1 Departamento de Biología Celular, Universidad de Valencia, 46100
Burjassot, Spain
2 Spanish National Cancer Center (CNIO), 28029 Madrid, Spain
Author for correspondence (e-mail:
isabel.farinas{at}uv.es)
Accepted 8 April 2004
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SUMMARY |
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Key words: Telomerase knockout, Neural progenitor, Neurogenesis, Differentiation
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Introduction |
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Telomeres are specialized chromatin structures at the ends of eukaryotic
chromosomes that consist of non-coding single G-rich DNA repeats (TTAGGG in
all vertebrates), bound to an array of associated proteins, and that play an
essential role in chromosome capping
(Greider, 1998;
Blackburn, 2000
). In most
somatic tissues, telomeric DNA undergoes progressive shortening with each
round of DNA replication, at a rate between 50 and 200 bp per cell division,
resulting from incomplete replication of linear chromosomes by cellular DNA
polymerases (see Blackburn,
2000
). Telomere dysfunction, caused by significant loss of TTAGGG
sequences or of telomere-binding proteins, leads to disruption of the telomere
structure resulting in end-to-end chromosome fusions and genomic instability
(de Lange, 2002
). The
non-homologous end-joining pathway for double strand break repair has been
recently shown to mediate these outcomes of telomere dysfunction, suggesting
that a dysfunctional telomere is signaled as damaged DNA (Espejel et al.,
2002a; Espejel et al., 2002b
;
Goytisolo and Blasco, 2002
;
Smogorzewska et al., 2002
;
d'Adda di Fagagna et al.,
2003
; Takai et al.,
2003
). Telomere shortening to a critical length, therefore, can
activate DNA damage-induced pathways that trigger cell cycle arrest or
apoptosis (Chiu and Harley,
1997
; Goytisolo and Blasco,
2002
). Thus, telomeric erosion limits the life-span of dividing
cells unless counteracted by elongation mechanisms, among which the best
characterized is that mediated by the ribonucleoprotein telomerase. In
telomerase-proficient cells, the reverse transcriptase component of the
telomerase enzyme (Tert, telomerase reverse transcriptase) adds telomere
repeat sequences to chromosome ends by using its RNA component (Terc,
telomerase RNA component) as a template
(Blackburn, 2000
). Telomerase
activation is an essential property of pluripotent embryonic stem cells and of
some tissue-specific long-term self-renewing stem cells
(Morrison et al., 1996
;
Thomson et al., 1998
). In
addition, high telomerase activity in germline cells, and telomerase activity
upregulation in tumor cells and immortalized cell lines probably accounts for
their unlimited lifespan (Kim et al.,
1994
; Chiu and Harley,
1997
; Bodnar et al.,
1998
). Most somatic cells, however, express low or undetectable
levels of telomerase activity resulting in progressive telomere attrition
coupled to cell division (Harley et al.,
1990
; Prowse and Greider,
1995
). The effects of telomere dynamics in vivo have been analyzed
in mice that carry a deletion in the mouse telomerase RNA component and that,
therefore, lack telomerase activity (Blasco
et al., 1997
). On a mixed C57BL6/129Sv (B6/Sv) genetic background
these Terc/ mice are viable and fertile up
to the fifth generation (G5) (Blasco et
al., 1997
; Lee et al.,
1998
; Herrera et al.,
1999b
). As telomeres shorten in
Terc/ mice, at a rate of around 5 kb per
generation, cytogenetic instability appears in multiple organ systems and
results in decreased proliferation and increased apoptosis in highly
proliferative tissues such as the reproductive and hematopoietic systems.
Consistently, telomerase-deficient mice of late generations exhibit strain
collapse due to increased infertility, reduced viability, and a wide spectrum
of premature aging pathologies (Blasco et
al., 1997
; Lee et al.,
1998
; Rudolph et al.,
1999
; Herrera et al.,
1999a
; Herrera et al.,
2000
; Samper et al.,
2002
; Leri et al.,
2003
) (reviewed by Goytisolo
and Blasco, 2002
).
The role of telomerase and of telomere dynamics on neurogenic subsets, and
in particular on NSC compartments, has not been addressed so far. High levels
of both Terc and Tert mRNA are present in the developing
neural tube as early as E10.5
(Martín-Rivera et al.,
1998; Herrera et al.,
1999b
). Later, both Terc and the Tert mRNA are
found in different regions of the developing murine CNS, in a complex pattern
of gene expression characterized by a temporal correlation with proliferation
of neural progenitors in different areas
(Prowse and Greider, 1995
;
Greenberg et al., 1998
;
Martín-Rivera et al.,
1998
; Fu et al.,
2000
; Haik et al.,
2000
; Ostenfeld et al.,
2000
; Klapper et al.,
2001
). These expression patterns suggest a role for telomerase in
neural precursor biology. More recently, telomerase activity has also been
demonstrated in neural precursor cells isolated from the adult SVZ and
hippocampus (Caporaso et al.,
2003
). By comparison with other stem cell populations, the current
working model holds that expression and activity of telomerase in actively
cycling neural progenitors may overcome the progressive proliferation-induced
telomere shortening and promote growth and survival of progenitors.
We have analyzed neural progenitor proliferation in embryos and adult
telomerase-deficient mice with shortened telomeres. We have found that cell
proliferation is severely impaired in the SVZ of late-generation
telomerase-deficient mice and that NSCs isolated from this region are not
capable of in vitro expansion under mitogenic stimulation (see also
Wong et al., 2003).
Surprisingly, NSCs from late-generation telomerase-deficient embryos
proliferated normally despite having shortened telomeres, cytogenetic
abnormalities and increased levels of nuclear p53. Altogether, these findings
reveal a fundamentally different response to telomere dysfunction and p53
activation in embryonic versus adult NSCs.
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Materials and methods |
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Measurement of telomere lengths and cytogenetic analysis
For quantitative fluorescence in situ hybridization (Q-FISH) analysis,
cultured cells were incubated in hypotonic buffer (0.56% KCl) for 5 minutes at
room temperature, fixed in methanol:acetic acid (3:1) and dropped onto wet
slides. After drying overnight, cells were fixed in formaldehyde, digested
with pepsin (1 mg/ml), dehydrated through graded ethanol and incubated with a
fluorescent telomeric peptic nucleic acid probe (Cy-3-(AATCCC)3),
as described previously (Blasco et al.,
1997). After washing, cells were dehydrated and mounted in
Vectashield with DAPI (Vector Laboratories, Burlingham, CA, USA). Images were
captured using a Leica microscope LEITZ DMRB equipped with a 100x/NA 1.0
objective lens and a COHU High Performance CCD camera, with a red fluorescence
filter (Leica I3-513808) for the Cy3-conjugated telomeric probe and a DAPI
fluorescence filter (Leica A-513808) for the nuclear imaging. For metaphase
analysis, 10 metaphases per sample were analyzed using Leica Q-FISH v2.1
software. Fluorescence beads (Molecular Probes, Eugene, OR, USA) were used as
quantification standards, as described previously
(Hande et al., 1999
). For
interphase cells, 50 interphase nuclei were captured. To translate arbitrary
fluorescence units to kb, two murine lymphoma cell lines of known telomere
length (length ratio, 7:1) were assayed in parallel and used to generate a
linear regression curve (McIlrath et al.,
2001
). After Q-FISH hybridization, 50 metaphases were captured at
100x magnification and evaluated for cytogenetic abnormalities, such as
aneuploidy and fusions. Chromosomes lacking telomeres were identified and
counted from 10 metaphases using Leica Q-FISH software.
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Results |
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Constitutively proliferating progenitor cells labeled with the BrdU
administration protocol used here
(Morshead et al., 1994)
include a population of transit-amplifying progenitor cells (type C cells) as
well as their progeny, a population of proliferating neuroblasts (type A
cells) (Doestch et al., 1997; Doestch et al., 1999) that migrate to the
olfactory bulbs (Luskin, 1993
;
Lois and Alvarez-Buylla, 1994
;
Galli et al., 2003
). Because
type A neuroblasts are more frequently found in their migratory route at the
dorsolateral corner of the anterior SVZ (see
Fig. 1A)
(Doetsch et al., 1999
), we
determined the proportion of BrdU-labeled cells specifically in this region
and found a reduction in mutant animals of around 70% relative to control
values [mean number of BrdU-labeled cells per section±s.e.m.; wild type
(WT): 53.6±16.7, n=3 animals; G4
Terc/: 15.1±4.7, n=2].
Consistent with this dramatic decline in proliferative activity in the SVZ and
the rostral migratory stream (rms), labeling of the migrating neuroblasts with
anti-PSA-NCAM antibodies appeared reduced
(Fig. 1D,E). The reduction in
migrating neuroblasts results in a significant decrease in the overall volume
of the olfactory bulb in G4 Terc/ animals to
more than 50% relative to those of wild type
(Fig. 1G). Interestingly,
striatal volume was not altered in G4 Terc/
mice, an indication that the reduction in the olfactory bulb was most probably
due to a specific reduction in the proliferation of neural progenitor cells in
the SVZ postnatally rather than to defects in proliferation or differentiation
during embryogenesis (Fig. 1G).
Together, these results indicate that proliferation potential of neural
progenitors in vivo is decreased in late-generation telomerase-deficient adult
mice. Although our data indicate that numbers of migrating neuroblasts (type A
cells) were lowered in the Terc-deficient mice, we cannot exclude a
decrease in the number of type C-cells as well.
Normal development of sensory ganglia and subcortical telencephalon in late generation telomerase-deficient embryos
Despite significant reductions in postnatal progenitor populations, the
brains of late-generation telomerase-deficient mice are normal in appearance
and overall size, suggesting that embryogenesis proceeds normally in these
mutants. To determine whether the proliferation, differentiation, and/or
survival of embryonic neural precursors is altered by telomere shortening in
Terc homozygous mutant mice, we analyzed the development of specific
neural structures of the PNS and of the CNS in late generation (G5)
Terc/ mice. Previous work had indicated that
telomerase is expressed in neuroepithelial cells of the developing neural tube
of mouse embryos and that telomerase-mediated telomere length maintenance
appears to be required for developmentally programmed closure of the neural
tube in a certain proportion of late generation
Terc/ embryos
(Herrera et al., 1999b). Thus,
we focused our analysis on the embryonic development of dorsal root ganglia
(DRG), since DRG neural and glial progenitors derive from neural crest cells,
a population that delaminates from the dorsal neural tube after its closure.
In developing DRGs, neurogenesis is initiated shortly before E10.5 and is
complete by E13.5 (Fariñas et al.,
1996
). We analyzed ganglia from wild-type and G5
Terc/ embryos at E12.5, when neurogenesis
peaks and precursor proliferation and differentiation are actively taking
place. Complete series of sections through lumbar 1 (L1) DRGs of G5
Terc/ and wild-type embryos were analyzed
(Fig. 2). Measurements of BrdU
incorporation after a one hour-administration revealed that the numbers and
proportions of labeled progenitor cells in G5
Terc/ and wild-type DRGs
(Fig. 2B,C,F) were not
significantly different. Similarly, there were no differences in the number of
differentiated, neurofilament-immunopositive neurons
(Fig. 2D-F), in the number or
proportion of pyknotic, degenerating cells, or in the total number of cells
(Fig. 2F). Together, these
results suggest that telomerase deficiency and telomere shortening in G5
Terc/ embryos do not appear to result in
defects in the development of neural crest-derived sensory ganglia.
|
Embryonic and adult telomerase-deficient NSCs behave differently under the same mitogenic conditions
Because proliferation of adult SVZ progenitors was severely impaired in the
Terc-deficient animals, we decided to analyze possible effects of the
deficiency in the capacity of SVZ cells to generate neurospheres under
mitogenic stimulation in vitro (Gritti et
al., 1996; Gritti et al.,
1999
). Although similar numbers of neurospheres were formed from
dissociated SVZ tissue of both genotypes (mean number of sphere-forming
units/brain±s.e.m.: WT, 304±63; G4
Terc/, 308±48, n=3
independent cultures), G4 Terc/ primary
spheres were 60% smaller after 10 DIV (Fig.
3A). Because each sphere originates from one cell and because most
cells in a neurosphere are not themselves sphere-forming NSCs, the number of
spheres formed after a passage can be taken as a reliable estimation of
self-renewal capacity. The number of spheres formed relative to the number of
cells plated in wild-type and G4 Terc/
secondary cultures were not significantly different (mean percentage ±
s.e.m.: WT, 2.1±0.4, n=3 independent cultures; G4
Terc/, 1.8±0.1, n=2).
However, secondary spheres were also significantly smaller in G4
Terc/ than wild-type spheres
(Fig. 3A). To analyze overall
growth we determined ratios of cell production after 7 DIV relative to number
of cells plated at passages 2 and 3. Growth ratios in the G4
Terc/ cultures were significantly lower than
in wild-type cultures (Fig.
3B). This reduction in cell yield is not the consequence of a
differential apoptotic response because the frequency of nuclei with apoptotic
condensed chromatin was the same in G4 Terc/
(1.3±0.4%) and wild-type (1.2±0.7%) neurospheres. Because these
data suggest that limited growth of mutant NSCs is caused by slower
proliferation, we sought to determine proliferation rates. BrdU incorporation
was significantly reduced in G4 Terc/
neurospheres compared with wild-type ones
(Fig. 3C,D; see
Fig. 5C for quantification).
Thus, NSCs isolated from the adult SVZ appear to proliferate more slowly than
wild-type NSCs.
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Because shortened dysfunctional telomeres appear to trigger signaling
pathways associated with damaged DNA (Espejel et al., 2002a;
Espejel et al., 2002b;
Goytisolo and Blasco, 2002
;
Smogorzewska et al., 2002
;
d'Adda di Fagagna et al.,
2003
; Takai et al.,
2003
), we examined whether telomere length reductions found in
adult NSCs resulted in the activation of DNA damage responses that were not
active in embryonic NSCs with longer telomeres. Telomere shortening and
associated chromosomal instability in late generation
Terc/ adult mice had been previously shown
to correlate with increased proportions of cells expressing p53 and,
therefore, p53 upregulation is considered to be an indication of the presence
of dysfunctional telomeres (Chin et al.,
1999
; Leri et al.,
2003
). Moreover, activation of p53 appears to mediate the adverse
effects of critically short telomeres on the proliferation and survival in
different cell types (Chin et al.,
1999
;
González-Suárez et al.,
2002
; Leri et al.,
2003
). To evaluate if telomeric erosion found in embryonic and
adult NSCs was equivalent in terms of p53 activation we immunostained
neurospheres for p53 (Fig. 5B).
Neurospheres derived from G4 Terc/ adult
mice showed an increase in the number of p53 immunopositive nuclei
(Fig. 5B). However, p53 protein
was undetectable in embryonic wild-type and G5
Terc/ neurospheres under the same conditions
(Fig. 5B). Therefore, it
appears that p53 is upregulated in early cultures of mutant G4 adult NSCs but
not of G5 embryonic NSCs, suggesting that telomere reductions in embryonic G5
mutant NSCs may not be sufficient to activate a DNA damage response.
Because activation of p53 induces either cell cycle arrest or apoptotic
cell death (for a review, see Vogelstein
et al., 2000), it is likely that the hypocellularity observed in
G4 Terc/ cultures is the result of a nuclear
increase in p53 levels. In agreement with this proposal, an inverse
correlation was observed between the percentage of p53-immnopositive cells and
the percentage of cells in S-phase (Fig.
5C). DAPI-stained chromatin condensations indicative of apoptosis
were rare in both wild-type and mutant cultures, an indication that in adult
NSCs telomere dysfunction results in cell cycle arrest but not cell death.
Embryonic NSCs with very short telomeres, cytogenetic abnormalities, and nuclear p53 accumulation proliferate extensively in vitro
In order to determine whether normal cellularity and absence of nuclear p53
accumulation was indeed related to the presence of sufficiently long telomeres
in NSCs isolated from mutant embryos, we analyzed embryonic cultures after
extensive proliferation in vitro. Studies in stem cells of other tissue types,
such as hematopoietic, indicate that telomerase deficiency may not impair
proliferation under steady-state conditions but that it is detrimental for
high demand proliferative challenges
(Samper et al., 2002). Mutant
NSCs were collected at different time points in long-term cultures and
telomere lengths were measured using Q-FISH during metaphase
(Fig. 6A,B). Using metaphasic
Q-FISH for telomeric sequences it is possible to determine telomere length
distribution as well as the frequency of chromosomes with very short,
undetectable telomeres (length <200 bp; see proportions in histograms and
in Fig. 6E) and chromosomal
abnormalities (Fig. 6C-E). With
time in culture, the telomeres of telomerase-incompetent cells shortened and
the percentage of undetectable telomeres and of cytogenetic abnormalities
increased significantly (Fig.
6).
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Discussion |
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Cell-intrinsic limitation of proliferative life-span to a finite number of
divisions is a characteristic of most somatic human cells and it is dependent
on progressive shortening of telomeres to the point of loss of chromosomal end
integrity (see Blackburn,
2000). Therefore, telomere maintenance is essential, particularly
in highly proliferative cell populations. In contrast to human primary cells,
cells from inbred mouse strains do not appear to undergo telomere-mediated
replicative senescence under normal conditions because they have very long
telomeres (40-60 kbp) compared to humans and to wild mice (5-10 kbp)
(Prowse and Greider, 1995
;
Blasco et al., 1997
).
Nevertheless, the effects of telomere shortening in different types of cells
can be analyzed in the Terc-deficient strain, in which critical
reductions in telomere lengths have been shown to result in deleterious
cellular phenotypes after extensive culture or after several generations of
mice (Blasco et al., 1997
;
Rudolph et al., 1999
;
Niida et al., 2000
; Espejel
and Blasco, 2002a; Espejel and Blasco, 2002b). After four to six generations
without telomerase activity in laboratory mice, telomeres approach the size of
those found in human cells and effects of telomere attrition can be
investigated. Proliferative defects have been observed in germinal centers,
skin and the hematopoietic system and fibroblasts of late generation
telomerase-deficient mice, which are consistent with telomere-dependent
premature senescent-like growth arrest or apoptosis
(Lee et al., 1998
;
Rudolph et al., 1999
;
Samper et al., 2002
; Espejel
and Blasco, 2002a; Espejel and Blasco, 2002b). As shown in the present study
adult neural progenitors are also sensitive to telomere shortening
(Wong et al., 2003
) but,
surprisingly, embryonic neural progenitors with critically short telomeres
proliferate normally.
The reduced capacity for proliferation of adult NSCs is found in the in
vivo natural steady-state condition, in the absence of any exogenous mitogenic
stimulation. Moreover, large significant reductions in the number of
proliferating cells in the SVZ is observed in the mutants as early as in the
second breeding generation, when most chromosomes are still sufficiently long.
A formal possibility to explain this result is that telomerase activity itself
contributes to cell cycle dynamics in adult NSCs. A possible role of
telomerase activity in the regulation of proliferation in cells with
sufficiently long telomeres has been suggested by the fact that G1
Terc/ are less prone to skin tumor formation
and that telomerase overexpression in epithelial cells makes them more
susceptible to mitogenic stimulation and tumor formation
(González-Suárez et al., 2000;
González-Suárez et al.,
2001;
González-Suárez et al.,
2002
; Artandi et al.,
2002
; Smith et al.,
2003
). In the nervous system, pharmacological inhibition of
telomerase activity decreases FGF2-induced proliferation of cortical precursor
cells (Haik et al., 2000
),
suggesting that telomerase activity may be linked to cell cycling regulation
in certain neural progenitors, although possible mechanisms for these actions
are presently unclear (for a review, see
Blasco, 2002
). The other
possibility is that adult NSCs might be extremely sensitive to erosion of
particular chromosomes (see Hemann et al.,
2001
). The involvement of telomere shortening in the growth
retardation of G4 mutant NSCs in vitro are supported by the observation that
proliferation deficits correlate with a rise in the detectable levels of
nuclear p53. Further support for a direct effect of telomere erosion, even at
G2, in the proliferation of these cells is provided by recent data showing
that BrdU incorporation in the SVZ of G1 Terc-deficient mice, which lack
telomerase activity but have longer telomeres, is not decreased
(Wong et al., 2003
).
Despite the fact that proliferation rates were substantially reduced in
telomerase-deficient brains we could recover the same number of
neurosphere-forming cells from the SVZ of adult telomerase-deficient mice as
from wild-type SVZ, an indication that stem cell state is not affected by the
lack of telomerase activity or by telomere shortening. Moreover, stem cell
renewal appears to be also preserved in culture, as the proportions of sphere
forming units at each passage are similar in wild-type and mutant cultures
despite reduced numbers of cells produced per neurosphere upon growth factor
stimulation. Thus, our results suggest that proliferation and maintenance of
the stem cell state are regulated intrinsically by distinct signals
(Tropepe et al., 1997;
Shimazaki et al., 2001
).
When telomeres become sufficiently short to compromise their interaction
with specific telomere-binding proteins, they are recognized as damaged DNA
(see d'Adda di Fagagna et al.,
2003; Takai et al.,
2003
). This results in the activation of the non-homologous
end-joining pathway for double strand break repair, which mediates some of the
outcomes of telomere dysfunction (Espejel et al., 2002a;
Espejel et al., 2002b
;
Goytisolo and Blasco, 2002
;
Smogorzewska et al., 2002
) and
the ARF/p53 and the p16/Rb signaling pathways for growth arrest or apoptosis
(Harley et al., 1990
;
Vaziri et al., 1994
;
Chin et al., 1999
). p53
immunoreactivity is generally low, as this tumor-suppressor protein is
relatively short-lived, but several types of DNA damage, including telomere
erosion, can activate p53 and result in a rapid increase in the level of
nuclear p53 and the transcriptional activation of genes that induce cell cycle
arrest or apoptosis depending on the cell types
(Vogelstein et al., 2000
). In
our NSC cultures, p53 up-regulation is observed in adult NSCs with shortened
telomeres, in agreement with the importance of the p53 pathways for the murine
response to telomere damage (Chin et al.,
1999
). In murine cells telomere damage signaling appears to be
transduced only through the p53 pathway because, in contrast to p53-deficient
human cells, mouse cells that lack p53 do not arrest, suggesting that other
pathways such as activation of p16 may be dispensable (Smogorwewska and de
Lange, 2002). Consistently, p53 deficiency can rescue many of the phenotypes
of late generation Terc/ mice and extend
strain viability and fertility into the eight generation
(Chin et al., 1999
). The
observation that culture itself is not inducing p53 in wild-type cells
suggests that its activation is specifically induced by DNA damage at
telomeres. The p53 upregulation is coincidental with growth impairment in the
adult NSCs, suggesting induction of a p53 checkpoint arrest.
The proliferation rate of mutant embryonic cells, however, remained
constant with continual passaging, despite the fact that aneuploidy and
chromosome rearrangements were frequently detected in these NSC cultures, and
p53 levels increased in virtually all nuclei. Cells can escape replicative
senescence by acquiring inactivating mutations in cell cycle checkpoint
regulating genes, most characteristically p53, that lead to continued
proliferation and, most frequently, to a final crisis associated with extreme
telomere erosion and massive chromosomal instability
(Sherr and DePinho, 2000).
Only rare survivors could emerge from crisis and proliferate indefinitely,
possibly by activation of alternative telomere elongation mechanisms such as
ALT (see Kass-Eissler and Greider, 2000). Nevertheless, we have not detected
any crisis events in our cultures and, moreover, all three independent
cultures behaved similarly suggesting that mutation of p53 is unlikely to
explain the normal growth of all G5 Terc/
mutant cultures.
The mechanisms that regulate p53 transcriptional activity are now beginning
to be understood and include both post-translational modifications and
alterations in p53 binding proteins
(Giaccia and Kastan, 1998;
Brooks and Gu, 2003
).
Interactions with coactivators such as p300/CBP, PCAF, Sp1 or Ets1 that are
required to form stable DNA-p53 transcription initiation complexes appear to
be modulated by phosphorylation. Moreover, activation of the complexes appears
to be subject to regulation by acetylation
(Giaccia and Kastan, 1998
;
Barlev et al., 2001
;
Xu et al., 2002
;
Brooks and Gu, 2003
).
Therefore, cell context differences in the expression of coactivators or in
the signaling pathways upstream of p53 modifications could underlie
ontogeny-related changes in the response of NSCs to alterations in telomerase
activity and telomere lengths. Interestingly, silencing of p53 transcriptional
activity by NAD+-dependent histone deacetylases of the Sir2 family
suggests that cell metabolism may influence final cell fate decisions during
cellular stresses, including DNA damage, and underscores the importance of
cell context and activity (Langley et al.,
2002
; Vaziri et al.,
2001
; Luo et al.,
2001
; Brooks and Gu,
2003
). Embryonic neural precursor cells have been shown to be
relatively resistant to deficits in DNA repair molecules that play a role in
nervous system development. Mouse strains deficient in members of the
non-homologous end-joining mechanism for double strand break repair, such as
Ku70, Ku80, XRCC4 or Lig4, are characterized by defective neurogenesis during
embryogenesis as a result of increased apoptosis in postmitotic neuronal
populations, but not in their progenitor populations (see
Gao et al., 1998
;
Gu et al., 2000
).
In conclusion, we have shown that proliferative potential of NSCs residing in the adult brain is subject to tight intrinsic regulation and, therefore, more knowledge will be needed on intracellular mechanisms of cycling control if we are to pursue the reactivation of latent NSC populations to engage endogenous neurogenesis for the treatment of brain disease. Moreover, embryonic NSCs can escape cellular check points and, therefore, methods to monitor genetic stability of ex vivo expanded neural stem cells will be necessary prior to any therapeutic intervention.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Laboratory of Molecular Neurobiology, Department of
Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm,
Sweden
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