Remyelination in the CNS is primarily performed by oligoden-
drocyte precursor cells (OPCs; Franklin and ffrench-Constant,
2008). Following demyelination, OPCs proliferate and differen-
tiate into myelinating oligodendrocytes. A number of growth
factors have been identified that regulate the proliferation and
differentiation phase of OPCs, such as fibroblast growth factor 2
(FGF-2) and insulin-like growth factor 1 (IGF-1; Frost et al.,
2003; Hsieh et al., 2004). In addition, cytokines such as tumor
necrosis factor-? (TNF-?) and interleukin-1? (IL-1?) have also
been implicated in remyelination (Arnett et al., 2001; Mason et
sis (Todorich et al., 2009). In addition, iron plays a role in
proliferation and differentiation of cells via enzymes involved in
energy metabolism and DNA synthesis (Cazzola et al., 1990).
Iron deficiency during development leads to hypomyelination
(Lozoff and Georgieff, 2006). However, not much is known
iron is made available to OPCs. Iron is thought to enter the CNS
from the circulation via capillary endothelial cells (Moos et al.,
2007). As ?95% of the capillary surface is covered by astrocytic
circulation and distribute it to other cells in the CNS. Astrocytes
possess the iron influx and efflux mechanisms required for cell-
to-cell transport of iron (Dringen et al., 2007). Iron efflux from
astrocytes is mediated by the ubiquitously expressed iron ex-
porter ferroportin (Fpn), which partners with ceruloplasmin
(Cp), a ferroxidase that oxidizes ferrous iron (Fe2?) transported
through Fpn to its ferric form (Fe3?; Jeong and David, 2003).
We investigated whether iron released from astrocytes plays a
role in remyelination. To study this, we generated mice deficient
in astrocytic iron efflux by deleting the iron exporter Fpn specif-
ically in astrocytes and induced demyelination in the spinal cord
matter of the spinal cord.
Care Committee of the Research Institute of the McGill University
Health Centre and followed the guidelines of the Canadian Council on
Animal Care. Astrocyte-specific conditional deletion of Fpn, the iron
efflux transporter, was generated by crossing GLAST::CreERT2 mice
(Mori et al., 2006) with Fpnflox/floxmice (Donovan et al., 2005) to gen-
erate GLAST::CreERT2;Fpnflox/floxmice. Tamoxifen dissolved in corn
oil (Sigma)/ethanol (9:1) at 10 mg/ml and 1 mg was administered intra-
peritoneally twice a day for 5 d to 6–8-week-old female GLAST::
CreERT2;Fpnflox/floxmice. Control female animals of the same genotype
Two weeks after the last tamoxifen injection, GLASTCre::
ERT2;Fpnflox/floxmice or vehicle-injected controls were anesthetized
performed at the 11th thoracic vertebral level (T11). One microliter of
the dorsal column white matter.
PCR genotyping and Western blotting. DNA was extracted from brain,
PCR was performed using the Advantage 2 PCR Kit (Clontech Labora-
tories). GLAST::CreERT2;Fpnflox/floxmice were genotyped with one
PCR that had one forward primer (5?-CTA CAC GTG CTC TCT TGA
GAT-3?) and two reverse primers (5?-GGT TAA ACT GCT TCA AAG
G-3? and 5?-CCT CAT ATG TGA GTC AAA GTA TAG-3?). The Fpn
wild-type allele generated a 355 bp band, and the Fpn floxed allele gen-
This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) to S.D. K.S. has
Correspondence should be addressed to Dr. Samuel David, Centre for Research in Neuroscience, The Research
TheJournalofNeuroscience,April4,2012 • 32(14):4841–4847 • 4841
tissue using rabbit anti-Fpn IgG (1:1500; Alpha Diagnostics) was per-
formed as described previously (Jeong and David, 2006).
under deep anesthesia with 0.5% paraformaldehyde and 2.5% glutaral-
ing the lesion site were postfixed overnight in the same fixative and then
with 2% osmium tetroxide for 2 h and embedded in Epon. Cross-
sections (1 ?m thick) of the spinal cord were stained with 1% toluidine
blue and examined by light microscopy. Ultrathin sections of the lesion
site were stained with lead citrate and viewed with a Philips CM10 elec-
tron microscope. The ratio of the axon diameter over the fiber (axon ?
of LPC injections was measured from electron micrographs. Briefly, EM
images of cross-sections of the lesion site were taken at 1550? magnifi-
cation (10–15 images per animal). Because the axons were not always
circumference and the fiber circumference (i.e., outside the myelin
sheath) based on the following formula: d ? p/? (where d ? diameter;
p ? perimeter measurement). One hundred axons per animal were an-
alyzed (n ? 3 animals per group) and statistical significance was deter-
mined by two-way repeated-measures (RM) ANOVA.
maldehyde in 0.1 M phosphate buffer, pH 7.4. Spinal cord segments
containing the lesion site were removed, and processed for cryostat sec-
tioning (14 ?m) and double immunofluorescence was performed using
rat anti-GFAP (1:200; Invitrogen) and rabbit anti-Cre recombinase (1:
400; Covance) or goat anti-olig2 (1:200; R&D Systems) and rabbit anti-
anti-IL-1? (1:100; R&D Systems) or rat anti-GFAP (1:200; Invitrogen)
and goat anti-IGF-1 (1:100; R&D Systems) as described previously
(Jeong and David, 2006). Images of the IL-1?/Mac-1 and IGF-1/GFAP
labeled cells were acquired using a confocal laser scanning microscope
(FluoView FV1000, Olympus) and prepared using FV10-ASW 3.0 soft-
ware (Olympus). Quantification of the number of IL-1? and IGF-1-
from an Axioskop 2 Plus microscope (Zeiss) using a QImaging Retiga
(IL-1? and IGF-1) and extrapolated to square millimeters.
Olig2- and Ki67-double-positive cells or olig2-positive cells were
counted at the rostrocaudal midpoint of the lesion and divided by the
area of the respective lesion. Counts were obtained from three sections
per animal and averaged (n ? 4 animals per group). Only animals with
comparable lesion size were analyzed (mean lesion site CTRL: 0.082 ?
0.001 mm2; KO: 0.085 ? 0.005 mm2).
Microglial purity examined using Mac-1 immunoreactivity was ?97%.
Microglia were incubated with 10 ng/ml lipopolysaccharide (LPS from
isonicotinoyl hydrazone (SIH) overnight. SIH is an analog of pyridoxal
isonicotinoyl hydrazone that shows high affinity and selectivity for iron
(Richardson and Ponka, 1998). Astrocyte cultures were prepared as de-
cells were incubated overnight with 40 ?M FeCl3together with
L-ascorbate (molar ratio of FeCl3to L-ascorbate was 1:44). The next day,
RT-PCR. RNA was isolated from astrocyte and microglia cultures us-
ing the RNeasy Minikit (Qiagen) and reverse transcribed to cDNA with
using the HotStarTaq PCR Kit (Qiagen) with the following primers:
TGF-?_reverse (rev): 5?-TGT ACT GTG TGT CCA GGC TCC AAA-3?;
5?-CAG GTT GCT CAA GCA GCA AAG GAT-3?; FGF-2_for: 5?-AAC
Conditional deletion of Fpn in astrocytes by GLASTCre::ERT2. A, The Fpn deletion band (398 bp) is only seen in brain (Br) and spinal cord (SC), but not in the liver (L) of the
4842 • J.Neurosci.,April4,2012 • 32(14):4841–4847 Schulzetal.•AstrocytesProvideIronforRemyelination
CCA CAC GTC AAA CTA-3?; IL-1?_for: 5?-AAG TTT GTC ATG AAT
GAT TCC CCT C-3?; IL-1?_rev: 5?-GTC TCA CTA CCT GTG ATG
5?-GAA GAC TCC TCC CAG GTA-3?.
Mice lacking the sole known iron exporter Fpn specifically in
astrocytes were generated by using the tamoxifen-inducible Cre-
ERT2/loxP system as described in the Materials and Methods
section. PCR analysis using primers that selectively recognize the
recombined Fpn allele shows Fpn deletion in DNA from brain
further confirmed at the protein level by Western blotting (Fig.
ment results in the translocation of Cre
cleus in astrocytes, while it remains local-
ized in the cytosol in vehicle-treated
confirm the excision of Fpn in astrocytes
using the tamoxifen-inducible Cre/LoxP
Injection of LPC into the spinal cord pro-
duces focal demyelination within 2–3 d
followed by remyelination starting after
5–7 d (Jeffery and Blakemore, 1995; Ous-
man and David, 2000). LPC was injected
into the dorsal white matter of the spinal
or vehicle injection. All control animals
mates that had been injected intraperitone-
ally with vehicle instead of tamoxifen.
ter (Fig. 2A,B). Remyelinated axons were
identified in electron micrographs by their
thin myelin sheath in relation to the axon
diameter. Remyelinating axons with thin
myelin sheaths were seen in the LPC-
injected control animals (Fig. 2C). In con-
trast, very few remyelinated axons were
detected in LPC-injected astrocyte-specific
Fpn KO mice (Fig. 2D). The percentage of
remyelinated axons present in the lesion
tio; i.e., the axon diameter divided by the
of 1 indicates demyelinated or unmyeli-
nated axons, whereas a g-ratio ?0.9 indi-
cates remyelinated axons. In control
animals, 51.03 ? 6.02% of the axons
within the lesion site show evidence of re-
myelination and only 33.76 ? 5.35% are
the astrocyte-specific Fpn KO mice, only
25.26 ? 3.53% of axons are remyelinated
and 66.24 ? 4.22% are still demyelinated (Fig. 2E), indicating
that remyelination is impaired in the astrocyte-specific Fpn KO
mice. These results suggest that iron efflux from astrocytes via
Fpn contributes to remyelination.
Remyelination involves the proliferation and differentiation of
OPCs into myelinating oligodendrocytes. We therefore assessed
KO mice may be due to a decrease in OPC proliferation. Double
immunofluorescence labeling for the proliferation marker Ki67
liferating OPCs in the astrocyte-specific Fpn KO mice compared
with control animals (Fig. 3A–M). The total number of olig2
labeled cells was also significantly reduced (Fig. 3N). Therefore,
E, The g-ratio analysis shows a significantly lower percentage of remyelinated axons (g-ratio ? 0.9) and a significant higher
percentage of demyelinated axons (g-ratio ? 1.0) in astrocyte-specific Fpn KO mice compared with wild-type mice (n ? 3,
Remyelination after LPC-induced demyelination is impaired in astrocyte-specific Fpn knock-out mice. A, Epon-
Schulzetal.•AstrocytesProvideIronforRemyelinationJ.Neurosci.,April4,2012 • 32(14):4841–4847 • 4843
reduced OPC proliferation may contrib-
ute to the impaired remyelination seen in
data also suggest that iron release from
ation of OPCs after LPC-induced demy-
elination in vivo.
Macrophages and microglia play an im-
portant role in remyelination by clearing
myelination and by secreting growth fac-
tors and cytokines that promote OPC
proliferation and differentiation (Kotter
et al., 2005; Neumann et al., 2009). The
lack of iron efflux from astrocytes in
astrocyte-specific Fpn KO mice may de-
prive not only OPCs but also microglia of
iron. We therefore investigated whether
iron deficiency alters the ability of micro-
glia to express cytokines. The expression
cated in remyelination, was assessed in
microglial cultures under iron-deficient
conditions. Purified microglia were treated
with the iron chelator SIH and stimulated
with LPS to induce cytokine expression.
LPS-activated microglia show high mRNA
expression of TNF-? and IL-1?, which are
reduced in cells rendered iron-deficient by
and IL-1?. TNF-? and IL-1? could either
entiation or activate astrocytes to produce
growth factors, which could in turn act on
remyelinating OPCs. In addition, there is a
macrophages in the lesion site in astrocyte-
specific Fpn KO animals 10 d after LPC
We next assessed whether TNF-? and
IL-1? can induce astrocytes to express
growth factors such as FGF-2 and IGF-1 that promote OPC pro-
cyte cultures were treated with either TNF-? or IL-1? and the
mRNA expression of FGF-2 and IGF-1 assessed. The expression
of FGF-2 and IGF-1 was significantly upregulated by IL-1? and
TNF-?, respectively (FGF-2: 3.39-fold ? 0.56, p ? 0.01; IGF-1:
TNF-? and IL-1? by iron-deprived microglia might result in
decreased expression of FGF-2 and IGF-1 by astrocytes. TGF-?
mRNA was also upregulated in astrocyte cultures with IL-1?
when astrocytes were iron-loaded (1.77-fold ? 0.19, p ? 0.05),
sion. TGF-? is thought to play a role in OPC differentiation
(McKinnon et al., 1993). Furthermore, there is a reduction in
IGF-1 expression in astrocytes in the lesion site in astrocyte-
specific Fpn KO animals 10 d after LPC injection (Fig. 4C,L–S).
iron supply or to indirect effects via iron-deficient microglia,
which could in turn affect astrocyte expression of growth factors
in Figure 4T.
Fpn KO mice, suggesting that iron efflux from astrocytes plays a
OPC proliferation after LPC-induced demyelination is reduced in astrocyte-specific Fpn knock-out mice. Double
4844 • J.Neurosci.,April4,2012 • 32(14):4841–4847 Schulzetal.•AstrocytesProvideIronforRemyelination
Schulzetal.•AstrocytesProvideIronforRemyelination J.Neurosci.,April4,2012 • 32(14):4841–4847 • 4845
due to decreased proliferation and/or differentiation of OPCs.
of cells, because it plays an important role in DNA synthesis as
well as in oxidative metabolism (Cazzola et al., 1990). Iron has
also been shown to be involved in proliferation of OPCs in vitro
(Morath and Mayer-Pro ¨schel, 2001) and in oligodendrogenesis
after intraspinal LPS injections (Schonberg and McTigue, 2009).
Since iron would be retained within astrocytes in the astrocyte-
specific Fpn KO animals, OPCs might be iron-deficient, which
may account for the reduced proliferation of OPCs after LPC-
induced demyelination in these mice. Our in vivo results suggest
Microglia/macrophages have also been suggested to deliver iron
to OPCs for oligodendrocyte genesis after intraspinal LPS injec-
tion (Schonberg and McTigue, 2009). However, in the LPS
model, increased iron accumulation was detected in macro-
in the LPC-induced demyelination model used in the current
major source of iron in the LPC model. The LPS-induced iron
accumulation in microglia/macrophages may be a reflection of
the response of macrophages to sequester iron in response to
bacterial infection (Nairz et al., 2011).
In addition to the direct effects of iron deprivation on OPCs,
iron may also have indirect effects on remyelination, such as
altering the levels of cytokines and growth factors that can influ-
ence OPC proliferation and differentiation. Iron retention in as-
trocytes in the astrocyte-specific Fpn KO mice could also render
and cytokine expression. Cytokines such as TNF-? and IL-1?
have been shown to play a role in remyelination (Arnett et al.,
2001; Mason et al., 2001). We found here that LPS-stimulated
IL-1? when treated with an iron chelator to render them iron-
deficient. These data suggest a role of iron in the expression of
TNF-? and IL-1? by microglia, which is consistent with a previ-
ous report showing decreased expression of these cytokines by
microglia treated with the iron chelator deferoxamine (Zhang et
al., 2006). TNF-? has been shown to directly promote OPC pro-
et al., 2001). One of the factors known to induce OPC prolifera-
tion is FGF-2 (Wolswijk and Noble, 1992). FGF-2 is predomi-
nantly expressed by astrocytes in response to myelin damage
(Messersmith et al., 2000). Furthermore, it is also upregulated in
reactive astrocytes after LPC-induced demyelination (Hinks and
Franklin, 1999). We found that stimulation of astrocytes with
IL-1? induced increased expression of FGF-2. TNF-? has a sim-
ilar effect on IGF-1 expression. IGF-1 not only promotes OPC
differentiation and survival but also affects OPC proliferation
(Mozell and McMorris, 1991). IGF-1 is also expressed by astro-
role after experimentally induced demyelination (Mason et al.,
lated by IL-1? and TNF-?, respectively. We further showed that
pression by astrocytes, was also reduced in the LPC lesion site of
astrocyte-specific Fpn KO mice, which may contribute to im-
paired OPC proliferation and differentiation. TGF-? has been
suggested to play a role in differentiation of OPCs and is also
upregulated after LPC-induced demyelination (Hinks and
Franklin, 1999). We show here that IL-1?-stimulated astrocytes
upregulate TGF-? expression, which is markedly reduced when
astrocytes are iron-loaded.
In summary, our findings suggest that iron efflux from astro-
for remyelination. Since iron may also be available from other
sources, the deletion of astrocyte Fpn may delay remyelination
As illustrated in Figure 4N, astrocytes may acquire iron from
brain capillary endothelial cells via their astrocytic end feet. Re-
lease of iron from astrocytes would make it available to OPCs,
which can then use the iron for enzymatic processes involved in
their proliferation and differentiation into myelin-forming cells.
In addition, astrocytes might also provide iron to microglia,
astrocytes to produce growth factors involved in OPC prolifera-
tion and differentiation such as FGF-2 and IGF-1. IL-1? can also
induce TGF-? expression by astrocytes, which could contribute
directly to OPC differentiation. These results suggest an impor-
tant role for astrocytes in taking up iron from endothelial cells at
the blood–brain barrier and distributing it to other cell types in
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