellular Ca2+ signaling
affects hemichannel activity in OPCs.
(A) Intracellular Ca2+ signaling ([Ca2+]i) in
oligodendroglial [OPC and
oligodendrocyte (OL)] cells is monitored by
Rhod-2 loading. Spontaneous
intracellular Ca2+ signals (ΔF/F) in OPCs
and oligodendrocytes are shown in a2,
respectively, and their morphologies are
shown in a1. (B) Percentages of cells with
‘oscillatory’-like [Ca2+]i in OPCs and
oligodendrocytes. **P<0.01 between
two groups (unpaired t-test). (C) Real-time
[Ca2+]i in OPCs with different conditions.
Arrowhead indicates the starting point of
the treatment. (D) Relative 2-NBDG uptake
per second. Note that the 2-NBDG uptake
rate is repressed by BAPTA-AM (25 µM)
but increased by ionomycin (1 µM) and that
this effect can be inhibited by CBX (50 µM).
(E) EtBr (red) uptake in OPCs with or
without BAPTA-AM (25 µM) treatment.
Values are the means±s.e.m., more than
10 cells were tested independently in each
group. *P<0.05, **P<0.01 compared to
vehicle; ##P<0.01 between two groups
(unpaired t-test).
RESEARCH ARTICLE
Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
mediated metabolic supply alternative pathway besides Glut- or
MCT-mediated pathways in the oligodendroglial lineage.
However, using 2-NBDG, we could not observe such gapjunction-mediated glucose transport between OPCs and adjacent
astrocytes. Instead, we showed for the first time that connexin-based
hemichannels in OPCs are responsible for glucose analog uptake
from the extracellular environment, though there is still a lack of
evidence to show which connexin protein plays the dominant role in
this process. These in vitro results imply that a functional gap
junction channel might not exist at early OPC developmental stages
until they differentiate into GalC-positive oligodendrocytes
(Venance et al., 1995). Consistently, it has been demonstrated that
cells positive for the cell surface ganglioside A2B5, and NG2 glial
cells do not electrically or dye couple with astrocytes whereas
mature oligodendrocytes do (Bergles et al., 2000; Lin and Bergles,
2004; Xu et al., 2014). Importantly, given that neither Gluts (i.e.
Glut1, Glut2 and Glut3) nor MCT1 was detectable in OPCs in our
studies and in studies by other (Lee et al., 2012), whereas 2-NBDG
uptake in OPCs can be blocked by CBX but not by Glut inhibitors
such as STF31 and Cytochalasin B, it is likely that connexinmediated hemichannels serve as a major metabolic supply pathway
in OPCs. Thus, the crucial developmental step of myelination might
depend on a connexin-channel-mediated glucose supply (Rinholm
et al., 2011; Yan and Rivkees, 2006).
Normally it is thought that glial hemichannels are either kept closed,
to maintain cellular integrity (Spray et al., 2006), or active, as in certain
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brain areas such as the hippocampus (Chever et al., 2014) and the
olfactory bulb (Roux et al., 2015). Moreover, they are triggered to
open by some stimulation, such as inflammation, mechanical stress
or ischemia (Batra et al., 2012; Johansen et al., 2011; Karpuk et al.,
2011). In our study, however, we demonstrated for the first time
that connexin-mediated hemichannels are responsible for glucose
uptake in OPCs, implying a role for oligodendroglial connexin
hemichannels under physiological conditions. We further revealed
that the activation of hemichannels in the oligodendroglial lineage
depends on intracellular Ca2+ signaling, which can trigger connexin
hemichannel opening in other cell types (De Vuyst et al., 2006).
Interestingly, we observed that OPCs exhibit stronger spontaneous
Ca2+ oscillations than oligodendrocytes do (Fig. 4) as recently reported
(Cheli et al., 2015), which might explain why more glucose uptake
through connexin hemichannels occurred in OPCs compared to
oligodendrocytes (Fig. 3). In this regard, our data shed light on
different energy substrate uptake mechanisms and pathways between
OPCs and oligodendrocytes. In OPCs, connexin hemichannels
provide a main route for glucose entry, whereas in oligodendrocytes,
glucose and lactate transporters, supplemented by the gap junctions
between astrocytes and oligodendrocytes, as well as their
hemichannels contribute to the energy substrate uptake and traffic.
As elegantly stated by A. Harris (2007), “Although connexinbased channels are considered as poorly selective channels leading
to an idea that they are ‘non-specific large conductance channels’,
numbers of studies demonstrate that there are dramatic,
Journal of Cell Science
Fig. 5. Inhibition of the hemichannel
activities impacts on OPC proliferation.
(A) CCK-8 assay shows the viable cell
numbers of OPCs in proliferation medium with
different concentrations of glucose at the
indicated time points. The lower glucose
concentrations (0 and 0.75 mg/ml)
significantly decreased the viable cell
numbers in comparison with the normal
medium containing 1.5 mg/ml glucose.
(B) The cell count of proliferating OPCs (Ki67+
and Olig2+) shows that reduced glucose
concentrations decrease OPC proliferation at
24 h. Values are the means±s.e.m., three
independent experiments were performed in
triplicate, *P<0.05, **P<0.01 compared to
normal conditions (unpaired t-test). (C) Pure
cultured OPCs were treated with CBX (20 µM)
or La3+ (200 µM) for 24 h; proliferating OPCs
are labeled with Ki67 (red) and Olig2 (green).
Arrows show the double-positive cells.
(D) Quantification of Ki67 and Olig2 doublepositive cells shows that the percentage of
proliferating OPCs is significantly decreased
in the CBX or La3+ treatment groups. (E) The
CCK-8 assay shows that a 12-h treatment
with CBX or La3+ significantly decreased the
viable cell numbers in comparison with
vehicle group. Values are the means±s.e.m.,
three independent experiments were
performed in triplicate. *P<0.05, **P<0.01
compared to vehicle (unpaired t-test).
RESEARCH ARTICLE
Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
unanticipated and connexin-specific differences in the channel
permeability to cytoplasmic molecules, particularly among those
thought to directly mediate intercellular signaling (i.e. cAMP,
IP3,…). The data strongly indicate that certain biological molecules
have highly specific interactions within connexin pores that enable
surprising degrees of selective permeability that cannot be predicted
from simple considerations of pore width or charge selectivity”
(Harris, 2007). Based on these statements, it seems reasonable to
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Journal of Cell Science
Fig. 6. Cx43 deletion in astrocytes decreases
glucose concentration in the extracellular
medium and inhibits OPC proliferation.
(A) Glucose assay of the astrocytic culture
medium. The concentration of glucose in the
Cx43-KO group is lower than that in the wild-type
group. (B) In pure cultured astrocytes, CBX
(50 µM) treatment reduces the glucose
concentration in the culture medium. (C) qPCR
shows the connexin mRNA levels in P7 mice
brain, there is no significant difference between
wild-type and Cx43-KO mice. (D) EtBr (red)
uptake assay and quantification in corpus
callosum from acute brain slices of P14 wild-type
or Cx43-KO mice. Arrows highlight OPCs in
corpus callosum. (E) EtBr (red) uptake assay and
quantification in astrocyte (AST) and OPC cocultures. OPCs are labeled by anti-PDGFRa
(PDGRaR, green). Note that hemichannel
function in OPCs is blocked by CBX and
increased with Ca2+-free solution but is not
impacted by Cx43 deletion in astrocytes.
(F) Quantification of PDGFRa+ (OPCs) or Ki67
and Olig2 double-positive cells. Cx43 deletion in
astrocytes significantly decrease the number of
OPCs and Ki67 and Olig2 double-positive
cells during the development of brain white
matter. (G) In astrocyte–OPC co-cultures, the
lack of Cx43 in astrocytes reduces the number of
OPCs, and the number of Ki67 and Olig2 doublepositive proliferating OPCs. Values are the
means±s.e.m., more than 4 mice (P14) were
tested in each group; for co-cultures three
independent experiments were performed in
triplicate. *P<0.05, **P<0.01 (unpaired t-test).
RESEARCH ARTICLE
Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
OPC hemichannel properties. Moreover, this effect can be rescued
by a compensatory extracellular glucose supply, indicating the
importance of astrocytes in maintaining the glucose levels of the
CNS extracellular matrix. Another point that needs caution is that
lactate is also an important metabolic substrate for the brain during
the early postnatal period (Barros, 2013; Rinholm et al., 2011).
Given the extensive permeability of hemichannels for various
metabolites (Giaume et al., 2013), there is no evidence to exclude
the uptake of lactate or other energy substrates through
hemichannels in OPCs. In fact, it has already been reported that
gap junction channels in astrocytes are permeable to lactate in
vitro and ex vivo (Tabernero et al., 1996; Rouach et al., 2008).
Given that various connexin channels exhibit different functional
properties, including size selectivity, charge selectivity and
voltage or chemical gating, we still have no evidence to show
that connexin channels in OPCs are permeable or impermeable to
other substrates, such as Ca2+, K+ and metabolites.
Because OPCs need to go through a series of highly energyconsuming developmental events (Barateiro and Fernandes, 2014)
and likely cannot get enough energy support from Glut1, MCT1 or
gap junctions as oligodendrocytes do, they might rely more on the
hemichannel-mediated energy supply pathway (Fig. 8). This
dependence might partly explain why OPCs are more vulnerable to
oxygen-glucose deprivation than oligodendrocytes (Fern and Moller,
2000; Ziabreva et al., 2010). Moreover, likely due to deficient Cx47based hemichannel properties, Cx47-null mice exhibit decreased
oligodendroglial lineage cell numbers at P14, but gene knockdown
does not influence myelination in adult mice (Tress et al., 2012).
Finally, abnormal connexin expression is involved in limiting OPC
recruitment and consequent remyelination failure in multiple sclerosis
patients and in autoimmune encephalomyelitis mice (a model of
multiple sclerosis) (Markoullis et al., 2012a,b, 2014).
Concluding remarks
consider that although we show here that astrocyte–oligodendrocyte
gap junction channels and OPCs hemichannels are permeable to
2-NBDG, it does not automatically imply that all cytoplasmic or
extracellular signaling molecules with small molecular mass can
permeate through these channels.
Finally, we used a conditional astrocytic Cx43 knockout (Cx43KO) mouse as a glucose-deprivation model to confirm the
importance of connexin-channel-based energy support on the
OPC proliferation in both in vivo and co-culture systems. In these
mice, glucose uptake in Cx43 lacking astrocytes is increased by
compensatory upregulation of Glut1, Glut3 and type I/II
hexokinase expression (Gangoso et al., 2012; Tabernero et al.,
2006). As predicted, we observed that OPC proliferation was
inhibited by Cx43 deletion in astrocytes without impacting on
1910
In this study, we focused on connexin-mediated energy supply
pathways in oligodendroglial lineage cells, providing direct
evidence that glucose can be delivered between astrocytes and
oligodendrocytes through gap junction channels, whereas
Fig. 8. Schematic of the hemichannel and gap junction contribute to
glucose supply in oligodendroglial lineage cells. Glucose can be
transferred between astrocytes and mature oligodendrocytes (OL) through gap
junction channels when functional gap junction channels are not formed at the
early stage of oligodendroglial development. However, a connexinhemichannel-based glucose supply from the extracellular microenvironment
maintains OPC proliferation. Deletion of Cx43 in astrocytes (Cx43-KO) leads to
glucose over consumption by compensatory upregulation of Glut1 and Glut3,
and results in the suppression of OPC proliferation.
Journal of Cell Science
Fig. 7. The reduced OPC proliferation caused by the lack of Cx43 in
astrocytes is compensated by increased glucose supply. (A) Quantification
of PDGFRa+ (PDGRaR, green) OPCs in different conditions. OPC proliferation
is reduced when grown with CX43-KO astrocytes (ASTCx43−/−), and
supplementary glucose significantly reversed this proliferation deficit. (B) The
diagram shows the sandwich co-culture system. OPCs are seeded on cover
slips placed on top of an astrocyte monolayer. (C) Proliferating OPCs in
sandwich co-cultures are double-labeled with anti-Ki67 (red) and anti-PDGFRa
(green) under different conditions. Arrows show the double-positive cells.
(D) Cell counting of Ki67 and PDGFRa double-positive OPCs illustrates that the
decrease of OPC proliferation in the ASTCx43−/−–OPC group can be reversed
by adding glucose (3 mg/ml) to the culture medium. Values are the means±
s.e.m., three independent experiments were performed in triplicate.*P<0.05,
**P<0.01 between the indicated groups (unpaired t-test).
spontaneous intracellular Ca2+ signaling triggers connexin
hemichannel activity that enables glucose uptake in OPCs that
supports their proliferation. Identification of the dual functions of
connexin-based channels in the oligodendroglial lineage, along
with their metabolic roles, might provide valuable insights into the
energy supply pathways of oligodendroglial cells and might result in
new therapeutic strategies for energy-related diseases.
MATERIALS AND METHODS
Animals
Cx43flox/flox and hGFAPCre/+ mice were purchased from the Jackson
Laboratory. These two mouse strains were paired to produce offspring,
either hGFAPCre/+:Cx43fl/fl (Cx43-KO) or hGFAP+/+:Cx43fl/fl (wild type).
The offspring were genotyped by PCR for Cre and floxed Cx43 alleles.
More than four animals were tested in each group. All animal studies were
conducted with the approval of the Laboratory Animal Welfare and Ethics
Committee of the Third Military Medical University (TMMU)
Administrative Panel on Laboratory Animal Care.
Acute brain slices, EtBr uptake and immunofluorescence
staining
Acute brain slices were prepared from brains of P14 Cx43-KO and
wild-type littermate mice using a vibroslicer (Thermo Scientific,
Microm HM650V) as previously described (Geiger et al., 2002).
Slices were incubated with the hemichannel-permeable fluorescent
tracer ethidium bromide (EtBr) (394.3 g/mol, 4 µM final concentration,
ThermoFisher, Grand Island, NY, USA) in oxygenated artificial
cerebro-spinal fluid (ACSF) for 10 min at room temperature. After
fixation with 4% paraformaldehyde (PFA), the slices were incubated
with anti-platelet-derived growth factor a receptor (PDGFRa) primary
antibody (1:200, cat. no. sc-338; Santa Cruz Biotechnology, Dallas,
TX, USA) overnight at 4°C after pre-incubation in the blocking buffer
for 1 h (containing 0.2% gelatin and 1% Triton X-100), followed by
incubation in fluorescent-conjugated secondary antibody at room
temperature for 1 h. Images were taken using a confocal laserscanning microscope (Olympus, IV1000) at the selected specific
wavelength (561 nm). More than eight images were captured in each
group for statistical analysis.
Astrocyte and oligodendroglial cell cultures
Primary astrocyte cultures were prepared from brain hemispheres of
Cx43-KO and wild-type littermate mice pups (P1–P3) as previously
described (Giaume et al., 2012). The purified astrocytes were plated into
six-well plates (105 cells per well). The OPC cultures were prepared as
previously described (Niu et al., 2012b). Briefly, the mixed glial cells
were isolated from cortex of P1–P3 animals and enriched in OPC growth
medium followed by a two-passage purifying processes. The purified
OPCs were induced to differentiate by using OPC differentiation medium
[DMEM/F12 + 1% N2 supplement + 5 g/ml NAC + 1% FBS; Dulbecco’s
modified Eagle’s media/F12 (DMEM/F12, cat. no. SH30023, Hyclone,
Logan, Utah, USA), N2 supplement (cat. no. 17502048, Life
Technologies), fetal bovine serum (FBS, cat. no. SV30087, Hyclone),
N-acetyl-l-cysteine (NAC, cat. no. 0LA0011, AMERSCO, Solon, OH,
USA)]. For co-cultures, OPCs were seeded either on an astrocyte
monolayer to set up astrocyte–OPC co-cultures or on poly-D-lysinecoated glass coverslips placed above the astrocytes to set up a sandwich
co-culture system. We used 4.5×104 cells per well in 24-well plates and
106 cells per well in 60-mm dishes.
Dye uptake and quantification
OPCs or oligodendrocytes were incubated with 4 µM EtBr or 500 µM
2-NBDG (342.3 g/mol, neutral, Life Technologies, Carlsbad, CA) for
30 min at room temperture in different conditions (Giaume et al., 2012),
including normal culture medium (OPCs are in OPC proliferation
medium and oligodendrocytes are in OPC differentiation medium) or
culture medium pre-incubated with carbenoxolone (CBX, Sigma,
Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
St Louis, MO, USA), La3+ ions (lanthanum, Sigma), Glut1 inhibitor
(STF31, Tocris Bioscience, Bristol, UK), Cytochalasin B (Sigma),
Cytochalasin D (Enzo Life Sciences, East Farmingdale, NY) or Ca2+free solution. Then, the cells were fixed with 4% PFA for 15 min at room
temperature. Immunofluorescence pictures were captured using a
confocal laser-scanning microscope (Olympus, IV1000) at an
appropriate wavelength (EtBr, 561 nm; 2-NBDG, 488 nm). The
EtBr fluorescent signal in the nuclei and the 2-NBDG fluorescent
signal in cell bodies were analyzed using Mac Imaris 7.4.0 software. The
mean fluorescence intensity per µm3 was quantified in arbitrary units
(AUs) (Giaume et al., 2012).
Immunofluorescence staining and quantification
Mouse pups of different ages (P7, P14, P21) were anesthetized with 1%
pentobarbital and transcardially perfused with 4% PFA. Brains were
dissected and cryoprotected in 30% sucrose at 4°C. Serial coronal
sections (20 μm) were obtained using a cryostat microtome (MS 1900,
Leica, Wetzlar, Germany). OPCs on the cover slips were fixed as
previously described (Niu et al., 2012a). Brain sections or cell cultures
were blocked with 0.5% bovine serum albumin (BSA) and 0.2% Triton
X-100 for 1 h and then incubated with primary antibodies overnight at 4°
C followed by the fluorescence-conjugated secondary antibodies at room
temperature for 1 h. Cell nuclei were stained with 4′,6-diamidino-2phenylindole (DAPI, 0.1 μg/ml, ThermoFisher). Rabbit anti-PDGFRa
(1:200, cat. no. sc-338; Santa Cruz, Dallas, TX, USA) or rat antiPDGFRa (1:100, cat. no. ab61219; Abcam, Cambridge, UK) antibodies
were used to label OPCs. Mouse anti-O4 (1:100, cat. no. O7139; Sigma)
antibody was used to label immature oligodendroglial cells, and goat
anti-MBP (1:300, cat. no. sc-13914; Santa Cruz Biotechnology) or
mouse anti-CC1 (1:1000, cat. no. OP80; Millipore, Billerica, MA)
antibodies were used to label mature oligodendrocytes. Mouse anti-Olig2
(1:200, cat. no. MABN50; Millipore) antibody was used to label all
oligodendroglial lineage cells. Rabbit anti-Ki67 (1:1000, cat. no. RM9106; Thermo Scientific) antibody was used to detect proliferative cells.
Rabbit anti-Cx29 (1:100, cat. no. sc-68377; Santa Cruz Biotechnology),
rabbit anti-Cx32 (1:100, cat. no. C3595; Sigma), rabbit anti-Cx47 (1:200,
cat. no. 364700; Life Technologies; or 1:100, sc-30335-R; Santa Cruz
Biotechnology), mouse anti-Glut1 (1:500, cat. no. ab40084; Abcam),
goat anti-Glut2 (1:100, cat. no. sc-7580; Santa Cruz Biotechnology) and
mouse anti-Glut3 (1:100, cat. no. ab41525; Abcam) antibodies were used
to detect connexin proteins and Glut in oligodendroglial cells. Mouse
anti-Cx43 (1:100, cat. no. 610062; BD, Erembodegem, Belgium)
antibody was used to label Cx43 in astrocytes. Appropriate AlexaFluor-conjugated secondary antibodies included donkey anti-mouse,
rabbit and goat IgG (1:1000, Life Technologies). The immunofluorescent
signal was determined using a fluorescence microscope (Olympus BX60) or a confocal laser-scanning microscope (Olympus, IV 1000) with
excitation wavelengths appropriate for Alexa Fluor 488 (488 nm, Life
Technologies), Alexa Fluor 568 (568 nm) or Alexa Fluor 647 (647 nm).
Cell counting and fluorescence intensity analyses were conducted on nine
randomly chosen fields under a 20× objective lens for each sample using
an Image Pro Plus image analysis system.
Western blotting
SDS-PAGE western blotting was performed as previously described (Niu
et al., 2010). Proteins were transferred to polyvinylidenedifluoride
membranes and visualized by chemiluminescence (ECL Plus, GE
Healthcare, Marlborough, MA, USA) after incubation with antibodies.
β-actin (1:1000, cat. no. sc- 47778; Santa Cruz Biotechnology) was used as
the loading control. Quantification of band intensity was analyzed using the
Image Pro Plus software. The primary antibodies included: rabbit antiPDGFRa (1:500, cat. no. sc-338; Santa Cruz Biotechnology), goat
anti-MBP (1:1000, cat. no. sc-13914; Santa Cruz Biotechnology), mouse
anti-Olig2 (1:1000, cat. no. MANB50; Millipore), rabbit anti-Cx29 (1:500,
cat. no. sc-68377; Santa Cruz Biotechnology), rabbit anti-Cx32 (1:500,
cat. no. C3595; Sigma), rabbit anti-Cx47 (1:1000, cat. no. 364700; Life
Technologies; 1:500, cat. no. sc-30335-R; Santa Cruz Biotechnology) and
mouse anti-Glut1 (1:2000, cat. no. ab40084; Abcam).
1911
Journal of Cell Science
RESEARCH ARTICLE
RESEARCH ARTICLE
CCK-8 assay
The number of viable cells was estimated using the CCK-8 assay, which
provides an effective and reproducible measurement of OPC proliferation
(Xiao et al., 2008). Briefly, OPCs were cultured at a density of 1.5×104
cells per well in poly-D-lysine-coated 96-well plates containing 100 µl
OPC proliferation medium [DMEM/F12 + 1% N2 supplement + 10 ng/ml
bFGF + 10 ng/ml PDGF-AA; PDGF-AA (cat. no. 100-13A, Peprotech,
Rocky Hill, NJ, USA), bFGF (cat. no. 100-18B, Peprotech)]. After 12 h,
the cells were subjected to different conditions, and at the indicated time
points, 10 µl of CCK-8 solution reagent (Dojindo, Cell Counting Kit-8)
was added to each well according to the manufacturer’s instructions,
followed by a 4 h incubation. The relative viable cell numbers were
determined by measuring the absorbance at 450 nm using a microplate
reader (Bio-Rad, Model 680). Three independent experiments were
performed in triplicate.
Glucose assay
Glucose concentrations were determined using a glucose assay kit (Abcam)
according to the manufacturer’s instructions. Supernatants from 48 h
cultured astrocytes in different conditions were collected for the glucose
assay. Treatment with CBX (50 µM, 48 h) was used to mimic Cx43 deletion
in astrocytes. The optical density (OD) value was measured according to the
absorbance at 570 nm using a microplate reader.
Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731
baseline 2-NBDG fluorescence intensity; S, time (seconds) from start point
of treatment to the end time point of experiment].
Quantification real-time polymerase chain reaction
Total ribonucleic acid (RNA) was isolated from brains or cultured astrocytes
of wild-type and Cx43-KO mice using TRIzol (Life Technologies) and the
RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Real-time quantitative
PCR (qPCR) was performed with the C1000 Touch™ Real-time PCR
Detection System (Bio-Rad) and GoTaq® qPCR Master Mix (Promega,
Sunnyvale, CA, USA). The oligonucleotide primers, amplification
procedure, and melt curve analysis were performed. For each sample, three
independent repeats were performed. Each sample was tested in triplicate.
Statistical analysis
Statistical significance between groups was determined with GraphPad
Prism 5 software. Statistical analyses were performed by one-way analysis
of variance (ANOVA) followed by Tukey’s post-hoc test. Comparisons
between two experimental groups were made using an unpaired t-test. A
probability of P<0.05 was considered statistically significant.
Acknowledgements
The authors wish to thank Jia Lou for her assistance in preparing the figures.
Competing interests
The authors declare no competing or financial interests.
Intercellular dye trafficking
Intracellular Ca2+ imaging and glucose analog uptake imaging
OPCs were cultured on glass-bottomed dishes in OPC proliferation medium
and then loaded with the fluorescent Ca2+-sensitive dye Rhod-2 (5 μM, Life
Technologies) for 30 min at 37°C. The loaded cells were rinsed three times
with modified Ca2+ imaging buffer solution before confocal imaging. OPCs
were incubated in fresh modified Ca2+ imaging buffer (NaCl 125 mM; KCl
5 mM; CaCl2 1 mM; MgSO4 1.2 mM; glucose 5 mM and HEPES 25 mM)
with 2-NBDG (500 µM) and were real-time recorded using a confocal laserscanning microscope (Olympus). During recording, cytoplasmic Ca2+
concentration and 2-NBDG uptake were measured by exciting Rhod-2 at
581 nm and exciting 2-NBDG at 488 nm, respectively. To chelate
intracellular Ca2+, OPCs were pretreated with medium containing
BAPTA-AM (25 µM, ThermoFisher) for 20 min before recording.
Ionomycin (1 µM, ThermoFisher), a Ca2+ ionophore that allows Ca2+ ions
to enter the cells through pores made in the plasma membrane, was applied
to increase the intracellular Ca2+. The real-time Ca2+ and 2-NBDG levels
were recorded using a confocal laser-scanning microscope (Olympus,
IV1000). Recordings were analyzed with Fluoview Image Processing
Software (V2.1) and GraphPad Prism 5 Software. Ca2+ fluorescence signals
and 2-NBDG fluorescence signals were analyzed in regions of interest
(ROIs) covering the somata of oligodendroglial cells. Normalized changes
of Rhod-2 fluorescence intensities were calculated as ΔF/F=(F−F0)/F0
(F, fluorescence intensity; F0, baseline intensity) (Otsu et al., 2015). And
the relative changes of 2-NBDG fluorescence intensities were calculated as
the (F−F0)/S [F, 2-NBDG fluorescence intensity at end time point; F0,
1912
Author contributions
L.X., C.G., J.N. and T.L. designed experiments and wrote the manuscript; J.N., T.L.,
C.Y., N.H. and C.W. conducted the experiments; C.L. and C.-J.Z. collected and
analyzed the data; A.K. reviewed and edited the manuscript.
Funding
This work is in part supported by the National Natural Science Foundation of China
(NSCF) [grant numbers 31171046, 31300906]; Chongqing Scientific and Technical
Innovation Foundation of China [grant number CSTCKJCXLJRC07]; and the
China-France Joint Program YUANPEI 2013 PROJECT [grant numbers 26038XE].
Supplementary information
Supplementary information available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.178731/-/DC1
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