Pigment epithelium-derived factor (PEDF) is a serine protease inhibitor (serpin) protein with well established neuroprotective and
anti-angiogenic properties. Recent studies have also shown that PEDF enhances renewal of adult subventricular zone (SVZ) neural
enhanced expressions of oligodendroglial lineage markers (NG2 and PDGFr?) and transcription factors (Olig1, Olig2, and Sox10).
Similarly, continuous PEDF administration into the lateral ventricles of adult glial fibrillary acidic protein:green fluorescent protein
droglial lineage markers and transcription factors. Notably, PEDF infusion also resulted in an induction of doublecortin- and Sox10
in the normal corpus callosum, and accelerated oligodendroglial regeneration in lysolecithin-induced corpus callosum demyelinative
lesions. Western blot analysis showed a robust upregulation of endogenous PEDF in the corpus callosum upon lysolecithin-induced
demyelination. Our results document previously unrecognized oligodendrotrophic effects of recombinant PEDF on the adult SVZ and
corpus callosum, demonstrate induction of endogenous CNS PEDF production following demyelination, and make PEDF a strong
Glial fibrillary acidic protein-positive (GFAP?) neural precur-
sors that reside in the subventricular zone (SVZ) in close prox-
imity to endothelium and ependyma constitute a neurogenic
niche in the adult forebrain (Shen et al., 2008; Danilov et al.,
2009). These cells are capable of both self-renewal, driven by
tricular infusion enhances the generation of oligodendroglial
progenitor cells (OPCs) from SVZ GFAP? neural precursors, and
accelerates remyelination of lysolecithin-induced corpus callosum
In a search for additional SVZ niche factors that enhance for-
mation of oligodendroglia in the adult forebrain, we evaluated
a neurotrophic, anti-angiogenic, and anti-tumorigenic mem-
ber of the serine protease inhibitor (serpin) protein family
(Tombran-Tink and Barnstable, 2003; Ek et al., 2006). PEDF has
previously been reported to be secreted by adult SVZ endothelial
astroglia and neurons (Sanagi et al., 2007; Yasuda et al., 2007),
and to enhance self-renewal of adult murine SVZ precursor cells
(Ramírez-Castillejo et al., 2006; Andreu-Agullo ´ et al., 2009).
In the present study, we found that addition of PEDF to EGF/
basic fibroblast growth factor (bFGF)-containing culture me-
dium elevated expression of Olig1/2, Sox10, and PDGFr? and
numbers of GFAP:GFP?NG2? cells in neurospheres derived
from the SVZ of adult GFAP:green fluorescent protein (GFAP:
eration. Corroborating these in vitro findings, PEDF intra-
ventricular infusion increased numbers of GFAP:GFP? cells
ingly, the oligodendroglial inductive effect of PEDF was also ev-
ident in SVZ GFAP:GFP? cells. Furthermore, PEDF infusion
the adult SVZ. PEDF infusion also enhanced survival and matu-
ration, but not mitotic cycling, of OPCs in the adult corpus cal-
losum, and accelerated oligodendroglial regeneration in the
12152 • TheJournalofNeuroscience,August29,2012 • 32(35):12152–12164
lysolecithin-demyelinated corpus callosum. PEDF was robustly
induced in corpus callosum after lysolecithin lesioning, suggesting a
Our studies are the first to report oligodendroglial morphogenic, sur-
son Laboratory. PEDF-null mice (Doll et al., 2003) were obtained from
Northwestern University. Two- to 4-month-old male and female adult
mice were used in this study. They were housed in a pathogen-free facil-
Animal Care and Use Committee of the University of California Davis,
and National Institutes of Health guidelines.
SVZ dissociation and neurosphere culture. SVZ tissue was microdis-
brains, minced with a razor blade, and incubated for 30 min at 37°C in
HBSS medium (Invitrogen) containing 0.05% trypsin (Invitrogen) and
60U/ml DNase I (Sigma). Cell suspensions were generated by gentle
trituration using a fire-polished Pasteur pipette and then centrifuged for
5 min at 400 g. Single cells were resuspended and plated in noncoated
culture flasks (BD Biosciences) containing neurosphere medium
vitrogen), 20 ng/ml EGF (Millipore) and 20 ng/ml bFGF (R&D Sys-
were then spun and enzymatically dissociated into single cells. The dissociated
Flow cytometry and fluorescence-activated cell sorting. Control and
PEDF-treated secondary GFAP:GFP neurospheres were dissociated into
single cells and positive GFP expression was determined using wild-type
of percentages of NG2? cells within the GFAP:GFP? cell population,
GFP neurospheres were incubated with anti-NG2 antibody (1:500) for
conjugated with allophycocyanin (1:1000, R&D Systems) for 15 min at
room temperature, and then analyzed using a Cyan flow cytometer
(Dako). For fluorescence-activated cell sorting (FACS) isolation of
GFP?NG2? and GFP?NG2? single cells, primary GFAP:GFP neuro-
spheres were dissociated into single cells, immunolabeled for NG2 ex-
pression as described above, and isolated using a FACS Aria II (BD
in neurosphere medium in the absence or presence of PEDF to generate
munocytochemical characterization of wild-type control and PEDF-
treated secondary neurospheres, the spheres were directly plated or
mechanically dissociated into single cells before plating onto poly-D-
cells were fixed with 4% paraformaldehyde (PFA) for 15 min, washed
min at room temperature. Cells were then incubated for 3 h at room
temperature or overnight at 4°C with primary antibodies as follows:
anti-nestin (1:250, Millipore), anti-NG2 (1:400, Millipore), anti-O4 (1:
200, Millipore), or anti-Tuj1 (1:400, Covance). Cells were washed three
times with PBS, and then incubated with Alexa Fluor-conjugated secondary
immunostaining, permeabilization was done with 0.1% Triton X-100 in PBS
dihydrochloride (DAPI; Invitrogen) and images were acquired using a Zeiss
rospheres derived from FACS-purified GFAP:GFP?NG2? or GFAP:
and GFAP:GFPtransgenic mice(FVB/N-Tg
GFP?NG2? cells, the spheres were plated on PDL (20 ?g/ml, Sigma)-
and laminin (5 ?g/ml, Sigma)-coated chamber slides in differentiation
bFGF, or PEDF) for 3 d. Cells were then fixed with 4% PFA and immu-
nostained for NG2, O4, or Tuj1 as described above.
For validation of GFAP expression in GFAP:GFP? or GFAP:GFP?
cell fractions, GFP? and GFP? cells that had been FACS-isolated from
GFAP:GFP neurospheres were plated on PDL-coated chamber slides for
Dako). The staining procedure was done in the same way as for the Tuj1
staining described above.
Proliferation assay in neurosphere cultures. To assess proliferation of
to control and PEDF-treated GFAP:GFP secondary neurospheres
for 12 h before cell dissociation and NG2 labeling. FACS-purified
GFAP?NG2? cells were plated on PDL-coated chamber slides for 1 h
before fixation with 4% PFA. Cells were then permeabilized with 0.1%
Triton X-100 in PBS (v/v) for 10 min and blocked with 8% normal goat
serum in PBS (v/v) for 30 min. DNA denaturation was done with 0.07N
then incubated with anti-BrdU (1:100, Dako) overnight at 4°C. After
incubated with Alexa Fluor-conjugated secondary antibodies for 1 h at
Control and PEDF-treated wild-type neurospheres were pulsed with
BrdU for 12 h before plating on a PDL-coated chamber slide and fixation
control and PEDF-treated wild-type neurospheres were directly plated on
PDL-coated chamber slides for 2–3 h and fixed with 4% PFA. Cells were
RNA isolation and reverse transcriptase-PCR. For RNA isolation, neu-
rospheres were spun and lysed with RNA lysis buffer, and total RNA was
extracted using an absolute microRNA prep kit (Stratagene). During
RNA extraction, DNase I was included to prevent genomic DNA con-
tamination. cDNA was synthesized from 0.5 ?g to 1 ?g of total RNA
kit (Invitrogen) in 20 ?l reactions. For detection of PEDF receptor, also
known as patatin-like phospholipase domain-containing protein 2
was used as a template, and PCR amplification was conducted using
AmpliTaq Gold PCR master mix (Applied Biosystems). The oligonucle-
otide primers for the PEDF receptor were: forward 5? ACAGTGTC-
CCCATTCTCAGG 3?; and reverse 5? TGGTGAAGGACACTGCACTC
3?. PCR conditions were as follows: 95°C for 5 min; 30 or 36 cycles of
(95°C for 15 s, 55°C for 15 s, 72°C for 1 min); 72°C for 10 min. The PCR
products were resolved on a 2% agarose gel and photographed using a
Kodak Gel Logic Digital Imaging System. For quantitative analysis of
oligodendroglial lineage-associated mRNAs, real-time PCR was per-
formed on a LightCycler 480 (Roche) using a SYBR Green qPCR kit
in the melting curve analysis ensuring the absence of false signaling.
internal control for normalization of mRNA levels. The oligonucleotide
primer sets used in this assay are listed as follows: Olig1 forward, 5?
CTTGCTCTCTCCAGCCAAAC 3?, reverse, 5? CAGAACTGGGAGTG-
GAGAGG 3?; Olig2 forward, 5? AGCAATGGGAGCATTTGAAG 3?, re-
verse, 5? TTCCATATCGGGACTTTTGG 3?; Sox10 forward, 5? AGG
CAGGAAGGGTTAGGGTA 3?, reverse, 5? GCGGAGAAAGGATCA-
GAGTG 3?; PDGFr? forward, 5? TGGCATGATGGTCGATTCTA 3?,
reverse, 5? CGCTGAGGTGGTAGAAGGAG 3?; GAPDH forward,
PEDF in vivo infusion and lysolecithin lesioning. PEDF (Bioproducts
MD) was dissolved in 0.9% saline and continuously administered (300
Sohnetal.•PEDF:AnOligodendroglialLineageMorphogenJ.Neurosci.,August29,2012 • 32(35):12152–12164 • 12153
and brain infusion kit #3; Durect). Control animals received 0.9% saline
alone. Stereotaxic coordinates for cannula implantation of the pumps
into the lateral ventricle were 0.2 mm posterior and 1.1 mm lateral to
SVZ (see Fig. 6), BrdU (100 mg/kg body weight, i.p.) was daily injected
saline or PEDF infusion. For the BrdU labeling followed by PEDF infu-
sion described below in Figures 7 and 8, adult wild-type mice first re-
ceived BrdU (100 mg/kg body weight, i.p.) three times at 6 h intervals to
label cycling OPCs in the corpus callosum. Twenty-four hours after the
the corpus callosum for 9 d (see Fig. 7), or 2 or 5 d (see Fig. 8). The
cannula was placed at the following coordinates for corpus callosum
infusion: 0.2 mm posterior and 1.1 mm lateral to bregma, and 1.5 mm
8, EdU (100 mg/kg body weight, i.p.) was injected into the animals 5 h
before they were killed to label proliferating cells.
For the lysolecithin-induced corpus callosum demyelination studies
(see Fig. 9), 1 ?l of 1% lysolecithin (Sigma) was stereotaxically injected
into the corpus callosum of adult mice at 0.2 mm posterior, 1.1 mm
lateral to bregma, and 1.7 mm deep from the skull surface. Saline or
12154 • J.Neurosci.,August29,2012 • 32(35):12152–12164Sohnetal.•PEDF:AnOligodendroglialLineageMorphogen
PEDF (300 ng per day) was then continuously infused via mini-osmotic
pumps into the corpus callosum for 3 or 7 d. The cannula was placed at
the following coordinates for saline or PEDF intracerebral infusion: 0.2
mm posterior and 1.1 mm lateral to bregma, and 1.5 mm deep from the
was stereotaxically injected into the corpus callosum of adult mice at 0.2
surface. Corpus callosum tissues were harvested at 2, 5, or 7 d postinjec-
tion. PEDF (300 ng per day) was infused into the corpus callosum of
intact, nonlesioned adult mice for 2 d at the following coordinates: 0.2
mm posterior and 1.1 mm lateral to bregma, and 1.5 mm deep from the
Tissue preparation, immunohistochemistry, and quantification. After
the saline or PEDF infusion, mice were anesthetized with ketamine (150
tion, and transcardially perfused with PBS, followed by 4% PFA in PBS.
Brain tissues were isolated and postfixed with 4% PFA in PBS overnight
tial immersions in 15% and 30% sucrose solutions (v/v) before embed-
ding in cryostat mounting medium (Tissue-Tek OCT, Sakura Finetek).
perature before blocking with 8% normal serum (v/v) and 0.1% Triton
X-100 (v/v) in PBS for 1 h at room temperature. Sections were then incu-
bated with primary antibodies overnight at 4°C in PBS containing 5% nor-
Alexa Fluor-conjugated secondary antibodies (1:800, Invitrogen) for 1 h at
room temperature. Primary antibodies were used as follows: anti-GFP (1:
500, Rockland), anti-PDGFr? (1:200, BD Biosciences), anti-NG2 (1:300,
Millipore), anti-Olig1 (1:500, Millipore), anti-Olig2 (1:100, R&D Systems),
anti-Sox10 (1:100, Santa Cruz Biotechnology), anti-BrdU (1:100, Santa
lipore), anti-MBP (anti-myelin basic protein; 1:200, Novus Biologicals), or
tissue sections were treated with 2N HCl for 30 min at 37°C to denature
DNA, followed by 10 min incubation with 0.1 M sodium borate, pH 8.5 for
neutralization. Subsequent immunostaining steps were performed as de-
(Invitrogen) following the manufacturer’s instructions. TUNEL (terminal
deoxynucleotidyl transferase dUTP nick end labeling) staining was per-
ufacturer’s instructions. For BrdU/TUNEL double labeling, TUNEL
staining was performed first, and tissues were then fixed for 8 min with 4%
PFA followed by BrdU staining as described above. For TUNEL/Olig2 and
TUNEL/NG2? staining, Olig2 or NG2 immunolabeling was first per-
All fluorescent images were captured by laser scanning confocal mi-
were analyzed for each marker, and cells were identified by their DAPI-
labeled nuclei. Cell counts were performed in the dorsal corner of the
SVZ (see Figs. 3, 6), with the total number of cells analyzed ranging
between 998 and 1484, in the ipsilateral corpus callosum (see Figs. 7, 8),
and in the lesion core determined by MBP immunostaining and cortical
needle tract (see Fig. 9).
Western blot analysis. Control and PEDF-treated secondary neuro-
spheres derived from the SVZ of adult wild-type mice (see Fig. 2) and
corpus callosum tissues (see Fig. 10) were collected and lysed in a
radioimmunoprecipitation assay buffer containing protease inhibitors
(Thermo Fisher Scientific). Protein concentration was determined by
BCA assay (Thermo Fisher Scientific). Protein separation was done on
staining for NG2, O4, or Tuj1. E, G, Greater numbers of NG2? and O4? oligodendroglial cells were produced from both GFP?NG2? and GFP?NG2? cell-derived neurospheres with PEDF
Sohnetal.•PEDF:AnOligodendroglialLineageMorphogenJ.Neurosci.,August29,2012 • 32(35):12152–12164 • 12155
SDS polyacrylamide gels (Invitrogen) and transferred to PDVF mem-
branes (Millipore). Membranes were blocked and then incubated with
primary antibodies: Olig1 (1:3000, Millipore), Olig2 (1:3000, R&D Sys-
tems), Sox10 (1:1000, Millipore), PDGFr? (1:500, Santa Cruz Biotech-
nology), and PEDF (1:100, R&D Systems). Protein bands were detected
using ECL (Thermo Fisher Scientific) with horseradish peroxidase-
conjugated secondary antibodies (1:5000, GE Healthcare Life Sciences).
Protein loading was determined by using antibody against GAPDH (1:
Statistical methods. Paired comparisons were analyzed by two-tailed
Student’s t tests, with p ? 0.05 required for statistical significance.
To examine the effect of PEDF on cellular phenotypes within
neurospheres, secondary neurospheres derived from the SVZ of
adult wild-type mice were grown in the absence or presence of
spheres were then subjected to immunocytochemical character-
ondary neurospheres by reverse transcriptase PCR (RT-PCR)
analysis (Fig. 1B). Before immunostaining, control and PEDF-
ciated into single cells, and then plated onto PDL-coated
chamber slides. Approximately 90% of the cells in both control
and PEDF-treated neurospheres were positive for nestin, a
marker for neural precursors (Frederiksen and McKay, 1988;
Doetsch et al., 1997) (Fig. 1C,D), indicating the vast majority of
cells within the neurospheres were at precursor stage. Interest-
ingly, PEDF treatment increased the proportion of cells that ex-
pressed the OPC surface marker NG2 (Aguirre and Gallo, 2004)
(Fig. 1C,D). Although present at low density, cells expressing the
were also significantly more frequent in PEDF-treated (3.1%)
against GFP and PDGFr?, NG2, or Olig1. Insets show magnified images of areas indicated by rectangles. Scale bar, 30 ?m. G–K, Percentages of DAPI? nuclei labeled with PDGFr??,
PEDF infusion increases the numbers of SVZ cells expressing early oligodendroglial lineage markers or oligodendroglial transcription factors. Saline or PEDF (300 ng per day) was
12156 • J.Neurosci.,August29,2012 • 32(35):12152–12164 Sohnetal.•PEDF:AnOligodendroglialLineageMorphogen
were rarely present in both control and PEDF-treated neuro-
sphere cells (Fig. 1D). PEDF treatment did not alter the propor-
tions of cells labeled with BrdU or expressing the proliferative
yses suggested a role for PEDF in enhancing oligodendroglial
lineage specification of SVZ neural precursors.
To further characterize the oligodendrogenic effect of PEDF on
SVZ GFAP? neural precursors, we used neurospheres prepared
from the SVZ of adult GFAP:GFP transgenic mice. Secondary
GFAP:GFP neurospheres cultured in the absence or presence of
in Figure 1A. This transgenic animal model has been well char-
acterized in previous studies showing that GFP reporter expres-
sion reliably identifies GFAP? cells (Zhuo et al., 1997; Pastrana et
al., 2009; Platel et al., 2009). To verify the specificity of GFP
expression in our cultures, GFP? and GFP? cell fractions were
isolated from secondary GFAP:GFP neurospheres by FACS,
plated, and subjected to GFAP immunostaining (Fig. 1F,G).
Close to 80% of the neurosphere cells were GFP? (Fig. 1F) and
GFAP immunoreactivity was specific to
GFP? cell fractions (Fig. 1G). PEDF
treatment induced a substantial increase
in the proportion of NG2? cells within
the GFAP:GFP? cell fraction (Fig. 1H)
without altering their proliferation (Fig.
1I). Activated caspase 3 immunostaining
indicated that cell death was very infre-
quent in both control and PEDF-treated
neurospheres maintained in EGF- and
bFGF-containing growth medium (data
treated neurosphere is likely to have been
lineage inductive action of PEDF on the
SVZ GFAP? cells.
To explore the target specificity of PEDF,
we FACS-separated GFAP:GFP? cells from
primary GFAP:GFP SVZ neurospheres into
ral precursors not specified to the oligoden-
droglial lineage, and GFAP:GFP?/NG2?
cells as precursors specified to the oligoden-
droglial lineage. As depicted in Figure 2A,
FACS-isolated GFP?NG2? and GFP?
NG2?cellswereculturedin the absence or
presence of PEDF for 5 d to generate sec-
of FACS-purified cells was low (i.e., 5
ondary neurospheres were then subjected
to quantitative RT-PCR (qRT-PCR; Fig.
2B,C), or plated on PDL- and laminin
double-coated chamber slides in the ab-
sence of EGF, bFGF, and PEDF for 3 d before performing NG2,
O4, or Tuj1 immunostaining (Fig. 2E–G). qRT-PCR results
showed that PEDF elevated steady-state levels of mRNAs encod-
the oligodendroglial lineage (Lu et al., 2001; Zhou et al., 2001;
Finzsch et al., 2008; Pozniak et al., 2010), as well as for the OPC
GFP?NG2? and GFP?NG2? cell-derived neurospheres (Fig.
2B,C). Western blot analysis of control and PEDF-treated sec-
ondary neurospheres derived from the SVZ of wild-type mice
further confirmed PEDF-induced elevation of these oligoden-
droglial markers at protein levels (Fig. 2D). Differentiation
oligodendrogenic potential than GFP?NG2? cells (Fig. 2E).
Importantly, PEDF treatment increased the numbers of
NG2? and O4? oligodendroglial lineage cells (Fig. 2E,G),
but reduced the number of Tuj1? neurons (Fig. 2F,G) differ-
entiated from both GFP?NG2? and GFP?NG2? cell-
derived neurospheres. In particular, the oligodendrogenic
effect of PEDF on GFP?NG2? cell-derived neurospheres ar-
gued that PEDF enhances specification of adult SVZ GFAP?
cells to the oligodendroglial lineage.
Sohnetal.•PEDF:AnOligodendroglialLineageMorphogenJ.Neurosci.,August29,2012 • 32(35):12152–12164 • 12157
droglial specification of SVZ cells in vivo,
we continuously infused saline or PEDF
GFP transgenic mice for 7 d. In the saline-
were positive for PDGFr? (Fig. 3A,G) and
proportions of both PDGFr?? (Fig. 3D,G)
and NG2? (Fig. 3E,H) cells were markedly
increased (39.5% and 12.1%, respectively) in
the PEDF-infused SVZ. We also immuno-
stained saline- and PEDF-infused SVZ sec-
tions for the oligodendroglial transcription
ther confirming the pro-oligodendroglial
effect of PEDF in vivo (Fig. 3C,F,I–K). This
PEDF effect was evident in both GFP? and
ing oligodendroglial markers, suggesting that
PEDF may act on multiple cell types in the
Immunohistochemical analysis revealed
that PEDFr was ubiquitously expressed in
the adult SVZ including in GFAP? cells
(Fig. 4A) and early neuronal lineage cells
tablished maker for SVZ neuroblasts
(Brown et al., 2003; Lagace et al., 2007;
Jablonska et al., 2010). To examine oligo-
dendroglial induction in SVZ neuroblasts
by PEDF, we double-immunostained the
mice for DCX and Sox10. In the saline-
infused control SVZ, we did not observe
DCX? neuroblasts that coexpressed Sox10
(Fig. 5A,B), whereas DCX?/Sox10? cells
5C,D). Together, our results indicate that
PEDF promotes oligodendroglial lineage
sors and DCX? neuroblasts. Consistent
fect of PEDF on neurosphere cells, total
numbers of BrdU? cells (Fig. 6A,B,E) and
supporting an oligodendroglial inductive
PEDFr immunoreactivity was also detected in corpus callosal
Olig2? oligodendroglial lineage cells (Fig. 4C–E) and GFAP:
GFP? astrocytes (Fig. 4F–H), suggesting possible PEDF actions
on the corpus callosum. Previous studies have shown that OPCs
in the adult brain proliferate, albeit at a much lower rate than in
the newborn, and generate mature oligodendrocytes (Dawson et
12158 • J.Neurosci.,August29,2012 • 32(35):12152–12164Sohnetal.•PEDF:AnOligodendroglialLineageMorphogen
al., 2003; Polito and Reynolds, 2005; Rivers et al., 2008). To de-
injection), a small number of BrdU? cells were present in the
corpus callosum. Greater than 90% of these BrdU? cells were
also Olig2? (Fig. 7B,G), but none expressed immunohistologi-
Sohnetal.•PEDF:AnOligodendroglialLineageMorphogenJ.Neurosci.,August29,2012 • 32(35):12152–12164 • 12159
cally detectible CC1, a marker for mature oligodendrocytes (Fig.
7C,H), indicating that they were immature oligodendroglial lin-
cells that were colabeled with Olig2 in the corpus callosum were
the results obtained at day 0 (Fig. 7G). We observed occasional
BrdU?/CC1? cells in the corpus callosum of the saline-infused
mice at day 9 (Fig. 7H), indicating that some OPCs initially labeled
hanced by PEDF infusion, as demonstrated by substantially greater
numbers of BrdU?/CC1? cells in PEDF-infused than in saline-
were found to be immunoreactive for the myelin sheath protein
CNP in saline-infused corpus callosum at this time-point (Fig.
7E,I), whereas ?20% of BrdU? cells expressed CNP in PEDF-
apoptosis. C, D, Confocal images of cells double-assayed for BrdU and TUNEL in the saline (C)- or PEDF (D)-infused corpus callosum. C, BrdU?/TUNEL? cell is indicated by white arrows. D,
12160 • J.Neurosci.,August29,2012 • 32(35):12152–12164Sohnetal.•PEDF:AnOligodendroglialLineageMorphogen
The observed increase in the number of BrdU?/CC1? cells by
vival of newly born OPCs in the corpus callosum. To test this
hypothesis, we designed an experiment (Fig. 8A) in which BrdU
was administered to wild-type adult mice, followed by saline or
PEDF infusion for 2 or 5 d, at the end of which time cells under-
going apoptosis were visualized by TUNEL assay. In addition,
5?-ethynyl-2?-deoxyuridine (EdU) was also administered 5 h be-
the corpus callosum. TUNEL? cells were detected in the corpus
callosum of the saline-infused corpus callosum at day 2 and 5
Fig. 8E). Some TUNEL? cells were also colabeled with BrdU?
(Fig. 8F), providing evidence for apoptosis of newly born OPCs.
We detected occasional TUNEL? cells that were colabeled with
Olig2 or NG2 in the corpus callosum (Fig. 8B), but in most
instances we were unable to immunohistologically classify these
apoptotic cells, presumably due to substantial cell degradation
and loss of protein expression (Fig. 8B, weak residual Olig2 ex-
pressions in TUNEL? cells indicated by arrows). Notably, the
frequencies of both total TUNEL? (Fig. 8E) and BrdU?/
TUNEL? (Fig. 8F) cells in the corpus callosum on both days 2
and 5 were sharply lower in the PEDF-infused mice than in the
motes cell survival in the adult corpus callosum, including sur-
vival of mitotically cycling OPCs. PEDF did not significantly
mitogenic activity by PEDF.
After eliciting focal demyelinative lesions in the corpus callosum
we infused saline or PEDF via osmotic pump into the lesion site
in the lesions of the saline-infused mice (Fig. 9A,M), but were
present in substantially higher numbers in the lesions of the
NG2? OPCs in the saline-infused controls had increased and
became comparable to that in the PEDF-treated mice (Fig.
Sohnetal.•PEDF:AnOligodendroglialLineageMorphogenJ.Neurosci.,August29,2012 • 32(35):12152–12164 • 12161
dendrocytes were present at significantly higher density in the
PEDF-infused than in the saline-infused corpus callosum (Fig.
(Fig. 9G,H,N) and, as judged by MBP immunohistology, remyeli-
Altogether, our results demonstrate that PEDF administration
accelerates oligodendroglial regeneration in the lysolecithin-
demyelinated corpus callosum.
Next, we compared PEDF expression in normal corpus callosum
We also determined PEDF accumulation in nonlesioned corpus
callosum under our intracerebral PEDF infusion paradigm (300
ng per day). As shown by Western blotting (Fig. 10A,B), there
was a robust induction of PEDF after lysolecithin lesioning, but
not after saline control injection. This post-lysolecithin induc-
tion was evident on day 2, peaked on day 5, and had partially
subsided by day 7 postlesioning. Recombinant PEDF infusion
into the nonlesioned corpus callosum for 2 d resulted in a sub-
comparable to that present on day 7 post-lysolecithin lesioning.
No PEDF immunoreactivity was detected in corpus callosum
extracts from PEDF-null mice (Doll et al., 2003), validating the
specificity of the PEDF antibody used in our Western blot analy-
regeneration of the oligodendroglial lineage and remyelination.
PEDF has well characterized anti-angiogenic and neuroprotec-
and has been shown to promote self-renewal of mouse SVZ
neural precursor cells (Ramírez-Castillejo et al., 2006; Andreu-
Agullo ´ et al., 2009), but the effects of this protein on the oligo-
dendroglial lineage have not previously been defined. In the
present studies, PEDF receptor was found to be ubiquitously
effects of PEDF on the germinal zone and the whiter matter. We
demonstrate that PEDF enhances (1) oligodendroglial lineage
induction of adult SVZ neural precursors both in vitro and in
vivo, (2) oligodendrocyte formation and survival in the normal
adult corpus callosum, and (3) oligodendroglial regeneration in
corpus callosum focal demyelinative lesions.
Our initial observation that PEDF treatment during expan-
sion of adult wild-type SVZ neurospheres augmented the
number of NG2? cells prompted us to investigate the oligoden-
drogenic effects of PEDF on SVZ GFAP? neural precursors. Us-
ing neurosphere cultures prepared from the SVZ of adult GFAP:
GFP transgenic mice, we showed that PEDF treatment enhanced
the proportion of GFAP:GFP? neurosphere cells coexpressing
promoting oligodendroglial specification of GFAP? neural pre-
cursors. This notion was further supported by our finding that
the oligodendrogenic effects (i.e., enhanced oligodendroglial
transcription factor expressions and oligodendroglial differenti-
ation, Fig. 2) of PEDF were demonstrable in neurospheres
derived from GFAP?NG2? (i.e., unspecified toward oligoden-
droglial lineage) neural precursors. Interestingly, these PEDF ef-
fects were also evident in GFAP?NG2? precursor-derived
neurospheres, suggesting that PEDF is an oligodendroglial mor-
phogen acting on overall GFAP? neural precursor populations.
In our neurosphere culture studies, however, we did not address
the oligodendrogenic effect of PEDF on SVZ GFAP:GFP? pop-
tained extremely few early neuronal lineage cells (Fig. 1D) and
(2) FACS-purified single GFAP:GFP? cells substantially lacked
neurosphere forming capability (data not shown).
Our analyses of the adult SVZ after PEDF infusion using os-
motic pumps further demonstrated the effects of PEDF on en-
hancing oligodendroglial induction in both SVZ GFAP? cells
and DCX? neuroblasts. It has previously been reported that a
subset of GFAP? cells in adult mouse SVZ expresses PDGFr?
(Jackson et al., 2006), suggesting the presence of SVZ GFAP?
neural precursor cells with early specification toward the oligo-
dendroglial lineage. In contrast, Chojnacki et al. (2011) reported
the absence of GFAP and PDGFr? double-positive cells in the
adult mouse SVZ. Employing GFAP:GFP transgenic mice al-
lowed us to visualize GFAP? cells and to firmly discriminate
ably confident that PDGFr?? immunoreactivity in the SVZ is
expressed by some GFAP? cells, and observed that intraventric-
ular PEDF infusion significantly augmented proportions of SVZ
lineage markers. Our results therefore indicate that increasing
corpus callosum. Mice received no treatment, saline or lysolecithin (LPC) injections to corpus
callosum (CC), or PEDF infusion (300 ng per day) to corpus callosum. Protein extracts from
tative Western blot showing robust upregulation of PEDF expression in the corpus callosum
after lysolecithin injection. Specificity of the PEDF antibody used for Western blotting was
injection, respectively. In nonlesioned mice, 2 d of intracerebral recombinant PEDF infusion
Regulation of PEDF protein expression in lysolecithin-induced demyelinative
12162 • J.Neurosci.,August29,2012 • 32(35):12152–12164 Sohnetal.•PEDF:AnOligodendroglialLineageMorphogen
PEDF level stimulates the prevalence of these oligodendrogenic
GFAP? cells in the adult SVZ.
Our SVZ findings are not fully in line with those previously
reported by another group (Ramírez-Castillejo et al., 2006;
Andreu-Agullo ´ et al., 2009), who, while not commenting on oli-
godendrotrophic effects of PEDF, observed that PEDF induced
self-renewal and multipotency of adult SVZ GFAP? neural pre-
cursor cells. These discrepancies may be at least in part attribut-
able to two factors: (1) they infused less than one tenth the
concentration of PEDF that we used; and (2) they appear to have
focused on the ventral lining of the lateral ventricles, whereas we
analyzed the dorsal SVZ, which is in close proximity to the over-
lying corpus callosum and hence may be more intrinsically
It should be also noted that unlike in the adult corpus callo-
sum, where all PDGFr?? OPCs were colabeled for NG2 (Rivers
et al., 2008), PDGFr? and NG2 expressions showed different
patterns in the adult SVZ. For example, the percentage of
PDGFr?? cells was substantially higher than that of NG2? cells
cells were mostly GFP? whereas the SVZ NG2? cells largely
lacked GFP expression. We reason that PDGFra? and NG2?
cells are at different states of oligodendroglial lineage in adult
at lower densities than PDGFr?? cells in the SVZ and were also
mostly GFP-. These findings suggest that PDGFra? cells are
likely earlier in oligodendroglial lineage than NG2?, Olig2 or
Sox10? cells in the adult SVZ.
Jablonska et al. (2010) have reported oligodendroglial lineage
myelination in the adult corpus callosum. Our findings that (1)
PEDF receptor is expressed by SVZ DCX? neuroblasts and (2)
PEDF infusion resulted in an induction of DCX?/Sox10? cells
in the adult SVZ, suggest that PEDF infusion can potentiate oli-
godendroglial lineage plasticity of SVZ neuroblasts even under
nondemyelinating condition. In sum, our data indicate that
(1) enhancing oligodendroglial specification of GFAP:GFP?
cells, thereby increasing the number of oligodendrogenic GFAP:
GFP? cells and (2) inducing specification of DCX? neuroblasts
to the oligodendroglial lineage.
We also determined that PEDF infusion enhanced differenti-
ation of mitotically labeled OPCs to mature oligodendroglia, as
clearly demonstrated by robust induction of BrdU?/CNP?
cells. Rivers et al. (2008) previously documented continu-
ous addition of oligodendroglial lineage cells derived from
PDGFr?? OPCs in the adult corpus callosum by using
PDGFr?-ERT2/Rosa-EYFP transgenic mice. Our data are in
line with that prior result, and add to it by demonstrating that
PEDF potentiates the production of mature oligodendroglial
cells by cycling OPCs in the adult corpus callosum. While a
survival effect of PEDF on neuronal cells was previously dem-
onstrated in vitro and in vivo (Yabe et al., 2005; Sanagi et al.,
2010), such actions have never been documented in the oligo-
dendroglial lineage. During development, OPCs actively pro-
liferate and considerable programmed cell death of these
proliferating OPCs occurs (Barres et al., 1992, 1993). In the
normal adult corpus callosum, we found that some mitotically
cycling OPCs also undergo apoptosis, and that PEDF infusion
robustly enhances the survival of these newly born OPCs. This
presumably contributed to the increased production of
BrdU?/CC1? mature oligodendrocytes we observed at day 9
(Fig. 7H). By performing EdU incorporation assays, we ruled
out enhanced OPC proliferation as another possible cause for
PEDF-induced augmentation of CC1? cell production. Alto-
gether, our studies indicate that PEDF promotes both matu-
ration and survival of OPCs in the normal adult corpus
We also evaluated the effects of PEDF infusion on regenera-
tion of the oligodendroglial lineage after lysolecithin-elicited fo-
cal corpus callosum demyelination, primarily focusing on early
postlesion phases (i.e., 3 and 7 dpl). PEDF-elicited enhancement
of oligodendroglial regeneration was evident by increased (1)
MBP immunoreactivity with PEDF treatment at 7 dpl. Notably,
treatment than in the saline controls at 3 dpl whereas the num-
control mice were comparable by 7 dpl, suggesting a role for
elucidate mechanisms underlying PEDF-mediated myelin repair
will require additional studies that determine the relative contri-
butions of: (1) increased specification of SVZ cells (i.e., GFAP?
neural precursors and neuroblasts) to the oligodendroglial lin-
erated terminal differentiation of OPCs, to this enhancing effect
To evaluate the possibility that endogenously synthesized
PEDF enhances oligodendroglial lineage regeneration and re-
myelination, we compared levels of PEDF in intact versus
lysolecithin-demyelinated corpus callosum, and demonstrated
substantial elevations in corpus callosum PEDF content during
the first week post-lysolecithin lesioning. In mice infused with
saline, rather than with recombinant PEDF, this PEDF elevation
was temporally correlated with the recruitment of NG2? OPCs
Lesional repopulation with OPCs and oligodendroglia, and re-
myelination, were accelerated in mice given recombinant PEDF
elinative effects of endogenous PEDF had been reinforced by the
lesional administration of exogenous PEDF.
In conclusion, we have documented oligodendrotrophic ef-
fects of PEDF on SVZ neural progenitors and corpus callosum
OPCs in the adult CNS. Corpus callosum PEDF expression is
sion of the demyelinated lesions with recombinant PEDF
accelerates repopulation by OPCs and oligodendroglia, and re-
myelination. These results provide a rationale for trials of PEDF
therapy in demyelinative diseases.
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