The Journal of Neuroscience, August 20, 2003, 23(20):7710-7718
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Insulin-Like Growth Factor (IGF) Signaling through Type 1 IGF Receptor Plays an Important Role in Remyelination
Jeffrey L. Mason,1
Shouhong Xuan,2
Ioannis Dragatsis,2
Argiris Efstratiadis,2 and
James E. Goldman1
1Department of Pathology and The Center for
Neurobiology and Behavior, and 2Department of Genetics
and Development, Columbia University, New York, New York 10032
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Abstract
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We examined the role of IGF signaling in the remyelination process by
disrupting the gene encoding the type 1 IGF receptor (IGF1R) specifically in
the mouse brain by Cre-mediated recombination and then exposing these mutants
and normal siblings to cuprizone. This neurotoxicant induces a demyelinating
lesion in the corpus callosum that is reversible on termination of the insult.
Acute demyelination and oligodendrocyte depletion were the same in mutants and
controls, but the mutants did not remyelinate adequately. We observed that
oligodendrocyte progenitors did not accumulate, proliferate, or survive within
the mutant mice, compared with wild type, indicating that signaling through
the IGF1R plays a critical role in remyelination via effects on
oligodendrocyte progenitors.
Key words: demyelination; oligodendrocyte; progenitors; apoptosis; macrophages; transgenic
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Introduction
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After exposure to chemical, mechanical, or autoimmune insults resulting in
demyelination of axons within the adult CNS, oligodendrocyte progenitors have
some capacity to initiate a restorative remyelination process
(Ludwin, 1978
;
Yao et al., 1995;
Gensert and Goldman, 1997).
Several of the signaling molecules that influence the migration,
proliferation, and differentiation of oligodendrocyte progenitors are
expressed within remyelinating lesions in the CNS
(Redwine and Armstrong, 1998;
Hinks and Franklin, 1999;
Mason et al., 2000a). However,
the factor(s) responsible for the recruitment and differentiation of these
progenitors in vivo is not known.
An important signaling system that has been implicated in the process of
myelination consists of IGF-1 activating the type 1 receptor (IGF1R)
(Liu et al., 1993). IGF-1 is a
survival factor for oligodendrocytes
(Barres et al., 1993;
Ye and D'Ercole, 1999;
Mason et al., 2000b) and a
differentiation factor for neonatal
(McMorris and Dubois-Dalq,
1988; Mozell and McMorris,
1991) and adult (Mason and
Goldman, 2002) oligodendrocyte progenitors in vitro. Its
levels are elevated within demyelinating and remyelinating lesions in the
adult CNS (Liu et al., 1994;
Yao et al., 1995;
Mason et al., 2000a). In
addition, the inability of the adult CNS to remyelinate after a demyelinating
lesion in interleukin-1
-/- mice coincides with a significant reduction
in IGF-1 expression (Mason et al.,
2001a). Thus, although the function of IGF-1 within demyelinating
and remyelinating lesions is not clear, it seems that it may be important for
inducing oligodendrocyte progenitors to differentiate and remyelinate the
demyelinated axons.
To address the role of IGF signaling in the remyelination process directly,
we used conditional mouse mutants, in which the expression of the
Igf1r gene was ablated by Cre-mediated recombination specifically in
the CNS. We assessed, in comparison with normal siblings, the consequences of
exposure of these animals to the neurotoxicant cuprizone
(Matsushima and Morell, 2001),
which induces a reversible demyelinating lesion in the corpus callosum of
adult mice. We observed that remyelination does not adequately occur in the
absence of IGF signaling and that oligodendrocyte progenitors do not
proliferate or survive as well without IGF1R.
Materials and Methods
Mouse crosses. CamKII
-cre transgenic mice (line R1ag#5)
(Dragatsis and Zeitlin, 2000)
were mated with Igf1r+/-
heterozygotes (Liu et al.,
1993) to derive among other progeny animals with a
CamKII-cre; Igf1r+/-
genotype. These offspring were then crossed with homozygous conditional
mutants carrying modified Igf1r alleles with loxP sites
flanking exon 3 (Igf1rflox/flox)
(Dietrich et al., 2000) to
generate the desired experimental animals (CamKII-cre;
Igf1rflox/-), the genotype of which was
converted by Cre-mediated recombination to
Igf1rflox/- specifically in the brain. The
CamKII-cre transgenic animals were also crossed with R26R
reporter mice carrying a LacZ transgene in the ROSA26 locus
(Soriano, 1999) that can be
activated by Cre-mediated recombination.
Detection of CaMKII-cre recombination. We used a
CaMKII-cre transgenic mouse and assessed cre expression using
a -gal reporter sensitive to Cre-mediated recombination in the brain
(Soriano, 1999). We perfused
postnatal day 1 (P1) transgenic mice intracardially with paraformaldehyde
(PFA), removed the forebrains, and processed the tissue for frozen sectioning.
Tissue sections were dried, rehydrated, blocked with 5% normal goat serum
(NGS), and then stained with the mouse IgM monoclonal O4 supernatant (1:5) or
the rabbit polyclonal NG2 antibody (1:100; a gift from Bill Stallcup, Scripps
Institute, San Diego, CA) to detect oligodendrocyte progenitors and a mouse
monoclonal IgG1 antibody directed against -gal (1:100;
Promega, Madison, WI) to detect Cre-mediated recombination overnight. The
sections were then incubated with the appropriate rhodamine-conjugated
(anti-rabbit IgG or anti-mouse IgM) and FITC-conjugated anti-mouse IgG1
secondary antibodies (1:100; Southern Biotechnology, Birmingham, AL),
cover-slipped, and examined using an Olympus (Lake Success, NY) BX60
microscope equipped with epifluorescent optics.
Southern and Western analyses. The degree of Cre-mediated DNA
excision was quantitated by Southern analysis, after determination of the
level of hybridization signal in diagnostic fragments using a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) and correcting for background
hybridization.
Western blot analysis was performed from the forebrain of 4-week-old mice,
as described previously (Di Cola et al.,
1997), using antisera against the carboxyterminal domain of IGF1R
(C20; Santa Cruz Biotechnology, Santa Cruz, CA) or against tubulin (Covance,
Berkeley, CA). Immunoreactive bands were visualized with ECL detection
reagents (Amersham Biosciences, Piscataway, NJ). Quantitation of luminescent
signals was performed on a Kodak (Rochester, NY) Digital Science 1D 440CF
Imaging Station.
Detection of IGF1R expression in adult oligodendrocyte
progenitors. Cycling progenitors were isolated from the corpus callosum
of adult wild-type or 
Igf1r mice, as described previously
(Gensert and Goldman, 2001;
Mason and Goldman, 2002).
Briefly, the corpus callosum was dissected from 8-week-old wild-type or
Igf1r mice, then mechanically and enzymatically dissociated
with trypsin (Sigma, St. Louis, MO) and collagenase (Worthington, Freehold,
NJ). The cellular suspension was filtered through sterile 0.74 µm mesh, and
the trypsin was neutralized with 10% heat-inactivated serum. Cells were
collected by centrifugation and resuspended in 0.9 M sucrose/MEM.
This cellular suspension was centrifuged, and the cycling progenitors (bottom
layer) were collected and resuspended in serum-free media (d-Biotin; 10
g/ml; Sigma), insulin (5 µg/ml; Collaborative Research, Bedford, MA),
progesterone (20 M; Sigma), putrescine (100 µM;
Sigma), selenium (5 g/ml; Collaborative Research), transferrin (50
µg/ml; Sigma), glutamine (2 mM; Invitrogen, Carlsbad, CA), HEPES
buffer (15 mM; Sigma), 3,3,5-triiodo-L-thyronine (15
M; Sigma), penicillin/streptomycin (100 U/100 µg/ml;
Invitrogen), and BSA (1 mg/ml; Sigma) in DMEM/F12 (Invitrogen).
Cells were stained for O4 and IGF1R immediately after they were plated. The
cells were fixed with 4% PFA, incubated with 20% NGS for 30 min to block
nonspecific binding, and then incubated with mouse monoclonal IgM O4
supernatant (1:5) and mouse monoclonal IgG1 anti-IGF1R (1:200;
Upstate Biotechnology, Lake Placid, NY) overnight at 4°C. The anti-IGF1R
antibody from Upstate Biotechnology was used for immunohistochemistry, because
the anti-IGF1R antibody from Santa Cruz Biotechnology used for Western blot
analysis does not work well for this type of analysis. The cells were then
incubated with a combination of FITC-conjugated goat anti-mouse IgM and
TRITC-conjugated goat anti-mouse IgG1 antibodies (1:100; Southern
Biotechnology) for 1 hr at room temperature, counterstained with
4,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR),
coverslipped, and examined using an Olympus BX60 microscope equipped with
epifluorescent optics. Cells were isolated, stained, and counted from the
forebrain of three separate wild-type and Igf1r mice. Two
hundred O4+ cells from each experiment were counted.
Induction of demyelination/remyelination. Igf1r
mice were bred and maintained in our animal facility at Columbia University.
At 8 weeks of age, Igf1r and littermate wild-type male mice
were fed a diet of milled Purina mouse chow containing 0.2% cuprizone (Sigma)
by weight for up to 6 weeks to induce demyelination. Subsequently, mice were
returned to a normal diet for another 3-6 weeks to allow for remyelination
(Hiremath et al., 1998). All
animal procedures were conducted in accordance with guidelines approved by the
Institutional Animal Care and Use Committee and the Columbia Division of
Laboratory Animal Medicine.
Immunohistochemistry. We anesthetized and intracardially perfused
mice with PFA, removed the brains, and processed the tissue for frozen
sectioning, as described previously (Mason
et al., 2000a). All comparative analyses were focused at the
fornix region of the corpus callosum corresponding to sections 220-260 of the
mouse brain atlas (Sidman et al.,
1971).
Tissue sections were dried, rehydrated, blocked with 5% NGS, and then
incubated overnight with the primary antibody(s) diluted in the blocking
solution. The sections were then incubated with the appropriate
FITC-conjugated secondary antibody (1:100; Southern Biotechnology),
counterstained with DAPI (Molecular Probes), coverslipped, and examined using
an Olympus BX60 microscope equipped with epifluorescent optics.
Antibodies. A mouse monoclonal IgG2B antibody directed
against MBP (1:1000; Sternberger Monoclonals) was used to detect myelin. A
mouse monoclonal IgG1 antibody directed against phosphorylated and
nonphosphorylated high molecular weight neurofilaments (NF-H; 1:1000;
Sternberger Monoclonals) was used to detect axons. The mouse monoclonal
IgG2B antibody CC1 (1:10; Oncogene, Uniondale, NY) was used as a
marker for mature oligodendrocytes
(Messersmith et al., 2000).
The rabbit polyclonal NG2 antibody (1:100) was used as a marker for
oligodendrocyte progenitors (Nishiyama et
al., 1996). The mouse monoclonal IgG2A antibody F4/80
(1:10; Serotec, Indianapolis, IN) was used to detect microglia/macrophages.
The rabbit polyclonal KI-67 antibody (1:1000; Santa Cruz Biotechnology) was
used to detect proliferating cells. The goat polyclonal anti-tumor necrosis
factor- (TNF-) antibody (1:100; R & D Systems, Minneapolis,
MN) was used to detect TNF-.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick
end labeling (TUNEL) assay. The TUNEL assay was used to detect cells
undergoing apoptosis using the NeuroTACS II assay kit (Trevigen, Gaithersburg,
MD). Frozen tissue sections were incubated with either the NG2 or CC1 antibody
as described above. The tissue sections were then incubated with 1x
terminal deoxynucleotidyltransferase labeling buffer for 5 min and then
processed for biotin-dNTP-labeling of fragmented DNA, according to the
manufacturer's instructions. The tissue sections were then incubated with a
fluorescein-conjugated rat anti-mouse IgG2b diluted in a 2%
streptavidin-Texas Red complex (Vector Laboratories, Burlingame, CA)/PBS,
counter-stained with DAPI, coverslipped, and examined using an Olympus BX60
microscope equipped with epifluorescent optics.
Cell number quantification. Immunopositive cells were obtained by
counting only those cells with an identified nucleus, observable by DAPI
staining, within the medial region of the corpus callosum. Individual cell
counts were conducted on both sides of midline, each corresponding to an area
of 0.033 mm 2. The cell counts from both areas were averaged to
give a total for each tissue section. Cell counts from three nonadjacent
tissue sections for each animal were then averaged to give a representative
cell count for each animal in the study. Because of the high density of NG2
+ cells and their processes at 4 weeks of cuprizone treatment, a
Leica (Nussloch, Germany) TCS-NT Laser Scanning microscope using 100x
oil objective and a pinhole size of 1.0 Airy disk units was used to obtain
cell counts for the NG2 + cells. The cell counts are presented in
the text as the mean and SEM from at least three to four mice at each time
point.
Statistical analysis. Statistical comparisons were made using a
one-factor between subjects ANOVA, followed by Tukey's test to assess
significance among groups.
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Results
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Ablation of IGF1R function in oligodendrocyte progenitors
Tissue-specific conditional mutagenesis using the cre/loxP
recombination system (Sauer,
1998; Rossant and McMahon,
1999) requires crosses between Cre-producing and Cre-responding
strains of mice. Responders used in this study, in which exon 3 of the
Igf1r gene is "floxed" (flanked by loxP sites in
direct orientation; Fig. 1),
have been described previously (Dietrich et
al., 2000). To drive cre expression in the
oligodendrocytes of producer mice, we used CaMKII-cre
transgenic mice (Dragatsis and Zeitlin,
2000). Southern and Western analyses
(Fig. 1B,C) yielded
results consistent with the information provided by the use of the LacZ
reporter. Thus, Cre-mediated DNA excision in Igf1r mice was
detected in forebrain (and at very low levels in the cerebellum and the
testis, in which the cre transgene is marginally expressed)
(Dragatsis and Zeitlin, 2000),
whereas in all other examined tissues the floxed allele had remained intact
(Fig. 1B). The near
absence of Cre-mediated recombination in the cerebellum suggests intrinsic
differences among neural cells within different regions of the brain in regard
to CaMKII expression. Quantitation (see Materials and Methods)
indicated that 
45% of the Igf1rflox allele
was converted to Igf1rflox (the
cre transgene is not expressed in all forebrain cell constituents).
In close agreement with the results of Southern analysis, Western blotting
showed that IGF1R protein was reduced to 50% of normal in the forebrain
of adult conditional mutants (Fig.
1C).
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Figure 1. Partial restriction map in the region of exons 3 and 4 (black rectangles)
of the mouse Igf1r locus. A, Targeted null and also
conditional mutant alleles before and after Cre-mediated excision of exon 3
and flanking sequences (Igf1rflox and
Igf1rflox, respectively) are shown. The
floxP sites (gray triangles) are not in scale. The position of a 0.8
kb EcoRV-NsiI (R-N) probe used for Southern
analyses and the sizes of the KpnI (K) DNA fragments
detectable by this probe are indicated. Brain-specific, Cre-mediated
recombination in heterozygous mice possessing floxed and null Igf1r
alleles and also carrying a CaMKII-cre transgene is shown. WT,
Wild-type. B, Example of Southern analysis of KpnI-digested
DNA that was extracted from various tissues of the same
Igf1rflox/-;
CaMKII-cre animal: forebrain (lane 1), cerebellum (lane 2),
heart (lane 3), lung (lane 4), liver (land 5), kidney (lane 6), spleen (lane
7), and testis (lane 8). Because the CaMKII-cre transgene is
not expressed in all forebrain cells, Cre-mediated recombination and
forebrain-selective appearance of a fragment characteristic for an
Igf1rDflox allele occurred in a fraction of all
floxed loci. C, Western blot analysis of IGF1R expression. The
examined protein extracts were from cultured fibroblasts prepared from
wild-type (lane 1) and Igf1r null (lane 2) embryos (positive and
negative control, respectively) or from mouse forebrains dissected from
wild-type (lane 3) or Igf1rflox/-;
CaMKII-cre (lane 4) animals. The positions of the IGF1R
precursor and -subunit detectable by the specific antibody used (see
Materials and Methods) are shown. The blot was reprobed with an anti-tubulin
antiserum (loading control; bottom). Quantitation of the results (see
Materials and Methods) indicated that the amount of IGF1R -subunit in
the mutant relative to wild type is 50%.
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Although it has been reported that, in contrast to neurons, glial cells do
not express CaMKII (Vallano et al.,
2000), we observed that in the particular
CaMKII-cre transgenic line used in our analysis (R1ag5), the
CaMKII promoter was active and able to expresses cre, not only
in 90% of neurons in the adult forebrain
(Dragatsis and Zeitlin, 2000),
but also in oligodendrocyte progenitors. This localization was demonstrated by
generating and analyzing bitransgenic mice carrying the
CaMKII-cre transgene and, in addition, a -galactosidase
(LacZ) transgenic reporter (Soriano,
1999) that becomes functional only after Cre-mediated
recombination (activity can be monitored by X-gal staining). When forebrain
sections from such bitransgenic neonates (P1) were stained to assess
-gal expression in O4-positive and NG2-positive oligodendrocyte
progenitors, we observed that the cells were also -gal positive
(Fig. 2A),
demonstrating that Cre is, indeed, expressed and is able to exert its
recombinogenic action in oligodendrocyte precursors during development. These
results suggested that, on expression of the recombinase from the
CaMKII-cre transgene, both oligodendrocyte progenitors and
mature oligodendrocytes should become devoid of IGF signaling by conversion of
the Igf1rflox/- genotype to
Igf1rflox/-. For simplicity,
these experimental mice will be referred to below as Igf1r
mice, although in all cells other than those expressing Cre the floxed allele
of the Igf1r gene remains intact.
As a test for Cre activity in oligodendrocyte progenitors of the adult CNS,
we isolated O4+ cells from the corpus callosum of
Igf1r mice and compared the expression of IGF1R in these mice
compared with O4+ cells from wild-type mice using
immunohistochemistry, as described previously
(Mason and Goldman, 2002). We
found that 91 ± 3.1% of the wild-type cells expressed IGF1R, whereas
only 6 ± 2.1% of the cells from Igf1r mice expressed
the receptor (Fig.
2B). Thus, the genetic strategy successfully excises the
IGF1R from oligodendrocyte progenitors in both neonates and adults.
In contrast to mice globally lacking IGF1R, which die at or soon after
birth because of respiratory failure (Liu
et al., 1993), the Igf1r mice survived to
adulthood and did not exhibit any overt morphological or behavioral
abnormalities. In particular, the degree of myelination appeared to be normal
in adult Igf1r mice (Fig.
3A), suggesting that IGF signaling may not play a
significant role in the myelination process during development. Alternatively,
any such role could have been compensated effectively by some other signaling
pathway. On the basis of these results, we decided that the
Igf1r mice are suitable models that would allow us to
investigate the functional significance of IGF signaling during demyelination
and remyelination.
IGF signaling plays a critical role in remyelination of the adult
murine CNS
Cuprizone induces a consistent and reversible demyelinating lesion within
the medial region of the corpus callosum of adult mice
(Mason et al., 2001b). This
demyelination occurred to the same extent in both wild-type and
Igf1r mice by 4 weeks, as assessed by the absence of MBP
(Fig. 3A). Subsequent
remyelination of the corpus callosum in wild-type mice begins at 6 weeks and
is completed by 9-10 weeks if the mice are returned to a normal diet after 6
weeks of cuprizone (Fig.
3A) (Mason et al.,
2001b). In contrast, remyelination was not observed in the middle
region of the corpus callosum in Igf1r mice by 9 weeks
(Fig. 3A) or 14 weeks
(data not shown). We noted, however, a thin zone of remyelination in the
extreme ventral and dorsal edges of the callosum (see Discussion). This
failure of remyelination in the middle region of the corpus callosum was a
reproducible phenomenon, observed in all four of the Igf1r
mice examined. The lack of remyelination at 14 weeks suggests that
remyelination is not simply delayed in the Igf1r mice.
To determine whether the lack of remyelination in Igf1r
mice might be because of a loss of axons within this region, we stained for
MBP (myelin) and NF-H (axons) at 9 weeks. The results from three separate mice
demonstrated that axons were still present within the middle region of the
demyelinated corpus callosum (Fig.
3B).
The accumulation of microglia/macrophages and TNF- corresponds
spatially with demyelination and persists in the absence of IGF signaling
Microglia/macrophages clearing myelin debris by phagocytosis accumulate
within the demyelinating corpus callosum in wild-type mice between 2 and 5
weeks (Hiremath et al., 1998).
Also present during the same period is the cytotoxic cytokine TNF-,
which is expressed by microglia/macrophages and astrocytes
(Arnett et al., 2001).
TNF- facilitates the continued activation of microglia/macrophages and
promotes the proliferation of oligodendrocyte progenitors to restore the
lesion (Arnett et al., 2001).
Thus, for further assessment of the demyelination process, we examined
histologically the demyelinated region for the presence of TNF- and of
microglia/macrophages, the latter identified by the F4/80 antibody. We
observed that the accumulation of these two indicators at 4 weeks was the same
in both wild-type and Igf1r mice
(Fig. 4). In contrast, a
significant difference was observed at 9 weeks. At this time, the
microglia/macrophages and TNF- had practically disappeared in wild-type
mice from the region that was previously damaged, whereas their presence
persisted in Igf1r mutants
(Fig. 4). Thus, counting of
F4/80+ cells at 9 weeks indicated that their number was at least
fourfold higher in the mutants than in the controls
(Fig. 4).
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Figure 4. Effects of IGF signaling on the accumulation of microglia/macrophages
within the demyelinating and remyelinating lesion. Large numbers of
microglia/macrophages (A; red), detected by the ant-F4/80 antibody,
and TNF- (B; green) accumulate within the demyelinating corpus
callosum in both wild-type (WT) and Igf1r mice, peaking at 4
weeks. However, although the microglia/macrophages and TNF- disappear
from the remyelinating lesion in WT mice, a large number of
microglia/macrophages and TNF-+ cells remain within the
demyelinated region of the corpus callosum in Igf1r mice at 9
weeks. d, Dorsal; m, middle; v, ventral. Scale bars, 50 µm. C, The
mean and SEM bars for the number of F4/80+
macrophages/mm2 are plotted. *p < 0.01.
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Mature oligodendrocyte regeneration is decreased in the absence of
IGF signaling
During cuprizone-induced demyelination, the mature oligodendrocyte
population, identified by the CC1 antibody, disappeared throughout the entire
depth of the corpus callosum in both wild-type and Igf1r mice
at 4 weeks (Fig. 5)
(Mason et al., 2000a).
Subsequently, mature oligodendrocytes reappeared in normal numbers in
wild-type mice after they were returned to a normal diet after 6 weeks of
cuprizone treatment (Fig. 5)
(Mason et al., 2000a). In
contrast, mature oligodendrocytes did not regenerate to normal numbers in
Igf1r mice at 9 weeks (Fig.
5). Counting of CC1+ oligodendrocytes at this time
indicated the number of mature oligodendrocytes in the mutants was 45% of
that found in controls. These cell counts reflect CC1+
oligodendrocytes throughout the entire depth of the corpus callosum (counting
cells within specific regions of the callosum is very difficult), and after
examining many sections, we can state that most of these oligodendrocytes
resided in the remyelinated ventral and dorsal regions of the callosum and not
in the demyelinated middle region. Additional analysis (TUNEL assay combined
with CC1 immunostaining) indicated that the paucity of oligodendrocytes in the
middle region was not because of the apoptotic death of newly generated
oligodendrocytes during recovery at 6 and 9 weeks (data not shown).
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Figure 5. Effects of IGF signaling on the regeneration of the mature oligodendrocyte
population after an acute demyelinating lesion. Mature oligodendrocytes (red),
detected by the anti-CC1 antibody, become depleted in both wild-type (WT) and
Igf1r mice by 4 weeks. However, the regeneration of mature
oligodendrocytes during recovery is dramatically reduced in the absence of IGF
signaling for the entire depth of the corpus callosum, and there is most
likely even a greater reduction in the middle region. d, Dorsal; m, middle; v,
ventral. Scale bar, 40 µm. The mean and SEM bars for the number of
CC1+ mature oligodendrocytes/mm2 are plotted.
*p < 0.01.
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Oligodendrocyte progenitors do not accumulate in the absence of IGF
signaling
In wild-type mice, NG2+ oligodendrocyte progenitors accumulate
within the demyelinated corpus callosum between 3 and 5 weeks of cuprizone
feeding and then decline in number during the reappearance of mature
oligodendrocytes and remyelination (Fig.
6A,B) (Mason et al.,
2000a). In contrast, few NG2+ oligodendrocyte
progenitors were observed within the middle region of the callosum in
Igf1r mice at 4 weeks (Fig.
6A,B). However, there was some accumulation of these
progenitors within the dorsal and ventral edges of the callosum, in which
regeneration of mature oligodendrocytes and remyelination occurred
(Fig. 6A). Thus, the
absence of mature oligodendrocytes and lack of remyelination in the middle
region of the callosum correlates spatially with the near absence of
progenitors at 4 weeks. However, by 9 weeks, there appeared to be a low
density of progenitors evenly distributed throughout the corpus callosum in
both wild-type and Igf1r mice
(Fig. 6A), even though
there was still a significant reduction in the number of oligodendrocyte
progenitors throughout the entire corpus callosum in Igf1r
mice (Fig. 6B). Under
our conditions, NG2 staining did not co-localize with F4/80+
microglia/macrophages as reported previously
(Nishiyama et al., 1997).
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Figure 6. Effects of IGF signaling on the accumulation of oligodendrocyte
progenitors. A, Oligodendrocyte progenitors (green), detected by NG2,
accumulate with the demyelinating corpus callosum in wild-type (WT) mice,
peaking at 4 weeks when demyelination is complete. However, oligodendrocyte
progenitors only accumulate within the ventral (v) and dorsal (d) edges of the
corpus callosum, with very few cells in the middle (m) region of the corpus
callosum in Igf1r mice. By 9 weeks, there is a dramatic
reduction in the number of NG2+ oligodendrocyte progenitors within
the corpus callosum of WT and Igf1r mice. B, The mean
and SEM bars for the number of NG2+ oligodendrocyte
progenitors/mm2 throughout the entire depth of the corpus callosum
are plotted. *.p < 0.01; **p <
0.05. C, Although a large number of NG2+ oligodendrocyte
progenitors (green) proliferate, detected by an anti-KI-67 antibody (red), at
4 weeks within the demyelinated corpus callosum in wild-type mice (arrowheads
indicate NG2+/KI-67+ cells), no proliferating
NG2+ oligodendrocyte progenitors were observed in the demyelinated
corpus callosum of Igf1r mice at 4 weeks. Three nonadjacent
sections from each of three different wild-type and Igf1r mice
were examined for proliferating NG2+ oligodendrocyte progenitors.
D, Apoptotic [detected by TUNEL assay (red)] NG2+
oligodendrocyte progenitors (green) were observed within the middle region of
the corpus callosum in Igf1r mice at 4 weeks (arrowhead
indicates apoptotic NG2+ cells), whereas no apoptotic progenitors
were detected in wild-type mice. Three nonadjacent sections from each of three
different wild-type and
CaMKII-cre/Igf1rflox/-
mice were examined for apoptotic NG2+ oligodendrocyte progenitors.
Scale bars: A, 50 µm; C, 15 µm; D, 10
µm.
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The lower number of NG2+ progenitors within the middle region of
the callosum in Igf1r mice could be because of increased cell
death and/or hypoproliferation. To address these issues, we used
immunohistochemistry to determine whether proliferating (using the KI-67
antibody) and/or apoptotic (TUNEL assay) NG2+ progenitors were
present in wild-type and Igf1r mice at 4 weeks. In contrast to
the number of proliferating NG2+ progenitors throughout the corpus
callosum in wild-type mice (88 ± 21 cells/mm2)
(Arnett et al., 2001), there
was a significant (p < 0.05) reduction in the number of
proliferating NG2+ progenitors throughout the corpus callosum in
Igf1r mice (22 ± 9 cells/mm2). All of the
proliferating NG2+ progenitors were observed within the ventral and
dorsal regions of the corpus callosum and not within the middle region in the
Igf1r mice (Fig.
6C). Apoptotic NG2+ progenitors were only
observed throughout the corpus callosum in Igf1r mice (55
± 17 cells/mm2) and not within wild-type mice (0
cells/mm2; Fig.
6D) (Arnett et al.,
2001). It seems, therefore, that the lower number of progenitors
within the demyelinating lesion is because of the inability of these cells to
survive and then possibly proliferate.
|
Discussion
|
|---|
Our observations in the Igf1r mice demonstrate the
importance of IGF signaling through IGF1R in remyelination. This signaling is
not required for myelination during development, as shown in
IGF-1-/- mice (Cheng et al.,
1998). In addition, we noted that the lack of IGFR1 in
oligodendrocyte progenitor cells does not appear to impede myelination during
CNS development. Although we have not examined the kinetics of myelination or
counted numbers of progenitors and mature oligodendrocytes in detail, the
adult mice appeared neurologically normal, both behaviorally and
morphologically. We suggest that other factors may compensate for the absence
of IGF signaling through IGFR1 to allow oligodendrocytes to survive and
myelinate appropriately. However, remyelination was impaired in these
Igf1r mice.
The absence of remyelination in the middle region of the corpus callosum in
the Igf1r mice could be because of the loss of IGF signaling
in neurons, oligodendrocytes, or both, because the CaMKII promoter
expresses cre in both neurons and oligodendrocyte progenitors in
these transgenic mice. Because normal-appearing axons (without swelling) were
observed within the demyelinated middle region, we presume that the absence of
remyelination was not because of axonal degeneration. Although we do not know
whether the Igf1r mice retain the full complement of axons
after cuprizone treatment, the degree of NF-H immunoreactivity appears similar
to that in wild-type mice. In addition, it would appear that the absence of
IGF signaling in neurons does not affect the ability of their axons to become
remyelinated, because remyelination was observed in the dorsal and ventral
regions. In contrast, the absence of oligodendrocyte progenitors and mature
oligodendrocytes within the middle region of the callosum correlates spatially
with the absence of remyelination in the Igf1r mice. Thus, we
postulate that IGF signaling within oligodendrocytes is needed for
remyelination.
The belief that IGF-1 promotes spontaneous remyelination of the adult CNS
following a demyelinating insult has been widely speculated. Systemic
administration of IGF-1 enhances remyelination by inhibiting the immune
response in some autoimmune models of demyelination
(Liu et al., 1997;
Li et al., 1998), but not in
others (Cannella et al., 2000).
Although IGF-1 induces the differentiation of oligodendrocyte progenitors
in vitro (McMorris and
Dubois-Dalq, 1988; Mozell and
McMorris, 1991; Barres et al.,
1993) and is upregulated within remyelinating lesions in
cuprizone-, experimental autoimmune encephalomyelitis-, and
lysolecithin-induced demyelination (Liu et
al., 1994; Hinks and Franklin,
1999; Mason et al.,
2000a), there has been no evidence to support the theory that
IGF-1 promotes remyelination by inducing the differentiation of
oligodendrocyte progenitors (O'Leary et
al., 2002). In contrast, increased levels of IGF-1 protect mature
oligodendrocytes within demyelinating lesions, thus facilitating a rapid
recovery (Mason et al.,
2000b). In addition, we show here that IGF signaling is required
for the survival, proliferation, and differentiation of oligodendrocyte
progenitors during remyelination.
We suggest that the increased death of oligodendrocyte progenitors in the
Igf1r mice is related to their inability to respond to IGF-1
as a survival factor (Barres et al.,
1993; Ness and Wood,
2002). Cytotoxic factors, such as TNF-, that normally
increase and then decrease with the cycle of demyelination and remyelination
(Arnett et al., 2001) remain
elevated within the middle region of the demyelinating lesion in
Igf1r mice, most likely because of the continued presence of
macrophages and some astrocytes (Arnett et
al., 2001). TNF- appears to have two opposite effects on
oligodendrocytes and their progenitors, each mediated by a distinct TNF
receptor (TNFR), both of which are upregulated during inflammatory conditions
(Tchelingerian et al., 1995;
Dopp et al., 1997).
TNF- produces cell death through TNFR1
(Haridas et al., 1998;
Weiss et al., 1998;
Ashkenazi and Dixit, 1999), but
it also induces the proliferation of oligodendrocyte progenitors through TNFR2
(Arnett et al., 2001). It is
possible that in the absence of IGF signaling, the balance of effects induced
by TNF- is tilted toward cell death, and, thus, oligodendrocytes
succumb to the toxic effects of TNF- rather than surviving and
manifesting the proliferative effects. In fact, IGF-1 inhibits the death of
oligodendrocyte progenitors induced by TNF- in vitro
(Ye and D'Ercole, 1999).
Interestingly, remyelination occurred in the dorsal and ventral zones of the
callosum, in which macrophages and their associated cytokines disappeared
according to the normal time schedule
(Arnett et al., 2001;
Matsushima and Morell, 2001).
It is, therefore, possible that there is a reduced level of TNF- in the
dorsal and ventral zones compared with the middle, and, thus, progenitor
survival and remyelination are better preserved at the edges of the callosum,
even in the absence of IGF signaling.
Although IGF1R expression is not disrupted within microglia and macrophages
(data not shown), these cells remain within the lesion in
Igf1r mice, even after the mice are returned to a normal diet.
The stimulus that keeps the macrophages within the lesion and the effect their
presence has on the inability of the lesion to remyelinate is not known.
Because we did not find evidence for recurrent remyelination and
demyelination, we do not believe that the macrophages remained to clear debris
from failed attempts at remyelination. It is possible that macrophages remain
in the area because of continued progenitor cell death. We did observe small
numbers of NG2+ cells in the middle region of the callosum after 9
weeks (Fig. 6A), and
although we did not detect TUNEL+ cells in that population, we
cannot rule out the possibility of a slow continuation of progenitor
death.
Although spontaneous remyelination occurs after a demyelinating insult to
the adult CNS, it is often incomplete, presumably because of the depletion of
oligodendrocyte progenitors (Lucchinetti
et al., 1996; Blakemore and
Keirstead, 1999), the loss of axons, the absence of an environment
to support progenitor differentiation
(Wolswijk, 1998;
Chang et al., 2002), or a
combination of the above events. The present work, when combined with previous
results demonstrating the ability of IGF-1 to prevent mature oligodendrocyte
death and depletion during acute demyelination
(Mason et al., 2000b),
suggests that IGF signaling is vital for not only facilitating recovery from
acute demyelinating insults, but that therapies incorporating increased levels
of IGF-1 may prevent the death and depletion of oligodendrocytes and formation
of chronic lesions. Further investigation of IGF signaling in preventing the
formation of chronic demyelinating lesions is warranted.
|
Footnotes
|
|---|
Received Apr. 10, 2003;
revised Jun. 11, 2003;
accepted Jul. 7, 2003.
This work was supported by Grant FG 13377-A-1 to J.L.M. from the National
Multiple Sclerosis Society and by National Institutes of Health Grants NS17125
to J.E.G. and CA75553 (Project 3) to A.E. We thank Bernetta Abramson for
technical help and Dr. Bill Stallcup for the NG2 antibodies.
Correspondence should be addressed to Dr. Jeffrey Mason, Department of
Pathology, Columbia University, 630 West 168th Street, P&S 15-420, New
York, NY 10032. E-mail:
jm1306{at}columbia.edu.
I. Dragatsis' present address: Department of Physiology, University of
Tennessee, Memphis, TN 38163.
Copyright © 2003 Society for Neuroscience
0270-6474/03/237710-09$15.00/0
|
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Top
Abstract
Introduction
