Available via license: CC BY 4.0
Content may be subject to copyright.
OPEN
ARTICLE
P53 regulates disruption of neuronal development in the adult
hippocampus after irradiation
Y-Q Li
1,4
, ZW-C Cheng
2,4
, SK-W Liu
1
, I Aubert
3
and CS Wong
1
Inhibition of hippocampal neurogenesis is implicated in neurocognitive dysfunction after cranial irradiation for brain tumors. How
irradiation results in impaired neuronal development remains poorly understood. The Trp53 (p53) gene is known to regulate cellular
DNA damage response after irradiation. Whether it has a role in disruption of late neuronal development remains unknown. Here
we characterized the effects of p53 on neuronal development in adult mouse hippocampus after irradiation. Different
bromodeoxyuridine incorporation paradigms and a transplantation study were used for cell fate mapping. Compared with wild-
type mice, we observed profound inhibition of hippocampal neurogenesis after irradiation in mice deficient in p53 despite the
absence of acute apoptosis of neuroblasts. The putative neural stem cells were apoptosis resistant after irradiation regardless of p53
genotype. Cell fate mapping using different bromodeoxyuridine incorporation paradigms revealed enhanced activation of neural
stem cells and their consequential exhaustion in the absence of p53 after irradiation. Both p53-knockout and wild-type mice
demonstrated similar extent of microglial activation in the hippocampus after irradiation. Impairment of neuronal differentiation of
neural progenitors transplanted in irradiated hippocampus was not altered by p53 genotype of the recipient mice. We conclude
that by inhibiting neural progenitor activation, p53 serves to mitigate disruption of neuronal development after irradiation
independent of apoptosis and perturbation of the neural stem cell niche. These findings suggest for the first time that p53 may
have a key role in late effects in brain after irradiation.
Cell Death Discovery (2016) 2, e16072; doi:10.1038/cddiscovery.2016.72; published online 3 October 2016
Radiotherapy is an important cancer treatment modality for
primary and secondary brain tumors. Unfortunately, cranial
irradiation may result in devastating late clinical consequences
including neurocognitive impairment.
1
Although recent advances
in radiation planning and delivery have allowed for a reduction in
the volume of normal brain irradiated, whole or large volume
brain irradiation remains the standard treatment for multiple brain
metastases and many intracranial tumors.
Multipotent neural progenitor cells (NPCs) or stem cells are
present in adult mammalian brain. They continuously generate
new neurons, a process termed neurogenesis. An area in adult
mammalian brain where neurogenesis has been characterized is
the dentate gyrus of the hippocampus. Radial glial cells, or type-1
cells, in the subgranular zone (SGZ) of the dentate gyrus are
thought to be the neural stem cells. Once activated, they undergo
asymmetric divisions to self-renew and generate proliferative
type-2 NPCs or intermediate neural progenitors (INPs). INPs give
rise to type-3 NPCs or neuroblasts, which differentiate into
immature and then mature neurons that become integrated into
the neuronal circuitry.
2,3
Neurogenesis is associated with hippocampal function of
learning and memory.
4–7
Irradiation is known to disrupt
neurogenesis,
8
a process implicated in neurocognitive decline
following cranial irradiation.
9
Damage of the vascular niche for
neurogenesis is thought to contribute to inhibition of neuronal
development after irradiation.
1
The Trp53 (p53) gene has a major role in regulating cellular
response after irradiation.
10
Alterations in the p53 gene have been
linked to tumor resistance to radiotherapy. There is evidence that
p53 has a role in regulating radiation injury in the gastrointestinal
tract and the heart.
11,12
Enhanced anticancer effects have also
been shown by genetic and pharmacologic inhibition of p53 in
tumor endothelium.
13
Whether and how p53 regulates inhibition
of adult neurogenesis after irradiation is unclear. Here we showed
that deficiency in p53 resulted in enhanced activation of neural
stem cells and NPCs, with consequential depletion of the neural
stem cell pool and profound inhibition of neurogenesis after
irradiation. These findings provide novel mechanistic insight into
the molecular regulation of disruption of hippocampal neuronal
development after irradiation.
RESULTS
DNA damage response is altered in p53-deficient NPCs
We first asked whether DNA damage response following
irradiation in NPCs was altered in the absence of p53. The kinetics
of formation and loss of γH2AX nuclear foci is associated with the
efficiency of repair of DNA strand breaks and radiosensitivity.
14
We
thus used γH2AX nuclear foci as a readout for DNA damage
response in NPCs cultured from mice, wild type (+/+) or knock
out (−/−), of the p53 gene. Consistent with the negative effects
of p53 on cell proliferation,
15,16
neurospheres generated from
p53 −/−mouse brain grew faster and were larger compared with
1
Department of Radiation Oncology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, Canada;
2
Institute of Medical Science, University of Toronto,
Toronto, ON, Canada and
3
Department of Laboratory Medicine and Pathobiology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, Canada.
Correspondence: CS Wong (shun.wong@sunnybrook.ca)
4
These authors contributed equally to this work.
Received 27 July 2016; accepted 19 August 2016; Edited by R Killick
Citation: Cell Death Discovery (2016) 2, e16072; doi:10.1038/cddiscovery.2016.72
Official journal of the Cell Death Differentiation Association
www.nature.com/cddiscovery
those derived from p53+/+ mice. Dissociated neurosphere
p53 −/−cells cultured in non-differentiation medium also
demonstrated a higher density compared with p53+/+ cells. These
cells were positive (+) for nestin and sex-determining region Y-box 2
(SOX2), markers of early NPCs (Supplementary Figures 1a–f).
NPCs cultured from dissociated neurosphere showed only the
occasional γH2AX nuclear foci. At 1 h after 5 Gy, there was a
marked increase in nuclear foci (Supplementary Figures 1g–j). The
number of foci per nucleus returned to non-irradiated level by
24 h. Compared with p53+/+ NPCs, there was delay in clearance of
γH2AX nuclear foci in p53 −/−NPCs at 3 h after irradiation, and
the effect of p53 was independent of time after irradiation
(number of foci per nucleus: time after irradiation, Po0.0001; p53
genotype, Po0.0001; interaction, P= 0.0001; % nuclei with foci;
time after irradiation, Po0.0001; p53 genotype, Po0.01; interac-
tion, P= 0.0001; two-way analysis of variance (ANOVA);
Supplementary Figures 1k–l). These results were consistent with
altered DNA damage response in NPCs in vitro after irradiation in
the absence of p53.
Deficiency in p53 results in profound inhibition of neurogenesis
after irradiation
Irradiation is known to inhibit hippocampal neurogenesis.
8
At
9 weeks after irradiation, a very apparent change in dentate
gyrus was the marked loss of cells immunoreactive for
doublecortin (DCX) and calretinin, markers of neuroblasts and
immature neurons, respectively (DCX+ cells, 315 ±104, 17 Gy
versus 9896 ± 483, 0 Gy, Po0.00001, t-test (Figures 1a–d);
calretinin+ cells, 423 ± 12, 17 Gy versus 910 ± 188, 0 Gy, Po0.05
(Figures 1e–j)).
To determine directly the effects of irradiation on neurogenesis,
mice were given bromodeoxyuridine (BrdU), 50 mg/kg daily
x7 days, 4 weeks after irradiation. Animals were killed 9 weeks
after irradiation for an analysis of the number of newborn neurons
or BrdU+ cells immunoreactive for the neuronal marker, neuronal
nuclei (NeuN) (Figures 1k–m). Irradiation resulted in a dose-
dependent decrease in the number of BrdU+/NeuN+ cells.
Consistent with the negative effect of p53 in cell
proliferation,
15,16
an increase in BrdU+/NeuN+ cells was associated
with p53 deficiency. In contrast, the number of BrdU+/NeuN+ cells
after irradiation demonstrated the opposite effect, highest in p53
+/+ mice, intermediate in p53 heterozygous (+/ −) mice and
lowest in p53 −/−mice (radiation dose, Po0.0001; p53 genotype,
Po0.0005; interaction, Po0.0001, two-way ANOVA; Figure 1n).
Results of pairwise comparisons are shown in Supplementary
Table 1. The number of BrdU+/NeuN+ cells after 5 Gy in p53+/+,
p53+/ −and p53 −/−mice decreased to 50.6%, 10.4% and 1.9%,
respectively, compared with their respective genotype controls
(Supplementary Figure 2). This profound inhibition of neurogen-
esis associated with p53 deficiency was also observed after a
clinically relevant irradiation schedule of 20 Gy in 5 daily fractions
(irradiation, Po0.0001; p53 genotype, Po0.005; interaction,
Po0.005; Figure 1o).
To determine whether an extra copy of p53 gene conferred
protection, neurogenesis in super-p53 (p53
S
) mice that have an
extra copy of p53 gene
17
was compared with their wild-type
littermates after irradiation. The number of BrdU+/NeuN+ cells
was significantly reduced in both p53
S
mice and wild-type controls
after 5 Gy, but there was no evidence of a protective effect
because of the extra copy of p53 gene (irradiation, Po0.005; p53
S
genotype, P-value not significant; Supplementary Figure 3).
P53 regulates impairment of neurogenesis after irradiation
independent of apoptosis of neuroblasts
NPCs in the SGZ of dentate gyrus are known to undergo apoptosis
within hours of irradiation.
18,19
It has been postulated that
apoptosis of NPCs contributes to impaired neurogenesis after
irradiation.
18
In non-irradiated p53+/+ mice, apoptotic cells were
rarely observed in the SGZ, a robust apoptotic response in the SGZ
within hours after irradiation as shown previously.
19,20
The peak
response, 9849 ± 622, of apoptotic cells based on the morphologic
criteria was observed at 8 h after irradiation, compared with
91 ± 27 in control (Po0.001, t-test). The response returned to non-
irradiated control level by 24 h. Similar results were observed
using terminal deoxynucleotidyl transferase dUTP nick-end label-
ing (TUNEL) and caspase-3 immunohistochemistry (data not
shown).
Of the apoptotic cells that showed characteristic nuclear
condensation and fragmentation, about a third expressed DCX.
Among the TUNEL+ and caspase-3+ cells, about a third also
expressed DCX (Figures 2a–h). None of the DCX+ apoptotic cells
expressed nestin. Consistent with DCX-expressing cells or
neuroblasts representing the apoptosis-susceptible population
after irradiation, a marked clearance of DCX+ cells was observed at
24 h after irradiation (5553 ±2126, 17 Gy versus 21 773 ± 1598,
0 Gy, Po0.005, t-test; Figures 2i–l).
Type-1 cells express glial fibrillary acidic protein (GFAP) and
nestin, and have a characteristic long radial process that spans the
entire granule cell layer and ramifies in the molecular layer.
2,3
Although the occasional apoptotic cells expressed nestin, no GFAF
+/nestin+ apoptotic cells were observed. At 24 h after irradiation,
the number of GFAP+/nestin+ cells remained unchanged
(1851 ± 179, 17 Gy versus 1743 ± 150, 0 Gy, t-test, P-value not
significant). These results provide no evidence that type-1 cells
undergo radiation-induced apoptosis.
Radiation-induced apoptosis of subgranular cells is known to be
p53 dependent.
21,22
It was extremely difficult to observe apoptotic
cells in p53 −/−mice after irradiation. Following irradiation, the
number of TUNEL+/DCX+ cells at 8 h was dose and p53 genotype
dependent (irradiation dose, Po0.001; p53 genotype, Po0.001,
two-way ANOVA; Figure 2m). Abrogation of radiation-induced
apoptosis in p53 −/−mice supports the notion that p53 regulates
inhibition of neurogenesis after irradiation independent of acute
apoptosis of neuroblasts.
Irradiation results in p53-dependent late ablation of proliferating,
newborn and total neural stem cells
We next asked if the profound late inhibition of neurogenesis in
the absence of p53 after irradiation could be due to increased
ablation of neural stem cells. We first characterized change in
type-1 cell population (nestin+/GFAP+ or SOX2+/GFAP+ cells) in
p53+/+ mice at 9 weeks after irradiation. Animals were given BrdU
daily for 7 days at 4 weeks after irradiation for cell fate tracing.
About half of the nestin+/GFAP+ cells (361 ± 38, 17 Gy versus
693 ± 30, 0 Gy; Po0.01, t-test) and SOX2+/GFAP+ cells (123 ± 10,
17 Gy versus 289 ± 530 Gy, 0 Gy; Po0.01) disappeared at 9 weeks
after 17 Gy. Newborn type-1 cells (BrdU+/nestin+/GFAP+ cells;
Figures 3a–d) showed a dose-dependent ablation after irradiation
(0 Gy, 70.0 ± 10.1; 5 Gy, 23.4 ± 11.6; 17 Gy, none observed,
Po0.005; one-way ANOVA).
We next performed a population analysis of type-1 cells in
p53+/+ and p53 −/−mice at 9 weeks after 0 and 5 Gy using the
same BrdU-labeling paradigm. A 5-Gy dose was used as it resulted
in the loss of approximately half of the number of newborn
neurons at 9 weeks, and was considered optimal to discern the
effect of p53 or the lack of it. In non-irradiated mice, p53 genotype
had no effect on the total number of type-1 cells, BrdU+
(newborn) type-1 cells and Ki67+ (proliferating) type-1 cells
(Figures 3e–h). Increased ablation of total, newborn and
proliferating type-1 cells was observed in p53 −/−mice com-
pared with p53+/+ mice after irradiation (total type-1 cells:
irradiation, Po0.001; p53 genotype, Po0.05; BrdU+ type-1
cells: irradiation, Po0.05; p53 genotype, Po0.001; Ki67+ type-1
cells: irradiation, Po0.001; p53 genotype, Po0.005, two-way
P53 regulates neuronal development after irradiation
Y-Q Li et al
2
Cell Death Discovery (2016) e16072 Official journal of the Cell Death Differentiation Association
ANOVA, Figures 3i–k). See Supplementary Table 1 for results of
pairwise comparisons.
We did not observe any BrdU+/nestin+ cells that were non-
type-1 cells in any control or irradiated p53+/+ and p53 −/−mice.
BrdU+ cells immunoreactive for Mash1, another marker of INPs,
were also not observed. These results were consistent with culling
and/or differentiation of INPs over the 5 weeks after they
incorporated BrdU.
20
Taken together, these results are consistent
with increased neural stem cell exhaustion in p53 −/−mice after
irradiation.
P53 regulates neural stem cell and progenitor cell fate after
irradiation
To determine if dysregulated neural stem cell and NPC fate
underlies the increased inhibition of neurogenesis associated with
p53 deficiency after radiation, a single dose of BrdU (150 mg/kg)
was given at 4 weeks after 0 or 5 Gy, and the number of type-1, -2
and -3 cells in p53+/+ and p53 −/−mice was determined at 2 h,
2 days, 1 week and 5 weeks after BrdU administration. Using these
schemas, BrdU+ cells at 2 h represented proliferating cells, those
at 2 days a blend of proliferating and newly divided cells and
Merged
mµ05mµ05 DCX DAPI DAPINeuN
Calretinin
NeuN
BrdU
10 µm
p53+/+ p53+/−p53−/−
p53+/+ p53+/−p53−/−
0
20
40
60
400
800
1200 0 Gy
5 Gy
10 Gy
17 Gy
0 Gy
20 Gy / 5
0
5
10
15
20
200
400
600
800
No. of BrdU+/NeuN+ cellsNo. of BrdU+/NeuN+ cells
Figure 1. Inhibition of hippocampal neurogenesis after irradiation is p53 dependent. There is loss of DCX+ (aand b, 0 Gy; cand d, 17 Gy; DCX,
green; DAPI, blue) and calretinin+ cells (e–g, 0 Gy, h–j, 17 Gy; calretinin cells, arrow, green; NeuN, red; DAPI, blue) in SGZ at 9 weeks after
irradiation. Arrowhead (e) denotes the normal band of calretinin+ nerve fibers at the inner molecular layer. Newborn neurons in dentate gyrus
demonstrate BrdU (k, arrows, green) and NeuN immunostaining (l, red; m, merged). The p53 genotype has an independent effect on the
number of BrdU+/NeuN+ cells at 9 weeks after single doses of cranial irradiation (n) or 20 Gy in 5 daily fractions (o). Mice were given BrdU
daily for 7 consecutive days 4 weeks after irradiation. Data are expressed as mean ±S.E.M. and analyzed with two-way ANOVA with three to
five mice per dose per genotype.
P53 regulates neuronal development after irradiation
Y-Q Li et al
3
Official journal of the Cell Death Differentiation Association Cell Death Discovery (2016) e16072
those at 1 and 5 weeks were principally cells born during the 1-
and 5-week interval, respectively, after BrdU administration.
In non-irradiated mice, the number of BrdU+ type-1 (BrdU+/
nestin+/GFAP+) cells declined over the 5 weeks after BrdU but p53
genotype had no effect on the cell numbers (time after BrdU,
Po0.0001; p53 genotype,P-value not significant; two-way
ANOVA; Figure 4a). In contrast, the number of BrdU+ type-1 cells
after 5 Gy was p53 genotype dependent (time after BrdU,
Po0.0001; p53 genotype, Po0.05; interaction, Po0.005;
Figure 4b). Irradiation resulted in a spike of BrdU+ type-1 cells in
p53 −/−mice at 2 days after BrdU compared with p53+/+ mice
(Po0.001, Bonferroni post hoc analysis (Figure 4b), see
Supplementary Table 1 for results of pairwise comparisons).
Hence, neural stem cell fate was not altered by p53 genotype in
the absence of irradiation, but there was enhanced activation in
the absence of p53 after irradiation.
The number of BrdU+ type-2 cells (BrdU+/nestin+/GFAP −cells)
decreased rapidly by 2 days and 1 week after BrdU in both non-
irradiated p53+/+ and p53 −/−mice (time after BrdU, Po0.0001;
p53 genotype,P-value not significant; Figure 4c). Irradiation
resulted in an increase in BrdU+ type-2 cells at 2 days in
p53 −/−compared with p53+/+ mice (Po0.01; Figure 4d), and
p53 genotype had a significant effect in the number of BrdU+
type-2 cells observed after irradiation (time after BrdU, Po0.005;
p53 genotype,Po0.05; interaction, Po0.05; Figure 4d). No BrdU+
type-2 cells were identified at 5 weeks after BrdU in control or
irradiated mice irrespective of p53 genotype.
In non-irradiated mice, BrdU+/DCX+ cells declined over 5 weeks
after BrdU and p53 genotype had no effect (time after BrdU,
Po0.0005; p53,P-value not significant; Figure 4e). After irradia-
tion, BrdU+/DCX+ cells also showed an increase at 2 days in
p53 −/−mice compared with p53+/+ mice after 5 Gy (Po0.001;
Figure 4f). This was followed by decline over the next 5 weeks with
p53 genotype demonstrating a significant effect (time after BrdU,
Po0.0001; p53 genotype, Po0.0001; interaction, Po0.0001;
Figure 4f).
For further evidence of enhanced NPC renewal in p53 −/−mice
after irradiation, we determined the number of BrdU doublets and
10 µm
0
1000
2000
3000
4000
1750
No. of TUNEL+/DCX+ cells
Irradiation dose (Gy)
p53+/+
p53−/−
50 µm IPADXCD
0 Gy
17 Gy
10 µm DCXCaspase 3 Merged
degreMLENUT
DAPI
DAPIDCX
Figure 2. Neuroblasts in SGZ undergo p53-dependent apoptosis after irradiation. DCX+ apoptotic cells are identified using TUNEL (a–d,
arrows) and caspase-3 immunohistochemistry (e–h, arrows). There is a marked loss of DCX+ cells at 24 h after irradiation (iand j, 0 Gy; kand l,
17 Gy; DCX, green; DAPI, blue). The number of DCX+/TUNEL+ apoptotic cells observed at 8 h is radiation dose and p53 genotype dependent.
Data are expressed as mean ±S.E.M. and analyzed with a two-way ANOVA with three to five mice per experimental group.
P53 regulates neuronal development after irradiation
Y-Q Li et al
4
Cell Death Discovery (2016) e16072 Official journal of the Cell Death Differentiation Association
type-1 (nestin+/GFAP+) BrdU doublets (Figure 4g) at 2 days after
BrdU. In the absence of irradiation, there was no difference in the
number of BrdU doublets in p53+/+ mice compared with p53 −/−
mice. After 5 Gy, the number of BrdU doublets decreased in
p53+/+ mice but increased in p53 −/−mice (Figure 4h). Similar
observations were noted for type-1 BrdU doublets (Figure 4i).
Thus, p53 does not alter neural stem cell fate in non-irradiated
hippocampus, but absence of p53 results in enhanced activation
and renewal after irradiation.
P53 deficiency does not alter neuroinflammation or neurovascular
niche dysfunction after irradiation
The fate of neural stem cells and NPCs is regulated by
neurovascular interactions.
23
Damage to the neurogenic niche
such as neuroinflammation is thought to contribute to the deficit
in neurogenesis after irradiation.
8,24–28
We thus asked whether the
increased inhibition of neurogenesis after irradiation in p53-
deficient mice could also be related to increased microglial
activation after irradiation. Newborn microglia (BrdU+/CD68+ and
BrdU+/Iba1+ cells; Figures 5a–i) have been extensively used as
surrogates for activated microglia.
26,29,30
Nine weeks after 5 Gy
(BrdU given daily for 7 days at 4 weeks after irradiation), there was
an increase in BrdU+/CD68+ and BrdU+/Iba1+ cells in dentate
gyrus, independent of p53 genotype (BrdU+/CD68+ cells: radia-
tion, Po0.0001; p53 genotype, P-value not significant; BrdU+/
Iba1+ cells: radiation, Po0.0001; p53 genotype, P-value not
significant; two-way ANOVA; Figures 5e and j).
To examine whether there was increased damage of the
neurogenic niche after irradiation in the absence of the p53, and
hence its ability to support neurogenesis, we asked if there could
be increased inhibition of neuronal differentiation of NPCs
transplanted into irradiated p53 −/−mouse hippocampus com-
pared with irradiated p53+/+ mouse hippocampus. P53+/+ and
p53 −/−mice were given 0 or 5 Gy. After 3 weeks, NPCs cultured
from the hippocampus of enhanced green fluorescent protein
(eGFP) mice were stereotactically transplanted into the
hippocampus.
19
At 5 weeks after transplantation, eGFP cells
immunoreactive for DCX or Prox1 could be seen in the
hippocampus (Figures 5k and m). Only the rare NeuN+/eGFP+
cells were found. The proportion of eGFP cells that expressed DCX
or Prox1 was decreased in irradiated hippocampus compared with
control, but p53 genotype had no effect (DCX+ cells: irradiation,
Po0.01; p53 genotype, P-value not significant; Prox1+ cells:
irradiation, Po0.005; p53 genotype, P-value not significant; two-
way ANOVA; Figures 5l and n). These results did not support the
notion that the irradiated microenvironment in p53 −/−hippo-
campus had further inhibitory effects on neuronal differentiation
compared with wild-type mice. Taken together, the increase in
disruption of neurogenesis in p53 −/−mice after irradiation is
unlikely to be due to increased microglial activation or increased
injury in the irradiated p53 −/−neurogenic niche.
DISCUSSION
The adult mammalian brain contains neural stem cells that have
the ability to proliferate and generate multipotential NPCs that
differentiate into neurons.
3,31
Although neural stem cells are able
to proliferate, their capacity for self-renewal is finite. Fate mapping
studies revealed that a type-1 cell upon exiting its quiescent state
undergoes only a few rounds of asymmetric divisions to produce
mature neurons and self-renew.
32
Division coupled production of
new neurons is thought to result in age-related depletion of the
neural stem cell pool.
33,34
We observed depletion of total, proliferating and newborn type-
1 cells after irradiation. Their ablation after irradiation was further
enhanced in the absence of p53. There was an increase in the
number of BrdU+ type-1 cells and type-1 BrdU doublets at 2 days
0
20
40
60
80
100
120
No. of Ki67+ type-1 cells
0 Gy
5 Gy
0
20
40
60
80
100
120
No. of BrdU+ type-1 cells
0 Gy
5 Gy
0
200
400
600
800
1000
No. of type-1 cells
0 Gy
5 Gy
p53−/−p53+/+ p53−/−p53+/+p53−/−p53+/+
MergedGFAPNestinKi67
20 µm
Merged
20 µm BrdU Nestin GFAP
Figure 3. Irradiation results in p53-dependent ablation of type-1 cells in mouse dentate gyrus. A representative newborn type-1 cell
(a–d, arrow) demonstrates BrdU incorporation (a, green), and is positive for nestin (b, red) and GFAP (c, yellow; d, merged), and has a
characteristic process that traverses the granule cell layer. A proliferating type-1 cell (e–h, arrow) demonstrates immunostaining for Ki67
(e, green), nestin (f, red) and GFAP (g, white; h, merged). At 9 weeks after irradiation, there is p53-dependent reduction of total (i), BrdU+ (j)
and Ki67+ type-1 cells (k). Data are expressed as mean ±S.E.M. and analyzed with two-way ANOVA with three to four mice per
experimental group.
P53 regulates neuronal development after irradiation
Y-Q Li et al
5
Official journal of the Cell Death Differentiation Association Cell Death Discovery (2016) e16072
0 1 2 3 4 5
0.1
1
10
100
Time after BrdU injection (week)
No. of BrdU+ type-1 cells
0 1 2 3 4 5
10
100
1000
Time after BrdU injection (week)
No. of BrdU+ type-2 cells
0 1 2 3 4 5
10
100
1000
Time after BrdU injection (week)
No. of BrdU+/DCX+ cells
0 1 2 3 4 5
10
100
1000
Time after BrdU injection (week)
No. of BrdU+/DCX+ cells
5 Gy0 Gy
5 Gy0 Gy
0 Gy 5 Gy
0 1 2 3 4 5
10
100
1000
Time after BrdU injection (week)
No. of BrdU+ type-2 cells
0 1 2 3 4 5
0.1
1
10
100
Time after BrdU injection (week)
No. of BrdU+ type-1 cells
p53−/−
p53+/+
p53−/−
p53+/+
p53−/−
p53+/+
p53−/−
p53+/+
p53−/−
p53+/+
p53−/−
p53+/+
0
100
200
300
400
500
No. of BrdU-doublets
at 2 days
p53−/−
p53+/+
0Gy 5Gy
20 µm *
0
10
20
30
40
50
No. of type-1
BrdU-doublets at 2 days
p53−/−
p53+/+
0 Gy 5 Gy
***,†
***
***
**
Figure 4. Deficiency in p53 alters neural stem cell and progenitor cell fate after irradiation. In non-irradiated mice, p53 genotype does not alter
the decline of BrdU+ type-1 cells over time after BrdU (a). After 5 Gy, the decrease in the number of BrdU+ type-1 cells over time is p53
dependent (b). The decline of BrdU+ type-2 cells over time is independent of p53 genotype in non-irradiated mice (c) and is p53 genotype
dependent after 5 Gy (d). The number of BrdU+/DCX+cells over time after BrdU is independent of p53 genotype in non-irradiated mice (e) but
p53 genotype dependent after 5 Gy (f). A type-1 BrdU-doublet is observed in SGZ of a p53 −/−mouse after irradiation (g, arrow; BrdU, green;
nestin, red; GFAP, white). The number of BrdU doublets and type-1 BrdU doublets in SGZ at 2 days after BrdU is p53 genotype dependent
following 5 Gy (hand i). BrdU was given at 4 weeks after 0 or 5 Gy, and cell populations determined at 2 h, 2 days, 1 and 4 weeks after BrdU.
Data are represented as mean ±S.E.M. and analyzed with two-way ANOVA and post hoc Bonferroni test, *Po0.05, **Po0.01, ***Po0.001,
p53 −/−versus p53+⧸+;
†
Po0.01, 5 Gy versus 0Gyinp53 −/−mice. There was a minimum of three to four mice per genotype per time point.
P53 regulates neuronal development after irradiation
Y-Q Li et al
6
Cell Death Discovery (2016) e16072 Official journal of the Cell Death Differentiation Association
0
100
200
300
400
500
No. of BrdU+/CD68+ cells
0 Gy
5 Gy
0
500
1000
1500
No. of BrdU+/Iba1+ cells
0 Gy
5 Gy
0
5
10
15
20
DCX+/eGFP+ cells (%)
0 Gy
5 Gy
0
5
10
15
20
Prox1+/eGFP+ cells (%)
0 Gy
5 Gy
DAPI Merged
BrdU CD68
20 µm
Iba1
BrdU
DAPI Merged
20 µm
DAPI
eGFP
DCX
DAPI
eGFP
Prox1
p53−/−
p53+/+
p53−/−p53+/+
p53−/−p53+/+
p53−/−p53+/+
***
*
**
***
*
*
Figure 5. Deficiency in p53 does not alter microglial activation or inhibition of neuronal differentiation after irradiation. An activated microglia
demonstrates nuclear BrdU incorporation and CD68+ (a–d, arrow) or Iba1+ (f–i, arrow). The increase in the number of BrdU+/CD68+ (e) and
BrdU+/Iba1+ (j) cells in the dentate gyrus at 9 weeks after cranial irradiation is independent of p53 genotype. An eGFP+ neural progenitor cell
transplanted in mouse hippocampus demonstrates immunoreactivity for DCX (k, arrow) and another one for Prox1 (m, arrow). The percentage
of eGFP+ cells that expresses DCX or Prox1 is reduced in mice given cranial irradiation before transplantation, independent of p53 genotype of
the recipient mice (l, DCX+/eGFP+ cells; n, Prox1+/eGFP+ cells). Data are expressed as mean ±S.E.M. and analyzed with two-way ANOVA and
post hoc Bonferroni test, *Po0.05, **Po0.01, ***Po0.001, 5 Gy versus 0 Gy; a minimum of three to five mice per experimental group (eand j)
and four to seven mice per experimental group (land n).
P53 regulates neuronal development after irradiation
Y-Q Li et al
7
Official journal of the Cell Death Differentiation Association Cell Death Discovery (2016) e16072
after BrdU given 4 weeks after 5 Gy, whereas the opposite effect
was seen in p53+/+ mice. Hence, the absence of p53 resulted in
enhanced neural stem cell activation after irradiation, whereas
neural stem cell fate did not appear to be altered by p53 in non-
irradiated mice.
During neurogenesis in adult dentate gyrus, only a few
newborn cells become mature neurons. The majority of newborn
die of apoptosis within a few days of birth before they transition
into DCX+ neuroblasts.
20
In non-irradiated mice, regardless of p53
genotype, we also observed a sharp decline in the number of
BrdU+ type-2 and BrdU+/DCX+ cells between 2 and 7 days
after BrdU.
A homeostasis of neural stem cell activation and quiescence
allows for the continuous generation of new neurons throughout
life. Disruption of signaling pathways that lead to excessive
activation of neural stem cells resulted in their subsequent
depletion and failure of neurogenesis.
33,35,36
Certain brain
pathologies such as seizures and trauma associated with
activation of stem cell division also demonstrated their acceler-
ated loss.
37,38
P53 is known to negatively regulate NPC prolifera-
tion in vitro. Based on the neurosphere assay, it was postulated
p53 might negatively regulate self-renewal of neural stem cells.
15
In a previous study, p53 −/−mice were noted to have accelerated
‘neurogenesis’in dentate gyrus within 2 weeks after irradiation
based on the expression of cyclin-dependent kinase 1.
39
Our
in vivo data here showed that p53 deficiency did not alter neural
stem cell fate in non-irradiated hippocampus. Enhanced neural
stem cell activation associated with p53 deficiency was only
observed after irradiation. Given the well-known effect of ionizing
radiation in mitotic-linked death, we propose that in the absence
of p53, increased cell cycle entry leads to enhanced division
coupled death and consequential depletion of neural stem cell
pool and profound inhibition of neurogenesis.
For the cell fate studies, our results were unlikely to be
confounded by the potential dilution of the BrdU labeling over the
5-week interval since the two genotypes were compared at the
same time point. It might be argued if p53 −/−NPCs undergo
more divisions than p53+/+ NPCs, there could be increased
dilution of the BrdU label below the level of detection to yield
lower counts of BrdU-retained cells in irradiated p53 −/−mice.
This is however not supported by the greater number of BrdU
+/NeuN+ cells in non-irradiated p53 −/−dentate gyrus compared
with wild-type mice. Recent studies on hippocampal neurogenesis
using similar BrdU paradigms reported negligible impact of label
dilution up to 30 days after BrdU injections.
20,34
How p53 regulates the differential DNA damage response in
neural stem cells and NPCs remains unclear. We showed here that
p53 −/−NPCs in vitro demonstrated a slower clearance of γH2AX
foci compared with p53+/+ cells. For hematopoietic and
mammary stem cells in vitro, DNA damage resulted in the
activation of p21 and inhibition of p53, which lead to cell cycle
entry and symmetric self-renewing divisions.
40
The increase in
BrdU-labeled type-1 cells at 2 days after BrdU in irradiated p53 −/−
mice compared with wild-type mice is consistent with stem cell
activation and symmetric division. Considerable heterogeneity,
however, exists in the DNA damage response of tissue-specific
stem cells.
41
Endothelium deficient of p53 gene has been noted to have
increased radiosensitivity.
13
P53-regulated responses mediated by
endothelium may modulate late normal tissue responses after
radiation treatment. Mice with endothelial cell-specific deletion of
p53 demonstrated increased 2-month lethality from gastrointest-
inal syndrome after subtotal body irradiation.
11
Endothelial cell-
specific deletion of p53 was also shown to result in increased
myocardial injury after whole-heart irradiation.
12
Endothelial cells
represent a key component of the neurogenic niche.
23
There is an
intimate association of hippocampal neurogenesis with
angiogenesis.
42
Inhibition of neurogenesis is associated with
increased microglial activity, and reducing neuroinflammation has
been shown to partially restore deficit in neurogenesis after
irradiation.
8,9,25–28
Disruption of the neurogenic niche is thought
to contribute to failure of NPCs to differentiate into neuroblasts
after irradiation. Here we observed no evidence of increased
microglial activation in irradiated p53 −/−mice compared with
p53+/+ mice. Similarly, results of the transplantation experiment
failed to demonstrate increased failure of neuronal differentiation
of NPCs in irradiated p53 −/−mice. These results were consistent
with the BrdU cell fate study, which showed an increase rather
than decrease in newborn/proliferating DCX+ cells in p53 −/−
dentate gyrus 4 weeks after irradiation compared with irradiated
wild-type mice. Hence, the increased disruption of neurogenesis
after irradiation in the absence of p53 is unlikely to be due to
increased damage of the p53 −/−neurogenic niche after
irradiation.
Abrogating apoptosis has been shown to augment adult
neurogenesis.
6
Pharmacologic approaches to suppress apoptosis
have thus been proposed as potential therapeutic strategies to
mitigate radiation-induced inhibition of neurogenesis.
43,44
Radiation-induced apoptosis of NPCs in the dentate gyrus is
abrogated in the absence of p53.
19,21,22
Here we observed
profound inhibition of neurogenesis in irradiated p53 −/−
hippocampus that failed to mount an apoptotic response in
NPCs. These results provide compelling evidence that p53
regulates neuronal development independent of apoptosis of
neuroblasts after irradiation.
In summary, deficiency in p53 resulted in profound inhibition of
adult neurogenesis after irradiation independent of apoptosis.
There was no evidence of increased neuroinflammation and
damage of the neurogenic niche in p53 −/−hippocampus after
irradiation. Rather, p53 deficiency resulted in increased activation
of neural stem cells and NPCs after irradiation, leading to
subsequent exhaustion of the neural stem cell pool. We propose
that p53 serves to mitigate disruption of neuronal development
after irradiation and may thus have a role in regulating late effects
in brain after irradiation.
MATERIALS AND METHODS
Animals
Ten-week-old male C57 mice +/+, +/ −or −/−for p53 (Jackson
Laboratory, Bar Harbor, ME, USA) were irradiated as described
previously.
19
P53
S
mice were generous gifts from Dr. Manual Serrano,
and have one extra copy of the normal p53 gene.
17
NPCs for
transplantation were cultured from the brain of Tg/CAG-EGFP/B5Nagy
mice (Jackson Laboratory) that express eGFP.
19
They were wild type for
p53. Mouse colonies were maintained by littermate inbreeding, housed
under a 12–12 h light–dark cycle at 21 °C and fed a standard rodent diet
with food and water ad libitum. Genotyping was performed by PCR as
described previously.
19
Only male mice were used to avoid the potential
confounding influence of sex and estrous cycles on neuronal
development.
45
All animal protocols were approved by the institutional
animal care committee in accordance with the Canadian Council on
Animal Care guidelines.
Irradiation
Animals were anesthetized using an intraperitoneal injection of ketamine
(75 mg/kg) and xylazine (6 mg/kg), immobilized in a customized jig, and
the entire hippocampus was irradiated using an anterior–posterior and
posterior–anterior pair of 160 kV X-ray beam (CP160, Faxitron X-ray)
defined by an 8-mm diameter lead cut-out.
19
BrdU incorporation
Various BrdU incorporation schedules were used for cell fate mapping as
described in the Results section. BrdU was administered by intraperitoneal
injection.
P53 regulates neuronal development after irradiation
Y-Q Li et al
8
Cell Death Discovery (2016) e16072 Official journal of the Cell Death Differentiation Association
Primary culture of NPCs
Neurospheres were cultured from 8-week-old p53+/+, p53 −/−and eGFP
mouse hippocampus.
19
After 10 days in culture, mechanically dissociated
neurosphere cells were plated onto culture slips precoated with poly-L-
ornithine (Sigma-Aldrich, St Louis, MO, USA) and fed with DMEM/F12
medium containing penicillin/streptomycin, B27 supplement, basic fibro-
blast growth factor and epidermal growth factor. The non-differentiation
medium was changed every other day until cells grew to confluence on
day 8. NPCs cultured from p53+/+, p53 −/−and eGFP mice demonstrated
multipotential properties as reported previously.
19
Transplantation of eGFP-NPCs
eGFP-NPCs after 8 days in culture were dissociated into single-cell
suspensions in DMEM/F12 medium, and stored in ice before transplanta-
tion. Transplantation was carried out within 3 h following cell harvesting.
eGFP-NPCs were transplanted into the hippocampus of p53+/+ and p53 −/−
mice, which had received 0 or 5 Gy of cranial irradiation 3 weeks
previously. The cranium was fixed in a stereotactic frame (Kopf Small
Animal Stereotaxtic 900) during transplantation with the animals under
anesthesia using a cocktail of ketamine and xylazine.
19
Two craniotomies
were performed to allow cell transplantation into the right dentate gyrus in
two locations: first location, 1.8 mm laterally to the right, 1.1 mm caudally
and 3.3 mm ventrally; second location, 2.6 mm laterally to the right,
1.6 mm caudally and 3.6 mm ventrally, all with reference to the bregma.
A suspension of 2.5 μl of eGFP cells (50 000 cells per μl) in DMEM/F12
medium was introduced at 1 μl/min into each transplantation site, and for
an additional 2 min to allow pressure equalization. The scalp was closed
with synthetic suture monofilament after transplantation. Subcutaneous
buprenorphin (0.05–0.1 mg/kg) was given as applicable. Antibiotics were
not used.
19
Histopathology and immunohistochemistry
Under anesthesia with ketamine and xylazine, mice were perfused with
0.9% saline followed by 4% paraformaldehyde in PBS. Mouse brains were
retrieved, postfixed for 2 days and cryoprotected in a 30% sucrose solution.
Coronal sections between 1.3 and −3.5 mm caudal to the bregma were
cut at 40-μm thickness, collected in tissue cryoprotectant solution in 96-
well plates and stored at −20 °C before immunohistochemistry.
As morphological characterization remains the gold standard for
identification of apoptotic cells,
20
cells that showed nuclear condensation
and fragmentation upon 4′,6-diamidino-2-phenylindole (DAPI) staining
were considered apoptotic cells.
19
Apoptotic cells were further identified
and quantified using TUNEL and caspase-3 (1 : 1000; Cell Signaling
Technology, Beverly, MA, USA) immunohistochemistry.
19
NPCs, immature and mature neurons and microglia were identified by
different phenotypic markers using antibodies listed in Supplementary
Table 2. Secondary antibodies were conjugated to Cy2, Cy3 (1 : 200;
Jackson ImmunoResearch, West Grove, PA, USA) or Alexa Fluor 647 (1 : 200;
Invitrogen, Waltham, MA, USA). Colocalization of BrdU (1 : 200; Abcam,
Toronto, ON, Canada), Ki67 (1 : 1000; Novocastra, Newcastle upon Tyne, UK )
and phenotypic markers in selected sections were evaluated using a
confocal laser scanning microscope (Zeiss LSM700, Carl Zeiss AG
Corporate, Oberkochen, Germany). A BrdU-doublet was defined as two
abutting DAPI-stained nuclei that demonstrated nuclear BrdU
immunoreactivity.
Stereological analysis
Apoptotic cells and cells labeled using different phenotypic markers were
counted within the dentate gyrus including a 50-μm hilar margin of the
SGZ.
19
Cell counting was performed using a Zeiss Imager M1 microscope
(Carl Zeiss AG Corporate) with the Stereo Investigator software (MBF
Bioscience, Williston, VT, USA). The observers were blinded to the
experimental groups. Apoptotic cells were counted using a counting
frame and a sampling grid of 75 × 75 μm
2
, NPCs using counting frame of
20 × 20 μm
2
and sampling grid of 180 × 180 μm
2
, and microglia, counting
frame and sampling grid of 75 × 75 μm
2
, all at a magnification of × 630.
Every seventh section was used as the periodicity of sections sampled.
For the transplantation study, 10 coronal sections containing the
hippocampus at 5-section intervals from each mouse were used for
exhaustive cell counting of eGFP cells with a 100 × 100 μm
2
sampling grid.
The coefficient of error for all the stereology data was between 0.03
and 0.06.
Assessment of DNA damage repair foci
NPCs from p53+/+ and p53 −/−mice were cultured in non-differentiation
medium for 8 days before they were given a single dose of 0 or 5 Gy. At
various time intervals up to 24 h after irradiation, cells were fixed with 4%
paraformaldehyde for 10 min at room temperature. After treatment with
0.5% nonylphenoxypolyethoxylethanol in PBS, sections were incubated
with mouse anti-phospho-histone H2AX IgG1 antibody (1 : 200; Millipore,
Billerica, MA, USA) at 4 °C overnight followed by donkey anti-mouse Cy3
for 45 min at room temperature, and counterstained with DAPI. A
minimum of 50 nuclei from a minimum of five independent experiments
per treatment group was used to determine the number of γH2AX foci per
nucleus. As the occasional non-irradiated NPC nuclei contained up to six
foci, the nuclei with ≥5 foci were considered foci+.
Statistical analysis
All cell population analysis represented data from three to five mice per
genotype per dose per time point, except for the cell fate experiments
where there were three to four mice per genotype per dose group. There
were four to seven mice per experimental group in the transplantation
experiment. All data were expressed as mean ± S.E. Comparison of cell
numbers after irradiation to controls was performed using t-test. Dose–
response analysis for cell numbers was performed by one-way ANOVA. The
effect of variables, namely irradiation and p53 genotype, or p53 genotype
and time after BrdU on cell numbers, was determined using two-way
ANOVA. Pairwise comparisons were based on post hoc Bonferroni
correction for multiple comparisons. Differences were considered sig-
nificant for Po0.05. Statistical analyses were performed with the
GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
ABBREVIATIONS
ANOVA, analysis of variance; BrdU,bromodeoxyuridine; DAPI, 4′, 6-diamidino-
2-phenylindole; DCX, doublecortin; eGFP, enhanced green fluorescent
protein; GFAP, glial fibrillary acidic protein; INP, intermediate neural
progenitors; NeuN, neuronal nuclei; NPCs, neural progenitor cells; +, positive;
SGZ, subgranular zone; SOX2, sex-determining region Y-box 2; TUNEL,
terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
ACKNOWLEDGEMENTS
The work was supported by funding from the Canadian Cancer Society Research
Institute (CSW) and Cancer Research Society (CSW).
AUTHOR CONTRIBUTIONS
Conception and design: ZC Cheng, Y Li, CS Wong; development of
methodology: ZCC, YL, CSW; acquisition of data: YL, ZCC; analysis and
interpretation of data: IA, ZCC, YL, SL, CSW; writing, review and/or revision of
the manuscript: IA, ZCC, YL, SL, CSW; administrative, technical or material
support: YL, CSW; study supervision: CSW; other (oversight of every aspect of
the research): CSW.
COMPETING INTERESTS
The authors declare no conflict of interest.
REFERENCES
1 Greene-Schloesser D, Moore E, Robbins ME. Molecular pathways: radiation-
induced cognitive impairment. Clin Cancer Res 2013; 19: 2294–2300.
2 Zhao C, Deng W, Gage FH. Mechanisms and functional implications of adult
neurogenesis. Cell 2008; 132:645–660.
3 BonaguidiMA,SongJ,MingGL,SongH.Aunifyinghypothesisonmammalianneural
stem cell properties in the adult hippocampus. Curr Opin Neurobiol 2012; 22:754–761.
4 Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E. Neurogenesis in the
adult is involved in the formation of trace memories. Nature 2001; 410:372–376.
5 Feng R, Rampon C, Tang YP, Shrom D, Jin J, Kyin M et al. Deficient neurogenesis in
forebrain-specific presenilin-1 knockout mice is associated with reduced clear-
ance of hippocampal memory traces. Neuron 2001; 32:911–926.
6 Sahay A, Scobie KN, Hill AS, O'Carroll CM, Kheirbek MA, Burghardt NS et al.
Increasing adult hippocampal neurogenesis is sufficient to improve pattern
separation. Nature 2011; 472: 466–470.
P53 regulates neuronal development after irradiation
Y-Q Li et al
9
Official journal of the Cell Death Differentiation Association Cell Death Discovery (2016) e16072
7 Akers KG, Martinez-Canabal A, Restivo L, Yiu AP, De Cristofaro A, Hsiang HL et al.
Hippocampal neurogenesis regulates forgetting during adulthood and infancy.
Science 2014; 344:598–602.
8 Monje ML, Mizumatsu S, Fike JR, Palmer TD. Irradiation induces neural precursor-
cell dysfunction. Nat Med 2002; 8: 955–962.
9 Monje ML, Palmer T. Radiation injury and neurogenesis. Curr Opin Neurol 2003; 16:
129–134.
10 Gudkov AV, Komarova EA. The role of p53 in determining sensitivity to radio-
therapy. Nat Rev Cancer 2003; 3: 117–129.
11 Kirsch DG, Santiago PM, di Tomaso E, Sullivan JM, Hou WS, Dayton T et al. P53
controls radiation-induced gastrointestinal syndrome in mice independent of
apoptosis. Science 2010; 327:593–596.
12 Lee CL, Moding EJ, Cuneo KC, Li Y, Sullivan JM, Mao L et al. P53 functions in
endothelial cells to prevent radiation-induced myocardial injury in mice. Sci Signal
2012; 5: ra52.
13 Burdelya LG, Komarova EA, Hill JE, Browder T, Tararova ND, Mavrakis L et al.
Inhibition of p53 response in tumor stroma improves efficacy of anticancer
treatment by increasing antiangiogenic effects of chemotherapy and radio-
therapy in mice. Cancer Res 2006; 66: 9356–9361.
14 Banath JP, Macphail SH, Olive PL. Radiation sensitivity, H2AX phosphorylation,
and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines.
Cancer Res 2004; 64: 7144–7149.
15 Meletis K, Wirta V, Hede SM, Nister M, Lundeberg J, Frisen J. P53 supp-
resses the self-renewal of adult neural stem cells. Development 2006; 133:
363–369.
16 Armesilla-Diaz A, Bragado P, Del Valle I, Cuevas E, Lazaro I, Martin C et al. P53
regulates the self-renewal and differentiation of neural precursors. Neuroscience
2009; 158: 1378–1389.
17 Garcia-Cao I, Garcia-Cao M, Martin-Caballero J, Criado LM, Klatt P, Flores JM et al.
'Super p53' mice exhibit enhanced DNA damage response, are tumor resistant
and age normally. EMBO J 2002; 21: 6225–6235.
18 Mizumatsu S, Monje ML, Morhardt DR, Rola R, Palmer TD, Fike JR. Extreme sen-
sitivity of adult neurogenesis to low doses of X-irradiation. Cancer Res 2003; 63:
4021–4027.
19 Lu F, Li YQ, Aubert I, Wong CS. Endothelial cells regulate p53-dependent apop-
tosis of neural progenitors after irradiation. Cell Death Dis 2012; 3:e324.
20 Sierra A, Encinas JM, Deudero JJ, Chancey JH, Enikolopov G, Overstreet-Wadiche
LS et al. Microglia shape adult hippocampal neurogenesis through apoptosis-
coupled phagocytosis. Cell Stem Cell 2010; 7: 483–495.
21 Chow BM, Li YQ, Wong CS. Radiation-induced apoptosis in the adult central
nervous system is p53-dependent. Cell Death Differ 2000; 7: 712–720.
22 Limoli CL, Giedzinski E, Rola R, Otsuka S, Palmer TD, Fike JR. Radiation response of
neural precursor cells: linking cellular sensitivity to cell cycle checkpoints, apop-
tosis and oxidative stress. Radiat Res 2004; 161:17–27.
23 Shen Q, Goderie SK, Jin L, Karanth N, Sun Y, Abramova N et al. Endothelial cells
stimulate self-renewal and expand neurogenesis of neural stem cells. Science
2004; 304: 1338–1340.
24 Pineda JR, Daynac M, Chicheportiche A, Cebrian-Silla A, Sii Felice K, Garcia-
Verdugo JM et al. Vascular-derived TGF-beta increases in the stem cell niche and
perturbs neurogenesis during aging and following irradiation in the adult
mouse brain. EMBO Mol Med 2013; 5: 548–562.
25 Monje ML, Toda H, Palmer TD. Inflammatory blockade restores adult hippocampal
neurogenesis. Science 2003; 302: 1760–1765.
26 Lee SW, Haditsch U, Cord BJ, Guzman R, Kim SJ, Boettcher C et al. Absence of
CCL2 is sufficient to restore hippocampal neurogenesis following cranial irradia-
tion. Brain Behav Immun 2013; 30:33–44.
27 Belarbi K, Jopson T, Arellano C, Fike JR, Rosi S. CCR2 deficiency prevents neuronal
dysfunction and cognitive impairments induced by cranial irradiation. Cancer Res
2013; 73: 1201–1210.
28 Jenrow KA, Brown SL, Lapanowski K, Naei H, Kolozsvary A, Kim JH. Selective
inhibition of microglia-mediated neuroinflammation mitigates radiation-induced
cognitive impairment. Radiat Res 2013; 179:549–556.
29 Song J, Zhong C, Bonaguidi MA, Sun GJ, Hsu D, Gu Y et al. Neuronal circuitry
mechanism regulating adult quiescent neural stem-cell fate decision. Nature
2012; 489:150–154.
30 Pereboeva L, Harkins L, Wong S, Lamb LS. The safety of allogeneic innate lym-
phocyte therapy for glioma patients with prior cranial irradiation. Cancer Immunol
Immunother 2015; 64:551–562.
31 Garcia AD, Doan NB, Imura T, Bush TG, Sofroniew MV. GFAP-expressing pro-
genitors are the principal source of constitutive neurogenesis in adult mouse
forebrain. Nat Neurosci 2004; 7:1233–1241.
32 Bonaguidi MA, Wheeler MA, Shapiro JS, Stadel RP, Sun GJ, Ming GL et al. In vivo
clonal analysis reveals self-renewing and multipotent adult neural stem cell
characteristics. Cell 2011; 145: 1142–1155.
33 Kippin TE, Martens DJ, van der Kooy D. P21 loss compromises the relative
quiescence of forebrain stem cell proliferation leading to exhaustion of their
proliferation capacity. Gene Dev 2005; 19: 756–767.
34 Encinas JM, Michurina TV, Peunova N, Park JH, Tordo J, Peterson DA et al. Division-
coupled astrocytic differentiation and age-related depletion of neural stem cells
in the adult hippocampus. Cell Stem Cell 2011; 8:566–579.
35 Mira H, Andreu Z, Suh H, Lie DC, Jessberger S, Consiglio A et al. Signaling through
BMPR-IA regulates quiescence and long-term activity of neural stem cells in the
adult hippocampus. Cell Stem Cell 2010; 7:78–89.
36 Guo JU, Ma DK, Mo H, Ball MP, Jang MH, Bonaguidi MA et al. Neuronal activity
modifies the DNA methylation landscape in the adult brain. Nat Neurosci 2011; 14:
1345–1351.
37 Segi-Nishida E, Warner-Schmidt JL, Duman RS. Electroconvulsive seizure and VEGF
increase the proliferation of neural stem-like cells in rat hippocampus. Proc Natl
Acad Sci USA 2008; 105: 11352–11357.
38 Gao X, Enikolopov G, Chen J. Moderate traumatic brain injury promotes pro-
liferation of quiescent neural progenitors in the adult hippocampus. Exp Neurol.
2009; 219:516–523.
39 Uberti D, Piccioni L, Cadei M, Grigolato P, Rotter V, Memo M. P53 is dispensable
for apoptosis but controls neurogenesis of mouse dentate gyrus cells following
gamma-irradiation. Brain Res Mol Brain Res 2001; 93:81–89.
40 Insinga A, Cicalese A, Faretta M, Gallo B, Albano L, Ronzoni S et al. DNA damage in
stem cells activates p21, inhibits p53, and induces symmetric self-renewing
divisions. Proc Natl Acad Sci USA 2013; 110: 3931–3936.
41 Blanpain C, Mohrin M, Sotiropoulou PA, Passegue E. DNA-damage response in
tissue-specific and cancer stem cells. Cell Stem Cell 2011; 8:16–29.
42 Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neuro-
genesis. J Comp Neurol 2000; 425: 479–494.
43 Yazlovitskaya EM, Edwards E, Thotala D, Fu A, Osusky KL, Whetsell WO Jr. et al.
Lithium treatment prevents neurocognitive deficit resulting from cranial irradia-
tion. Cancer Res 2006; 66: 11179–11186.
44 Zanni G, Di Martino E, Omelyanenko A, Andang M, Delle U, Elmroth K et al.
Lithium increases proliferation of hippocampal neural stem/progenitor cells and
rescues irradiation-induced cell cycle arrest in vitro.Oncotarget 2015; 6:
37083–37097.
45 Roughton K, Kalm M, Blomgren K. Sex-dependent differences in behavior and
hippocampal neurogenesis after irradiation to the young mouse brain. Eur J
Neurosci 2012; 36: 2763–2772.
This work is licensed under a Creative Commons Attribution 4.0
International License. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated
otherwise in the credit line; if the material is not included under the Creative Commons
license, users will need to obtain permission from the license holder to reproduce the
material. To view a copy of this license, visit http://creativecommons.org/licenses/
by/4.0/
© The Author(s) 2016
Supplementary Information accompanies the paper on the Cell Death and Discovery website (http://www.nature.com/cddiscovery)
P53 regulates neuronal development after irradiation
Y-Q Li et al
10
Cell Death Discovery (2016) e16072 Official journal of the Cell Death Differentiation Association
