NEUROENDOCRINOLOGY
Progesterone and Nestorone Facilitate Axon
Remyelination: A Role for Progesterone Receptors
Rashad Hussain, Martine El-Etr, Ouardia Gaci, Jennifer Rakotomamonjy,
Wendy B. Macklin, Narender Kumar, Regine Sitruk-Ware, Michael Schumacher,
and Abdel M. Ghoumari
Unité Mixte de Recherche 788 Institut National de la Santé et de la Recherche Médicale and
University Paris-Sud 11 (R.H., M.E.-E., O.G., R.J., M.S., A.M.G.), 94276 Kremlin-Bicêtre, France;
Department of Cell and Developmental Biology (W.B.M.), University of Colorado Denver Health
Science Center, Aurora, Colorado 80045; and Rockefeller University and Population Council (N.K.,
R.S.-W.), New York, New York 10065
Enhancing the endogenous capacity of myelin repair is a major therapeutic challenge in demyelinating diseases such as multiple sclerosis. We found that progesterone and the synthetic
19-norprogesterone derivative 16-methylene-17␣-acetoxy-19-norpregn-4-ene-3,20-dione
(Nestorone) promote the remyelination of axons by oligodendrocytes after lysolecithin-induced demyelination in organotypic cultures of cerebellar slices taken from postnatal rats or
mice. The intracellular progesterone receptors (PR) mediate the proremyelinating actions of
Nestorone, because they are not observed in slices from PR knockout mice. Notably, Nestorone
was less efficient in heterozygous mice, expressing reduced levels of PR, suggesting PR haploinsufficiency in myelin repair. Using mice expressing the enhanced green fluorescent protein
(EGFP) under the control of the proteolipid gene promoter, we showed that both progesterone
and Nestorone strongly increased the reappearance of cells of the oligodendroglial lineage in
the demyelinated slices. In contrast to Nestorone, the pregnane derivative medroxyprogesterone acetate had no effect. The increase in oligodendroglial cells by Nestorone resulted from
enhanced NG2⫹ and Olig2⫹ oligodendrocyte progenitor cell (OPC) recruitment. In cocultures
of lysolecithin-demyelinated cerebellar slices from wild-type mice apposed to brain stem slices
of proteolipid gene promoter-EGFP mice, Nestorone stimulated the migration of OPC towards
demyelinated axons. In this coculture paradigm, Nestorone indeed markedly increased the
number of EGFP⫹ cells migrating into the demyelinated cerebellar slices. Our results show that
Nestorone stimulates the recruitment and maturation of OPC, two steps which are limiting for
efficient myelin repair. They may thus open new perspectives for the use of progestins, which
selectively target PR, to promote the endogenous regeneration of myelin. (Endocrinology 152:
3820 –3831, 2011)
P
rogesterone, in addition to its well-known role in reproduction, also exerts marked influences on the function and viability of neural cells throughout the nervous
system (1–3). Because of its neuroprotective actions, documented by a series of experimental studies, progesterone
is now considered as a promising therapeutic candidate for
brain injuries (4).
A particular asset of progesterone is that it also promotes the formation of new myelin sheaths, necessary not
only for the saltatory conduction of action potentials but
also for axonal integrity. A role of progesterone in myelin
formation was first demonstrated in peripheral nerves and
dorsal root ganglia (5). Progesterone also accelerates axonal myelination by oligodendrocytes during brain devel-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/en.2011-1219 Received March 29, 2011. Accepted June 30, 2011.
First Published Online August 9, 2011
Abbreviations: DIV, Day in vitro; EGFP, enhanced green fluorescent protein; GR, glucocorticoid receptor; KO, knockout; MBP, myelin basic protein; MPA, medroxyprogesterone
acetate; Nestorone, 16-methylene-17␣-acetoxy-19-norpregn-4-ene-3,20-dione; OPC, oligodendrocyte progenitor cell; P, postnatal day; PBSGTA, PBS blocking solution [0.12 M (pH
7.4)] containing 0.9% NaCl, 0.25% Triton X-100, 0.1% gelatin, and 0.1% sodium azide;
PLP, proteolipid gene promoter; PR, progesterone receptor.
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Endocrinology, October 2011, 152(10):3820 –3831
opment, as shown in organotypic cultures of cerebellar
slices taken from postnatal rats, at a stage when the myelination of axons is very intense (6). The proremyelinating actions of progesterone involve the stimulation of oligodendrocyte progenitor cell (OPC) proliferation and
maturation and require the presence of progesterone receptors (PR), because they are not observed in PR knockout (KO) mice (6, 7). Consistent with a role of progesterone in oligodendrocyte maturation are the observations
that its addition to the medium of glial cell cultures prepared from neonatal rat brains increased the number of
oligodendrocytes (8) and that it also increased the branching of oligodendrocytes in purified cultures (9).
After destruction of the myelin sheath, as a consequence of neuronal injury, axonal dysfunction or immune
attacks of myelin, as occurs in demyelinating diseases such
as multiple sclerosis, oligodendrocytes are primed to repair to some extend the lost myelin sheaths. This process,
called remyelination or myelin repair, requires the recruitment of OPC present in the demyelinated lesions or recruited from undamaged areas, their proliferation, and
differentiation into mature oligodendrocytes (10, 11). Unfortunately, the endogenous capacity of myelin repair has
its limits, and remyelination efficiency progressively decreases during relapsing/remitting multiple sclerosis.
Thus, boosting this process would be of great therapeutic
benefit.
Studying the process of remyelination is facilitated by
the availability of cellular markers allowing to identify
OPC, e.g. NG2 proteoglycan, or the more mature stages of
oligodendrocytes (12, 13). However, it is important to
distinguish between developmental myelination and remyelination. Indeed, although some developmental events
are recapitulated during myelin repair (14), some of the
intracellular signaling mechanisms involved in both processes may differ (15, 16).
Progesterone has also been shown to have a beneficial
influence on the remyelination of axons after toxin-induced demyelination of the cerebellar peduncle of
9-month-old male rats, an age when the endogenous capacity of myelin repair is already much reduced (17). In an
experimental model of rat spinal cord injury, resulting in
the death of oligodendrocytes and demyelination, the administration of progesterone increased the proliferation of
OPC and their maturation (18).
Thus, besides its potential for neuroprotective interventions, progesterone may also be considered as a promising proremyelinating agent. In addition to the use of
natural progesterone, synthetic progestins may offer an
interesting option. Indeed, they show greater and prolonged activity when compared with progesterone and
may be devoid of some of the side effects associated with
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the natural hormone. On the other hand, progestins have
been designed to target the so called “classical” intracellular PR for contraceptive purposes or to protect the uterus
during hormone replacement therapies, and they do not
necessarily mimic all the actions of progesterone (1). Indeed, progesterone and its neuroactive metabolites have
been demonstrated to bind to multiple target proteins,
including neurotransmitter receptors and membrane receptors of progesterone, which are not necessarily targeted
by the synthetic analogs (19, 20).
The aim of the present study was to determine whether
16-methylene-17␣-acetoxy-19-norpregn-4-ene-3, 20-dione (Nestorone), a 19-norprogesterone derivative, efficiently stimulates remyelination. Nestorone has been designed to selectively target PR, does not possess any
significant androgenic, estrogenic, or mineralocorticoidlike activity, and it is about 100 times more potent than
natural progesterone in bioassays (21, 22). In addition, it
binds to glucocorticoid receptor (GR) with low affinity
but does not manifest glucocorticoid-like effects, possibly
because of rapid metabolism in the liver. We examined the
role of the PR in mediating the effects of Nestorone on
remyelination, and we characterized its effects at the level
of oligodendroglial cells. We also compared the actions of
Nestorone with those of another synthetic progestin, medroxyprogesterone acetate (MPA). Although progesterone and Nestorone stimulate neuroprotective responses,
this is not the case for MPA (23). For these studies, we used
an experimental system of lysolecithin-induced demyelination in organotypic slice cultures of the rat and mouse
cerebellum, a powerful experimental system to study the
effects of proremyelinating factors and to explore the underlying mechanisms (24 –26).
Materials and Methods
Slice cultures and cocultures
Newborn postnatal day (P)10 Sprague Dawley male rats (Janvier, Le Genest-Saint-Isle, France) and transgenic animals, male
mice expressing the enhanced green fluorescent protein (EGFP)
under the control of the proteolipid gene promoter (PLP) (PLPEGFP mice) and PRKO mice were used. PRKO mice are here
referred to as wild-type PR⫹/⫹, heterozygous PR⫹/⫺, and homozygous KO PR⫺/⫺ mice, generated by inserting the lacZ reporter and neomycin resistance genes into exon 1 of the PR gene
to effectively disrupt its transcription (27). Newborn pups of P0
(the day of birth) from PLP-EGFP mice were used in the coculture
experiment. After decapitation, brains were dissected out into
cold Gey’s balanced salt solution containing 5 mg/ml glucose,
and meninges were removed. Cerebellar parasagittal slices (350
␮m thick) were cut on a MacIlwain tissue chopper and transferred onto membranes of 30-mm Millipore culture inserts with
a 0.4-␮m pore size (Millicell; Millipore, Bedford, MA). Slices
were maintained in culture on top of the membranes in six-well
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Nestorone Effect on remyelination
plates containing 1 ml of medium at 35 C in an atmosphere of
humidified 5% CO2. The medium was composed of 50% basal
medium with Earle’s salts (Invitrogen, Gaithersburg, MD), 25%
Hanks ’ balanced salt solution (Life Technologies, Grand Island,
NY), 25% horse serum (Life Technologies), L-glutamine (1 mM),
and 5 mg/ml glucose.
To induce demyelination, the medium was removed from
the wells after 7 d in vitro (DIV), and fresh medium containing
lysophosphatidylcholine (lysolecithin 0.5 mg/ml; Sigma, St.
Louis, MO) was added to the cultures for approximately 17 h.
The medium was then changed, and the slices incubated for
four additional days (4 DIV) in the absence or presence of
progesterone, Nestorone, or MPA. Steroids were dissolved in
ethanol (0.01%), and control cultures were treated with vehicle alone. The medium with the respective steroids was replaced once after 2 d.
Cocultures were prepared as follows. After lysolecithin treatment, P10 cerebellar slices were transected with a glass knife
through lobules III and VIII under a dissecting microscope to
remove deep nuclei zone. The two parts were gently separated to
ensure complete axotomy. The dorsal parts, containing the upper parts of lobules and the fiber tract area, were apposed with
portions of P0 brain stem slices from PLP-EGFP⫹ mice. This
coculture system allowed us to study the migration of PLPEGFP⫹ oligodendroglial cells into lysolecithin-demyelinated
slices. Cocultures were treated with Nestorone or vehicle alone
for 4 d, the medium being replaced after 2 d, and after 10 d, the
number of EGFP⫹ cells that had migrated into the demyelinated
slice and the distance of their migration from the limit between
the two slices were quantified. The migrating cells were also
analyzed for the expression of the oligodendrocyte transcription
factor Olig2.
Steroids
Steroids were diluted at a final concentration of 0.1% ethanol. Control media contained the same amount of vehicle. Doseresponse curves were determined by treating cerebellar slices
with different concentrations (1–100 ␮M) of each compound,
and we show here only results for the dose with maximal efficiency (20 ␮M).
Steroids used were progesterone (Sigma) and synthetic progestins (gifts of the Population Council, New York, NY): the
19-norprogesterone derivative Nestorone and 17-OH progesterone derivative MPA.
Antibodies and staining procedures
The following primary antibodies were used: rabbit polyclonal antibody against Calbindin D-28K (1/10,000 dilution;
Swant, Bellinzona, Switzerland) to visualize Purkinje cells,
monoclonal antibodies against myelin basic protein (MBP) (1/
1000 dilution; Millipore) to examine the extent of myelination,
Olig2 (1/200, rabbit polyclonal; Millipore), and rabbit anti-NG2
chondroitin sulfate proteoglycan polyclonal antibody (1/1000
dilution; Millipore). Primary antibodies were detected, respectively, with secondary goat antirabbit cyanine 3-labeled antibody (1/250 dilution; Jackson ImmunoResearch Laboratories,
Inc., West Grove, PA) and antimouse Alexa Fluor 488-labeled
antibody (1/1000 dilution; Molecular Probes, Leiden, Netherlands). 4⬘,6-Diamidino-2-phenylindole (Sigma) was used to label
cell nuclei. Slice cultures were fixed in 4% paraformaldehyde in
phosphate buffer (0.1 M) (pH 7.4), for 1 h at room temperature.
Endocrinology, October 2011, 152(10):3820 –3831
After washing in PBS, slices were taken off the Millicell and
processed for immunohistochemistry. They were incubated for
1 h in a PBS blocking solution [0.12 M (pH 7.4)] containing 0.9%
NaCl, 0.25% Triton X-100, 0.1% gelatin, and 0.1% sodium
azide (PBSGTA) and lysine (0.1 M). Incubations with primary
antibodies were performed in PBSGTA overnight at 4 C. Secondary antibodies were incubated for 2 h at room temperature
in PBSGTA. Slices were washed in PBS and then mounted in
Fluoromount-G mounting medium.
Quantification of myelination, OPC density, and
OPC migration
To determine the number of PLP-EGFP⫹ cells in the slices,
fluorescent cells were directly visualized and counted under a
fluorescence microscope (Carl Zeiss, Inc., Oberkochen, Germany). Under these conditions, we counted the total number of
surviving PLP-EGFP⫹ cells in three different cerebellar lobules
and calculated the means. MBP staining was quantified in these
areas with NIH image software [Ghoumari et al. (6)] to evaluate
the extent of remyelination and oligodendrocyte density. Images
of the immunostained oligodendrocytes and myelin fibers in organotypic slice cultures of rat or mice cerebella were acquired
using the image analyzing system Axiovision 4 (Carl Zeiss, Inc.).
Quantification of intact myelin segments (MBP⫹ fibers) per field
was performed by numbering the myelin segments (without interrupted area) from three random fields per slice. Half of the
apical fields (⬃105 ␮m2) of the lobule of interest were used for
this quantification, by calibrating ⫻20 objective images, under a
fluorescence microscope (Carl Zeiss, Inc.). At least 18 slices were
used per treatment. The thickness of axons and fibers (axon ⫹
myelin) and the g ratios (axon diameter/fiber diameter) were also
evaluated by calibrating ⫻100 objective images, using a confocal
Zeiss LSM 410 (Carl Zeiss, Inc.). The NG2 staining density was
measured using NIH image software. This staining density was
quantified on a continuous scale of 0 –255 (darkest). To minimize differences between different measurements, we set as control an arbitrary level of staining at the value of 100. NG2 staining density was evaluated as percentage (light pixels/light ⫹ dark
pixels).
In cocultures, only the EGFP-PLP⫹ cells visualized in the
white matter of the demyelinated P10 cerebellar slices were
counted within a surface of 2.105 ␮m2. The mean lengths of the
longest distances of EGFP-PLP⫹ cell migration, from the limit
between the two slices and invading the white matter of P10
cerebellar slices, were measured using a confocal Zeiss LSM 410
(Carl Zeiss, Inc.) image analyzing system. For each measurement, at least four cocultures, each prepared from a different
wild-type and transgenic animal, were used.
Western blot analysis
Cerebellar slices treated with lysolecithin were cultured in the
absence or presence of 20 ␮M Nestorone for 3 h. Slices were then
washed with Gey’s balanced salt solution containing 5 mg/ml
glucose and dissolved in a Triton lysis buffer containing 50 mM
Tris (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 100
mM NaF, 5 ␮M ZnCl2, 1% Triton X-100, 10% glycerol, and a
cocktail of protease inhibitors (Sigma). After homogenization,
extracts were clarified by centrifugation (14,000 ⫻ g for 10 min
at 4 C). For baseline (time 0), cerebellum was directly homogenized in the lysis buffer. The concentrations of soluble proteins
in the supernatants were quantified by the Bradford method
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MA). After washing with Tris-buffered saline
containing 0.1% Tween 20, membranes were
incubated for 1 h at room temperature with
peroxidase-conjugated AffiniPure goat antirabbit (1/20,000 dilution; Jackson ImmunoResearch Laboratories, Inc.). After addition of chemiluminescence reagent (GE
Healthcare, Buckinghamshire, UK), blots
were exposed to G:box iChemi System (Syngene, Cambridge, UK) for developing, and
images were captured using Genesnap software (Syngene). Results were quantified using
Genetools software (Syngene).
Statistical analysis
Data were expressed as means ⫾ SEM of
at least n ⫽ 18 cerebellar slices from four
animals. The significance of differences between means was evaluated by NewmanKeuls tests after two- or one-way ANOVA
for multiple group comparisons. The level
of significance was set at P ⬍ 0.05.
Results
Progesterone and Nestorone
promote remyelination in
organotypic cerebellar slice
cultures
To determine whether progesterone
promotes the remyelination of axons,
cerebellar slices taken from P10 rats were
cultured for 7 DIV allowing axons to become myelinated (Fig. 1A). They were
then treated with 0.5 mg/ml of lysoleciFIG. 1. Progesterone and Nestorone promote remyelination after lysolecithin-induced
thin for approximately 17 h, resulting in
demyelination. Confocal images of whole mount immunostaining for the myelin marker, MBP
(green), and the Purkinje cell marker (Calbindin) (red) were performed in slice cultures of P10
marked demyelination but sparing the
rat cerebellum in absence (A, control) or presence of 0.5 mg/ml lysolecithin for approximately
axons of Purkinje neurons (24). Four
17 h (B). Control cultures were exposed to vehicle (ethanol) alone. C and D, Slices treated
days after lysolecithin-induced demyeliwith 20 ␮M progesterone or Nestorone, respectively, after removal of lysolecithin. Slice
nation, immunostaining for the myelin
cultures treated by lysolecithin show broken myelin staining (B), suggestive of damaged
myelin. Four-day (4 DIV) treatment with progestin enhances (MBP)-positive cell staining. In
marker MBP still appeared sparse and ircontrast to progesterone and Nestorone, the 17-OH progesterone derivative MPA (20 ␮M)
regular when slices were cultured in mehad no significant effect on MBP-immunoreactive myelin (same as B). E, Quantification of
dium without the addition of progestins
MBP staining intensity under different treatment, measured using NIH image software to
determine the degree of MBP⫹ remyelinated axons (calculated as staining intensity of myelin
(Fig. 1B). However, in slices cultured for
MBP). F, Analysis of MBP expression by Western blotting after slice treatment with lysolecithin
4 d in the presence of progesterone (20
alone or with lysolecithin and thereafter with Nestorone (mean ⫾ SEM; **, P ⬍ 0.01; *, P ⬍
␮M), MBP immunoreactive processes
0.05, when compared with the lysolecithin-treated condition using Newman-Keuls tests after
one-way ANOVA). Nes, Nestorone; Ctr, control; Lyso, lysolecithin; Prog, progesterone. Scale
were aligned, and there was a 1.5-fold
bar in A–D, 100 ␮m.
increase in MBP staining (P ⬍ 0.05) (Fig.
1, C and E). Adding a micromolar con(Bio-Rad, France). Extracts were resolved (20 ␮g) by SDS-PAGE
centration of progesterone to the culture medium is necessary
(10% polyacrylamide gel) and electrophoretically transferred to
to stimulate myelin formation, because the hormone does
polyvinylidene fluoride membranes (Millipore). Membranes were
not easily penetrate the 350-␮m-thick cerebellar slices, which
incubated with 5% nonfat dry milk in Tris-buffered saline containare
cultured on top of a microporous membrane and not
ing 0.1% Tween 20 at room temperature for 1 h and probed oversubmerged by the culture medium (6). When slices were exnight at 4 C with an anti-MBP (see above) or with antitotal p38
antibodies (total MAPK; Cell Signaling Technology, Inc., Danvers,
posed to the same concentration of the potent synthetic pro-
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thin myelin as shown by double-label
immunostaining (Fig. 2, B1–B3). When
slices were treated with Nestorone (20
␮M), Purkinje axons were surrounded
by thick myelin. Consistent with a proremyelinating effect of Nestorone, g ratios (axon diameter/fiber diameter)
were significantly decreased by the progestin (controls, 0.71 ⫾ 0.02; lysolecithin, 0.80 ⫾ 0.01; and lysolecithin ⫹
Nestorone, 0.64 ⫾ 0.01; P ⬍ 0.05). We
also observed more MBP-positive oligodendrocytes close to the remyelinated axons in response to Nestorone
treatment (Fig. 2, C1 and C3).
The proremyelinating action of
Nestorone requires the
intracellular PR
Nestorone is a 19-norprogesterone
derivative, designed to efficiently target
the intracellular PR. Although Nestorone does not exert androgenic, estrogenic, or glucocorticoid activity in different in vivo assays, it has been shown
FIG. 2. Action of progestin Nestorone on myelin sheath thickness. P10 rat cerebellar slices
to bind to the GR and to cause thymus
were not treated (control, A), treated with lysolecithin (B) alone or with lysolecithin, and 20
involution, although only at high doses
␮M Nestorone (C) for 4 DIV. MBP and Calbindin were used for immunostaining of myelin
(21, 22). To determine whether Nesto(green) and axon (red), respectively. Fibers (axon ⫹ myelin) at high magnification show thick
rone promotes myelin repair via PR, we
or thin myelin surrounding axons. Arrowheads indicate myelinating oligodendrocytes. Images
were analyzed using confocal microscopy. Scale bar, 10 ␮m.
used adult male wild-type PR⫹/⫹,
heterozygous PR⫹/⫺, and homozygous
gestin Nestorone, dense networks of well-organized MBP- KO PR⫺/⫺ mice. Cerebellar slices of PR⫹/⫹, PR⫹/⫺, and
positive myelinated fibers were observed, and MBP PR⫺/⫺ mice were sampled at P10. As in the previous experimmunostaining was increased 2-fold when compared with iments, slices were cultured for 7 DIV, then demyelinated by
slices grown in the absence of exogenous progestins (P ⬍
0.01) (Fig. 1, D and E). Nestorone treatment increased MBP
staining, reaching the level of controls. No significant difference was seen between these two groups. The marked increase in MBP expression in response to Nestorone treatment was confirmed by Western blot analysis (Fig. 1F).
Progesterone (20 ␮M) also enhanced remyelination, but its
effect remained statistically lower than the one of Nestorone
(56.8 ⫾ 5.1 vs. 74.48 ⫾ 4.3% staining intensity). In contrast
to Nestorone, the 17-OH progesterone derivative MPA (20
␮M) had no significant effect on MBP-immunoreactive my- FIG. 3. The intracellular progesterone receptor (PR) is necessary for
elin (Fig. 1E).
Nestorone effect on remyelination. Cerebellar slices from P10 wild-type
We examined the proremyelinating effect of Nestorone (WT) or PRKO mice (Htz, Heterozygous; Hz, homozygous) were
cultured for 7 d and treated with lysolecithin (0.5 mg/ml) for
by confocal microscopy at high magnification. In control approximately 17 h. Then, Nestorone (20 ␮M) was added to cultures
cerebellar cultures, thick Purkinje axons were observed, for 4 DIV. The apical fields (⬃105 ␮m2) of the lobule of interest were
most of which were myelinated throughout (Fig. 2, A1– used for this quantification. Results expressed as means ⫾ SEM; ***,
A3). In contrast, 4 d after lysolecithin-induced demyelina- P ⬍ 0.001; **, P ⬍ 0.01; *, P ⬍ 0.05 when compared by NewmanKeuls test after two-way ANOVA (genotype ⫻ treatment). Nes,
tion, some axons were still completely devoid of myelin, Nestorone; ctr, control; lyso, lysolecithin; WT, wild-type; htz,
whereas myelinated axons were only surrounded by very heterozygous; hz, homozygous.
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(P ⬍ 0.001) and treatment (P ⬍ 0.001) on the
number of remyelinated MBP⫹ segments, and
a significant interaction between both factors
(P ⬍ 0.01). Treatment with Nestorone markedly stimulated remyelination in cerebellar
slices of PR⫹/⫹ mice, and slightly in PR⫹/⫺
slices, and it was without effect in PR⫺/⫺ animals (Fig. 3). Nestorone more efficiently stimulated myelination in PR⫹/⫹ slices when compared with PR⫹/⫺ slices (P ⬍ 0.001). Also, no
statistically significant difference was observed
between control and Nestorone-treated slices
(P ⬎ 0.05). Together, these results strongly
suggest that PR expression levels are a limiting
factor for efficient remyelination in response to
progesterone and Nestorone.
FIG. 4. Progestins restore the number of oligodendrocyte PLP-GFP⫹ cells after
lysolecithin treatment. PLP-EGFP mice (GFP under the control of the PLP) were used
to label oligodendrocytes in the white matter tracts of the cerebellum. A, Nontreated
slices (controls). Slices showing lower expression of PLP-EGFP when treated with
lysolecithin (B) and high expression when incubated with progesterone or Nestorone
(C and E). However, no increase in PLP-EGFP was seen when slices were treated with
20 ␮M MPA (D). F, The number of PLP-EGFP⫹ cells was markedly increased after
progesterone and Nestorone treatment in comparison with slices treated with MPA.
Areas of 0.6 mm2 in cerebellar lobule were used for quantification, and images were
acquired with an image analyzing system, Axiovision 4 (Carl Zeiss, Inc.) (mean ⫾ SEM;
**, P ⬍ 0.01; *, P ⬍ 0.05. ns, Nonsignificant, when compared with the lysolecithintreated groups by Newman-Keuls tests after one-way ANOVA). Nes, Nestorone; Ctr,
control; Lyso, lysolecithin. Scale bar, 100 ␮m.
an overnight treatment with lysolecithin and cultured for 4 d
in the absence or presence of Nestorone (20 ␮M).
As for rats, mouse cerebellar slices were abundantly
myelinated after 7 DIV, and transient treatment with lysolecithin produced a marked demyelination of Purkinje
axons, independent of the PR genotype (Fig. 3). Two-way
ANOVA revealed highly significant effects of PR genotype
Progesterone and Nestorone, but not
MPA, increase the number of
oligodendroglial cells
We next investigated whether the stimulation
of axonal remyelination by progestins after lysolecithin-induced demyelination is accompanied
by a replenishment of lost oligodendrocytes. We
prepared cerebellar slices from P10 transgenic
mice expressing EGFP driven by the mouse myelin PLP gene promoter (28). In these mice, EGFP
expression is high in all cells of the oligodendrocyte lineage and can be easily detected in the postnatal brain. After 7 DIV, PLP-EGFP⫹ oligodendroglial cells are largely distributed in the
cerebellar white matter tracts, and both cell bodies and myelinated processes were intensely
stained (Fig. 4A). Four days after overnight exposure to lysolecithin, the number of oligodendroglial cells was reduced by 70% when cerebellar slices were treated with vehicle (Fig. 4, B and
F). However, in the presence of progesterone or
Nestorone (both at 20 ␮M), oligodendroglial cells
were significantly replenished (Fig. 4, C, E, and
F). In contrast, MPA (20 ␮M) failed to increase
the number of PLP-EGFP⫹ cells (Fig. 4, D and F).
Effects of Nestorone on oligodendrocyte
progenitors
The increase in oligodendroglial cells by
Nestorone after the exposure of cerebellar slices to lysolecithin suggests a stimulation of OPC recruitment to demyelinated areas. In EGFP-PLP mice, the fluorescent protein is expressed at all stages of the oligodendrocyte
lineage. Thus, an additional marker is required to specifically label OPC. The most often used one is the proteoglycan NG2 (11). We have previously shown that progester-
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Nestorone Effect on remyelination
FIG. 5. Oligodendrocyte progenitor marker NG2 and EGFP-PLP⫹ cells
increase after lysolecithin exposure followed by Nestorone treatment
for 24 h. A and B, Representative fluorescent Axioskop-FS microscope
images of NG2⫹ cells and EGFP-PLP⫹ cells in P10 cerebellar slices
treated by lysolecithin or by lysolecithin followed by 20 ␮M Nestorone,
respectively. C and D, Asterisks indicate triple-positive cells, 4⬘,6diamidino-2-phenylindole (DAPI) (blue), EGFP-PLP (green), and NG2
(red), indicating an increase in the density of OPC. E, Quantification of
NG2 staining in control, lysolecithin and lysolecithin ⫹ Nestoronetreated slices (error bars represent SEM; **, P ⬍ 0.01, when compared
with the lysolecithin-treated condition using Newman-Keuls tests after
one-way ANOVA). Nes, Nestorone; Ctr, control; Lyso, lysolecithin.
Scale bar, 50 ␮m (A and B) and 10 ␮m (C and D).
one treatment stimulates the proliferation of NG2⫹
oligodendrocyte precursors in P7 cerebellar slices (7).
Here, we show that Nestorone treatment rapidly increases
the density of NG2⫹ cells after lysolecithin-induced demyelination. In cerebellar slices of PLP-EGFP⫹ mice, cultured for 7 DIV, we observed a more than 60% reduction
in the number of PLP-EGFP⫹ oligodendroglial cells 24 h
after lysolecithin exposure. However, adding 20 ␮M
Nestorone for 24 h to the culture medium after the removal of lysolecithin caused an approximately 2- and
3-fold increase in the number EGFP⫹ and NG2⫹ cells,
respectively (Fig. 5, A, B, and E).
Importantly, Nestorone also markedly increased the
density of NG2⫹ processes surrounding EGFP⫹ cells, indicating an increase in OPC density (Fig. 5, A–E). The
EGFP⫹ cells were indeed OPC responding to demyelination, because they also expressed Olig2, a basic helix-loophelix transcription factor required for the specification of
the oligodendrocyte lineage (29, 30). Nestorone treatment
significantly increased the density of Olig2⫹ and Olig2⫹/
Endocrinology, October 2011, 152(10):3820 –3831
FIG. 6. Progestins promote oligodendrocyte differentiation. Olig2, a
basic helix-loop-helix transcription factor, increases after lysolecithin
exposure and Nestorone treatment for 24 h. A–C, Quantifications of
EGFP-PLP⫹, Olig2⫹, and EGFP-PLP⫹/Olig2⫹ cells in control, lysolecithin,
and lysolecithin ⫹ (20 ␮M) Nestorone-treated slices, respectively. Error
bars represent SEM; **, P ⬍ 0.01, when compared with the lysolecithin-treated condition using Newman-Keuls tests after one-way
ANOVA. Nes, Nestorone; Ctr, control; Lyso, lysolecithin.
EGFP-PLP⫹ cells (Fig. 6). Thus, the progestin promotes
the remyelination of axons by stimulating the recruitment
of OPC and their differentiation into mature oligodendroglial cells.
Migration of oligodendrocytes and remyelination
in the presence of Nestorone
The recruitment of OPC after a demyelinating lesion
can result not only from their proliferation and specification but also their migration toward the demyelinated axons. Indeed, the vicinity of demyelinated axons becomes
depleted in oligodendrocytes and their progenitors, eventually resulting in the formation of chronically demyelinated lesions (31). We thus investigated whether Nestorone also promotes the migration of OPC toward a
demyelinated area. As an experimental system, we set up
cocultures to observe the migration of OPC from P0 brain
stem slices of PLP-EGFP mice into P10 cerebellar slices of
wild-type mice, demyelinated by exposure to lysolecithin
as described above. The demyelinated P10 cerebellar slices
Endocrinology, October 2011, 152(10):3820 –3831
endo.endojournals.org
FIG. 7. Migration of oligodendrocyte progenitors and remyelination in presence of
Nestorone. A, Model of axotomy and coculture of cerebellar and brain stem slices. Some
cultures were transected with a glass knife through lobules III and VIII under a dissecting
microscope to remove deep nuclei zone. The two parts were gently separated to ensure a
complete axotomy. The dorsal parts were apposed with portions of brain stem from P0-PLPEGFP mice, which allowed us, in coculture slices, a more precise analysis of the fate of the
migrating PLP-EGFP⫹ oligodendrocytes. Dashed lines indicate the limit between the two
cultures. B, After lysolecithin treatment, axotomized P10 cerebellar slices (Purkinje cells in red)
were cocultured with portions of P0 brain stem slices from PLP-EGFP⫹ oligodendrocytes
(green), showing low number of migrating oligodendrocytes, which invade P10 cerebellar
slices. However, treatment of the same cocultures with Nestorone (20 ␮M) highly increases
the number of oligodendrocytes and their migration onto transected and lysolecithin-treated
P10 cerebellar slices (C and D). Analysis of migrating oligodendrocytes was performed at 10
DIV after lysolecithin removal, and Nestorone allows immature oligodendrocytes to migrate
far into P10 cerebellar slices, represented by the mean lengths of the longest distances of
EGFP-PLP⫹ cell migration (E). F, Under Nestorone treatment, cocultures show increased
process extension and complexity, normally a feature of oligodendrocyte maturation. PLPEGFP⫹ extensions wind up Purkinje axons, showed at high magnification (G). Arrows indicate
migrating EGFP-PLP⫹ oligodendrocytes in B and C and 10 ␮m (F and G).and surrounding
Purkinje axons in F and G. For each group, 16 cocultures from four different mice were used.
Mean ⫾ SEM; **, P ⬍ 0.01. Nes, Nestorone; Ctr, control; Lyso, lysolecithin. Scale bar, 100 ␮m
(B and C).
were transected with a glass knife through lobules III and
VIII under a dissecting microscope to remove the deep
nuclei zone. The dorsal part of the slices, containing Purkinje neuron soma with their proximal portions of demyelinated axons, were then apposed to the P0 brain stem
slices containing EGFP⫹ OPC, and slices were then cocultured for 10 d (Fig. 7A, inset).
3827
Under these conditions, only a few
EGFP⫹ cells migrated into the lysolecithin-demyelinated cerebellar slices
(Fig. 7B). However, when slices were
cocultured in the presence of Nestorone
(20 ␮M), there was a nearly 3-fold increase in the number of EGFP⫹ cells migrating into the cerebellar slices (Fig. 7,
C and D). Moreover, treatment with
Nestorone doubled the OPC migration
distance (Fig. 7, C and E). Importantly,
the EGFP⫹ cells that migrated toward
demyelinated Purkinje axons extended
long processes, and some of the EGFP⫹
extensions tightly interacted with Purkinje cell axons (Fig. 7. F and G). These
are clearly features of oligodendrocyte
maturation.
We confirmed that coculturing brain
stem slices of EGFP-PLP mice with demyelinated cerebellar slices of wildtype mice for 10 d in the presence of 20
␮M Nestorone markedly increased the
number of migrating EGFP⫹ cells (Fig.
8). Moreover, in the presence of Nestorone, a significantly greater proportion
of migrating EGFP⫹ cells coexpressed
the oligodendrocyte transcription factor Olig2 (vehicle, 40.2 ⫾ 5.7%; Nestorone, 63.2 ⫾ 2.9%; **, P ⬍ 0.01).
Thus, Nestorone promotes both the recruitment of OPC toward demyelinated axons and their maturation into
oligodendrocytes.
Discussion
Our results show that progesterone and
Nestorone, but not MPA, increase remyelination in cerebellar slice cultures
after a demyelinating insult with lysolecithin. The proremyelinating effect of
Nestorone requires the presence of PR,
because it is not observed in cerebellar
slices from PRKO mice. This observation is consistent with the high affinity and selectivity of
Nestorone for PR and points to a potential role for PR in
myelin repair. Importantly, Nestorone stimulates the recruitment and maturation of OPC, which are both needed
for efficient axonal remyelination (11).
Progesterone has previously been shown to exert a beneficial influence on myelin repair in different in vivo par-
3828
Hussain et al.
Nestorone Effect on remyelination
Endocrinology, October 2011, 152(10):3820 –3831
in progesterone-driven myelin repair.
It is interesting to note that decreased
PR expression in hypothalamus or
uterus of PR⫹/⫺ mice does not result
in a particular reproductive phenotype (35).
This raises the important question of
the cellular targets of progesterone and
progestins, which has been previously
addressed. Thus, immunohistochemical
analyses showed that PR within the developing cerebellum is expressed in postnatal Purkinje neurons (37). This suggests that the proremyelinating effects of
progesterone may mainly involve neuronal PR, consistent with the key role
played by neuron-derived factors in the
maturation of oligodendrocytes and the
formation of myelin sheaths (38). However, PR expression has also been demonstrated in brain glial cells (39, 40). In
particular, PR signaling in astrocytes
may be involved in myelination by
stimulating growth factor production
FIG. 8. Increased number and differentiation of oligodendrocytes in presence of Nestorone.
(41, 42). Whether oligodendrocytes
Coculture of cerebellar slices from P10 wild-type mice and brain stem slices from P0
also express PR and are a direct target
transgenic PLP-EGFP mice were treated by lysolecithin for approximately 17 h and thereafter
for the actions of progesterone reby vehicle (A–C) or Nestorone (20 ␮M) (D–F) for 3 d. Ten days later, cocultures were
immunolabeled with Olig2 transcription factor (red). A and D, EGFP-PLP⫹ cells; B and E,
mains to be clarified (9, 43). On the
Olig2⫹ cells; C and F, EGFP-PLP⫹/Olig2⫹ cells. G and H, Quantification of these different
contrary, microglial cells have been
markers in untreated and Nestorone-treated cocultures. Treatment of cocultures with
reported to be devoid of PR (44).
Nestorone highly increases the number and differentiation of immature migrating
PR may thus be promising targets for
oligodendrocytes. Mean ⫹ SEM. Scale bar, 10 ␮m.
promoting myelin repair in demyelinating diseases such as multiple sclerosis.
adigms of demyelination/remyelination (17, 18, 32).
Consequently, our results open the way to a new theraMoreover, progesterone treatment attenuated the severity
peutic indication for some synthetic progestins, designed
of disease symptoms in mice with experimental autoimto highly target PR and already used in contraception,
mune encephalomyelitis, an experimental model of mulmenopausal hormone therapy, and the treatment of gytiple sclerosis. It improved spinal axon myelination and
necological diseases (22). However, it is important to
had protective effects on axons (33, 34). However, the
keep in mind that not all progestins are the same in terms
signaling mechanisms involved in the promyelinating actions of progesterone are not well defined. This is an im- of their actions on various targets. They belong to difportant question, because progesterone acts on multiple ferent classes, and even small changes in their structure
may result in considerable differences in their pharmatargets within the nervous system (1).
Our present findings identify PR as key mediators of cological actions (22, 45). It is thus important to deterthe proremyelinating actions of progesterone, because mine which synthetic progestin(s) efficiently stimulate
they were not observed in PR⫺/⫺ mice. Importantly, remyelination and whether their proremyelinating efprogesterone was also much less efficient in stimulating fects involve PR.
We report here that Nestorone is a potent proremyelithe remyelination of axons in PR⫹/⫺ mice, in which PR
nating
agent. This progestin belongs to the class of synbinding has been shown to be reduced by half in progesterone target tissues, including brain and uterus (35, thetic 19-norprogesterone derivatives, which show great
36). Together, these results point to an influence of PR selectivity for PR and may provide therapeutic benefits for
deficiency and haploinsufficiency on remyelination, relapsing/remitting multiple sclerosis by promoting enand they also suggest that PR may be a limiting factor dogenous myelin repair. This result gains significance in
Endocrinology, October 2011, 152(10):3820 –3831
light of an ongoing phase II clinical trial testing another
19-norprogesterone derivative, nomegestrol acetate, in
combination with estradiol, for the prevention of postpartum relapses in multiple sclerosis (46).
By contrast, the progestin MPA failed to promote the
replenishment of oligodendrocytes and the formation of
new myelin after lysolecithin-induced demyelination of
axons. Thus, not all progestins may be useful for the treatment of demyelinating diseases. MPA was the progestin
used in the Women’s Health Initiative study, a large prospective clinical trial aimed to test the efficacy of hormone
replacement therapy in postmenopausal women. MPA
probably contributed to the negative outcomes of the
Women’s Health Initiative trial (1, 47). Recent experimental studies have shown that MPA lacks neuroprotective activity, antagonizes the neuroprotective and promnesic effects of estrogen and exacerbates excitotoxic
damage to neurons (23, 48, 49).
Would the presence of the GR in cerebellar slices (50)
have a mediating Nestorone effect on remyelination? Several points may argue for the nonimplication or at the least
for a very small role of the GR in this effect:
1) MPA, which exerts a strong glucocorticoid effect, did
not induce the same positive response as Nestorone did; 2)
dexamethasone, known to bind with very high affinity to
GR, also did not influence remyelination in cerebellar slices
(data not shown); and 3) a previous study by Chari et al. (51)
showed that glucocorticoid delay oligodendrocyte-mediated remyelination in toxin-induced demyelinating in
vivo model. Therefore, it is very unlikely that the remyelinating action seen relates to the GR binding. However, further studies with a selective blocker of the GR
without blocking the PR (because mifepristone does
both) would be needed to fully exclude this possibility.
Progestins are particularly interesting for treating degenerative diseases of the nervous system, not only because they easily cross blood-brain barrier and rapidly
diffuse throughout nervous tissues but also exert multiple beneficial effects. Thus, progesterone and Nestorone are efficient neuroprotective agents to promote the
formation of new myelin sheaths and to modulate neuroinflammatory responses (23, 52). Likewise, Nestorone stimulates the formation of new myelin sheaths by
acting on multiple key steps of the remyelination process: the recruitment and maturation of OPC. These are
encouraging findings, because impaired recruitment of
OPC and failure of their differentiation may be a major
cause of remyelination failure, and agents stimulating
both processes may be of great interest for multiple
sclerosis patients (11, 53).
Nestorone is a fourth-generation synthetic progestin,
and it is currently undergoing testing as a contraceptive in
endo.endojournals.org
3829
both women and men (54, 55). It may thus become useful
as a proremyelinating agent in both female and male multiple sclerosis patients. Its strong progestational activity,
combined with the lack of androgenic, estrogenic, and
glucocorticoid-like activities, confers special advantages
to the therapeutic use of Nestorone.
Acknowledgments
We thank John P. Lydon and Bert W. O’Malley (Department of
Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX) for generous gifts of PR-KO mice and Philippe
Leclerc for image analysis.
Address all correspondence and requests for reprints to:
Dr. Abdel M. Ghoumari, Unité Mixte de Recherche 788 Institut National de la Santé et de la Recherche Médicale and
University Paris-Sud 11, 80 Rue du Général Leclerc, 94276
Kremlin-Bicêtre, France. E-mail: [email protected];
or Dr. Michael Schumacher. E-mail: michael.schumacher@
inserm.fr.
R.S.-W. is supported by a grant from BARUS for this research.
Disclosure Summary: M.E.-E., N.K., R.S.-W., M.S., and
A.M.G. are inventor on United States Patent WO/2011/040048.
R.H., O.G., R.J., and W.B.M. have nothing to disclose.
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