近日各媒体报道了美国哈佛大学用端粒酶激活实验，证明老鼠的器官可激活活力——“返老还童现象”，我们高兴，终于出现了重复我们人体再生复原科学的研究者。我们早就公布了大白鼠两倍年龄再生实验的结果，在两倍年龄时所有器官仍是年轻的。现在我们已经完成了五年期的老年人再生复原研究。人类年轻健康延寿的时代已经到来。下面是美国报告的原文。虽然其中很多解释是错误的，但他的实验是真实的。

LETTER doi:10.1038/nature09603
Telomerase reactivation reverses tissue degeneration
in aged telomerase-deficient mice
Mariela Jaskelioff1, Florian L. Muller1, Ji-Hye Paik1, Emily Thomas1, Shan Jiang1, Andrew C. Adams2, Ergun Sahin1,
Maria Kost-Alimova1, Alexei Protopopov1, Juan Cadin˜anos1, James W. Horner1, Eleftheria Maratos-Flier2 & Ronald A. DePinho1
An ageing world population has fuelled interest in regenerative
remedies that may stem declining organ function and maintain
fitness. Unanswered is whether elimination of intrinsic instigators
driving age-associated degeneration can reverse, as opposed to
simply arrest, various afflictions of the aged. Such instigators
include progressively damaged genomes. Telomerase-deficient
mice have served as a model system to study the adverse cellular
and organismal consequences of wide-spread endogenous DNA
damage signalling activationin vivo1. Telomere loss and uncapping
provokes progressive tissue atrophy, stem cell depletion, organ
system failure and impaired tissue injury responses1. Here, we
sought to determine whether entrenched multi-system degeneration
in adult mice with severe telomere dysfunction can be halted
or possibly reversed by reactivation of endogenous telomerase
activity. To this end, we engineered a knock-in allele encoding a
4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-
oestrogen receptor (TERT-ER) under transcriptional control
of the endogenous TERT promoter. Homozygous TERT-ER
mice have short dysfunctional telomeres and sustain increased
DNAdamage signalling and classical degenerative phenotypes upon
successive generational matings and advancing age. Telomerase
reactivation in such late generation TERT-ER mice extends telomeres,
reduces DNA damage signalling and associated cellular
checkpoint responses, allows resumption of proliferation in quiescent
cultures, and eliminates degenerative phenotypes across
multiple organs including testes, spleens and intestines. Notably,
somatic telomerase reactivation reversed neurodegeneration with
restoration of proliferating Sox21 neural progenitors, Dcx1 newborn
neurons, and Olig21 oligodendrocyte populations. Consistent
with the integral role of subventricular zone neural progenitors in
generation and maintenance of olfactory bulb interneurons2, this
wave of telomerase-dependent neurogenesis resulted in alleviation
of hyposmia and recovery of innate olfactory avoidance responses.
Accumulating evidence implicating telomere damage as a driver of
age-associated organ decline and disease risk1,3 and the marked
reversal of systemic degenerative phenotypes in adult mice observed
here support the development of regenerative strategies designed to
restore telomere integrity.
Accelerating structural and functional decline across diverse organ
systems is observed in the aged1,3,4. The loss of genome integrity and
associated DNA damage signalling and cellular checkpoint responses
are well-established intrinsic instigators that drive tissue degeneration
during ageing5. Of particular relevance to this study, age-progressive
loss of telomere function in mice has been shown to provoke widespread
p53 activation resulting in activation of cellular checkpoints of
apoptosis, impaired proliferation and senescence, compromised tissue
stem cell and progenitor function, marked tissue atrophy and physiological
impairment in many organ systems1,6.
Mounting evidence in humans has also provided strong association of
limiting telomeres with increased risk of age-associated disease7 andwith
onset of tissue atrophy and organ system failure in degenerative diseases
such as ataxia-telangiectasia, Werner syndrome, dyskeratosis congenita
and liver cirrhosis, among others1,3. In cell-based models of ataxiatelangiectasia
andWerner syndrome, enforced TERTcan restore normal
cellular proliferative potential8. These findings build on seminal cell culture
studies showing that enforced TERT expression can endow primary
human cells with unlimited replicative potential9. Importantly, TERT
overexpression in epithelial tissues of cancer-resistant mice leads to
extended median lifespan10. In addition, intercrossing wild-type and late
generation mTerc2/2 mice with severe degenerative phenotypes results
in healthy offspring11, indicating that viable late generation mTerc2/2
germ cells can be restored to normal telomere function on introduction
of a wild-type mTerc allele at the time of fertilization. However, to our
knowledge, there are no genetic or pharmacological studies showing
somatic reversal of age-related degenerative phenotypes driven by endogenous
genotoxic stresses in adult mammals. Here, in telomerasedeficient
mice experiencing severe tissue degeneration, we investigated
whether endogenous telomerase-mediated restoration of telomere function
throughout the organism would quell DNA damage signalling and
either arrest, or possibly reverse, cellular checkpoint responses and associated
tissue atrophy and dysfunction.Notably, the mice enlisted into this
study are adults exhibiting significant progeroid phenotypes.
Construction and functional validation of the germline TERT-ER
knock-in allele are detailed in Supplementary Fig. 1. In the absence of
4-OHT, ER fusion proteins remain in an inactive misfolded state12 and
thus we first sought to verify whether mice homozygous for TERT-ER
recapitulated the classical premature ageing phenotypes ofmice null for
mTerc or mTert.To that end,mice heterozygous forTERT-ER(hereafter
G0TERT-ER) were intercrossed to produce first generation mice homozygous
for TERT-ER (G1TERT-ER) which were then intercrossed to produce
successiveG2,G3 andG4TERT-ER cohorts. G1–G4TERT-ER cells have
no detectable telomerase activity (Fig. 1a). Accordingly, G4TERT-ER
primary splenocytes had hallmark features of short dysfunctional telomeres,
including decreased telomere-specific fluorescence in situ
hybridization (FISH) signal and Robertsonian fusions (Fig. 1b, e, f).
Moreover, G4TERT-ER fibroblasts failed to divide after five to six
passages and adopted a flat, senescence-like morphology (Fig. 1c, d).
AdultG4TERT-ER mice showedwidespread tissue atrophy, particularly in
highly proliferative organs including extreme testicular atrophy and
reduced testes size due to apoptotic elimination of germ cells, resulting
in decreased fecundity (Fig. 2a, d and Supplementary Fig. 2a), marked
splenic atrophy with accompanying increased 53BP1 (also known as
Trp53bp1) foci consistentwithDNAdamage (Fig. 2b, e, h) and intestinal
crypt depletion and villus atrophy in conjunction with numerous apoptotic
crypt cells and increased 53BP1 foci (Fig. 2c, f, i and Supplementary
Fig. 2b). Finally, median survival of G4TERT-ER mice is significantly
decreased relative to that of telomere intactmice (43.5 versus 86.8weeks,
***P,0.0001, Supplementary Fig. 2f). Thus, G4TERT-ER mice phenocopy
late generation mTert2/2 and mTerc2/2 animals13,14, indicating
that TERT-ER is inactive in the absence of 4-OHT.
1Belfer Institute for Applied Cancer Science and Departments of Medical Oncology, Medicine and Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
2Division of Endocrinology, Diabetes & Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
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Next, we assessed the impact of telomerase reactivation on telomere
dysfunction-induced proliferative arrest.On passage of adult G4TERT-ER
fibroblast cultures, cells adopted flat senescent-like morphology at
approximately five population doublings (Fig. 1d, upper panel). These
quiescent cultures showed prominent G0/G1 accumulation in the cell
cycle by fluorescence-activated cell sorting (FACS) analysis and rare cell
division events by time-lapse video microscopy (not shown). However,
upon replating these cells inmedia containing 100nM4-OHT, telomerase
reactivation led to elongated telomeres, prompt resumption of proliferation
over greater than eight additional passages tested, and
reduction in the G0/G1 phase fraction (Fig. 1c; data not shown).
Coincidently, high levels of cyclin-dependent kinase inhibitor, p21CIP1
(also known as Cdkn1a), declined upon 4-OHT treatment of the
G4TERT-ER cultures, allowing cell cycle re-entry (Supplementary Fig.
2e). This pattern of p21CIP1 regulation aligns with previous work documenting
its role as a key mediator of cell cycle arrest induced by
telomere dysfunction inmouse tissues15. Parallel G0 orG4TERT-ER fibroblastsmaintained
in 4-OHT at initial isolation did not undergo passageinduced
senescence and instead showed sustained proliferation (.20
passages; Fig. 1c, d).
These cell-based studies prompted systemic analyses of the impact of
4-OHT-mediated telomerase reactivation in the setting of entrenched
tissue degeneration. At the end of 4 weeks of continuous 4-OHT exposure,
documentation of telomerase-mediated telomere restoration and
function in G4TERT-ER tissues included increased telomere-FISH signal
in primary splenocytes (Fig. 1b, e, f), decreased p53 activation and
expression of p21CIP1 in liver (Supplementary Fig. 2d, e), and marked
decrease in 53BP1 foci in splenocytes (Fig. 2b, e) and intestinal crypt
cells (Fig. 2c, f). These molecular changes paralleled striking tissue
rejuvenation including reduced apoptosis of testes germ cells (data
not shown) and intestinal crypt cells (Supplementary Fig. 2b, i),
reduced tissue atrophy with restoration in normal testes and spleen
size (Fig. 2d, h and Supplementary Fig. 2a) and, most strikingly,
increased fecundity (Fig. 2g). Moreover, median survival increased in
G4TERT-ER mice treated with a 4-week course of 4-OHT (**P,0.005,
Supplementary Fig. 2f). Sustained 4-OHT treatment had no effect on
G0TERT-ER age- and gender-matched controls which were included in
all experiments. Together, these data indicate that, despite an
entrenched degenerative state, endogenous telomerase reactivation
results in marked extinction of DNA damage signalling, alleviation of
cellular checkpoint responses and reversal of tissue atrophy in highly
proliferative organ systems of the late generation TERT-ER mice.
Although the marked impact of telomerase reactivation on highly
proliferative organs is encouraging, we sought to assess more intensively
the potential benefits on brain health, which is a prime determinant
of age-progressive declining health in humans. Along these
lines, it is worth noting that the ageing mammalian brain shows accumulating
DNA damage16 and a progressive restriction of neurogenesis
and impaired re-myelination due to a decline in neural stem and
progenitor cell proliferation and differentiation17. As neural stem/
progenitor cells (hereafter NSCs) support neurogenesis, particularly
in the subventricular zone (SVZ), we first examined the properties of
NSCs derived from adult G0 and G4TERT-ER mice. As reported previously
for late generation mTerc2/2 mice6,14,18, vehicle-treated
G4TERT-ER NSC cultures showed decreased self-renewal activity relative
to G0TERT-ER controls and this defect was partially corrected with
4-OHT treatment (Fig. 3a, d). G4TERT-ER neurospheres were not only
rarer but also smaller in diameter than G0TERT-ER controls, and their
average diameter was restored to normal by 4-OHT treatment (Fig. 3a
and Supplementary Fig. 2c). These self-renewal profiles tracked with
activated p53-mediated DNA damage signalling in vehicle-treated
G4TERT-ER NSC cultures, which was extinguished with 4-OHT treatment
and absent in the G0TERT-ER controls (Fig. 3b, e). Examination of
NSC differentiation capacity revealed significant (twofold) reduction
in G4TERT-ER NSC capacity to generate neurons relative to 4-OHTtreated
G4TERT-ER cultures and 4-OHT- or vehicle-treated G0TERT-ER
controls (Fig. 3c, f). Consistent with previous work14,18, there was no
impact on astrocyte differentiation (data not shown).
On the basis of these cell culture observations, we examined the
SVZ, a region where NSCs reside and have an active role in adult brain
physiology. In adult mice, NSCs give rise to transit-amplifying progenitor
cells that divide rapidly and contribute to generation of neuroblasts,
astrocytes and myelinating oligodendrocytes. Consistent with
previous reports of an SVZ proliferation defect in mTerc2/2 mice6,14,18
and wild-type aged mice19, vehicle-treated G4TERT-ER mice show a
profound decrease in proliferating (Ki671) cells in the SVZ relative
to G0TERT-ER controls. Notably, 4-OHT-treated G4TERT-ER mice show
a striking, albeit partial, restoration of proliferation following only
0
5
10
15
20
25
0 10 20 30 40 50 60 70
Time (days)
Population doublings
100 nM 4-OHT
a b
c
e
G0/vehicle
G4/vehicle
G4/4-OHT
0
1
2
3 *** ***
Mean telomeric/
centromeric signal
G4/vehicle G4/4-OHT
G4/vehicle
G4/4-OHT
d
G0/vehicle
Heat
G0/4-OHT
Heat
G4/vehicle
Heat
G4/4-OHT
Heat
*
0
1,000
2,000
3,000
4,000
f
G4/vehicle G4/4-OHT 0
2
4
6
*
Percentage of
signal-free ends
Figure 1 | 4-OHT-dependent induction of
telomerase activity in TERT-ER cells.
a, Telomerase activity in eNSCs (*, telomerase
products) (top); real-time quantification of
reactions above (bottom). b, Representative
G4TERT-ER splenocyte metaphases. c, Proliferation
of adult G4TERT-ER fibroblasts (n53) in media
with vehicle (black) or 4-OHT (red).
d, Representative image of G4TERT-ER fibroblasts
(passage 6) in media with 4-OHT (bottom) or
vehicle (top). e, Signal-free ends in primary
splenocyte metaphases, 15 metaphases per sample,
n52 (*P,0.05). f, Mean telomere-FISH signal in
primary splenocyte interphases, normalized to
centromeric signal, n53 (***P,0.0001). Open
bars correspond to vehicle-treated and filled bars to
4-OHT-treated, error bars represent s.d.
RESEARCH LETTER
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©2010 Macmillan Publishers Limited. All rights reserved
4 weeks of treatment (Fig. 4, first row). This resumed SVZ proliferation
mirrors well restoration of Sox21 cells, a marker of NSCs (Fig. 4,
second row), and doublecortin (Dcx)-positive cells, an early neuronal
lineage marker, together demonstrating preservation of neural stem/
progenitor reserves and their neurogenic capacity in vivo (Fig. 4, third
row). Finally, quantitative FISH analysis shows telomere elongation in
the SVZ after 4 weeks of 4-OHT treatment (Supplementary Fig. 3).
Thus, the markedly constrained neural progenitor proliferation and
neurogenesis profile associated with telomere dysfunction can be ameliorated
by reactivation of endogenous telomerase activity.
To test the hypothesis that telomerase reactivation leads to tissue
rejuvenation, we conducted detailed morphological and functional
fitness analyses of different brain structures upon telomerase reactivation.
First, we examined the white matter of the corpus callosum and
observed that aged G4TERT-ER mice have far fewer Olig21 mature
oligodendrocytes (Fig. 4, fourth row). This cellular deficiency is associated
with reduced brain weight (Fig. 5a, b) and significantly thinner
myelin sheathing of neurons with g ratios (numerical ratio between the
diameter of the axon proper and the outer diameter of the myelinated
fibre) of 0.775660.0054 forG4TERT-ER mice versus 0.703260.0049 for
G0TERT-ER (mean6s.e.m., ***P,0.0001) (Fig. 5c, d). Remarkably,
endogenous telomerase reactivation reinstates normal numbers of
mature oligodendrocytes (Fig. 4) and reverses the hypomyelination
phenotype at the level of mean myelin sheath diameters (with g ratios
of 0.705860.0006 and 0.716460.0063 for 4-OHT-treated G4 and
G0TERT-ER mice, respectively) (Fig. 5c, d). Furthermore, a 4-OHT treatment
course of only 4weeks is sufficient to cause significant partial
reversion of the brain size defect, with G4TERT-ER brain weights increasing
from 77.363.3% of G0TERT-ER brain weights in the vehicle group to
89.764.0% in the 4-OHT group (Fig. 5a, b). Importantly, telomere
elongation can be detected in the corpus callosum after 4weeks of telomerase
reactivation (Supplementary Fig. 3c). Thus, endogenous telomerase
reactivation exerts a swift impact on oligodendrocyte proliferation
and differentiation, and promotes repopulation of white matter structures
with mature oligodendrocytes and active myelin deposition.
Lastly, we investigated the physiological effect of telomere dysfunction
and telomerase reactivation on olfactory function. Age-associated hyposmia,
as evidenced by an increased olfactory threshold and a reduced
ability in odour identification and discrimination, is a well established
phenomenon in aged humans20. In rodents, ageing is associated with
diminished olfactory neurogenesis and deficits in fine olfactory discrimination19,21.
Olfactory interneurons in the olfactory bulb that receive and
process information from the olfactory sensory neurons in the olfactory
epithelium derive from SVZ stem cells2. Rodents demonstrate avoidance
responses towards predators’ odorants as well as spoiled smells like
aliphatic acids, aliphatic aldehydes and alkyl amines, which are
0
G0 G4
G0 G4 G0 G4
1
2
3 *** *
Percentage
self-renewal
125 100
80
60
40
20
0
100
75
50
25
0
53BP1 foci
per 100 cells
** **
Percentage
multipotency
d e f
aVehicle 4-OHT b 4-OHT c 4-OHT
G4
G0
G4
G0
G4
G0
G4
G0
G4
G0
G4
G0
DAPI 53BP1 DAPI GFAP TUJ1
Vehicle Vehicle Figure 3 | Neural stem cell function following
telomerase reactivation in vitro.
a–c, Representative images of experimental and
control mice-derived NSCs. a, Secondary
neurospheres. b, Differentiated NSCs stained with
53BP1 or c, GFAP and TUJ1 antibodies. d,
Self-renewal capacity of secondary neurospheres
(n54) ***P,0.0001, *P,0.001. e, 53BP1
nuclear foci per 100 cells (.400 nuclei per culture,
n53). f, Multipotency (GFAP1/TUJ11) of NSCs
(n54; 308 wells per culture condition)
**P50.0066. Scale bar, 100 mm. Open bars
correspond to vehicle-treated and filled bars to
4-OHT-treated groups, error bars represent s.d.
c
G4/OHT
0
2
4
6
8 ** *
Pups
per litter
0.25
0.20
0.15
0.10
0.05
0.00
*** ***
Testes weight
(g)
d
g
a
Vehicle 4-OHT Vehicle 4-OHT Vehicle 4-OHT
Testes
120
100
80
60
40
20
0
100
75
50
25
0
** *
53BP1 foci
per 100 cells
b
e
h ** **
Spleen weight
(mg)
Spleen
*** ***
Apoptotic cells
per 100 crypts
G4
G0 G4 G0 G4
G0 G4 G0 G4
G0 G0
G4 G4
G0 G0
G4 G4
G0 G0
G4
G0 G4
G0 G4
150
100
80
60
40
20
0
100
50
0
*** ***
53BP1+ cells
per 100 crypts
f
i
Intestinal crypts Figure 2 | Telomerase activation in adult TERTER
mice. a–c, Representative images of tissues
from experimental and control mice.
a, Haematoxylin and eosin-stained sections of
testes. b, Primary splenocytes stained for 53BP1.
c, Small intestine sections stained for 53BP1.
d, Testes weight of adult males (30–50-week-old,
n$10). e, 53BP1 nuclear foci per 100 nuclei
(n53). f, 53BP1 nuclear foci per 100 crypts (n54).
g, Litter sizes (n53); h, Spleen weights (n$6).
i, Apoptotic cells per 100 intestinal crypts (n$20).
***P50.0001, **P,0.005, *P,0.05. Open bars
correspond to vehicle-treated and filled bars to
4-OHT-treated groups, error bars represent s.d.
LETTER RESEARCH
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©2010 Macmillan Publishers Limited. All rights reserved
processed in the olfactory bulb22. Given the marked decrease in SVZ
neurogenesis of G4TERT-ER mice and the fact that the olfactory bulb
retains high telomerase activity in adult wild-type mouse brains23, we
sought to determine whether telomere dysfunction results in a functional
deficit of these mice to detect and process odorants for elicitation
of instinctive avoidance/defensive behaviours.
Pathology within the olfactory epitheliumwhichmay be considered a
basis of age-related olfactory dysfunction, was ruled out by confirmation
of grossly normal histology of the olfactory epithelium in both
cohorts (Supplementary Fig. 4). Next, we ruled out alterations in
exploration behaviour and overall locomotion by monitoring total distance
travelled by the animals in the absence of odorants, which was
similar for all experimental groups (Supplementary Table 1; Fig. 5e).
We then performed innate avoidance tests using serially diluted
2-methylbutyric acid (2-MB), an odorant that rouses innate aversive
responses in mice. Whereas G0TERT-ER mice demonstrated avoidance
responses at all 2-MB concentrations tested (1.8731024M to
1.8731026 M), G4 mice showed attraction/neutral behaviours at concentrations
lower than 1.8731024M (Fig. 5e, f). Strikingly, following
only 4 weeks of 4-OHT treatment, the performance of G4TERT-ER mice
was markedly improved, with avoidance behaviours being apparent at
all 2-MB concentrations (Fig. 5e, g).Accordingly, the frequency of entry
into the odour zone was higher for vehicle-treated G4TERT-ER mice than
for the other three experimental groups (Supplementary Table 2).These
findings are consistent with significant alleviation of the olfactory defect
stemming from the documented wave of telomerase-mediated SVZ
neurogenesis and oligodendrocyte maturation which would promote
repopulation of olfactory bulbs with functional interneurons and
improve olfactory neuron function via remyelination.
Here, we report the generation of a novel mousemodel to explore the
impact of physiological telomerase reactivation across diverse adult cell
types and organ systems. InG4TERT-ERmice with advanced degenerative
G0
100
250
200
150
100
50
0
250
200
150
100
50
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80
60
40
20
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80
60
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G4
G0 G4
G0 G4
G0 G4
*** **
Ki67+ cells
per section
*** ***
Sox2+ cells
per section
*** ***
DCX+ cells
per section
Dcx Sox2 Ki67
G0/vehicle
Olig2
G0/4-OHT G4/vehicle G4/4-OHT
** **
Percentage of
Olig2+ cells
Figure 4 | NSC proliferation and differentiation
following telomerase reactivation in vivo. NSC
proliferation and neurogenesis were measured by
Ki67, Sox-2 and Dcx expression in SVZ from
experimental and control mice. Mature
oligodendrocytes in the corpus callosum were
stained with anti-Olig2 antibody. Equivalent
coronal sections (n.10) were scored in a blinded
fashion by laser scanning and plotted on the right
panels. 320 (SVZ) or 340 (corpus callosum)
objectives were used. *** P,0.0001,
**P50.0022. Open bars correspond to vehicletreated
and filled bars to 4-OHT-treated groups,
error bars represent s.d.
500
400
300
200
100
0
0.80
0.75
0.70
0.65
G0 G4
G0 G4
*** *
Brain weight (mg)
*** ***
g ratios
d
a
Vehicle 4-OHT
G0
G4
b
f
0
1.7 x 10–6
1.7 x 10–5
1.7 x 10–4
60 60
40
20
0
50
40
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20
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0
G0/vehicle
G4/vehicle
2-MB concentration (M)
0
1.7 x 10–6
1.7 x 10–5
1.7 x 10–4
2-MB concentration (M)
Time in scent zone (s)
Time in scent zone (s)
G0/4-OHT
G4/4-OHT
g
c
G0 G0
Vehicle 4-OHT
G4 G4
Water
G0/vehicle G4/vehicle
2-MB acid Water 2-MB acid
3 2 1
e
G0/4-OHT G4/4-OHT
Figure 5 | Brain size, myelination, and olfactory
function following telomerase reactivation.
a, Representative brains from age-matched
experimental and control animals. b, Brain
weights, n$10, ***P50.0004, *P50.02.
c, Representative electron micrographs of
myelinated axonal tracts in corpus callosum, arrow
heads indicate myelin sheath width (312,000).
Scale bars, 200 nm. d, g ratios (inner/outer radii)
(n52, .150 axons per mouse)
***P,0.0001. e, Representative tracings of
experimental and control mice during 3-min
exposure to water or 2-MB. f, g, Time spent in scent
zone 3 with water or 2-MB for vehicle- or 4-OHTtreated
G0TERT-ER (squares) and G4TERT-ER
(circles) mice; n54. Error bars represent s.d.,
except in (d) (s.e.m.).
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phenotypes, short-term telomerase reactivation restored telomere
reserves, quelled DNA damage signalling, and alleviated cellular checkpoint
responses in several high-turnover organ systems with significant
functional impact including increased fecundity. Fromthis, wespeculate
that some tissue stem/progenitor cells are retained in a quiescent and
intact state yet can be enlisted to resume normal repopulating function
upon elimination of genotoxic stress at telomeres.Despite chromosomal
instability, the brief course of telomerase reactivation was not sufficient
to promote carcinogenesis (data not shown), a finding consistent with a
role for telomerase in promotingprogressionof established neoplasms24.
However, it remains possible that more prolonged telomerase reactivation
schedules or applications in later life may provoke carcinogenesis.
As noted, age-associated compromise inmammalian brain function
is associated with extensive accumulation of DNA damage and progressive
reduction in neurogenesis and myelination. Indeed, many
aspects of this central nervous system decline are accelerated and
worsened in the setting of telomere dysfunction (refs 25, 26, this study).
Our data establish that telomerase reactivation in adult mice with
telomere dysfunction can restore SVZ neurogenesis and, consistent
with its role in sustaining new olfactory bulb neurons, can ameliorate
odour detection with improved performance in innate odour avoidance
tests. These results are consistent with previous studies showing
that prolonged inhibition of neurogenesis in the SVZ has a negative
effect on odour detection thresholds27. In conclusion, this unprecedented
reversal of age-related decline in the central nervous system and
other organs vital to adult mammalian health justify exploration of
telomere rejuvenation strategies for age-associated diseases, particularly
those driven by accumulating genotoxic stress.
METHODS SUMMARY
TERT-ER mice were generated with traditional knock-in methods and following
standard breeding protocol of successive generations of telomerase-deficientmice13.
All studies were performed on adult males. 4-OHT time-release pellets (2.5 mg;
Innovative Research of America) were inserted subcutaneously to reach steady state
blood levels of 1 ngml21 4-OHT. For neurosphere assays, SVZs were dissected,
dispersed into a single-cell suspensionand plated inneurobasalmedia supplemented
with EGF, bFGF and 100nM4-OHT or vehicle. Formultipotentiality assays, neurospheres
were transferred to differentiation media (1% FBS). For histological studies,
mice were perfused with10%formalin; equivalent coronal sections were stainedwith
indicated antibodies following standard immunohistochemistry protocol. Laser
scanning cytometric quantification was performed with an iCys Research Imaging
Cytometer (Compucyte). For innate olfactory avoidance tests, mice were fasted for
20 h and habituated for 20min to the test cage where their responses were recorded
on a video cameramounted above the test chamber.Afilter paper scentedwithwater
or progressively higher concentrations of 2-methylbutyric acid was placed in the
cage and mouse behaviour was recorded for 3min. NoldusEthovision v3.1 behavioural
analysis software was used to determine innate avoidance behaviour (time
spent in the third of the cage containing the scented filter paper).
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 8 May; accepted 26 October 2010.
Published online 28 November 2010.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements The authors would like to thank R. Segal for critical comments,
R. Bronson, K. Ligon and C. Maire for histological advice, S. S. Chae for assistance with
neurospheremeasurement studies and L.Cameron for time-lapse microscopy studies.
M.J. was supported in part by a Susan G. Komen for the Cure fellowship (PDF060881).
F.L.M. was supported by ACS fellowship PF-08-261-01-TBE. This work and R.A.D. was
supported by R01CA84628 and U01CA141508 grants from the NIH National Cancer
Institute and the Belfer Foundation. R.A.D. was supported by an American Cancer
Society Research Professorship.
Author Contributions M.J. and R.A.D. designed and guided the research; M.J., F.L.M.,
J.-H.P., E.S., E.T., S.J. and M.K.-A. performed research. J.C. and J.W.H. generated the
TERT-ER mouse. M.J., F.L.M., A.C.A., A.P., E.M.-F. and R.A.D. analysed data. M.J. and
R.A.D. wrote the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to R.A.D. (ron_depinho@dfci.harvard.edu).
LETTER RESEARCH
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©2010 Macmillan Publishers Limited. All rights reserved
METHODS
Generation of TERT-ER mice.Aknock-in targeting vector containing the ERT2-
LBD domain upstream and in frame with the mTert genomic sequence (exon 1
through intron 2) and a Lox-pgk-Neo-Lox fragment was introduced into ES cells.
Neomycin-resistant clones yielded five independent lines, two of which were
injected into C57BL/6 blastocysts and implanted into surrogate mothers, yielding
10 high-percentage chimaeras. Germline transmission was confirmed by crossing
the chimaeras to C57BL/6 females. Heterozygous TERT-ERneo animals were
crossed to EIIa-Cre animals to delete the NeoR cassette and further intercrossed
to homozygosity.The EIIa-Cre allele was then bred out of the line and heterozygous
animalswere backcrossed toC57BL/6 at least three times. Fromthis point, standard
breeding protocol of successive generations of telomerase-deficient mice was followed13,28.
All studies were performed on adult (30–35-week-old) males, heterozygous
(G0TERT-ER) or homozygous (G4TERT-ER) for this allele, unless otherwise
noted. 4-OHT time-release pellets (2.5mg; Innovative Research of America) were
inserted subcutaneously to reach steady state blood levels of 1 ngml21 4-OHT.
Mice were maintained in specific pathogen-free (SPF) conditions at Dana-Farber
Cancer Institute. All manipulations were performed with IACUC approval.
Histology and electron microscopy. Brains from animals perfused with 10%
formalin were further fixed for 24 h and coronally sectioned using a brain matrix
(Electron Microscopy Sciences). Equivalent sections were used for chromogenic
immunohistochemistry, which was performed according to standard procedures.
Antibodies used include anti-Ki67 (Dako), anti-53BP1 (Bethyl Labs), anti-Sox-2
and anti-Dcx (Santa Cruz Biotechnology) and anti-Olig-2 (Chemicon). For
immunofluorescence studies, cells were fixed in 4% paraformaldehyde (PFA) in
phosphate-buffered saline for 10 min, permeabilized (50mMNaCl, 3mMMgCl2,
200mMsucrose, 10mMHEPES pH7.9, 0.5%TX-100) for 5 min, and then stained
with primary antibodies against 53BP1 (Bethyl Labs), TUJ1 (Chemicon), GFAP
(Dako) and secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-
568 (Molecular Probes). Cells were mounted in DAPI-containing antifade solution
(Vector). Foci were scored by eye from a minimum of 300 randomly chosen
nuclei by using a 340 objective, and scoring was performed in a blinded manner
with respect to genotype. Immunofluorescence images were captured in greyscale
for each fluorophore and were merged by compilation in respective red-green-blue
(RGB) channels usingAdobe PhotoshopCS 8.0. For apoptosis assays, sections from
paraffin-embedded testes were deparaffinized and processed for apoptotic staining
(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling,
TUNEL) according to the manufacturer’s instructions (Chemicon). For electron
microscopy studies, animals were perfused for 30 min in Karnovsky’s solution B,
brains were further fixed for 24 h and delivered to the Harvard Medical School
Electron Microscopy Facility for embedding, sectioning and staining. Electron
micrographs were generated using a JEOL 1200EX microscope and analysed with
ImageJ software29. Inner and outer diameters were analysed as per ref. 30.
Assessment of telomerase activity. Telomeric repeats amplification protocol
(TRAP) was combined with real-time detection of amplification products to
determine telomerase activity with a Quantitative Telomerase Detection kit (US
Biomax) following the manufacturer’s recommendations. Total protein extract
(0.5 mg) was used in each reaction. End products were resolved by PAGE in a
12.5% non-denaturing gel, stained with Sybr Green Nucleic Acid gel stain
(Invitrogen) and visualized with a Bio-Rad Molecular Imager ChemiDoc System.
Cell culture and cytogenetic analysis. Ear skin fibroblasts were isolated as
described previously31. Proliferation assays were carried in triplicate on 6-well
plates. Cells were grown in RPMI-10% fetal calf serum-50 mMb-mercaptoethanol
with the addition of 100nM 4-OHT or vehicle (ethanol). Cells were counted and
replated at a density of 1,000 cells per well every 4 days. Splenocytes were isolated
by generating single-cell suspensions from whole spleen, stimulated for 48 h with
2.5 mgml21 concavalin A and 20 mgml21 LPS (Sigma) and treated with
KaryoMAX Colcemid solution (Invitrogen) for 2 h before collection. Telomere
fluorescence in situ hybridization (FISH) was performed on metaphase nuclei as
described previously28. At least 15 metaphases from harvested cell cultures were
analysed for telomere integrity by telomere-specific peptide nucleic acid (PNA)-
FISH. Telomere signal was normalized using a Pacific Blue centromeric PNA
probe. For telomere-tissue-FISH, frozen tissue sections (8-mm thick) were fixed
in 2% PFA for 15 min and permeabilized in 0.5% Triton X-100 for 10 min. Two
PNA probes, telomere-specific FITC-00-T2AG3 and Pacific Blue-centromerespecific,
were hybridized after 4 min denaturing at 83 uC under the following
conditions: 70% formamide, 0.063SSC, 0.2% BSA, 0.5 ng ml21 tRNA, 0.5 ng ml21
PNA probe; overnight at 25 uC. To achieve uniform hybridization we used MAUI
Mixer (BioMicro) with 40 ml chamber. Nuclei were stained with TOTO3
(Invitrogen) far red stain. Telomere signal was normalized using the centromeric
PNA probe. For neurosphere assays, subventricular region of brain from 3- to
6-week-old mice was dissected, dispersed into a single-cell suspension and plated
in neurobasal media (StemCellTechnologies) supplemented with EGF and bFGF
(20 ng ml21 each) for 4 days, in the presence of 100nM4-OHT or vehicle (ethanol).
Primary neurosphereswere dissociated and seeded at 2 cells per ml density inmultiwell
plates. After 7–10 days, cultures were monitored for the formation of neurospheres.
Alternatively, single cells were sorted into individual wells on 384-well
plates at a density of 10 cells per well on Dako MoFlo high-speed cell sorter and
grownfor 3weeks. Neurosphereswere transferred to culture wells coated with poly-
L-ornithine (15 mgml21) and fibronectin (1 mgml21) and differentiated in 1% FBS
in neurobasal media to measure their multipotentiality. Quantification of neurosphere
numbers and diameterswere performed by bright-fieldmicroscopy coupled
with an in-house semi-automated segmentation algorithm generated with
MATLAB software (The Mathworks). Formultipotentiality assays, cellswere fixed,
stained with GFAP and TUJ1 antibodies and quantified as described previously32.
Laser scanning analysis for the quantification of IHC and FISH. Laser scanning
cytometry quantification was performedwith an iCys Research Imaging Cytometer
(Compucyte) as described earlier32, 33, 34 with a fewmodifications. Counts of Ki671,
Dcx1 or Sox21 cells (DAB positive) were carried out within the subventricular
zones that were predefined by a certified pathologist with the haematoxylin and
eosin-stained brain architecture. The target number for each sample was approximately
500 cells counted. Olig21 cell within the corpus callosumwere counted in a
similar manner.
RT–PCR, Southern blotting and western blotting. DNase-treated total RNA
extracted from fresh liver samples with the RNeasy kit (Qiagen) was used to
prepare oligo-dT complementary DNA with Superscript III (Invitrogen). RT–
PCR primers are described in Supplementary Table 3. Southern and western blots
were performed following standard techniques. For western blots, 40 mg protein
were loaded per lane. Antibodies used include phospho-p53 (Ser15, Cell Signaling
Technologies), p21 (SantaCruz Biotechnology), Actin (Biolegend) and horseradish
peroxidase-conjugated secondary antibodies (Pierce/ThermoScientifics).
Innate olfactory avoidance test. Animals were kept in a 12-h light/dark cycle and
tested in the second half of the light cycle. Male mice (30–35-week-old, n54 per
experimental condition) were fasted for 20 h before testing. To avoid confounding
of data owing to learning, mice were used only once. To habituate to the experimental
environment, mice were placed individually in a cage that was identical to
the test cage (25934763209mm) for 20 min before the onset of testing.
Following acclimation, mice were placed in an identical test chamber where their
responses were recorded on a video camera (30 frames per second, 6403480
pixels) mounted above the test chamber. Confounding environmental/spatial
cue effects were ruled out by monitoring total time spent in different zones of
the chamber in the absence of odorants. For innate avoidance tests, circular filter
paper (2.5 cm diameter) was scented with 40 ml of either water or progressively
higher concentrations of 2-MB (1.7431026M to 1.7431024 M) and mouse
behaviour was recorded for 3 min. Videos were transferred to computer for subsequent
analysis using NoldusEthovision v3.1 behavioural analysis software35.
First the cage was divided into thirds and then time spent in each third for the
duration of the recording was determined using the software. Total distance
travelled, frequency of entry into the third containing the filter paper treated with
the odorants (zone 3) as well as time spent investigating in this zone (to determine
innate avoidance behaviour) was collated for each treatment and genotype.
Statistical analysis. All the data were analysed by one way ANOVA with
Bonferroni’s post test (significantly different at P,0.05). Survival curves were
analysed with Mantel–Cox test.
28. Maser, R. S. et al. DNA-dependent protein kinase catalytic subunit is not required
for dysfunctional telomere fusion and checkpoint response in the telomerasedeficient
mouse. Mol. Cell. Biol. 27, 2253–2265 (2007).
29. Abramoff, M. D., Magelhaes, P. J. & Ram, S. J. Image Processing with ImageJ.
Biophotonics Int. 11, 36–42 (2001).
30. Potzner, M. R. et al. Prolonged Sox4 expression in oligodendrocytes interferes with
normal myelination in the central nervous system. Mol. Cell. Biol. 27, 5316–5326
(2007).
31. Shao, C. et al. Mitotic recombination produces the majority of recessive fibroblast
variants in heterozygous mice. Proc. Natl Acad. Sci. USA 96, 9230–9235 (1999).
32. Paik, J. H. et al. FoxOs cooperatively regulate diverse pathways governing neural
stem cell homeostasis. Cell Stem Cell 5, 540–553 (2009).
33. Mahoney, J. E. et al. Quantification of telomere length by FISH and laser scanning
cytometry. Proc. SPIE 6859, 1–9 (2008).
34. Gorczyca,W. et al. Analysis ofhuman tumors by laser scanning cytometry.Methods
Cell Biol. 64, 421–443 (2001).
35. Spink, A. J. et al. The EthoVision video tracking system–a tool for behavioral
phenotyping of transgenic mice. Physiol. Behav. 73, 731–744 (2001).
RESEARCH LETTER
©2010 Macmillan Publishers Limited. All rights reserved