Department of Neurology and Program for Neuroscience, University of Michigan Medical Center, Ann Arbor, MI 48109-0585, USA
* Author for correspondence (e-mail: parent{at}umich.edu)
Accepted 18 April 2005
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SUMMARY |
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Key words: Neural stem cell, Neuronal migration, Retinoid signaling
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Introduction |
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Although retinoid signaling components persist in the mammalian forebrain
(Zetterstrom et al., 1999),
its role in postnatal brain development is poorly understood. Accumulating
data suggest that retinoid signaling influences neurogenesis in persistent
germinative zones of the neonatal and adult rodent forebrain. Neurogenesis
continues throughout life in the mammalian subventricular zone (SVZ)-olfactory
bulb pathway and hippocampal dentate gyrus
(Altman and Das, 1965
;
Altman, 1969
;
Luskin, 1993
;
Lois and Alvarez-Buylla, 1994
;
Eriksson et al., 1998
). In
neonatal and adult rodents, the forebrain SVZ generates neuroblasts that
migrate along the rostral migratory stream (RMS) to the olfactory bulb and
differentiate into granule and periglomerular neurons
(Luskin, 1993
,
Luskin, 1998
;
Lois et al., 1996
). Recent
work suggests that RA modulates cell proliferation or neurogenesis in regions
where neural progenitors persist postnatally. RA exposure stimulates
neurogenesis in neural stem cell cultures isolated from the embryonic striatal
SVZ or adult hippocampus (Wohl and Weiss,
1998
; Takahashi et al.,
1999
). Prolonged oral RA administration also increases adult rat
SVZ cell proliferation (Giardino et al.,
2000
). Moreover, the lateral ganglionic eminence (LGE), which
gives rise to the striatum under the influence of RA, evolves into the
postnatal forebrain SVZ and is a source of olfactory bulb neurons
(Anderson et al., 1997
;
Wichterle et al., 1999
;
Toresson and Campbell, 2001
;
Stenman et al., 2003
).
The presence of retinoid signaling components in the SVZ-olfactory bulb pathway and evidence that RA influences neural development in persistent germinative zones led us to explore the role of retinoid signaling in postnatal SVZ-olfactory bulb neurogenesis. We found that RA and its precursor retinol increased SVZ neurogenesis in dissociated and explant cultures. Moreover, blockade of retinoid signaling decreased SVZ cell proliferation in vitro and in vivo, and inhibited SVZ neuroblast migration to the olfactory bulb in explants. These findings suggest that RA signaling regulates multiple aspects of postnatal forebrain SVZ neurogenesis.
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Materials and methods |
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Reverse transcriptase polymerase chain reaction (RT-PCR) for retinoid receptors
mRNA was isolated from secondary NS after 6 DIV using Trizol reagent
(Invitrogen). RT-PCR was performed with the Titan one-step kit (Roche
Molecular) using a PCR cycler (RoboCycler Gradient 40; Stratagene). RT-PCR was
carried out at 55°C (50 minutes) for cDNA synthesis followed by 36 cycles
at 94°C (30 seconds), 58°C (45 seconds), 72°C (90 seconds) and a
final extension step at 72°C (7 minutes). Specific primer pairs were (1)
RA receptor (RAR
) (forward
5'-agagcagttccgaagagatag-3', reverse
5'-cgactgtccgcttagagtgtccaa-3'); (2) RARß
(5'-gcaggaatgcacagagagctatgagat-3' and
5'-ggtgactgactgactccactgttctccact-3'); (3) RAR
(5'-tgtatgcaatgacaagtcttctgg-3' and
5'-atgaggcagatagcactgagtagc-3'); (4) retinoid X receptor
(RXR
) (5'-cactgaggatatcaagccgccact-3' and
5'-ggtgtcacaccagctctgctatgc-3'); and (5) RXR
(5'-tgtgtacagctgtgaaggttgc-3' and
5'-tctgagaagtgggggatgc-3'). PCR products were analyzed using 1%
agarose gel electrophoresis. RT-PCR for actin served as an internal control;
no PCR product was detected without RT in the RNA mixture.
Explant culture and electroporation
Neonatal CD-1 mouse brains were hemisectioned, embedded in 4% LMP agarose
(Gibco), vibratome-sectioned into 250 µm-thick parasagittal slices, and
cultured on polycarbonate membrane filters (Whatman nucleopore 25 mm) floating
on 1 ml of serum-free medium (Neurobasal A/pen-strep/glutamax/B27 supplement,
all from Gibco). Only sections containing SVZ-olfactory bulb pathway were
used; typical yields were two explants per hemisphere. After 1 hour, RA [10
µM in dimethyl sulfoxide (DMSO)] or vehicle was added. BrdU (5 µM) was
added on day 2 for 24 hours and explants fixed 48 hours later (4 DIV), or
slices were incubated with BrdU for 2 hours prior to washout and fixation at 2
or 4 DIV. Pilot studies were performed using explants from P2-P10 mice
(Fig. 3). P2 mice were used for
subsequent experiments.
Electroporation was performed on hemispheres prior to explant preparation
(Fig. 3). DNA (1 µg
µl-1) was electroporated as follows: (1) co-electroporation of
enhanced green fluorescent protein (EGFP) or dsRed plasmids under the control
of an ubiquitin promoter (US2-EGFP, US2-dsRed; gifts from D. Turner); or (2)
electroporation of an EGFP reporter under the control of a simian CMV promoter
(pCS2+EGFP, gift of D. Turner) (Farah et
al., 2000) alone or combined with dominant-negative (dn)
RAR
or dnRXR
constructs (gifts from P. Chambon)
(Feng et al., 1997
;
Xiao et al., 1999
). DNA was
injected into the anterior portion of the open lateral ventricle, hemsipheres
incubated in D-PBS on ice (10 minutes), and electroporation (85 V, 10 pulses,
50 milliseconds per pulse, 1 second inter-pulse interval) performed in a
platinum petridish plate electrode (Protech International) using a square-wave
electroporator (BTX). For additional experiments, dnRAR
was subcloned
into a CMV-IRES-EGFP vector (BD Biosciences). CMV-dnRAR
-IRES-EGFP or
CMV-IRES-EGFP control construct was electroporated into slices as above.
Hemispheres were embedded, sectioned sagittally at 250 µm and cultured for
4 DIV.
Immunofluorescence histochemistry and TUNEL stain
Cultured cells were fixed at 4 or 7 DIV for 30 minutes in 4%
paraformaldehyde (PFA). After washes and blocking, cells were incubated
overnight at 4°C with the following primary antibodies: mouse
anti-ß-tubulin (TuJ1, diluted 1:1000; Covance); rabbit anti-calretinin
(1:2000; Chemicon); rabbit anti-glial fibrillary acidic protein (GFAP; 1:500;
Sigma); rat anti-myelin basic protein (MBP; 1:1000; Chemicon) or mouse
anti-activated caspase-3 (1:1000; BD PharMingen). Secondary antibodies (all
from Molecular Probes) included: anti-rat Alexa Fluor 594; anti-rabbit Alexa
Fluor 594 or 488; or anti-mouse Alexa Fluor 488 at 1:400 dilution.
Bis-benzimide (Molecular Probes) was used for counterstaining. To detect
double labeling for BrdU and ß-tubulin, GFAP, or MBP, cells were
incubated with the first antibody overnight at 4°C, washed and post-fixed
in 4% PFA for 30 minutes, and denatured as above. After blocking, cells were
incubated with rat anti-BrdU antibody (1:200; Serotec) overnight at 4°C,
and then with secondary antibodies as described above. Immunofluorescence was
analyzed using a DMIRB inverted epifluorescence microscope
(Leica).
Explant cultures were fixed with 4% PFA for 3 hours and vibratome-resectioned at 40-50 µm thickness. For immunofluorescence, sections were rinsed, blocked and incubated with the following primary antibodies (48 hours, 4°C): rabbit anti-calretinin (1:2000); mouse anti-nestin (1:10; rat-401 clone, University of Iowa); or mouse IgM anti-PSA-NCAM (1:500; 5A5 clone, University of Iowa). Rabbit anti-GFP antibody (1:2000; Molecular Probes) was used for double-labeling with nestin or PSA-NCAM. After washes, sections were incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200; for single labeling), or for double-labeling with Texas Red-conjugated anti-mouse IgG or IgM and FITC-conjugated anti-rabbit IgG (all 1:200; Jackson Laboratories) for 1 hour, washed, mounted on slides and coverslipped with anti-fade medium (ProLong, Molecular Probes). For BrdU immunohistochemistry, sections were denatured, incubated with rat anti-BrdU antibody (1:200; 4°C, 48 hours), and then processed as above. For BrdU/calretinin or PSA-NCAM double-labeling, tissue was incubated with non-BrdU primary antibody, washed, post-fixed with 4% PFA for 20 minutes, denatured and processed for BrdU labeling as above.
For Tdt-mediated dUTP nick end labeling (TUNEL), sections were treated with 0.3% Triton X-100 and incubated with 0.3 mg ml-1 proteinase K (37°C, 20 minutes). Sections were then rinsed, incubated in 0.25% acetic anhydride and treated with 0.2% H2O2 in methanol. The manufacturer's protocol for ApopTag Fluorescein Kit S7110 (Intergen) was then used.
In vivo disulfiram injections and BrdU labeling
P7 mice were injected intraperitoneally (i.p.) with the RALDH inhibitor
disulfiram (5 mg kg-1 in DMSO/olive oil) daily for 4 days. BrdU
(100 mg kg-1) was injected i.p. once on P7 (6 hours after
disulfiram). Animals gained weight normally and appeared healthy. On P10, mice
were perfused with 4% PFA. Brains were post-fixed, cryoprotected with 20%
sucrose and sectioned coronally at 40 µm. All animal experimentation was
approved by the University of Michigan Committee on Use and Care of
Animals.
Image analysis and quantification
Microscopic images were acquired with a SPOT-RT digital camera (SPOT
Diagnostic Instruments). For differentiated NS cell counts, labeled and total
cells were counted in five random fields (20x objective). Explants were
randomly assigned to treatment condition by a blinded observer. BrdU-positive
cells within a defined SVZ area (0.0676 mm2;
Fig. 4) were counted in three
resectioned slices/hemisphere (two explants/condition; four replicates) with a
grid-lined reticle (20x objective) without knowledge of treatment
condition. TUNEL-positive cells in two resectioned explants/treatment (three
slices/explant; three replicates) were counted from the SVZ to a point in the
RMS bisected by a line drawn from the rostral edge of frontal cortex,
perpendicular to the RMS (see inset, Fig.
7H). Maximum migration distance of GFP-labeled cells was
calculated as the linear distance from the anterior border of the ventricle to
the most rostral GFP-positive cell (see inset,
Fig. 7G) using NIH Image v.1.63
software. GFP-positive cells in the RMS and olfactory bulb were counted
rostral to a point in the line bisecting the RMS when drawn perpendicularly
from the rostral edge of the frontal cortex
(Fig. 7H). Data were obtained
from two explants/condition in 3-4 separate experiments. For in vivo BrdU
labeling, percentage area of BrdU-immunoreactivity in a 0.05 mm2
region of dorsolateral striatal SVZ (10x objective) was measured using
NIH Image (Parent et al.,
2002). Statistical analyses were performed using StatView 4.1
(Abacus Concepts). Multiple comparisons were analyzed by one-way ANOVA
(analysis of variance) with post hoc test (Fisher's PLSD); two-tailed
Student's t-test was used for two-group comparisons. Results are
shown as means±s.e.m.; significance level was P<0.05.
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Results |
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To directly test whether RA influences neurogenesis in forebrain SVZ NS
cultures, we differentiated primary NS for 7 days in the presence of different
concentrations of RA or vehicle. RA exposure significantly increased the mean
percentage and numbers of ß-tubulin-immunoreactive neurons in a
concentration-dependent manner (Fig.
1B,C; Fig. 2A,B).
Similar results were obtained when a subset of calretinin-expressing neurons
was analyzed (data not shown). The enhancement of neurogenesis by RA treatment
occurred at the expense of astrocyte differentiation, as the percentage of
GFAP-immunoreactive astrocytes decreased after RA exposure
(Fig. 2A). The percentage of
oligodendrocytes was small (<2% on average) and did not vary with RA
treatment (Fig. 2A). We next
examined whether the RA precursor retinol would increase neurogenesis in the
cultures. P15 mouse SVZ-derived NS were exposed to RA, retinol or vehicle for
7 days. Retinol significantly increased neuronal differentiation in the
cultures, although to a lesser extent than RA
(Fig. 2B). This effect was
blocked by the RALDH inhibitor disulfiram
(Fig. 2B). These findings
suggest that retinoid signaling increases postnatal forebrain SVZ
neurogenesis. Moreover, SVZ NS appear capable of synthesizing RA from its
precursor retinol, consistent with the reported expression of the
RA-synthesizing enzyme RALDH3 in the postnatal murine striatal SVZ
(Wagner et al., 2002).
RA may stimulate neurogenesis through a number of mechanisms. To examine whether RA increases the proliferation or survival of neuronal progenitors, NS cultures were treated with RA or vehicle for 4 days. BrdU incorporation and activated caspase-3 expression were assessed to examine proliferating and dying cells, respectively. Cultures were exposed to BrdU 2 hours prior to washout and fixation on day 4, and were double-immunostained for BrdU and neuronal or glial antigens. Compared with control cultures, RA exposure significantly increased BrdU-positive neurons, both as a percentage of total or BrdU-labeled cells (Fig. 1D-G and Fig. 2C,D). Conversely, RA treatment significantly decreased the proportion of BrdU-labeled cells that expressed GFAP; a trend towards decreased BrdU/GFAP double-labeled cells as a percentage of total cells was observed. The mean percentage of total cells incorporating BrdU (Fig. 2C) also significantly increased after RA treatment (12.8±1.4% RA vs. 9.0±0.7% vehicle). The absolute number of BrdU-immunoreactive cells, however, was slightly but non-significantly increased (mean cells/5 hpf: RA-treated 161±5; control: 118±28; P=0.20). RA did not influence cell death, as measured by activated caspase-3-immunoreactivity at 4 DIV (Fig. 2E). Moreover, total cell number (per 5 hpf) was not influenced by RA treatment at either 4 (RA 1 µM: 1471±152 cells vs. Control: 1328±224 cells; P=0.63) or 7 DIV (vehicle: 711±49; RA 0.2 µM: 698±48; RA 1 µM: 701±26; RA 5 µM: 772±32; P=0.53). The lack of altered cell numbers after RA exposure despite increased BrdU labeling likely indicates that RA produced only a small percentage increase in overall cell proliferation; i.e. stimulation of neuroblast proliferation largely was offset by reduced astrocytic proliferation.
|
SVZ-olfactory bulb neurogenesis persists in slice cultures
To study the effects of RA on postnatal neurogenesis in an in vitro system
that more closely resembles in vivo conditions, we developed a SVZ-olfactory
bulb slice culture preparation. Parasagittal, 250 µm-thick slices derived
from neonatal (P2-10) mice were cultured for up to 4 days in serum-free
medium. SVZ-olfactory bulb neurogenesis has been observed in similar cultures
(De Marchis et al., 2001;
Alifragis et al., 2002
). We
first confirmed SVZ neural precursor proliferation and migration by BrdU and
DiI labeling. DiI-labeled SVZ cells migrated to the olfactory bulb over
several days (Fig. 3A). BrdU
administered in vivo prior to slice preparation or in the culture medium
mainly labeled proliferating cells in the SVZ-olfactory bulb pathway
(Fig. 3B,B' and data not
shown). Less concentrated BrdU labeling also appeared in the corpus callosum,
cortex and striatum (Fig. 3B
and data not shown), probably reflecting proliferating glial precursors
(Levison and Goldman, 1993
;
Zerlin et al., 2004
).
Immunohistochemistry for neuroblast markers, such as doublecortin, showed
expression patterns in the SVZ, RMS and olfactory bulb similar to that seen in
vivo (Fig. 3C,C').
Neuroblasts in this pathway also expressed calretinin, PSA-NCAM and
neuron-specific ß-tubulin (Figs
5,
8 and data not shown).
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RA increases neurogenesis in SVZ-olfactory bulb explants
We next examined whether RA influences SVZ cell proliferation or survival
in slices. P2 mouse explants containing the SVZ-olfactory bulb pathway were
cultured with 10 µM RA for 2 days, and BrdU (5 µM) was added for 2 hours
prior to fixation at 2 DIV. RA treatment increased SVZ BrdU-labeled cell
numbers by about 33% above control values (mean cells/0.0676 m2:
RA-treated: 473±23; control: 354.5±17; P=0.004;
Fig. 4A-G). Similar results
were found after 4 DIV (data not shown). A portion of the neural progenitors
in the neonatal and adult rodent SVZ-olfactory bulb pathway normally undergoes
cell death (Biebl et al.,
2000). To examine potential survival effects of RA, P2 slices were
cultured with 10 µM RA for 4 DIV and apoptotic cells were identified by
TUNEL staining. No difference in apoptotic cell numbers were found in the SVZ
or RMS between RA- and vehicle-treated cultures (mean cell numbers:
112±13 for RA; 101±6 for controls; P=0.42;
Fig. 4H,I). These results
suggest that RA increases SVZ cell proliferation but not survival.
To explore whether RA specifically stimulates SVZ neuroblast proliferation,
we cultured slices in the presence of 10 µM RA or vehicle for 4 days and
examined the expression of calretinin, which labels SVZ-olfactory bulb
neuroblasts (Kato et al.,
1999). RA treatment markedly increased calretinin immunoreactivity
in the SVZ and RMS of explants (Fig.
5A,B). To confirm that the calretinin-positive cells were
proliferative, BrdU was added to the medium for 24 hours during the second DIV
and slices were cultured for 2 additional days (4 DIV total). Most
calretinin-positive cells in the SVZ and RMS incorporated BrdU
(Fig. 5C,D). Similar to the NS
culture data, these results indicate that RA treatment augments neurogenesis
in the neonatal SVZ.
The RA precursor retinol stimulates SVZ neurogenesis in explants
RA is synthesized from retinol by the successive actions of alcohol
dehydrogenase and RALDH enzymes (Duester
et al., 2003). The RALDH3 isoform persists in the postnatal
SVZ-olfactory bulb pathway (Wagner et al.,
2002
); therefore, RA is likely synthesized locally in these
regions. To test whether retinol has effects similar to RA, we exposed P2
explants to retinol for 2 days and labeled proliferating cells with BrdU. We
found that 10 µM retinol increased cell proliferation and neuroblast
numbers similar to RA treatment (Fig.
6A,B,I). To test whether this effect was mediated via RA
synthesis, some retinol-treated cultures were simultaneously exposed to the
RALDH inhibitor disulfiram. Disulfiram treatment blocked the proliferative
effects of retinol (Fig.
6A-D,I). Retinol also mimicked the effect of RA on SVZ
neurogenesis, as calretinin expression in the SVZ-olfactory bulb pathway of
the vehicle, disulfiram, or disulfiram plus retinol groups was less than in
retinol-treated slices (Fig.
6E-H). Compared with control, disulfiram treatment alone did not
significantly decrease BrdU incorporation in SVZ cells. This lack of effect
may be due to a relative paucity of `endogenous' retinol remaining in the
cultures for conversion to RA, and also suggests that the disulfiram was not
toxic to SVZ cells. Alternatively, RA may be unnecessary for basal SVZ cell
proliferation, although this idea is not supported by our in vivo studies
(below). Taken together, these results suggest that RA is produced locally in
the SVZ from retinol and augments postnatal neuroblast proliferation.
|
To inhibit retinoid signaling in the SVZ, we electroporated dnRAR or
dnRXR
into P2 mouse SVZ and prepared explants (see
Fig. 3D). A GFP reporter was
co-electroporated to identify transfected cells, or electroporated alone as a
control. Expression of dominant-negative RA receptors, alone or in
combination, into the SVZ of P2 slices markedly affected SVZ cell migration.
After 4 DIV, most SVZ cells expressing dnRAR
or both dnRAR
and
dnRXR
failed to migrate toward the olfactory bulb
(Fig. 7A-D). Expression of
dnRAR
or both dominant-negative receptors significantly shortened
migration distances (Fig. 7G),
and fewer GFP-labeled cells expressing dominant-negative receptors appeared in
the distal RMS or olfactory bulb (Fig.
7H). Indeed, many cells expressing dominant-negative RA receptors
appeared to migrate into the septum (Fig.
7B,D), and displayed a more undifferentiated or disorganized
morphology than SVZ cells in control explants
(Fig. 7E,F;
Fig. 8).
The vast majority of GFP-labeled cells likely expressed dominant-negative receptors given that we found an extremely high degree of dual expression when two reporter plasmids were co-electroporated (Fig. 3F). To ensure that GFP-positive cells also expressed a dominant-negative receptor construct, however, we electroporated a CMV-dnRAR-IRES-EGFP construct or CMV-IRES-GFP alone into P2 slices and cultured them for 4 days. After electroporation of the CMV-IRES-GFP control, GFP-positive cells migrated along the SVZ-olfactory bulb pathway (Fig. 7I). In contrast, GFP-labeled cells failed to migrate into the RMS after electroporation of the dnRAR-IRES-EGFP construct (Fig. 7J). Most GFP-positive cells appeared caudal and ventral to the SVZ/RMS in a pattern similar to that seen in the co-electroporation experiments (Fig. 7B).
In addition to the altered morphology and impaired migration of
dominant-negative retinoid receptor-expressing cells, double labeling for GFP
and the neural precursor marker nestin revealed that more GFP-positive cells
in dnRAR-, dnRXR
- and dnRAR
/dnRXR
-transfected
cultures co-expressed nestin than in control cultures
(Fig. 8A-F and data not shown).
Also, fewer GFP-positive cells in the RMS expressing dominant-negative
constructs co-expressed calretinin or PSA-NCAM
(Fig. 7A-D; Fig. 8G-L). These results
suggest that, in addition to stimulating SVZ neurogenesis, retinoid signaling
is required for the normal differentiation and olfactory bulb migration of SVZ
neuroblasts.
Inhibition of RA synthesis decreases SVZ cell proliferation in vivo
The results of our in vitro experiments indicate that RA has multiple
effects on postnatal SVZ progenitors. To examine the influence of RA on
postnatal neurogenesis in vivo, mice received disulfiram (5 mg
kg-1, i.p.) or vehicle daily from P7-10 to inhibit endogenous RA
synthesis. Proliferating SVZ cells were labeled with BrdU on P7, 6 hours after
the first disulfiram injection. Four days of disulfiram treatment
significantly decreased BrdU labeling in the dorsolateral SVZ
(Fig. 9). Double-label
immunofluorescence for BrdU and PSA-NCAM showed that, as expected, most
BrdU-labeled cells in the SVZ also expressed PSA-NCAM (insets in
Fig. 9A,B). These results
therefore suggest that RA regulates the proliferation of forebrain SVZ
neuroblasts in vivo.
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Discussion |
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The role of RA in postnatal neurogenesis
Recent work suggests that the striatum and olfactory bulb share similar
developmental mechanisms. The LGE contains two distinct progenitor populations
that give rise to striatal projection neurons and olfactory bulb interneurons
(Stenman et al., 2003). LGE
cells transplanted into the adult SVZ migrate to the olfactory bulb
(Wichterle et al., 1999
), and
mutations of DLX1/2 or GSH1/2 homeobox genes cause abnormal development of
both the striatum and olfactory bulb
(Anderson et al., 1997
;
Toresson and Campbell, 2001
).
Because embryonic striatal development is regulated in part by RA
(Wohl and Weiss, 1998
;
Toresson et al., 1999
), the
common LGE origin of striatal and postnatally-derived olfactory bulb
interneurons suggests that RA may influence persistent SVZ-olfactory bulb
neurogenesis. Our data indicate that RA does indeed modulate postnatal
forebrain development, and that it regulates multiple steps in SVZ-olfactory
bulb neurogenesis. Taken together with in vivo evidence that RA-responsive
cells persist in the adult rodent SVZ-olfactory bulb pathway
(Thompson Haskell et al.,
2002
) and findings that RA stimulates neurogenesis in adult
hippocampal neural stem cell cultures
(Takahashi et al., 1999
),
these results suggest that RA promotes neurogenesis throughout life in the
rodent forebrain.
We examined the influence of RA on SVZ neurogenesis in NS cultures as well as an explant culture system that better reflects the in vivo environment. Our data show that RA treatment of SVZ-olfactory bulb slices stimulates SVZ neuroblast proliferation and expands the SVZ-olfactory bulb pathway without influencing cell survival. Moreover, we found that retinol stimulated neurogenesis in vitro while the RA synthesis inhibitor disulfiram decreased SVZ cell proliferation in vivo, suggesting that RALDH3 in the postnatal SVZ synthesizes RA to regulate SVZ neural precursors. In addition to demonstrating a mitogenic effect of RA, the results of the dominant-negative retinoid receptor experiments indicate that inhibition of RA signaling alters the morphology and migratory behavior of SVZ cells. Because the morphology and antigen expression patterns suggest that dominant-negative retinoid receptor expression inhibits SVZ progenitor differentiation, the altered migration may be a secondary consequence of impaired differentiation. Indeed, a differentiation effect of RA on postnatal SVZ progenitors is supported by our finding of premature differentiation of NS expanded in the presence of RA (Fig. 2). RA also may directly influence both differentiation and migration of SVZ neuroblasts. These data indicate that RA regulates multiple steps in postnatal SVZ-olfactory bulb neurogenesis, and underscore the utility of combining NS, slice culture and in vivo approaches.
Potential mechanisms underlying RA-induced SVZ neurogenesis
The precise SVZ cell type(s) and stage(s) of neurogenesis influenced by RA
remain unclear. Postnatal SVZ-olfactory bulb neurogenesis involves multiple
steps and progenitor cell states: the generation of transit-amplifying
progenitors from neural stem cells
(Doetsch et al., 1997,
Doetsch et al., 1999
;
Johansson et al., 1999
); the
differentiation of transit-amplifying cells into neuroblasts
(Doetsch et al., 2002b
); the
migration of neuroblasts to the olfactory bulb
(Luskin, 1993
;
Lois and Alvarez-Buylla, 1994
;
Doetsch and Alvarez-Buylla,
1996
; Lois et al.,
1996
; Hack et al.,
2002
); and the differentiation, survival and integration of
adult-generated olfactory interneurons
(Bayer, 1983
;
Betarbet et al., 1996
;
Brunjes and Armstrong, 1996
;
Biebl et al., 2000
;
Petreanu and Alvarez-Buylla,
2002
; Belluzzi et al.,
2003
). Prior work using RARE-ß-gal transgenic reporter mice
showed that RA-responsive cells persist in the adult mouse SVZ-olfactory bulb
pathway, but the cell types expressing the reporter were not defined
(Thompson Haskell et al.,
2002
).
|
Our slice culture results suggest that altered RA signaling directly affects neuronal- or glial-restricted progenitors to modify SVZ cell proliferation and migration. RA synthesized in the SVZ-olfactory bulb pathway may act on neuroblasts to keep them from migrating out of the pathway, perhaps by regulating the expression of migration factor receptors. Alternatively, blockade of RA signaling could act on the transit-amplifying cells to inhibit their differentiation or change the identity of their progeny, perhaps from neurons to astrocytes, and thereby alter their migratory behavior. This latter mechanism is suggested by the altered morphology of SVZ cells expressing dominant-negative receptors that fail to migrate into the RMS in explants (Fig. 7). The finding that RA treatment inhibits astrocyte generation while increasing neurogenesis in differentiating NS cultures also is consistent with this idea. Further study is needed to determine which specific cell types synthesize RA, which are transcriptionally activated by retinoid signaling, and what downstream genes are influenced to regulate neurogenesis.
|
|
RA and injury-induced neurogenesis in the SVZ-olfactory bulb pathway
Prior studies of SVZ neurogenesis in adult rat stroke models suggest that
new striatal medium spiny neurons are generated from SVZ neural precursors
after focal ischemia (Arvidsson et al.,
2002; Parent et al.,
2002
). Our finding that RA regulates postnatal neurogenesis in the
SVZ-olfactory bulb pathway, together with previous data showing that RA
regulates the differentiation of embryonic striatal neurons
(Valdenaire et al., 1998
;
Wohl and Weiss, 1998
;
Toresson et al., 1999
), raise
the possibility that RA may be useful for augmenting injury-induced striatal
neurogenesis. Retinoid signaling is also a potential mediator of
ischemia-induced striatal neurogenesis. RA increases neurogenesis from adult
hippocampal-derived neural stem cells, in part by upregulating their
expression of neurotrophin receptors
(Takahashi et al., 1999
).
Several groups have shown that increasing forebrain levels of brain derived
neurotrophic factor (BDNF) in adult rats induces striatal neurogenesis
(Benraiss et al., 2001
;
Pencea et al., 2001
).
Therefore, increased retinoid signaling after stroke may induce the expression
of trkB, the BDNF receptor, on SVZ progenitors to stimulate striatal
neurogenesis. Such a role fits with the regenerative effects of RA in other
systems (reviewed in Maden and Hind,
2003
). A better understanding of how RA regulates postnatal
neurogenesis may therefore offer regenerative strategies to treat brain injury
or degeneration.
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ACKNOWLEDGMENTS |
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REFERENCES |
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