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The Journal of Neuroscience, July 15, 2002, 22(14):6041-6051
Myelination Is Altered in Insulin-Like Growth Factor-I
Null Mutant Mice
Ping
Ye1,
Liqin
Li1,
R. Gregg
Richards2,
Richard P.
DiAugustine2, and
A. Joseph
D'Ercole1
1 Department of Pediatrics, The University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and
2 Hormones and Cancer Group, National Institute of
Environmental Health Sciences, Triangle Research Park, North Carolina
27709
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ABSTRACT |
Increasing evidence indicates that insulin-like growth factor-I
(IGF-I) has an important role in oligodendrocyte development. In this
study, we examined myelination during postnatal development in IGF-I
knock-out (KO) mice by assessing myelin staining, the expression of
myelin basic protein (MBP) and proteolipid protein (PLP), two
major myelin-specific proteins, and the number of oligodendrocytes and
their precursors. For comparison, we also measured the expression of
median subunit of the neuron-specific intermediate filament, M-neurofilament (M-NF), to obtain an index of the effects of
IGF-I deficiency on neurons. We found that myelin staining, MBP and PLP
expression, and the percentage of oligodendrocytes and their precursors
are significantly reduced in all brain regions of developing IGF-I KO
mice but are similar to controls in adult IGF-I KO mice. In contrast,
the abundance of M-NF was decreased in both the developing and adult
brain of IGF-I KO mice. We also found that IGF-II protein abundance is
increased in the brains of IGF-I KO mice. Our data indicate, therefore,
that myelination during early development is altered in the absence of
IGF-I by mechanisms that involve a reduction in oligodendrocyte
proliferation and development. Although neuronal actions cannot be
excluded in the myelin normalization, the reduced axonal growth
suggested by the reduced M-NF expression makes a role for neuronal
factors less compelling. These data suggest that IGF-I plays a
significant role in myelination during normal early development and
that IGF-II can compensate in part for IGF-I actions on myelination.
Key words:
IGF-I; IGF-II; oligodendrocytes; oligodendrocyte
precursors; myelination; development
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INTRODUCTION |
In the CNS, myelination is
characterized by proliferation and differentiation of oligodendrocyte
precursors and accumulation of myelin-associated proteins and lipids.
In rodents, myelination begins in the first postnatal week and peaks at
~3-4 weeks (Morell, 1984 ; Jacobson, 1991). Increasing evidence
indicates that insulin-like growth factor-I (IGF-I) has an important
role in myelination. In culture, IGF-I increases the number of
oligodendrocytes and their precursors by mechanisms involving promotion
of oligodendrocyte precursor proliferation, differentiation, and
survival (McMorris et al., 1986; McMorris and Dubois-Dalcq, 1988;
Saneto et al., 1988; Mozell and McMorris, 1991; Barres et al., 1992; Ye
and D'Ercole, 1999). Consistent with in vitro data, we (Ye
et al., 1995a,b; Mason et al., 2000) and others (Carson et al., 1993)
have demonstrated that brain IGF-I overexpression in transgenic (Tg)
mice increases myelination by increasing oligodendrocyte number, myelin
protein gene expression, and myelin sheath thickness. Conversely,
inhibition of IGF-I bioactivity by ectopic expression of IGF binding
protein-1 (IGFBP-1) in brain decreases myelination (Ye et al.,
1995a,b).
Like IGFBP-1 Tg mice, mice with ablated IGF-I gene expression [IGF-I
knock-out (KO) mice] exhibit a decreased number of oligodendrocytes and myelinated axons in corpus callosum (CC) and anterior
commissure (Beck et al., 1995). More recently, Cheng et al.
(1998) reported that myelin-associated galactocerebroside and sulfatide
are reduced by ~25% in the brains of adult IGF-I KO mice but that
the concentration of myelin and myelin-specific proteins does not
differ from those in wild-type (WT) controls. Based on these findings,
Cheng et al. concluded that IGF-I does not have a major role in
myelination during development and that axonal factors other than IGF-I
determine oligodendrocyte survival and myelination. These
investigators, however, only examined myelination in adult IGF-I KO
mice, leaving open the possibility that myelination is altered during
development in the absence of IGF-I expression. In addition to IGF-I,
IGF-II and insulin (in high concentrations) can transduce signals
through interaction with the type 1 IGF receptor. It seems possible,
therefore, that IGF-II (and possibly other agents) can serve as
surrogates when IGF-I is deficient and mask the normal role of IGF-I in
myelination in IGF-I KO mice.
To investigate these possibilities, we examined myelination and IGF-II
expression during early postnatal development. We also measured the
expression of the neuron-specific intermediate filament neurofilament
(NF) to obtain an index of the effects of IGF-I deficiency on neurons.
Here we provide evidence that myelination is significantly reduced in
developing IGF-I KO mice. We also show that IGF-II protein abundance is
increased in the brains of IGF-I KO mice, suggesting that the increased
IGF-II expression may compensate for the loss of IGF-I and its
functions. Although we cannot exclude a role for neurons in the myelin
normalization, the reduced axon growth suggested by the reduced median
subunit of the neuron-specific intermediate neurofilament (M-NF)
expression makes this less compelling. These data suggest, therefore,
that IGF-I plays a significant role in myelination during normal early development.
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MATERIALS AND METHODS |
Mice. IGF-I KO mice (Liu et al., 1993) were a
generous gift from Dr. Argiris Efstratiadis (Columbia University, New
York, NY) and were housed at National Institute of Environmental Health Sciences (Research Triangle Park, NC). Homozygous IGF-I KO mice were
obtained by breeding heterozygous males with heterozygous females, and
only male IGF-I KO mice were studied. Consistent with a previous report
(Liu et al., 1993), ~20% of homozygous IGF-I KO mice survived
postnatally. Homozygous, heterozygous, and WT mice were routinely
identified by PCR of tail genomic DNA, using specific
oligonucleotide primers, as reported previously (Richards et al.,
2001). Mice were maintained with 12 hr light/dark cycles at
22°C. All procedures used were consistent with the guidelines of
National Institutes of Health and approved by institutional review
committees of the University of North Carolina at Chapel Hill and the
National Institute of Environmental Health Sciences.
Northern blot hybridization analysis. Total RNA was
extracted using the acidic guanidinium thiocyanate-phenol-chloroform
method (Chomczynski and Sacchi, 1987). Aliquots of 15-20 µg of total RNA were electrophoresed, transferred onto nylon membranes, and UV
cross-linked. After staining with 0.02% methylene blue, membranes were
photographed to quantify the amount of RNA transferred and then
hybridized with 32P-labeled
single-stranded DNA (ssDNA) probes in Church's buffer (0.5 M sodium phosphate, pH 7.1, 7% SDS, and 0.1 mM EDTA) and washed at high stringency with 40 mM sodium phosphate and 1% SDS at 55°C for 60 min. After autoradiography, membranes were stripped and hybridized with
a second probe.
Quantification was performed using a computer-assisted image analysis
system (Image-Pro; Media Cybermetics, Silver Spring, MD). To ensure the
accuracy of changes in mRNA abundance and equal loading of RNA, the
mRNA levels were normalized to cyclophilin (CYC) mRNA abundance or to
the amount of 18 S rRNA on the membrane visualized by methylene blue
staining. The abundance of CYC mRNA closely paralleled the amount of 18 S rRNA transferred.
Probes. Proteolipid protein (PLP), myelin basic protein
(MBP), and CYC DNA fragments were amplified by PCR and used as
templates for probes, as described in our previous report (Ye et al.,
1995a). The PLP DNA fragment corresponded to base pairs (bp) 268-903
of mouse PLP cDNA (Hudson et al., 1987), the MBP DNA fragment
corresponded to bp 58-490 of the mouse MBP cDNA (Newman et al., 1987),
and the CYC DNA fragment corresponded to bp 106-517 of the rat CYC cDNA (Danielson et al., 1988). ssDNA hybridization probes were generated from these templates by linear PCR (Ye et al., 1992, 1995a)
using their respective 3' end primers and
32P-labeled dCTP (Amersham
Biosciences, Arlington Heights, IL).
Western blot analysis. Protein from each brain region was
extracted as described previously (Klugmann et al., 1997). Briefly, pulverized tissues were lysed in 1% SDS and 1× PBS and sonicated, and
the resultant homogenates were boiled for 3-5 min. Supernatants were
collected by centrifugation at 12,000 rpm for 5 min at 4°C and stored
at  80°C until use. Protein concentration was determined with the
BCA protein kit (Pierce, Rockford, IL) using bovine serum albumin as a
standard. Aliquots of 15-30 µg of protein (80 µg of protein for
IGF-II) were separated on 7.5 or 12.5% polyacrylamide gel and
transferred onto polyvinylidene difluoride membrane (Amersham Biosciences). Specific proteins were detected using their specific antibody and visualized using ECL kits (Amersham Biosciences) or ABC
colometric kit (Vector Laboratories, Burlingame, CA). Primary antibodies were used and obtained as follows: anti-MBP antibody (1:1500), anti-M-NF antibody (1:1000), anti-H-NF antibody (1:1000), and
anti-glial fibrillary acidic protein (GFAP) (1:3000) were obtained from
Chemicon (Temecula, CA); anti-PLP antibody (1:500) was obtained from
Biogenesis (Brentwood, NH); and anti-IGF-II antibody (1:200),
anti-IGF-IR  subunit (1:1000), anti-insulin receptor substrate 1 (IRS1) antibody (1:1000), anti-IRS2 (1:1000), and anti-IRS4
antibody (1:1000) were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA). Human recombinant IGF-II (Eli Lilly, Indianapolis, IN) was a
gift from Dr. Marsh Davenport (University of North Carolina at Chapel
Hill) or obtained from Santa Cruz Biotechnology. Quantification of
specific protein abundance was performed using a computer-assisted
image analysis system (Image-Pro; Media Cybermetics). To ensure the
equal loading and accuracy of changes in protein abundance, the protein
levels were normalized to actin abundance. The abundance of actin
closely paralleled the amount of total protein loaded.
Histochemical and immunohistochemical staining. Brains were
perfused by transcardial infusion of 4% paraformaldehyde in PBS and
further fixed in the same fixative overnight. Brains were split along
the middle sagittal line. Left hemispheres were paraffin embedded and
sagittally sectioned at a thickness of 6 µm for myelin staining using
the Luxol Fast Blue-Periodic Acid Schiff (LFB-PAS) method and
glutathione S-transferase (GST)- (1:500) immunostaining. Right hemispheres were cryoprotected with 20% sucrose and sagittally frozen sectioned at a thickness of 10 µm. Sections were washed with
1× PBS and incubated with a polyclonal antibody specific for IGF-II
(1:200; Santa Cruz Biotechnology), NG2 (1:200; a gift from Dr. Bill
Stallcup, The Burnham Institute, La Jolla, CA), PLP (1:500), or MBP
(1:250; Chemicon). Antibody-antigen complexes were detected using
either a rhodamine-conjugated anti-rabbit IgG antibody or an ABC kit
and peroxidase (Vector Laboratories) and visualized by incubation with DAB.
Quantification of oligodendrocytes. To quantify the number
of oligodendrocytes and their precursors, sections were immunostained with GST- antibody or NG2 antibody, respectively, and a
peroxidase-conjugated secondary antibody. They then were counterstained
with hematoxylin. Images of labeled cells (blue hematoxylin-stained
nuclei and yellowish brown-immunostained cells) were digitally
captured using a microscope and a Spot Jr. digital camera (Diagnostic
Instruments, Sterling Heights, MI). Only immunolabeled cells with clear
nuclei were scored. The number of GST--positive cells or
NG2-positive oligodendrocyte precursors was calculated as percentage of
total number of cells as judged by the number of nuclei. In each
section, 300-500 cells in CC and brainstem (BS) and 400-600
cells in cerebral cortex (CTX) were counted. Overestimates of cell
number attributable to splitting of nuclei were corrected using
Abercrombie's methods (Abercrombie, 1946).
Statistics. Student's t test was used with
assistance of SigmaStat software for Widows (SPSS, Chicago, IL).
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RESULTS |
Compared with those of WT littermate controls (Fig.
1), brains of IGF-I KO mice were
significantly reduced in weight from the earliest time measured (1 week
of age). These results are consistent with data reported previously
(Beck et al., 1995; Cheng et al., 1998). To determine whether there are
regional alterations in brain growth during development, brains from
developing IGF-I KO mice, as well as WT controls, were dissected into
five distinct regions [CTX, hippocampus (HIP), diencephalon (DIE),
cerebellum (CB), and BS] and weighed. Compared with WT controls, the
weights of all five regions were significantly decreased in IGF-I KO
mice at all ages examined (Fig. 1). There was no significant difference in brain weight between WT and heterozygous IGF-I KO mice, and, therefore, the data from WT and heterozygous IGF-I KO mice were grouped
for additional analyses and referred to as littermate control or as
control mice.
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Figure 1.
Weights of whole brain and brain regions in IGF-I
KO and control mice during development. Brains were collected from
IGF-I KO mice (open circles) and their littermate
controls (filled circles), dissected, and
weighed. Values are means ± SE from 6-10 mice. Absence of error
bars indicates that the variance is smaller than the area encompassed
by the symbol. WB, Whole brain. At
each age studied, the weights recorded for IGF-I KO and control mice
were significantly different (p < 0.01).
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We first tested whether blunting of IGF-I expression during early
development affects myelination using
LFB-PAS staining as an index. As Figures 2 and
3 show, myelin is stained light
blue, cell nuclei is stained dark blue, and
hydrocarbonates are stained pink/purple. In control mice,
the abundance of light blue-stained myelin gradually increased during
the developmental period studied. At 1 week of age, myelin was readily
observed in BS and CB. Light blue-stained myelin became evident in CTX
of 2-week-old mice, and bundles of myelinated fibers were observed in
CC, BS, DIE, olfactory bulb, and other brain regions at this time.
Myelin staining significantly increased during the next several weeks
in all brain regions. Representative microphotographs of LFB-PAS
staining in developing CTX and BS are shown in Figures 2 and 3,
respectively. Myelin staining in IGF-I KO mice followed a similar
developmental pattern. Compared with control mice, however, myelin
staining in IGF-I KO mice was significantly reduced in the first 3 weeks of life. Myelin staining in most brain regions of IGF-I KO mice gradually increased and became similar to that in control mice at 10 weeks of age (Figs. 2, 3). It appears, however, that the myelin content
in CC of IGF-I KO mice remained less than that of littermate controls
(Fig. 2). Immunostaining of MBP and PLP, two major myelin-specific
proteins, exhibited similar patterns of change during development (data
not shown).
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Figure 2.
Myelin staining of CTX from IGF-I KO and
littermate control mice during development. Brains of IGF-I KO mice
(E-H) and their littermate controls
(A-D) were perfusion fixed with 4%
paraformaldehyde, sectioned, and stained using the LFB-PAS method.
Myelin is stained light blue, nuclei are dark
blue, and hydrocarbonates are pink/purple.
Sections from mice at 1 week of age are shown in A and
E, 2 weeks are shown in B and
F, 3 weeks are shown in C and
G, and 10 weeks are shown in D and
H. Scale bar, 50 µm.
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Figure 3.
Myelin staining of brainstem from IGF-I KO and
littermate control mice during development. Brains of IGF-I KO mice
(E-H) and their littermate controls
(A-D) were perfusion fixed with 4%
paraformaldehyde, sectioned, and stained using the LFB-PAS method.
Sections from mice at 1 week of age are shown in A and
E, 2 weeks are shown in B and
F, 3 weeks are shown in C and
G, and 10 weeks are shown in D and
H. Scale bar, 50 µm.
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To quantify changes in myelination during development, we examined the
expression of MBP and PLP, using Western immunoblot and Northern blot
hybridization analysis. At 2 weeks of age, four major MBP bands
(~14.4, ~17, ~18, and ~21 kDa) and a single PLP band (~30
kDa) are readily detected by Western immunoblot analysis (Fig.
4). The abundance of both MBP and PLP
proteins in IGF-I KO mice was significantly decreased in all five
regions examined compared with littermate controls. When the abundance
of all four MBP bands is combined for quantification, MBP protein in
IGF-I KO mice was decreased by ~80% in CTX, ~85% in HIP, ~75%
in DIE, ~60% in BS, and ~65% in CB. PLP protein abundance was
also significantly reduced in IGF-I KO mice, being ~30% of
littermate controls in CTX, ~45% in HIP, ~60% in DIE, ~50% in
BS, and ~55% in CB.
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Figure 4.
MBP and PLP protein abundance in 2-week-old IGF-I
KO and littermate control mice. A, Representative
Western immunoblot of MBP and PLP protein in brain regions of
2-week-old IGF-I KO and littermate control mice. The IGF-I KO mouse is
indicated by KO. After detection with anti-MBP antibody,
the blot was stripped and reprobed with antibodies to PLP and actin.
B, Quantitative analysis of MBP and PLP protein
abundance in 2-week-old IGF-I KO and control mice. The abundance of MBP
and PLP is expressed as percentage of that in control mice. A
line is drawn at 100% to facilitate comparison. Values
represent mean ± SE of three to five samples.
*p < 0.05; **p < 0.01;
***p < 0.001 compared with controls.
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As reported previously (Ye et al., 1995a), although a single band of
MBP mRNA at size of ~3 kb was detected, doublet bands of PLP mRNA at
~2.4 and ~3.2 kb were clearly observed (Fig.
5), and a third PLP mRNA at ~1.6 kb was
detected only after prolonged exposure. Consistent with the changes in
protein abundance, mRNA for MBP and PLP in 2-week-old IGF-I KO mice
also was decreased (Fig. 5). In IGF-I KO mice, the abundance of MBP
mRNA was ~10, ~50, and ~30% of control mice in CTX, BS, and DIE,
respectively. Similarly, when compared with those in control mice, PLP
mRNA abundance (quantified by combining both the ~2.4 and ~3.2 kb
mRNAs) in the CTX, BS, and DIE of IGF-I KO mice was decreased by ~60, ~50, and ~60%, respectively. A single experiment also showed that MBP and PLP mRNA abundance in HIP and CB was significantly decreased in
IGF-I KO mice (data not shown).
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Figure 5.
MBP and PLP mRNA abundance in the cerebral cortex,
brainstem, and diencephalon of 3-week-old IGF-I KO mice and littermate
controls. A, Representative Northern blot hybridization
analysis of MBP and PLP mRNA abundance in 3-week-old IGF-I KO and
control mice. Each lane was loaded with 20 µg of total RNA. The IGF-I
KO mouse is indicated by KO. The bottom
row shows methylene blue (MB) staining of the 18 S rRNA bands. B, Quantitative analysis of MBP and PLP
mRNA abundance in 3-week-old IGF-I KO mice and control mice. The
abundance of MBP and PLP mRNA is expressed as percentage of control
mice. A line is drawn at 100% to facilitate comparison.
Values represent mean ± SE of three to five samples.
*p < 0.05; **p < 0.01 compared with controls.
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Next we determined whether the reduced expression of MBP and PLP
observed in 2-week-old IGF-I KO mice persisted at later development stages. In this experiment, we focused on CTX and BS, because these
regions undergo myelination at different times, with myelination occurring earlier in BS than CTX. As Figure
6A shows, the abundance of MBP and PLP protein in both CTX and BS of control mice rapidly increases during the first 3 weeks of life and increased only modestly thereafter. Also as expected, accumulation of these proteins begins later in CTX than in BS. The expression of MBP and PLP proteins
in the CTX and BS of IGF-I KO mice followed a similar developmental
pattern. When compared with control mice, the abundance of MBP and PLP
proteins in both CTX and BS of IGF-I KO mice was significantly reduced
during their first 3 weeks of postnatal life (Fig.
6B). With increasing age, however, the MBP and PLP protein abundance in IGF-I KO mice gradually increased and became similar to that in controls by 10 weeks of age. The abundance of
MBP mRNA in CTX, DIE, and BS exhibited a similar pattern of changes
(Fig. 6C).
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Figure 6.
MBP and PLP expression in developing IGF-I KO mice
and their littermate controls. A, Representative Western
blots of MBP and PLP protein abundance in CTX and BS of IGF-I KO and
control mice during development. After detection with anti-MBP and
anti-PLP antibodies, the blot was stripped and incubated with
anti-actin antibody. IGF-I KO mouse is indicated by KO.
The age of each mouse in weeks (w) is indicated
at the top. B, Quantitative analysis of
MBP and PLP protein abundance in CTX and BS of IGF-I KO and control
mice during development. The abundance of MBP and PLP is
expressed as percentage of littermate control mice. C,
Quantitative analysis of MBP mRNA abundance in CTX, BS, and DIE in
IGF-I KO and control mice during development. MBP mRNA abundance is
expressed as percentage of control mice. Detection of MBP mRNA in DIE
from 10-week-old mice was not done. A line is drawn at
100% in B and C to facilitate
comparison. Values represent mean ± SE from three to five
samples. *p < 0.05; **p < 0.01; ***p < 0.001 compared with littermate
controls.
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To determine whether the alterations in the MBP and PLP expression in
the IGF-I KO mice were attributable to changes in the development of
oligodendrocytes, we quantified the number of oligodendrocytes and
their precursors during development. Mature oligodendrocytes and their
precursors in the frontal CTX (layers 2-6), CC and BS were identified
using antibody against the cell body markers GST- and NG2,
respectively. Because blunting IGF-I expression significantly increases
cell density (Liu et al., 1993; Beck et al., 1995; Cheng et al., 1998),
we measured oligodendrocytes and precursors as percentage of total cells.
In 1-week-old control mice, NG2-positive oligodendrocyte precursors
were relatively abundant, being ~9.9% of total cells in CTX, as
judged by nuclear staining, ~49% in CC, and ~17% in BS. With age,
the number of NG2-positive oligodendrocyte precursors gradually
decreased in all brain regions. Compared with control mice, the number
of NG2-positive cells was significantly reduced in 3-week-old and
10-week-old IGF-I KO mice (Fig. 7). In
3-week-old IGF-I KO mice, the percentage of NG2-positive cells in CTX,
CC, and BS was ~63, ~78, and ~68% of controls, respectively, and
was ~64, ~76, and ~80% of controls at 10 weeks of age.
One-week-old IGF-I KO mice also exhibited 10-12% decreases in the
three brain regions examined, but the reduction was not statistically
significant (Fig. 7), possibly attributable to large variation among
samples.
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Figure 7.
Number of oligodendrocytes and their precursors in
the CTX, CC, and BS of developing IGF-I KO mice and their littermate
controls. The numbers of NG2-positive precursor and GST--positive
oligodendrocytes is expressed in percentage of total cells. Values
represent mean ± SE from three samples. *p < 0.05; **p < 0.01; ***p < 0.001 compared with littermate controls.
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As reported previously (Tansey and Cammer, 1991; Cammer and Zhang,
1992; Mason et al., 2000), GST--positive cells exhibit a small cell
body with a few of the processes. In contrast to NG2-positive
precursors, GST--positive oligodendrocytes were rarely observed in
1-week-old control mice (data not shown), and their number gradually
increased with age in all brain regions. In 3-week-old control mice,
the percentage of GST--positive oligodendrocytes was ~6, ~40,
and ~20% of total cell number in CTX, CC, and BS, respectively. The
oligodendrocyte number reaches ~12% of the total cell number in CTX,
~55% in CC, and ~26% in BS at 10 weeks. These values are similar
to those determined using PLP in situ hybridization (Ye et
al., 1995a). Consistent with Western immunoblot analysis, the number of
GST--positive cells in 3-week-old IGF-I KO mice is reduced in all
three regions examined, being ~60, ~67, and 71% of control CTX,
CC, and BS, respectively (Fig. 7). At 10 weeks, the number of
GST--positive oligodendrocytes was moderately decreased in the three
regions of IGF-I KO mice. The decreases, however, were not
statistically significant (Fig. 7).
To examine the relationship between myelination and axon development in
IGF-I KO mice, we determined the abundance of NF protein in developing
and adult IGF-I KO mice and their littermate controls. NF is a
neuron-specific intermediate filament and plays an important role in
determining axon caliber. As Figure 8
shows, M-NF protein, a medium subunit of NF, was abundant in the CTX
and BS of 1-week-old controls. Its abundance gradually increased during
development, as reported previously (Julien et al., 1986; Hoffman et
al., 1987). Compared with control mice, the abundance of CTX and BS
M-NF protein was significantly reduced in the developing IGF-I KO mice,
being 60-80% of control mice (Fig. 8B).
Unlike the changes in myelin-specific proteins, the changes in
abundance of M-NF did not recover and remained reduced by ~40% in
10-week-old IGF-I KO mice (Fig. 8C). The abundance of
another neurofilament subunit, H-NF, in CTX of IGF-I KO mice exhibited
a similar pattern of change and was significantly reduced when compared
with control mice (data not shown).
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Figure 8.
NF and GFAP abundance in developing IGF-I KO and
their littermate controls. A, Representative Western
immunoblot of M-NF protein and GFAP abundance in CTX of IGF-I KO and
control mice during development. IGF-I KO mouse is indicated by
KO. The age of each mouse in weeks
(w) is indicated at the top.
B, Quantitative analysis of NF protein abundance in CTX
and BS of IGF-I KO and control mice during development. The abundance
of M-NF is expressed as percentage of control mice. C,
Correlation of the abundance of M-NF and of MBP and PLP protein in CTX
and BS of IGF-I KO and control mice. NF data are superimposed on the
data for MBP and PLP protein abundance shown in Figure
6B. M-NF, MBP, and PLP abundance is expressed as
percentage of that in control mice. A line is drawn at
100% in B and C to facilitate
comparison. Values represent mean ± SE from three to five
samples. ^p = 0.06; *p < 0.05; **p < 0.01; ***p < 0.001 compared with controls.
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The abundance of GFAP, an astrocyte-specific filament protein, was also
quantified. Similar to NF abundance, the abundance of GFAP gradually
increased during development (Fig. 8A). Its abundance
in IGF-I KO mice, however, did not significantly differ from that in
control mice (Fig. 8A).
We next investigated possible mechanism(s) for the "catch-up" in
myelination that we observed in IGF-I KO mice. Because IGF-II is
capable of interacting with the IGFIR (Kiess et al., 1987; Gustafson
and Rutter, 1990) and promoting oligodendrocyte development in culture
(McMorris et al., 1990; Masters et al., 1991; Barres et al., 1992), we
asked whether there is an increase in IGF-II expression that might
compensate for the IGF-I deficit. We also examined the abundance of
IGFIR and IRS-1, IRS-2, and IRS-4, the three IRSs expressed in brain,
to determine whether there is any alteration in their expression.
Western immunoblots of protein extracts from the CTX using IGF-II
antibody identified a band migrating at an apparent mass of ~7.5 kDa
and a more abundant doublet band of ~12 kDa. Recombinant human IGF-II
migrated identically to the ~7.5 kDa band. Recombinant IGF-I was not
recognized by the IGF-II antibody when similar amounts were applied to
the gel. When the antibody was preincubated with recombinant IGF-II,
neither the 7.5 kDa band nor the 12 kDa doublet band was observed (Fig. 9A). These data confirm that
the antibody is specific for IGF-II and likely recognizes both the
mature (7.5 kDa) and precursor forms (12 kDa), with no or very little
cross-reactivity with IGF-I. We then determined and compared IGF-II
protein abundance in the brains of IGF-I KO mice and their controls.
When the quantity of each IGF-II band was combined, the abundance of
IGF-II was increased in the CTX of IGF-I KO mice (Fig. 9B).
In control mice, IGF-II abundance gradually decreased with age, being
~61% of its level in 1-week-old mice at 2 weeks of age and ~44%
at 10 weeks of age. IGF-II abundance in IGF-I KO mice was relatively
constant through the period studied. In 1-week-old IGF-I KO mice,
IGF-II abundance was ~50% greater than in littermate control mice,
and its relative abundance increased to ~190% of controls at 2 weeks of age (p < 0.05 compared with its level in
1-week-old IGF-I KO mice), ~182% at 3 weeks of age, and ~160% at
10 weeks of age. The abundance of IGFIR, as well as that of IRS-1,
IRS-2, and IRS-4, was similar in IGF-I KO mice and littermate controls
(Fig. 9C,D).
View larger version (26K):
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Figure 9.
IGF-II expression in CTX of IGF-I KO mice.
A, Representative Western immunoblot of IGF-II protein
abundance in IGF-I KO and control mice. CTX protein (80 µg) from a
2-week-old IGF-I KO mouse and a littermate control, recombination human
IGF-II (II; 200 ng), and IGF-I (I; 200 ng) were, respectively, loaded in each lane. IGF-I KO mouse is
indicated by KO. The right lane
represents protein (80 µg) from CTX of a 2-week-old control mouse
reacted with the anti-IGF-II antibody that was preincubated with 5 µg/ml IGF-II for 1 hr at room temperature
(Pre-II). B, Quantitative analysis
of IGF-II abundance in the CTX of IGF-I KO and control mice. IGF-II
protein abundance is expressed as percentage of control mice. A
line is drawn at 100% to facilitate comparison. Values
represent mean ± SE from three to four samples.
*p < 0.05; **p < 0.01 compared with controls. C, Representative Western
immunoblots of IGFIR, IRS-1, IRS-2, and IRS-4 in IGF-I KO and control
mice. CTX protein (30 µg) from a 2-week-old IGF-I KO mouse and a
littermate control were loaded in each lane. IGF-I KO mouse is
indicated by KO. D, Quantitative analysis
of IGFIR, IRS-1, IRS-2, and IRS-4 abundance in the CTX of IGF-I KO and
control mice. Values represent mean ± SE from three to five
samples.
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DISSCUSION |
Our data show that myelin staining, expression of myelin-specific
protein genes, and oligodendrocyte number are significantly reduced in
IGF-I KO mice during the first 3 weeks of postnatal life. This finding
is consistent with our previous reports that myelination is decreased
in the brain of Tg mice that ectopically express IGFBP-1, an inhibitor
of IGF action, although it is increased when IGF-I is overexpressed in
brain (Ye et al., 1995a,b). The myelin content, expression of
myelin-specific proteins, and oligodendrocyte number in IGF-I KO mice,
however, increases after the weanling period, resulting in a normal
concentration in adult IGF-I KO mice. These data demonstrate,
therefore, that myelination is developmentally altered in IGF-I KO
mice. Our finding that IGF-II abundance is increased in the brains of
IGF-I KO mice suggests that IGF-II may partially compensate for the
loss of IGF-I expression and function.
These findings are consistent with those of Beck et al. (1995) who
showed that myelinated axons and myelin sheath thickness are
significantly reduced in adult IGF-I KO mice. The data reported by
Cheng et al. (1998) also are consistent with our findings, although
they reached different conclusions. Studying whole brains from young
adult IGF-I KO mice, they found a 25% reduction in myelin-associated
galactocerebroside and sulfatide but a similar concentration of several
myelin-specific proteins. They also reported that the number of
oligodendrocytes in the CB and anterior commissure of IGF-I KO mice
approximately correlated with the number of projection neurons. For
these reasons and because they did not study parameters of myelination
before adulthood, they concluded that neuronal factors govern
myelination and that IGF-I does not have a major role in myelination
during development. Although we found that the myelin in adult IGF-I KO
mice was similar to that in normal control mice, our data clearly
demonstrate significant decreases in myelin staining, the expression of
MBP and PLP proteins and their mRNAs, and the number of
oligodendrocytes and their precursors before adulthood. These data
indicate that IGF-I has a significant role in stimulating proliferation
and maturation of oligodendrocytes and promoting myelin protein
expression during development, albeit it can be subserved by IGF-II
and/or other agents, when IGF-I expression is deficient (see below).
IGF-II, an IGF-I homolog, also interacts with the IGFIR. Because IGF-II
is highly expressed in craniofacial mesenchymal structures (Rotwein et
al., 1988; Hepler and Lund, 1990; Rotwein, 1991), the brain is exposed
to high IGF-II levels during prenatal and early postnatal life. In
rodents, IGF-II expression persists in the choroid plexus,
leptomeninges, and parenchymal microvasculature through adulthood
(Rotwein et al., 1988; Rotwein, 1991; Cavallaro et al., 1993; Logan et
al., 1994). Recently, Logan et al. (Logan et al., 1994; Walter et al.,
1999) reported that, in the brain of adult rats, IGF-II and IGFBP-2
immunoreactivity colocalizes to the membrane of cells with
oligodendrocyte characteristics and to myelin sheaths. Together with
the data reviewed above showing that IGF-II is expressed in the choroid
plexus, leptomeninges, and parenchymal microvasculature in adult
animals, their study suggests that IGF-II is transported to myelin
tracks from its sites of synthesis. Although the precise function of
IGF-II in postnatal rodent brains is not clear, its pattern of
association with oligodendrocytes and myelin suggests that it may play
a role in promoting and/or maintaining myelination (Logan et al., 1994; Walter et al., 1999). The latter speculation is supported by the data
that IGF-II promotes development and survival of oligodendrocytes and
their precursors in culture (McMorris et al., 1990; Masters et al.,
1991; Barres et al., 1992).
Using Western immunoblot analysis, we found that IGF-II expression is
increased in IGF-I KO mice. We observed increases in IGF-II-immunoreactive bands at ~7.5 and ~12 kDa in CTX and BS. The
~7.5 kDa comigrates with recombinant IGF-II and represents mature
IGF-II, whereas the more abundant bands migrating at ~12 kDa likely
represent precursor forms. Precursor IGF-II forms of 10-18 kDa have
been reported in multiple tumor cells lines (Schmitt et al., 1997; Bae
et al., 1998) and in the serum of normal and tumor patients (Hoeflich
et al., 1995; Bae et al., 1998; Christofilis et al., 1998). The
expression of IGF-II in developing and adult rodent CNS has been well
studied using a variety of methods, including immunohistochemistry
(Logan et al., 1994; Walter et al., 1999), in situ
hybridization (Cavallaro et al., 1993; Logan et al., 1994; Walter et
al., 1999), and RNA protection assay (Rotwein et al., 1988); however,
only a few studies have documented its molecular forms in brain
(Haselbacher et al., 1985). Although the physiologic significance of
the expressed IGF-II precursor is not clear, the precursor derived from
tumors has been shown to have biologic activity, including promotion of
angiogenesis and hexosaminidase secretion (Hoeflich et al., 1995; Bae
et al., 1998; Christofilis et al., 1998). We speculate that the
increase in IGF-II during rapid myelination (2-4 weeks of age) and in
early adulthood is responsible for the recovery of myelin-specific
protein expression in adult IGF-I KO mice. Other studies of mouse CNS
myelination support this conclusion. Transgenic mice expressing high
levels of IGFBP-1 in brain exhibit a marked decrease in myelination
throughout life (Ye et al., 1995a,b; Ni et al., 1997). IGFBP-1 has a
similar affinity for both IGF-I and IGF-II (Bach et al., 1993; Oh et
al., 1993) and inhibits the actions of both IGF-I and IGF-II. Thus, when the actions of both IGFs are inhibited, myelination is decreased in the adult brain. These data argue that an IGF, either IGF-I or
IGF-II, is necessary for normal myelination. Our data, however, do not
exclude the possibility that other factors, alone or in concert, may
also compensate for the IGF-I loss. In culture, FGF and PDGF, alone or
synergistically with IGF-I, are capable of promoting oligodendrocyte
proliferation and survival (Barres et al., 1992, 1993; Jiang et al.,
2001).
Myelination is closely associated with axon growth, and, therefore, we
sought to assess whether there is a correlation between deficient axon
growth and delayed myelination in IGF-I KO mice. To provide an index of
axon growth during development, we measured the expression of NF. NF, a
class IV intermediate filament, is composed of three subunits with
molecular masses of ~200 kDa (high), ~150 kDa (medium), and ~68
kDa (low). The expression of each of the three NF subunits increases
during development, and their abundance correlates with the axon growth
(Friede and Samorajski, 1970; Lasek et al., 1983; Hoffman et al., 1985,
1987). In addition, loss of NF protein results in failure of axon
growth (Zhu et al., 1997; Elder et al., 1998). We found that, whereas
the expression of M-NF and H-NF proteins have a similar developmental
profile in both IGF-I KO and control mice, the expression of M-NF and H-NF in IGF-I KO mice is significantly reduced throughout the brain
during development and remains decreased in adult IGF-I KO mice,
suggesting a decreased axonal growth and/or axon maturation in adult
IGF-I KO mice. The expression of NF, therefore, does not parallel the
changes in abundance of myelin-specific proteins during development in
IGF-I KO mice. Although decreased axon growth could contribute to the
alteration in myelination in these mice, axon influences cannot by
themselves explain the pattern of myelination in IGF-I KO mice.
Beck et al. (1995) and Cheng et al. (1998) also assessed neuron number
in specific regions of IGF-I KO mice. Beck et al. (1995) reported that
the numbers of striatal parvalbumin-positive neurons and hippocampal
granular neurons were significantly reduced, whereas the numbers of
cortical projection neurons in CTX layers III and V and dopaminergic
neurons do not differ in IGF-I KO and control mice. Cheng et al. (1998)
indicated that there is significant decrease in olfactory bulb neurons
but not in Purkinje cells in IGF-I KO mice. Together, it seems likely
that IGF-I deficiency reduces overall neuron number but that the
influence of IGF-I differs among brain regions. This conclusion is
consistent with our findings that there are significant increases in
neuron number in some but not all medullary nuclei in
IGF-I-overexpressing transgenic mice (Dentremont et al., 1999).
Taken in aggregate, evidence for an important role for IGF-I in
oligodendrocyte development and in myelination is strong. We believe
that IGF-I is a major component of the signaling mechanisms that
regulate myelination during normal development. IGF-II also may have a
role in regulating myelination, and it can, at least in part, serve as
an IGF-I surrogate in IGF-I-deficient states. It is clear, however,
that many of signaling mechanisms are involved in the regulation of myelination.
| |
FOOTNOTES |
Received Oct. 10, 2001; revised April 18, 2002; accepted April 24, 2002.
This work was supported by National Institute of Neurological Disorders
and Stroke Grant NS38891 and National Institute of Child Health and
Human Development Grant HD08299 (A.J.D.).
Correspondence should be addressed to Dr. A. Joseph D'Ercole,
Department of Pediatrics, CB 7220, The University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599-7220. E-mail: joseph_d'ercole{at}unc.edu.
| |
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V. C. Russo, P. D. Gluckman, E. L. Feldman, and G. A. Werther
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O. Tschopp, Z.-Z. Yang, D. Brodbeck, B. A. Dummler, M. Hemmings-Mieszczak, T. Watanabe, T. Michaelis, J. Frahm, and B. A. Hemmings
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Development,
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M. J. E. Walenkamp, M. Karperien, A. M. Pereira, Y. Hilhorst-Hofstee, J. van Doorn, J. W. Chen, S. Mohan, A. Denley, B. Forbes, H. A. van Duyvenvoorde, et al.
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L. Y. Sun, M. S. Evans, J. Hsieh, J. Panici, and A. Bartke
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H. Shan, M. L. Messi, Z. Zheng, Z.-M. Wang, and O. Delbono
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C. P. Turner, M. Seli, L. Ment, W. Stewart, H. Yan, B. Johansson, B. B. Fredholm, M. Blackburn, and S. A. Rivkees
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P. Ye, R. Bagnell, and A. J. D'Ercole
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TOP
ABSTRACT
INTRODUCTION
Gene
