www.elsevier.com/locate/ymcne
Mol. Cell. Neurosci. 35 (2007) 261 – 271
Neuronal nitric oxide synthase (NOS-I) knockout increases the
survival rate of neural cells in the hippocampus independently
of BDNF
Sabrina Fritzen, a,1 Angelika Schmitt, a,1 Katharina Köth, a Claudia Sommer, b
Klaus-Peter Lesch, a and Andreas Reif a,⁎
a
Molecular and Clinical Psychobiology, Department of Psychiatry and Psychotherapy Josef-Schneider-Str. 11, Julius-Maximilians-University Würzburg,
Füchsleinstr. 15, D-97080 Würzburg, Germany
b
Department of Neurology, Josef-Schneider-Str. 11, Julius-Maximilians-University Würzburg, Füchsleinstr. 15, D-97080 Würzburg, Germany
Received 27 November 2006; revised 27 February 2007; accepted 28 February 2007
Available online 13 March 2007
Investigations regarding the regulation of adult neurogenesis, i.e., the
generation of new neurons from progenitor cells, have revealed a high
degree of complexity. Although the pleiotropic messenger molecule
nitric oxide (NO) has been suggested to modulate adult neurogenesis,
the evidence is inconclusive due to the presence of different NO synthase
isoforms in the brain. We therefore investigated whether stem cell
proliferation or survival is altered in mice lacking neuronal nitric oxide
synthase (NOS-I) or both endothelial and neuronal NOS (NOS-I/-III
double knockout). While proliferation of neural stem cells was only
numerically, but not significantly increased in NOS-I knockdown
animals, the survival of newly formed neurons was substantially higher
in NOS-I-deficient mice. In contrast, NOS-I/-III double knockout had
significantly decreased survival rates. QRT-PCR in NOS-I-deficient
mice revealed neither NOS-III upregulation compensating for the loss of
NOS-I, nor alterations in VEGF levels as found in NOS-III-deficient
animals. As changes in BDNF expression or protein levels were observed
in the cortex, cerebellum and striatum, but not the hippocampus, the
increase in stem cell survival appears not to be due to a BDNF mediated
mechanism. Finally, NOS-I containing neurons in the dentate gyrus are
rare and not localized close to progenitor cells, rendering direct NO
Abbreviations: AN, adult neurogenesis; ARP, acidic ribosomal phosphoprotein; BDNF, brain derived neurotrophic factor; BrdU, 5-bromo-2′deoxyuridine; BSA, bovine serum albumin; cc, corpus callosum; cor, cortex;
ec, external capsule; DG, dentate gyrus; GAPDH, glycerin aldehyde
phosphate dehydrogenase; GCL, granular cell layer; GFAP, glial fibrillary
acidic protein; gr, granular zone; hi, hilus; ht, heterozygous; ko, knockdown;
G
L-NAME, N -nitro-L-arginine methyl ester; NeuN, neuron-specific nuclear
protein; NHS, normal horse serum; 7-NI, 7-nitroindazole; NO, nitric oxide;
NOS, nitric oxide synthase; PBS, phosphate buffered saline; PFA,
paraformaldehyde; QRT-PCR, quantitative real-time polymerase-chainreaction; SGZ, subgranular zone; SSC, saline sodium citrate; st, striatum;
SVZ, subventricular zone; TBS, tris buffered saline; VEGF, vascular
endothelial growth factor; wt, wild-type.
⁎ Corresponding author. Fax: +49 931 201 76403.
E-mail address: a.reif@gmx.net (A. Reif).
1
Both authors contributed equally to this work.
Available online on ScienceDirect (www.sciencedirect.com).
1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.mcn.2007.02.021
effects on these cells unlikely. In conclusion, we suggest that NO
predominantly inhibits the survival of new-born cells, by an indirect
mechanism not involving BDNF or VEGF. Together, these results emphasize the important role of the different NOS isoforms with respect to
adult neurogenesis.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Nitric oxide synthase, NOS; NO; Adult neurogenesis;
Neurotrophic factors; BDNF; VEGF; Dentate gyrus; Hippocampus
Introduction
The generation of new functional neurons from neural
progenitor cells in the hippocampus has attracted much attention
in the recent years (Kempermann, 2002). This process, termed
adult neurogenesis (AN), has been implicated in the pathogenesis
of various psychiatric disorders (discussed in detail by Henn and
Vollmayr, 2004; Duman, 2004). AN has a potential role in the
formation of memory traces (Prickaerts et al., 2004), especially
with regard to the encoding of temporal information (Aimone
et al., 2006). Furthermore, AN was suggested to be involved in
depression (Kempermann and Kronenberg, 2003). This hypothesis,
however, has been questioned, as animals displaying an antidepressant phenotype surprisingly exhibit lower levels of AN
(Vollmayr et al., 2003) and as reduced neural stem cell proliferation
could not be demonstrated in humans (Reif et al., 2006). Most
notably, however, we could demonstrate by Ki67 staining of
human post-mortem tissue that neural stem cell proliferation is
significantly and specifically reduced in schizophrenia (Reif et al.,
2006), probably contributing to a hippocampal pathology of this
disease. Quite recently, mice deficient in NPAS3 (neuronal PAS
domain protein 3) were shown to display several schizophrenialike behavioral abnormalities, such as diminished PPI and deficits
in learning tests (Erbel-Sieler et al., 2004), accompanied by a
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significant reduction of hippocampal AN (Pieper et al., 2005). This
correlated also with the thickness of the granule cell layer (GCL)
and was reversible upon electroconvulsive treatment. As disruption
of NPAS3 co-segregated with schizophrenia in a family (Kamnasaran et al., 2003), there are thus two clear and independent links
between adult AN and schizophrenia (reviewed by Reif et al., in
press). Further mechanisms by which AN is regulated therefore are
of utmost interest, as this process represents a novel drug target, yet
the underlying molecular systems remain elusive.
The process of AN is divided into several distinct steps which
are probably regulated by different mechanisms: stem cell
proliferation, survival of the newly formed neural cells and their
migration from the subgranular zone (SGZ) to the GCL and finally
their differentiation to functional neurons. As a consequence of this
complex mechanism, as little as 5–10% of all new-born cells
finally differentiate into functional neurons and account for net
neurogenesis. The dissection into these different steps has therefore
to be taken into account when AN is investigated in in vivo systems
(Prickaerts et al., 2004).
The gaseous molecule nitric oxide (NO) does not only serve as
an intercellular messenger (Snyder and Ferris, 2000), but also as an
atypical neurotransmitter/neuromodulator. Moreover, NO has the
ability to regulate cell division and also cell death, the latter by
promoting cytoxicity in higher concentrations (Contestabile, 2000).
The regionally specific, biphasic pattern of postnatal NOS-I induction implicates NO in synaptogenesis and plasticity during
Fig. 1. Proliferation, survival and migration of new-born cells in NOS-I knockout and wild-type mice. Representative immunohistochemical labeling (BrdU)
of a frontal section of the dentate gyrus obtained from a wild-type (A) or knockdown (B) mouse sacrificed 48 h after BrdU injection (proliferation paradigm).
(C) Staining of a wild-type or knockdown (D) mouse DG section for analysis of stem cell proliferation with antibodies against Ki67. (A–D) Stained cells
often appear in clusters in the SGZ between the hilus and the GCL of the dentate gyrus (indicated by arrows). Evidently, proliferation level and pattern are not
different in knockdown animals as compared to their respective controls. (E, F) Representative photomicrographs of frontal sections through the dentate gyrus
of wild-type (+/+, E) and NOS-I knockout (−/−, F) mice, showing an increased number of BrdU-labeled nuclei (indicated by arrows) in the dentate gyrus of
NOS-I-deficient (−/−) mice. In these experiments, designed to analyze the survival rate of newly born neural cells, mice were sacrificed 28 days after they
received BrdU injections. At that time point, some of the BrdU-positive cells have already migrated in the granular cell layer (GCL, indicated by black
arrows), while others persist in the subgranular zone (SGZ, white arrows) of the DG as shown at higher magnification in the inset (G). Scale bars represent
50 μm in panels A–D, and 12.5 μm in panel E; hi, hilus; GCL, granule cell layer; SGZ, subgranular zone.
S. Fritzen et al. / Mol. Cell. Neurosci. 35 (2007) 261–271
263
the cells into the GCL and differentiation into different neural cell
types. Additionally, QRT-PCR and ELISA was used to determine
NOS-III as well as VEGF and BDNF expression level differences
in NOS-I ko mice.
Proliferation of neural stem cells in NOS-I knockdown mice
The proliferation rate of progenitor cells in NOS-I (−/−)
knockdown mice was examined by means of Ki67 immunohistochemistry (Kee et al., 2002) as well as the routine method, i.e.,
BrdU administration. Almost all detected newborn cells were
aligned along the SGZ of the DG and sometimes appeared in
clusters (Figs. 1A–D). BrdU or Ki67-labeled newborn cells in the
SGZ of the DG were quantified (Fig. 2). NOS-I ko mice exhibited
nominally increased levels of BrdU-positive cells (1818 ± 321
BrdU-labeled cells/mm3 of the granular zone; n = 6) compared to
wt control animals (1609 ± 91 cells/mm3; n = 9) and heterozygous
NOS-I (+/−) mice (1500 ± 145 cells/mm3; n = 7), which failed to
reach significance. Also by investigating Ki67-labeled cells in the
DG, no significant differences between NOS-I ko (6032 ± 1876
Ki67-labeled cells/mm3 of the granular zone; n = 5) and wt mice
(6907 ± 1374 Ki67-labeled cells/mm3 of the granular zone; n = 5)
were evident.
Fig. 2. Proliferation of progenitor cells is not altered in NOS-I knockout (−/−)
mice. Stem cell proliferation, expressed as the number of BrdU-labeled
cells/mm3 (A) and the number of Ki67-labeled cells/mm3 (B) of the GCL of
the dentate gyrus, is not significantly changed in hetero- (+/−; hatched bar)
and homozygous NOS-I knockout (−/−; open bar) mice compared to control
littermates (+/+) (solid bar), indicating that NOS-I does not play a role in
proliferation of progenitor cells. Results are given as mean ± SEM.
neuronal development (Ogilvie et al., 1995; Contestabile, 2000).
Thus, NOS-I has also been shown to be involved in the regulation
of AN by means of genetic (Packer et al., 2003) and pharmacological (Moreno-Lopez et al., 2004; Packer et al., 2003; Park
et al., 2001, 2002, 2003) approaches. NOS-I appears to inhibit AN
in a BDNF-mediated manner, at least in neurospheres and the
subventricular zone (SVZ; Cheng et al., 2003). With regard to
isoforms other than NOS-I, we recently reported that NO from
NOS-III, which is located in endothelial cells in close vicinity to
neural stem cells, increases the proliferation of progenitor cells in
the dentate gyrus (DG; Reif et al., 2004). This increase in
proliferation is mediated by VEGF which was shown to stimulate
adult neural stem cells (Cao et al., 2004; Fabel et al., 2003; Jin
et al., 2002; Schanzer et al., 2004; Sun et al., 2003). Similar data
could be shown for the SVZ following ischemic lesioning (Chen
et al., 2005). As these results point towards different roles of NO in
AN, we sought to re-evaluate the role of the different NO synthase
isoforms in the regulation of AN with respect to the distinct steps
of AN and the effects of NOS-I knockout on the growth factors
BDNF and VEGF.
Results
Adult neurogenesis in the dentate gyrus of NOS-deficient mice
was investigated focusing on two distinct developmental steps:
stem cell proliferation in the SGZ and survival of these newly
generated cells. The latter step is in part paralleled by migration of
Fig. 3. Survival of newborn cells in NOS knockout and littermate wild-type
mice. (A) A significant increase in BrdU-labeled cells can be observed in
NOS-I ko mice compared to littermate wild-type and heterozygous controls,
arguing for an influence of NOS-I on the survival of these cells. (B) In NOS
double-knockout mice (dko), the amount of newborn cells surviving and
migrating into the GCL is reduced about 50% compared to control (wt)
animals. Results are given as mean ± SEM; statistical differences were
calculated using Student's t-test (A), or ANOVA followed by Student's
t-test (B) and are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
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S. Fritzen et al. / Mol. Cell. Neurosci. 35 (2007) 261–271
Survival of newborn cells
To examine the survival rate of newborn cells in NOS
knockdowns, mice were sacrificed 28 days after BrdU injections.
Newly generated, surviving cells in NOS-I ko, as well as in NOSdko mice, as detected by BrdU-immunohistochemistry, were
distributed throughout the entire GCL. Some of those cells had
already migrated into the GCL, whereas others were still localized
in the SGZ (Figs. 1E–G, representative staining pattern).
Quantification of BrdU-labeled cells in the SGZ of the DG and
analysis of their migratory pattern via counting cells in the GCL
revealed that the number of BrdU-labeled cells in NOS-I ko mice
(n = 7; 1594 ± 306 BrdU-labeled cells/mm3 of the GCL) was
significantly increased compared to heterozygous (n = 9; 660 ±
128 cells/mm3) and wt mice (n = 6; 571 ± 78 cells/mm3, Fig. 3A).
The same holds true for the dko study (Fig. 3B). Here, NOS-I and
NOS-III single-ko mice were additionally analyzed. As expected,
there was a significant increase in the number of surviving cells in
NOS-I ko, but no difference between NOS-III ko and wt mice (cp.
with Reif et al., 2004). Furthermore, disruption of both NOS genes
resulted in a significant loss of more than 50% (n = 6; 231 ± 36
cells/mm3) of surviving cells compared to each of the three
control groups (wt: n = 7467 ± 51 cells/mm3; NOS-I ko: n = 5678 ±
79 cells/mm3; NOS-III ko: n = 7490 ± 54 cells/mm3).
NOS-I immunohistochemistry
To examine whether NOS-I positive cells in the DG are located
in the vicinity to dividing neural cells and thus are spatially capable
to influence AN, NOS-I immunohistochemistry was performed.
Fig. 4. Representative immunohistochemical labeling of NOS-I in littermate control mice (+/+, A–C) vs. heterozygous (+/−, D–F) and homozygous (−/−, G–I)
NOS-I knockout mutants. Frontal sections (40 μm) at the level of the striatum (A, D, G), cortex (B, E, H) and hippocampus (C, F, I) were labeled with a NOS-I
antibody. Intense labeling of neuronal cell bodies and cell processes (indicated by arrows) was seen in the striatum and cortex of wild-type and heterozygous
mice, while there was only background staining in knockouts. In the dentate gyrus only a few neurons in the hilus and SGZ of (+/+) and (+/−) mice stained for
NOS-I. In NOS-I (−/−) mice, no NOS-I expression could be observed in the DG. Scale bar represents 85 μm; st, striatum; ec, external capsule; cor, cortex; GCL,
granular cell layer; hi, hilus.
S. Fritzen et al. / Mol. Cell. Neurosci. 35 (2007) 261–271
The staining revealed that NOS-I is highly expressed in cell bodies
and processes of cells in the striatum, all parts of the cortex, several
parts of the hypothalamus (e.g., paraventricular nucleus and lateral
hypothalamus) and the mammillary nucleus (Fig. 4). In comparison, there are only few neurons expressing NOS-I in the DG and
the CA1–3 regions of the hippocampus (see Fig. 4C, wt mice). In
NOS-I ht animals, the distribution of NOS-I staining was
unchanged and of similar intensity compared to wt, arguing
against gene-dose effects (Figs. 4D–F). In contrast, only very few
and weakly labeled neurons could be observed in NOS-I ko mice,
which might be due to the expression of alternatively spliced NOS-
265
I isoforms such as βNOS-I and γNOS-I observed in previous
studies (Brenman et al., 1996a,b) (Figs. 4G–I). Double labeling of
NOS-I expressing cells with the astrocytic marker GFAP and the
neuronal marker NeuN demonstrated that NOS-I is exclusively
expressed in neurons, but not in astrocytes (Figs. 5A–F). As
depicted in Figs. 5G–I, NOS-I positive neurons in the dentate
gyrus do not co-express the proliferation marker BrdU and are
located distantly (approximately 45 μM, compare the scale bar in
Figs. 5A) to new-born cells. Thus, the notion of NOS-I expression
in neural stem cells or newly formed neurons, as suggested by in
vitro studies (Xiong et al., 1999) is not supported by these findings.
Fig. 5. Double labeling experiments demonstrating NOS-I expression in neurons, but not in astrocytes or new-born cells. (A–C) Representative confocal laser
scanning images displaying a striatal section labeled for NOS-I (A, red, arrows) and NeuN (B, green, arrowheads). The merged image (C) shows that all NOS-I
labeled cells are NeuN expressing neurons. (D–F) Representative confocal laser scanning images of a cortex section labeled for NOS-I (D, red, NOS-I positive
cells indicated by arrows) and GFAP (E, green, immunoreactive cells accentuated by arrowheads). The merged image (F) shows that astrocytes (arrowheads) do
not express NOS-I (arrows). (G–I) Immunohistochemical double-labeling for NOS-I and BrdU. The representative sections show a single NOS-I expressing cell
in the SGZ of the DG (G, red, indicated by an arrow) and a BrdU-positive, new-born cell (H, green, arrowhead). The merged image (I) reveals that this new-born
cell does not express NOS-I and is located remotely to the NOS-I-labeled neuron. Scale bar in panels A–I, 50 μm; cor, cortex; cc, corpus callosum; st, striatum;
hi, hilus; GCL, granular cell layer.
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and NOS-I ko mice. Regarding BDNF mRNA levels (determined
by QRT-PCR), no differences in the cortex and hippocampus of the
ko strain could be detected. Interestingly, however, a significant
reduction of BDNF mRNA expression in the cerebellum and in the
striatum was found (Fig. 8A). To investigate whether the same
holds true for BDNF protein levels, we performed BDNF ELISA
assays on NOS-I ko mice. Again, there was no significant
difference of BDNF protein levels in the hippocampus of ko mice,
while there appears to be less mRNA translation in the cortex. In
the striatum and the cerebellum, the reduced amounts of BDNF
mRNA do not impact on the protein levels, as no differences
between genotypes were observed (Fig. 8B).
Discussion
Fig. 6. NOS-III mRNA concentrations in hippocampus and cerebellum of
NOS-I-deficient and wild-type control mice, as measured by quantitative
real-time PCR. NOS-III mRNA expression is not altered in the cerebellum
and hippocampus of NOS-I (−/−) mice compared to wild-type controls.
Absolute cDNAvalues were normalized to the house-keeping genes GAPDH
and 18S ribosomal; relative cDNA values were calculated using the
geNORM software as described in Experimental methods. Open bars,
NOS knockout mice; closed bars, wild-type controls. Data are expressed as
means ± SEM.
Quantitative PCR of NOS-III transcripts
The main finding of the present study is that NOS-I has a major
regulatory role in the migration and survival of newly formed
neuronal cells, while its effect on stem cell proliferation is less
pronounced. This might help to resolve some of the inconsistencies
of previous studies on NO and AN. A number of studies employed
pharmacological techniques to examine the impact of NOS-I, or
NO, on AN. Systemic administration of a NO donor resulted in
elevated levels of neurogenesis (Zhang et al., 2001), which,
however, is discrepant to studies using NOS inhibitors. Decreased
AN upon NOS inhibition was demonstrated in cultured neuro-
As it was previously postulated that NO synthase isoforms
functionally compensate for disrupted isoforms (Kurihara et al.,
1998; Sanz et al., 2001), we examined by means of quantitative
real-time PCR (QRT-PCR) whether NOS-III mRNA expression
levels in NOS-I ko mice are altered. To do so, the hippocampus
was compared to the cerebellum, as NOS-I here shows the highest
activity (Barjavel and Bhargava, 1995). Contrasting the hypothesis,
there was neither a difference in cerebellar nor in hippocampal
NOS-III mRNA transcripts (Fig. 6), arguing against a compensation of NOS-III for the lacking NOS-I isoform on an expressional
level.
Expression of growth factors in NOS-I knockdown animals: VEGF
and BDNF
Vascular endothelial growth factor (VEGF) promotes adult
neurogenesis (Cao et al., 2004; Fabel et al., 2003; Jin et al., 2002;
Schanzer et al., 2004; Sun et al., 2003) and stimulates stem cells.
With respect to NOS, we recently showed that NOS-III impacts on
hippocampal neural progenitor cells probably in a VEGF-mediated
manner (Reif et al., 2004). We therefore examined whether the
same applies for NOS-I. Quantification of VEGF transcripts by
QRT-PCR demonstrated that the expression of all examined VEGF
isoforms (VEGF 120, 164, and 188) was unaltered in the
hippocampus (Fig. 7A) and cerebellum (not shown) of NOS-I ko
mice, as opposed to wt mice. Furthermore, no differences in VEGF
protein levels in the hippocampus and cerebellum were found by
ELISA (Fig. 7B) indicating that the effect of NOS-I on the survival
of newly formed cells is not mediated by VEGF.
As BDNF has an important role in AN (Lee et al., 2002;
Sairanen et al., 2005; Scharfman et al., 2005), and as NO has
complex interactions with the BDNF signaling cascade (Cheng
et al., 2003; Mantelas et al., 2003; Riccio et al., 2006; Xiong et al.,
1999), we sought to further elucidate these interactions. Therefore,
we analyzed cortex, striatum, hippocampus and cerebellum of wt
Fig. 7. VEGF expression in NOS-I knockout and control mice. (A) For the
most abundantly expressed VEGF isoforms (VEGF120, VEGF164 and
VEGF188), no significant changes in mRNA levels were detected in the
hippocampus of knockout animals (open bars) compared to littermate wildtype controls (solid bars). Absolute cDNA values were normalized to the
house-keeping genes GAPDH and 18S ribosomal. Relative cDNA values
were calculated using geNORM software as described in Experimental
methods. (B) VEGF120/164 protein levels measured by ELISA in the
hippocampus and cerebellum of NOS-I (+/+) and (−/−) mice likewise do not
show significant differences between genotypes, indicating that NOS-I
affects neither VEGF mRNA nor protein levels. Data are expressed as
means ± SEM.
S. Fritzen et al. / Mol. Cell. Neurosci. 35 (2007) 261–271
Fig. 8. BDNF expression in NOS-I knockout and wild-type control mice.
(A) BDNF mRNA expression levels are significantly decreased in the
striatum and cerebellum of NOS-I (−/−) mice, but remain unchanged in the
cortex and hippocampus. Absolute cDNA values were normalized to the
house-keeping genes GAPDH, 18 s ribosomal and ARP. Relative cDNA
values were calculated using the geNORM software as described in
Experimental methods. (B) BDNF protein levels, as measured by ELISA, in
the striatum, hippocampus and cerebellum of NOS-I (+/+) and (−/−) mice do
not show significant differences between genotypes. Exclusively in the
cortex of NOS-I ko mice, BDNF protein levels were significantly decreased.
Open bars represent knockout mice; closed bars wild-type controls. Data are
expressed as means ± SEM. *Indicates statistical difference with p < 0.05
(Student's t-test).
spheres of the SVZ (Cheng et al., 2003; Moreno-Lopez et al.,
2004). Park and associates demonstrated that only chronic NOS
inhibition results in marked elevation of stem cell proliferation
(Park et al., 2001, 2002, 2003), arguing for adaptive changes upon
NOS inhibition and against direct NO effects on stem cell
proliferation. These data were replicated by a recent study
employing a similar BrdU injection regime (Zhu et al., 2006). In
line with our findings, a recent study demonstrated an increase in
stem cell proliferation (cumulative BrdU labeling, 7.5 h) only in
the SVZ, but not in the dentate gyrus upon 7 days of continuous
NOS inhibition (Moreno-Lopez et al., 2004). A slightly different
protocol, however, was used by Packer and colleagues, who
allowed mice to survive for 6 days after the last BrdU injection.
Thus, a mixture between stem cell proliferation and cell survival
was determined and found to be increased by 32% (Packer et al.,
2003). This correlates well to our data showing only a numerical
increase of stem cell proliferation (13%), but a significant increase
in the number of surviving cells (279%) in NOS1 knockdown
mice. Together, pharmacological data are inconclusive arguing for
either increased or unchanged stem cell proliferation upon NOS
267
inhibition, while survival and migration of the newborn cells
appear to be inhibited by NOS-I (Zhu et al., 2006, and the present
study).
Knockout models have the advantage that isoform-specific
effects can be observed. Using this approach, it was demonstrated
that NOS-III-deficient mice feature a reduction in stem cell
proliferation (Chen et al., 2005; Reif et al., 2004), yet survival rates
remain unchanged. Two different NOS1-deficient models exist: in
a recently generated KOex6 knockout, exon 6 is disrupted, so that
absolutely no catalytically active NOS-I is present. An approximately 30% increase of stem cell proliferation in the DG was
demonstrated in these animals (Packer et al., 2003). In contrast to
the KOex6 animals, the animals used in the present and all other
studies feature a targeted deletion of exon 1 resulting in a loss of
the PDZ binding domain. Therefore, subsequent residual NOS-I
expression of up to 7% is present, making them actually NOS-I
knockdown animals (Brenman et al., 1996a) with predominant
expression of trace amounts of β- and γ-splice variants localized at
the membrane of the endoplasmatic reticulum (Rothe et al., 1999).
The use of different knockout models probably explains the
difference between Packer’s and our study, i.e., that we did not find
significantly increased stem cell proliferation while KOex6 mice had
markedly elevated numbers of BrdU positive cells. Likewise,
another study published while this paper was in preparation reported
increased stem cell survival in NOS1 knockdown animals (Zhu
et al., 2006). As a similar experimental protocol was used, the reason
for this is as yet unclear. The stress level of the animals might,
however, be a crucial factor as it was shown that corticosterone
treatment reversed L-NAME induced stimulation of neurogenesis
(Pinnock et al., 2007), which might confound proliferation levels in
genetically modified animals as well. This is especially noteworthy
as the animals examined in the present study feature a more than
two-fold higher expression of the glucocorticoid receptor in the
hippocampus (Wultsch et al., in press). While there is thus still
uncertainty about the effect of NOS-I on stem cell proliferation, i.e.,
early stages of AN, its inhibitory influence on late stages of adult
neurogenesis, namely migration and survival, is corroborated by the
present body of evidence. This fits current concepts on the general
effect of NO on stem cells (Thum et al., 2007) and is underscored by
a recent study which failed to detect a reduction of cultured stem cell
proliferation upon treatment with NO, but demonstrated impaired
neuronal differentiation (Covacu et al., 2006). Accordingly, NOS-I
slows down cell proliferation in vitro (Ciani et al., 2004) and signals
surviving cells to switch to terminal neuronal differentiation (Cheng
et al., 2003; Ciani et al., 2004). As this signal is lacking in NOS-I ko
animals, the survival of new-born cells is grossly increased (Fig. 3).
With regard to neurotrophic growth factors, the effects of NOSI and NOS-III knockout seem to be quite different. While
disruption of NOS-III results in significantly decreased levels of
VEGF in the hippocampus (Reif et al., 2004), the expression of
either VEGF isoform was unchanged in NOS-I ko animals (Fig. 7).
This is not surprising, considering that vasculature, which is
lacking NOS-I, is the major source of VEGF. As our and others
findings consistently converge to the notion of a connection
between vasculature, NOS-III/VEGF and neurogenesis, this
mechanism might be crucial in the repair of ischemic brain lesions
(Chen et al., 2005).
Recently, the interaction of BDNF – an important mediator of
neural stem cell survival (Lee et al., 2002; Sairanen et al., 2005) –
and NO came into the focus of several studies. Xiong et al. (1999)
showed that BDNF and NO production is co-regulated in rodent
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neocortical neurons, in that BDNF stimulates NOS-I activity as
well as expression while on the other hand NOS inhibition results
in an increase in BDNF mRNA concentrations. Cheng et al. (2003)
postulated a positive feedback loop between NO and BDNF
regulating neural progenitor cell proliferation and differentiation in
the mammalian brain. It thus was suggested that the stimulatory
effect of BDNF on the proliferation of neural progenitor cells is
NO-dependent (Cheng et al., 2003). This might be accomplished
by binding of CREB to CRE-responsive transcriptional enhancers,
as recent data demonstrated that BDNF signaling influences
CREB-mediated gene expression by NO signaling (Riccio et al.,
2006). Upon BDNF binding to TrkB, PLC-γ1 can be activated
leading to IP3 production and subsequent, NOS-activating Ca2+
release. NOS-I impacts on CREB in a dual way: first, its expression goes along with increased phospho-CREB (Mantelas et al.,
2003); second, nuclear proteins are S-nitrosylated enabling binding
of CREB initiating gene expression. The latter mechanism appears
to be crucial for BDNF-CREB mediated gene expression (Riccio
et al., 2006). Among the genes which are activated by CREB is
NOS-I itself (Riccio et al., 2006), while NO seems to inhibit BDNF
expression (Xiong et al., 1999). Thus, BDNF-NOS-I-CREB
comprises a negative feedback loop. This loop seems to be imbalanced in NOS-I ko in the striatum, cerebellum and cortex (Fig. 8);
in the hippocampus, however, we could neither detect changes in
BDNF mRNA nor protein. The marked increase in neural stem cell
survival thus appears not to be due to an increase in BDNF expression as initially reasoned.
NOS dko mice in contrast to NOS1 knockdown mice displayed
significantly reduced survival rates (Fig. 3), while having a residual
total NOS activity (i.e., all NOS isoforms together) of only 0.6% of
the respective controls (Son et al., 1996). On a functional level,
NOS-III thus appears to counter-regulate the effect of NOS-I. Also
in other tissues, both NOS isoforms can exert antagonistic effects,
which is known e.g., for cardiac structure and functioning
(Barouch et al., 2002). The underlying mechanisms, however,
are as yet unclear and do not include upregulation of the NOS-III
gene per se, nor do they involve VEGF or BDNF. Upregulation of
NOS-III on the protein level cannot be excluded by the present
study; however, we could never detect NOS-III in cells other than
endothelium arguing against NOS-III compensation for the loss of
NOS-I. Spatial relationships argue for indirect effects of NOS-I on
stem cell survival rather than direct cell–cell signaling. In the SVZ
as well as the rostral migratory stream, many NOS-I positive
neurons are found in close vicinity to BrdU-incorporating cells
(Moreno-Lopez et al., 2000, 2004), allowing direct NO flux from
NOS-I expressing cells to those stem cells. This is, however, not
true for the DG. Here, NOS-I positive cells are very sparse and
localized within the diffusion radius of NO, yet more distantly to
neural stem cells (Figs. 5G–I) than NOS-III positive endothelial
cells (Reif et al., 2004). Direct influence of NOS-I on stem cell
proliferation in the DG seems therefore unlikely: the NO-initiated
downstream signal seems to form a diffusion gradient influencing
cell migration and survival.
Finally, the question remains whether the marked increase in
stem cell survival in NOS-I knockdown mice is paralleled by
behavioral changes especially with regard to hippocampal functions. Interestingly, NOS-I-deficient mice show worse performance
in the Morris Water Maze, a hippocampus-dependent learning task
(Weitzdoerfer et al., 2004) which is in good agreement with data
demonstrating that mice with high de novo rates of AN are
deficient in spatial learning in this test (Dobrossy et al., 2003).
Depression-like behaviors (i.e., Learned Helplessness, Forced
Swim Test), which are also suggested to be mediated by AN, are
unchanged in these mice (Wultsch et al., in press).
In conclusion, NOS-I and NOS-III seem to exert antagonistic
effects on the different phases of AN, in both cases probably
mediated by indirect mechanisms. While NOS-III stimulates AN in
a VEGF-mediated manner, NOS-I appears to indirectly impede to
hamper the survival of new-born cells, probably by switching these
young neural cells from survival to differentiation (Cheng et al.,
2003; Ciani et al., 2004). As BDNF expression levels are unaltered
in the hippocampus of NOS-I-deficient mice, its contribution to
this mechanism appears to be minor in the DG and needs to be
further clarified. Together, these results emphasize the important
role of the different NOS isoforms with respect to adult
neurogenesis.
Experimental methods
Animals and BrdU administration
Neural stem cell proliferation and adult neurogenesis were investigated
in NOS-I knockdown (ko) as well as NOS-I/NOS-III double knockout
(dko) mice, as opposed to wild-type animals (wt) or in some cases
heterozygous NOS-I+/− mice (ht), by means of 5-bromo-2-deoxyuridine
(BrdU) administration and Ki67 labeling. Furthermore, we performed QRTPCR and ELISA on NOS-I ko animals. Table 1 provides an overview on the
investigated mice with regard to number and age.
NOS-I ko breeder pairs, which have been derived from the mutant strain
from Dr. Huang in which exon 1 has been disrupted, have been obtained from
Jackson Laboratories (Bar Harbor, MA, USA) and were backcrossed for at
least 8 generations onto C57BL/6J genetic background. For the generation of
NOS-I/NOS-III dko mice, NOS-I ko mice were crossed with NOS-III ko
animals, also resulting in NOS-I or NOS-III single knockouts, respectively,
which were used as control animals in experiments on AN in dko. All animals
were housed under identical conditions. The genotype was confirmed in each
animal by PCR or Southern blot analysis (experimental details available on
request). All animal protocols were reviewed and approved by the review
board of the Government of Lower Franconia and the University of
Würzburg and were in accordance with international guidelines on animal
testing.
For quantification of adult neurogenesis by means of BrdU administration, either for the proliferation or the survival paradigm, mice were injected
i.p. four times every 2 h (i.e., the total labeling time was 6 h; 100 mg BrdU/kg
Table 1
Mice used for the experiments
Mouse strain
Genotype
Amount of
animals (n)
Mean age
NOS-I, for BrdU
immunohistochemistry
ko
ht
wt
ko
wt
ko
wt
ko
wt
ko
wt
dko
NOS-I ko
NOS-III ko
wt
15
16
13
5
5
7
7
5
5
6
6
8
8
8
8
5–7 months
NOS-I, for Ki67
immunohistochemistry
NOS-I,
for QRT-PCR
NOS-I, for VEGF ELISA
NOS-I, for BDNF ELISA
NOS-dko, for BrdU
immunohistochemistry
3 months
2.5 months
4 months
4–5 months
4–5 months
S. Fritzen et al. / Mol. Cell. Neurosci. 35 (2007) 261–271
269
body weight, dissolved in 0.9% NaCl Roche, Mannheim, Germany). To
investigate the effect of different NOS genotypes on stem cell proliferation,
mice were sacrificed 48 h after the last BrdU injection by CO2 intoxication.
For the determination of cell survival and cell phenotype, mice were allowed
to survive 28 days.
Jackson ImmunoResearch Laboratories, PA, USA) and Cy™ 3-conjugated
anti-rabbit IgG (1:400; Jackson ImmunoResearch Laboratories, PA, USA)
were used. The stained sections were visualized with a Bio-Rad MRC 1024
laser-scanning confocal imaging system (Bio-Rad, Cambridge, MA, USA)
mounted onto a DMRBE Leica microscope (Leica, Wetzlar, Germany).
Immunohistochemistry
Quantification
For BrdU immunohistochemistry, the brains of NOS-I ko and wt mice
were dissected and fixed in 4% freshly prepared paraformaldehyde (pH 7.4)
at 4 °C for 4 days. After fixation, brains were washed in phosphate buffered
saline (PBS; 4 × 15 min) and subsequently immersed in 10% and 20% sucrose
in PBS. In the case of NOS dko, mice were perfused transcardially with 4%
PFA dissolved in PBS. After dissection, the brain tissue was postfixed 24 h in
4% PFA and washed in PBS as described above. The tissue was then frozen in
isopentane (cooled down with dry ice) and stored at −80 °C until use. Serial
sections (40 μm) were cut through the entire hippocampus using a
microtome. Every sixth section was processed for BrdU immunohistochemistry by the peroxidase method as reported (Kuhn et al., 1996) using a
monoclonal anti-BrdU antibody (Roche, Mannheim, Germany). Altogether,
about 10–12 slices per brain were investigated.
Ki67 immunohistochemistry was performed on 30 μm thin brain slices
cut with a microtome from frozen unfixed mice brain. Every ninth slice was
mounted on a Superfrost slide (Superfrost Plus; Menzel, Braunschweig,
Germany) and air-dried for at least 15 min. Thereafter sections were fixed
in 4% PFA for 5 min and after a short rinse in Tris buffered saline (TBS)
they were pretreated in 0.01 M citrate buffer heated to 95 °C for 5 min.
Sections were then incubated with 0.6% H2O2 in TBS for 30 min and
incubated in blocking solution (3% Normal Horse Serum in TBS) for 1 h,
followed by an overnight incubation with a mouse anti-Ki67 monoclonal
antibody at 4 °C (1:200 in TBS, Novocastra Laboratories Ltd., United
Kingdom). For the detection of the primary antibody, a biotinylated antimouse IgG antibody was used (1:400, Vector Laboratories, Burlingame,
CA) in combination with the avidin–biotin–peroxidase complex (Vector
Laboratories, Inc., USA) and 3,3′-diaminobenzidine (Roche, Mannheim,
Germany).
For NOS-I immunohistochemistry, tissue sections (postfixation analogous to BrdU labeling) were mounted on glass slides and pretreated with
0.01 M citrate buffer and 0.6% H2O2 as described above. Thereafter, slices
were incubated in blocking solution (5% NHS, 0.25% Triton X-100, 2%
BSA in TBS) for 1 h followed by an overnight incubation with rabbit antiNOS-I polyclonal antibody (1:4000, Chemicon International, Inc.,
Temecula, CA) diluted in blocking solution. The detection of the primary
antibody was performed using a biotinylated goat anti-rabbit IgG antibody
(1:300, Vector Laboratories, Burlingame, CA) followed by a labeling with
the avidin–biotin–peroxidase complex (Vector Laboratories, Inc., USA) and
3,3′-diaminobenzidine (Roche, Mannheim, Germany).
For the analysis of the phenotype of NOS-I-positive cells, free-floating
sections from animals sacrificed 48 h after the BrdU injections (fixed as
described above) were double-labeled for NOS-I and BrdU as well as for
NOS-I and the neural markers NeuN (neuron-specific nuclear protein) or
GFAP (glial fibrillary acidic protein) and analyzed by confocal microscopy.
For simultaneous detection of NOS-I and BrdU, sections were pretreated for
DNA denaturation as described above. Pretreatment with formamide and
SSC was skipped as it resulted in higher background staining. Pretreatment
with H2O2 was also not necessary, as no peroxidase staining method was used
(see below). Antibodies were diluted 1:400 (mouse anti-BrdU, Roche,
Mannheim, Germany) and 1:2000 (rabbit anti-NOS-I, Chemicon International, Inc., Temecula, CA).
For simultaneous detection of NOS-I and GFAP or NeuN, free floating
sections were rinsed in TBS and incubated in blocking solution as described.
Afterwards, sections were incubated overnight at 4 °C with either rabbit antiNOS-I (1:5000) and mouse anti-GFAP (1:2000; Sigma, Santa Louis, USA),
or rabbit anti-NOS-I and mouse anti-NeuN (1:4000; Chemicon International,
Inc., Temecula, CA). All primary antibodies were diluted in blocking
solution. To detect the primary antibodies in double labeling experiments, the
fluorescent secondary antibodies Cy™ 2-conjugated anti-mouse IgG (1:400;
For quantification of newborn cells in the subgranular zone (SGZ) and
granular cell layer (GCL) of the dentate gyrus, sections were examined
using a brightfield Leica microscope (DMRBE, Wetzlar, Germany) using a
40× objective. For GCL volume determinations, the microscope was
additionally equipped with a Hitachi digital camera. Cell counts were
performed blindly with respect to genotype. Results were expressed as the
average number of newborn cells per mm3 of the (sub-) granular zone of
both dentate gyri and reported as the mean ± SEM. The volume of the GCL
was determined using the DIGITRACE software package (Borland
International Inc., IMATEC Bildanalysesysteme, Neujahrn, Germany).
Differences between means were determined by Student’s t-test (NOS-I ko
mice) or ANOVA followed by Student’s t-test (dko mice), with p < 0.05
considered significant.
Quantitative real-time (QRT) PCR
Total RNA was isolated from hippocampus, cerebellum, cortex and
striatum dissected from NOS-I-deficient (ko) as well as wild-type control
(wt) mice using the RNeasy RNA isolation kit (Qiagen, Hilden, Germany)
and the RNase-free DNase Set (Qiagen) following the protocol of the
manufacturer. 0.5 μg of total RNA was reverse transcribed using the
ThermoScript™ RT-PCR System (Invitrogen, Karlsruhe, Germany). QRTPCR was performed using an iCycler iQ™ Real-Time Detection System
(BIO-RAD Laboratories, Hercules, USA) in the presence of SYBR-green.
The optimization of the QRT-PCR reaction was performed according to
the manufacturer’s instructions but scaled down to 25 μl per reaction.
Standard PCR conditions were used (iQ™ SYBR® Green Supermix
protocol) and all reagents were provided in the iQ™ SYBR® Green
Supermix, including iTaq DNA polymerase (BIO-RAD Laboratories,
Hercules, USA). The primers used are listed in Table 2. Ribosomal 18 s,
glycerin aldehyde phosphate dehydrogenase (GAPDH) and acidic
ribosomal phosphoprotein (ARP) were used to normalize each template.
Normalization factors were calculated with the aid of geNORM normalization software (http://medgen.ugent.be/~jvdesomp/genorm). Two or
three series of experiments, respectively, were performed with similar
results; PCR reactions of each series were run in duplicate. Standard
curves for each amplification product were generated from 10-fold
dilutions of pooled cDNA amplicons.
Table 2
Sequences of nucleotide primer pairs used for quantitative real-time PCR
Primer name
Sequence
VEGF forward
VEGF 120 reverse
VEGF 164 reverse
VEGF 188 reverse
BDNF forward
BDNF reverse
NOS-III forward
NOS-III reverse
GAPDH forward
GAPDH reverse
18S forward
18S reverse
ARP forward
ARP reverse
5′-GCC AGC ACA TAG AGA GAA TGA GC-3′
5′-CGG CTT GTC ACA TTTT TCT GG-3′
5′-CAA GGC TCA CAG TGA TTT TCT GG-3′
5′-AAC AAG GCT CAC AGT GAA CGC T-3′
5′-TGC CGC AAA CAT GTC TAT GAG G-3′
5′-GCT GTG ACC CAC TCG CTA ATA C-3′
5′-CCT TCC GCT ACC AGC CAG A-3′
5′-CAG AGA TCT TCA CTG CAT TGG CTA-3′
5′-AAC GAC CCC TTC ATT GAC-3′
5′-TCC ACG ACA TAC TCA GCA C-3′
5′-GAA ACT GCG AAT GGC TCA TTA AA-3′
5′-CCA CAG TTA TCC AAG TAG GAG AGG A-3′
5′-CGA CCT GGA AGT CCA ACT AC-3′
5′-ATC TGC TGC ATC TGC TTG-3′
270
S. Fritzen et al. / Mol. Cell. Neurosci. 35 (2007) 261–271
VEGF protein quantification by ELISA
For the determination of VEGF protein expression levels, brain tissue
(hippocampus, cortex, cerebellum and striatum) was placed in 2 volumes
of homogenization buffer (Cat. No. C3228, Sigma, MO, USA) containing
freshly dissolved protease inhibitors (Cat. No. 11873580001, Roche,
Penzberg, Germany). Samples were homogenized at 4 °C using a Qiagen
Tissue Lyser and then centrifuged at 10,000×g for 30 min at 4 °C.
Supernatants were aliquoted and frozen at -80 °C until use. The total
soluble protein concentration was measured by the Bradford method (Cat.
No. B6916, Sigma Missouri, USA). VEGF protein levels were determined using the DuoSet ELISA Development System for mouse
VEGF isoforms 120 and 164 (R&D Systems, MN, USA) following the
manufacturer’s instructions. Samples and standards were run at least in
duplicates.
BDNF protein quantification by ELISA
To determine BDNF protein expression levels, brain tissue (hippocampus, cortex, cerebellum and striatum) was placed in 2 volumes of
homogenization buffer containing 137 mM NaCl, 20 mM Tris–HCl (pH
8), 1% NP40, 10% glycerol, 1 mM PMSF, 0.5 mM Sodium vanadate and
freshly dissolved protease inhibitors (Cat. No. 11873580001, Roche,
Penzberg, Germany). Sample homogenization and measurement of total
protein concentration were performed as described above. BDNF protein
levels were determined using the BDNF Emax ImmunoAssay System from
Promega (Madison, USA) following the manufacturer’s instructions.
Samples and standards were run at least in duplicates.
Acknowledgments
We thank T. Töpner for excellent technical assistance, as well as
Dr. B. Holtmann and H. Brünner for their kind help in animal
housing. Dr. Kuhlencordt is acknowledged for his kind help and
assistance in establishing the dko Southern Blot assay. This study
was supported by the Deutsche Forschungsgemeinschaft (Grant
RE1632/1-1 and 1-3 to A.R., KFO 125/1-1 D to A.R. and K.P.L.,
and SFB 581 to K.P.L.), BMBF (IZKF 01 KS 9603) and the
European Commission (NEWMOOD LSHM-CT-2003-503474).
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