ARTICLE
DOI: 10.1038/s41467-018-03770-3
OPEN
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Targeting of NAT10 enhances healthspan in a
mouse model of human accelerated aging
syndrome
Gabriel Balmus 1,2, Delphine Larrieu 1,9, Ana C. Barros1,2, Casey Collins 2, Monica Abrudan 2,
Mukerrem Demir1, Nicola J. Geisler1,2, Christopher J. Lelliott 2, Jacqueline K. White2, Natasha A. Karp2,3,
James Atkinson 4, Andrea Kirton2, Matt Jacobsen 4, Dean Clift5, Raphael Rodriguez 6,7,8,
Sanger Mouse Genetics Project, David J. Adams2 & Stephen P. Jackson1
Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, but devastating genetic disease
characterized by segmental premature aging, with cardiovascular disease being the main
cause of death. Cells from HGPS patients accumulate progerin, a permanently farnesylated,
toxic form of Lamin A, disrupting the nuclear shape and chromatin organization, leading to
DNA-damage accumulation and senescence. Therapeutic approaches targeting farnesylation
or aiming to reduce progerin levels have provided only partial health improvements. Recently,
we identified Remodelin, a small-molecule agent that leads to amelioration of HGPS cellular
defects through inhibition of the enzyme N-acetyltransferase 10 (NAT10). Here, we show the
preclinical data demonstrating that targeting NAT10 in vivo, either via chemical inhibition or
genetic depletion, significantly enhances the healthspan in a LmnaG609G HGPS mouse model.
Collectively, the data provided here highlights NAT10 as a potential therapeutic target for
HGPS.
1 The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, UK. 2 The
Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. 3 Discovery Sciences, IMED Biotech Unit, AstraZeneca, Cambridge CB4 0WG, UK.
4 Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Cambridge CB2 23AT, UK. 5 Laboratory of Molecular Biology, Cambridge CB2 OQH, UK.
6 Institut Curie, PSL Research University, Paris Cedex 05, France. 7 CNRS UMR3666, 75005 Paris, France. 8 INSERM U1143, 75005 Paris, France. 9Present
address: Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK. These authors
contributed equally: Gabriel Balmus, Delphine Larrieu. Correspondence and requests for materials should be addressed to Sanger.Mouse.Genetics.Project,
D.L. (email: dl437@cam.ac.uk) or to S.P.J. (email: s.jackson@gurdon.cam.ac.uk)
A full list of consortium members appears at the end of the paper.
NATURE COMMUNICATIONS | (2018)9:1700
| DOI: 10.1038/s41467-018-03770-3 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03770-3
T
he nuclear envelope (NE) provides a dynamic boundary
between the inner nuclear mass and the cytoplasm, and is
critical for normal functioning of the eukaryotic cell. Key
factors for NE function as a compartmental border are the
nuclear lamins, scaffold proteins that specify nuclear architecture
and provide mechanical strength to the nucleus and the cell1.
Notably, over the past few years, lamins have emerged as significant players in many other critical cellular functions, including
differentiation, intracellular signaling, chromatin organization,
transcription, as well as DNA replication and repair2,3. In
mammals, the nuclear lamins are categorized in two distinct
classes: A-type lamins (Lamin A, Lamin C, Lamin C2, and Lamin
AΔ10; all encoded by the LMNA gene) and B-type lamins (Lamin
B1 encoded by LMNB1, and Lamin B2 that, together with Lamin
B3, are encoded by the LMNB2 gene)4. In accord with their
important roles, the loss-of-function mutations in lamin genes
result in genetic syndromes with severe presentations called
laminopathies (OMIM #150330; #150340; #150341). These
include muscular dystrophies (for example, Emery-Dreyfus
Muscular Dystrophy), peripheral neuropathies (for example,
Charcot-Marie Tooth-Disease), leukodystrophy, lipodystrophy, as
well as premature aging (progeria) syndromes, such as Atypical
Werner Syndrome (AWS), Restrictive Dermopathy (RD), and
Hutchinson Gilford Progeria Syndrome (HGPS)2,5. Of all these
syndromes, HGPS is the one with the most striking presentation.
After onset, usually within the first year of life, HGPS patients start
to display short stature, low body weight (BW), hair loss, lipodystrophy, scleroderma, decreased mobility and osteoporosis, as well
as facial features that resemble accelerated aspects of normal ageing6. While the cognitive development is normal, cardiovascular
abnormalities—characterized by medial smooth-muscle cell loss
and secondary maladaptive vascular remodeling (intimal thickening, disrupted elastin fibers, and deposition of atherosclerotic plaques)—are the main reasons for death, with the median life
expectancy at birth for HGPS patients being 14.6 years7–10.
HGPS arises from a heterozygous G608G point mutation of
LMNA exon 11, leading to cryptic mRNA splicing and expression
of a shorter, dysfunctional form of Lamin A, called progerin11–13.
Similar to wild-type Lamin A, progerin undergoes several posttranslational modifications, including the addition of a farnesyl
group required for its targeting to the nuclear envelope. However,
unlike Lamin A, progerin remains permanently farnesylated,
causing it to accumulate on the inner nuclear membrane. In HGPS
cells, progerin acts as a dominant-negative protein, aggregating the
wild-type lamins, disrupting nuclear shape and chromatin organization, and leading to the increased genomic instability and rapid
cell senescence14,15. Although farnesyl transferase inhibition (FTI)
16–20 is being explored as a therapeutic approach for HGPS and has
provided certain health improvements in patients21, there is a clear
need for additional therapeutic regimes22.
Recently, we discovered that a small-molecule compound,
which we named Remodelin, can ameliorate HGPS cellular
phenotypes. Remodelin acts in a progerin- and FTI-independent
pathway, by targeting and inhibiting the N-acetyltransferase
NAT1023. Here, we assess NAT10 inhibition as a potential
therapeutic strategy for HGPS by using an established mouse
model (Lmna—G609G allele) that exhibits premature-aging
phenotypes similar to those of HGPS patients24. Critical for
translating NAT10 inhibition toward human patients, we show
that chemical or genetic targeting of NAT10 decreases the
genomic instability, and improves age-related phenotypes of both
homozygous and heterozygous HGPS mice.
Results
Remodelin ameliorates age-dependent weight loss in HGPS
mice. To determine the effects of Remodelin on HGPS mouse
2
NATURE COMMUNICATIONS | (2018)9:1700
cells, we derived skin fibroblasts from LmnaG609G/G609G and wildtype (WT) littermates. As observed in human HGPS fibroblasts25,26, LmnaG609G/G609G fibroblasts displayed nuclear shape
defects and increased genomic instability, as reflected by a higher
level of the DNA double-strand break (DSB) marker gamma
H2AX (γH2AX, Ser-139 phosphorylated histone H2AX), in a
manner that was abrogated by Remodelin treatment (Fig. 1a, b).
These results showed that Remodelin treatment can reverse the
HGPS induced genomic instability and nuclear shape defects in
mouse cells, and provided us with encouraging preliminary evidence to proceed with in vivo studies.
To assess Remodelin’s suitability for in vivo studies, we initially
defined its pharmacokinetic properties in WT mice. Overall, the
oral (PO) delivery appeared to be the best route of administration
(Fig. 1c; Supplementary Fig. 1a; Supplementary Tables 1–2), with
bioavailability of ~44% (Fig. 1c,) and significant accumulation in
heart and skeletal muscle (Fig. 1e). Based on these data, WT and
LmnaG609G/G609G mice were treated with a Remodelin dose of
100 mg per kg orally, on a daily schedule from 3 weeks of age
onward, until the end-point. This treatment was well-tolerated by
both genotypes, with no weight loss (Supplementary Fig. 1b) and
with a significant amount of Remodelin being present in the
skeletal muscle, liver, and brain of the LmnaG609G/G609G mice
(Supplementary Fig. 1c). Furthermore, in line with a previous
report that NAT10 promotes melanogenesis27, we found that
Remodelin treatment led to hair graying (Supplementary Fig. 1d).
As described before, we found that LmnaG609G/G609G mice had
dramatically shorter healthspans, compared to WT controls,
associated with accelerated body-weight loss (Fig. 1f; Supplementary Table 3) and premature-aging phenotypes, resembling the
human syndrome24. Notably, Remodelin led to a 25% increase in
Kaplan–Meier area under the curve, based on 20% BW loss, for
the treated vs. the vehicle-treated mice (Fig. 1f; Supplementary
Table 3). Together, these data established that Remodelin is welltolerated in vivo and delays the age-dependent weight decline in
HGPS mice.
Remodelin ameliorates cardiac pathology of HGPS mice. HGPS
clinical presentation includes the loss of subcutaneous adipose
tissue7 and cardiovascular abnormalities, such as adventitial
fibrosis and medial smooth-muscle cell loss, with depletion of
smooth-muscle actin in the remaining cells8,9,28,29. These cardiovascular features represent major pathologies that contribute to
morbidity and lethality in HGPS2. We analyzed all these parameters at the level of the skin, aorta, and coronary heart arteries
in Remodelin-treated HGPS mice, as compared to the vehicletreated controls at their respective end-points. Importantly,
Remodelin treatment significantly reduced the loss of subcutaneous adipose tissue that is seen in the HGPS mouse model
(Fig. 2a). Moreover, it led to the dramatic amelioration of HGPS
cardiac pathologies, including reduction of adventitial fibrosis of
the aorta (Fig. 2b), rescue of vascular smooth muscle cell loss
(Fig. 2c), and salvage of smooth muscle actin (SMA) loss, both in
the aorta and the coronary arteries (Fig. 2d, Supplementary
Fig. 2a). By contrast, Remodelin treatment had no significant
effect on WT mice at the similar age (Supplementary Fig. 2b).
Furthermore, as observed in mouse and human fibroblasts,
Remodelin reduced the markers of genome instability in both
heart and lung tissues of LmnaG609G/G609G mice (Fig. 2e,f).
Together, these data highlighted how Remodelin treatment
delayed the onset of cardiovascular pathologies that represent the
most debilitating aspect of HGPS.
Engineering and characterization of a Nat10 mouse model. To
validate NAT10 as the relevant pharmacological target mediating
| DOI: 10.1038/s41467-018-03770-3 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03770-3
Nat10+/− mice (Supplementary Fig. 3b, c, Supplementary Fig. 4
and, Supplementary Data 1). In most regards, Nat10+/− mice
were indistinguishable from WT mice (blue color Supplementary
Fig. 3b), with the exception of BW, which was consistently lower
than that of WT mice, despite similar tail-to-nose lengths
associated with changes in the lean and fat mass, triglycerides,
and cholesterol levels (Supplementary Fig. 3b, Supplementary
Fig. 4a). Additional parameters, including potassium levels and
neutrophil numbers, showed evidence of sexual dimorphism
the in vivo effects of Remodelin, we engineered a Nat10 knockout
mouse model (Supplementary Fig. 3a). Bi-allelic Nat10 inactivation was lethal before embryonic day E14.5 (Fig. 3a), indicating
that NAT10 is critical for mouse development. However, heterozygous Nat10+/− mice were born healthy, and were observed
to express the Nat10 mRNA and protein products, at levels ~50%
of those in WT animals (Fig. 3b,c).
As NAT10 is largely uncharacterized and has not been studied
in mice before, we performed a broad phenotypic analysis of
a
n.s.
60
Remodelin
*
40
20
40
20
0
γH2AX
WT (n=3)
LmnaG609G/G609G
Vehicle (n=3)
Remodelin (n=3)
–
+
95
72
γH2AX
17
Tubulin
Concentration (ng per ml)
–
Lamin A
Progerin
Lamin C
0.4
0.2
IV - 1 mg per kg (n=3)
10
1
0
1
R
KDa
F = 43.5%
100
0.0
55
PO - 5 mg per kg (n=3)
1000
W
Ve T
hi
em cle
od
el
in
Remodelin:
Remodelin (n=3)
c
0.6
LmnaG609G/G609G
Relative γH2AX levels
(A.U.)
WT
T1/2 (h)
1.81
Tmax (h)
0.25
–1
Cmax (ng ml )
106
Cardiac muscle
Skeletal muscle
409
AUC0•t (ng h–1 ml–1)
235
AUC0•∞ (ng h–1 ml–1)
259
MRT_last (h)
0.84
Bioavailability (%)
f
e
43.5%
105
% Survival to
20% body weight loss
(end-point)
PO - 5 mg per kg
Concentration (nM)
d
*** **
60
0
WT (n=3)
LmnaG609G/G609G
Vehicle (n=3)
b
n.s.
80
*
% Misshapen nuclei
Vehicle
DAPI
Vehicle
% γH2AX positive cells
LmnaG609G/G609G
WT
2
Time (h)
3
4
100
*p <0.01
WT
Vehicle (n=4)
Remodelin (n=5)
50
LmnaG609G/G609G
Vehicle (n=18)
Remodelin (n=13)
0
0
104
300 mg
400 mg
25
50
75
Age (days)
100
125
Fig. 1 Oral administration of Remodelin decreases weight loss in progeria mice. a, b Cells were treated with DMSO or with 1 µM Remodelin for 7 days. a
Left: Representative immunofluorescence images of skin fibroblasts from LmnaG609G/G609G mice showing the accumulation of the DNA double-strand
break marker gamma H2AX (γH2AX) ( blue) and characteristic nuclear shape abnormalities, observed by DAPI staining. All images were acquired with the
same microscope intensity settings. Scale bar 20 µm. Right: Quantification of γH2AX positive cells and cells with misshapen nuclei (>100 cells/n = 3
independent cell lines; mean ± s.d.; n.s. not significant; *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t-test). b Western blotting analysis of γH2AX
levels (quantified in the right panel) in skin fibroblasts from the indicated genotypes. c,d Pharmacokinetic analyses of Remodelin in mice treated via oral
(PO; n = 3) or intravenous (IV; n = 3) delivery; mean ± s.e.m. F absolute bioavailability (%). e Tissues were collected after 2 weeks of daily PO
administration of the indicated Remodelin concentration and 1 h after the last dosing. Remodelin was quantified by mass spectrometry in the heart and the
skeletal muscle (n = 3); mean ± s.e.m. f Survival based on 20% body weight loss, showing a 25% increase in Kaplan–Meier area under the curve in
Remodelin-treated LmnaG609G/G609G mice, as compared to vehicle-treated mice (see Supplementary Table 3); (*Log-rank Mantel-Cox test; Chi-square
5.992). Due to animal welfare regulations, mice had to be killed when they had lost 20% of their body weight, compared to their individual weight maxima
(end-point). However, at this defined end-point, Remodelin-treated mice displayed considerably better health, compared to the vehicle-treated controls
(see supplementary movies and pathology assessments in Fig. 2)
NATURE COMMUNICATIONS | (2018)9:1700
| DOI: 10.1038/s41467-018-03770-3 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03770-3
(Supplementary Fig. 4b). Moreover, NAT10 reduction triggered
gene-expression changes in the heart (Fig. 3d, Supplementary
Data 2), with many deregulated genes being connected to cellular
pathways associated with longevity, such as responses to
starvation or insulin signaling30 (Supplementary Fig. 3c). Other
highly enriched pathways deregulated upon NAT10 depletion
included regulation of inclusion body assembly and protein
refolding. These data showed that, while complete NAT10 knockout leads to embryonic lethality, NAT10 haploinsufficient mice
b
Skin fat layer (μm)
+ Remodelin
200
100
40
30
20
10
0
0
H&E (skin)
H&E (aorta)
d
Wild type
n.s.
Nuclei per mm2
LmnaG609G/G609G
4
*
+ Remodelin
*
LmnaG609G/G609G
Wild type
+ Remodelin
3
2
1
0
DAPI (aorta)
e
Remodelin
f
150
100
50
Lung
Lmna
–
*
**** ***
0
G609G/G609G
–
200
SMA (aorta)
Heart
WT
–
+ Vehicle
+ Vehicle
LmnaG609G/G609G
+ Vehicle
LmnaG609G/G609G
+ Vehicle
SMA intensity
per 50 μm2
c
*
**** **
50
LmnaG609G/G609G
Wild type
LmnaG609G/G609G
+ Vehicle
Wild type
*
**** ***
300
+ Remodelin
+ Vehicle
LmnaG609G/G609G
+ Vehicle
LmnaG609G/G609G
+ Vehicle
Nat10 haploinsufficiency delays health decline of HGPS mice.
To explore the interaction between NAT10 and HGPS, we generated two cohorts of LmnaG609G/G609G mice: one on a WT Nat10
background (Nat10+/+) and the other carrying the Nat10
Adventitial width
(μm)
a
are born at expected frequencies and are overtly healthy, thereby
allowing us to explore the potential impacts of reducing Nat10
gene dosage in the context of a HGPS in vivo genetic model.
+
+
Lamin A
95
Lamin A
Progerin
Lamin C
γH2AX
Remodelin
WT
–
LmnaG609G/G609G
–
–
+
+
95
Progerin
72
Lamin C
γH2AX
17
72
17
H2AX
17
KDa
H2AX
0.6
17
KDa
0.15
4
A.U.
A.U.
0.4
0.10
0.2
0.05
0
0.00
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| DOI: 10.1038/s41467-018-03770-3 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03770-3
heterozygous deletion (Nat10+/−). Notably, as for Remodelin
treatment, reduced Nat10 gene dosage significantly extended
the timeframe of body-weight loss of LmnaG609G/G609G mice by
17% (Fig. 4a; Supplementary Table 3). Moreover, as compared to
LmnaG609G/G609G animals, LmnaG609G/G609GNat10+/− mice displayed a significant delay in the appearance of back curvature
(Fig. 4b–d) and enhanced fitness, as observed by lower incidence
of common LmnaG609G/G609G mouse pathologies, such as penile
prolapse and eye keratitis (Supplementary Fig. 5a). Additionally,
LmnaG609G/G609GNat10+/− mice were more active and overtly
healthier than their age-matched controls (Supplementary
Movies 1–3).
To better understand how HGPS phenotypes were counteracted by NAT10 depletion, we performed selected biochemical
analyses on mice at 9 and 12 weeks of age. While NAT10
depletion had no effects on parameters such as fat mass and
cholesterol levels of progeria mice, it significantly normalized
others, including glycerol and urea levels (Supplementary Data 3).
Furthermore, the decrease in heart rate observed in LmnaG609G/
G609G mice, compared to WT controls, was significantly
circumvented by Nat10 depletion at 9 weeks, but not at 12 weeks
(Fig. 4e), suggesting that the onset of heart abnormalities was
delayed. Notably, at 12 weeks, p21 expression was increased in the
hearts of LmnaG609G/G609G mice but not in LmnaG609G/
G609GNat10+/- mice, probably reflecting higher DNA-damage
loads (Fig. 4f).
While LmnaG609G/G609G mice were reported to be infertile24,
under our husbandry they were sub-fertile: LmnaG609G/G609G
males had decreased sperm counts (Supplementary Fig. 5b), but
these sperms were motile and able to fertilize wild-type oocytes
(Supplementary Fig. 5c). Similarly, LmnaG609G/G609G females
produced morphologically normal oocytes that were meiotically
competent (Supplementary Fig. 5d, e), and the super-ovulated
eggs from LmnaG609G/G609G females could be fertilized by WT
sperm (Supplementary Fig. 5c). However, while LmnaG609G/G609G
females never produced litters, LmnaG609G/G609G males could
father pups, albeit at a very low frequency and never more than
one litter. These data suggested that sub-fertility of HGPS mice
was caused by decreased reproductive fitness, rather than by a
specific physiological problem. Strikingly, global fitness/health
improvements associated with reduced Nat10 gene dosage in
LmnaG609G/G609GNat10+/− mice (Supplementary Movies 2–3)
correlated with an enhancement of male and female fertility
(Supplementary Fig. 5f; 45% in LmnaG609G/G609GNat10+/− vs.
21% in LmnaG609G/G609G).
While we, like others, have used homozygous LmnaG609G/
G609G to model HGPS, it is important to note that patients carry a
heterozygous Lmna mutation, leading to the expression of both
WT Lamin A and progerin. We therefore wished to study the
effect of reducing Nat10 gene dosage in heterozygous Lmna
+/G609G mice. Extending upon the previous report showing
reduced lifespan of such mice24, we performed an extensive
phenotypic analysis of Lmna+/G609G mice and found them to
display similar premature aging phenotypes as the homozygous
mutant (Supplementary Fig. 6, Supplementary Data 4), albeit
with delayed onset. When we assessed the impact of Nat10
heterozygosity in this human-disease model, we found that Lmna
+/G609GNat10+/− mice lived significantly longer before displaying
age dependent body-weight decline, compared to Lmna
+/G609GNat10+/+ mice. Indeed, there was a 90 day gap between
the longest lived (based on 20% BW loss) Lmna+/G609GNat10+/−
mouse and the longest lived Lmna+/G609GNat10+/+ mouse
(Fig. 4g; Supplementary Table 3). In addition, the Lmna
+/G609GNat10+/− mice displayed decreased back curvature
(Supplementary Fig. 7). Taken together, these data show that
Nat10 haploinsufficiency enhances health in both the homozygous
and the heterozygous HGPS mouse models.
Identification of readouts for NAT10 inhibition in HGPS. To
investigate the impacts of NAT10 inhibition in vivo, and as the
main cause of death in HGPS patients is due to heart dysfunction,
we performed global gene-expression analyses on tissues derived
from hearts of LmnaG609G/G609GNat10+/− and Remodelin-treated
LmnaG609G/G609G mice, compared to their respective controls
(Supplementary Data 5). This work identified a specific set of
genes (Supplementary Fig. 8a)—largely connected to metabolic
pathways (Supplementary Fig. 8b)—as significantly upregulated
(blue) or downregulated (red) in LmnaG609G/G609G mice, compared to WT (Supplementary Fig. 8a, lane 1). Interestingly, some
of these gene-expression differences (* in Supplementary Fig. 8a)
were counteracted by both Remodelin treatment (row 2) and
Nat10 depletion (row 3). Further investigation would be required
to establish whether these might comprise a gene-expression
signature for amelioration of HGPS pathologies by NAT10
inhibition. Collectively, these data highlighted a strong correlation between NAT10 chemical inhibition and its genetic depletion
on cellular imbalances, caused by the Lmna G609G/HGPS
mutation. They also suggested the potential for gene-expression
signatures as readouts for HGPS and its alleviation.
As acetylation of α-tubulin lysine 40 (K40) is a documented
NAT10 target31,32, we evaluated it as another potential readout
for NAT0 inhibition in vivo. Indeed, quantitative analyses
established that patient-derived HGPS fibroblasts, as well as
LmnaG609G/G609G mouse-derived skin cells and tissues, displayed
increased acetyl-α-tubulin K40, when compared to WT controls
(Fig. 5; Supplementary Fig. 9), probably associated with increased
microtubule stability, contributing to HGPS cellular phenotypes25. However, this was not associated with significant and
consistent increased NAT10 protein levels, suggesting that
NAT10 enzymatic activity might be elevated, leading to increased
α-tubulin acetylation. In line with these findings, Remodelin
Fig. 2 Remodelin ameliorates cardiac and other pathologies of HGPS mice. Pathological staining in panels a–d was carried out on the materials from endpoint mice (presented in Fig. 1f) of indicated genotypes (n = 6 per genotype) treated with vehicle or Remodelin 100 mg per kg per day (for detailed ages of
the mice, see Supplementary Table 4). All images are representative (scale bar 50 µm) and the correspondent bar graph quantifications are presented
(mean ± s.e.m.; individual data points represented; n.s. not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 two-tailed Student’s t-test). In WT
mice, Remodelin treatment has no significant effect, as compared to vehicle treatment; and for simplicity these animals have been pooled in one group; the
individual comparison is presented in Supplementary Fig. 2b. a Hematoxilin and eosin (H&E) staining of skin, indicating fat layer thickness (vertical bars
indicate the fat layer) and showing amelioration of the fat layer thickness upon Remodelin treatment in HGPS mice. b H&E staining of heart aorta, indicating
increased adventitial width in the HGPS mice, as compared to WT controls, which is rescued by Remodelin treatment (arrowheads demarcate the aorta
and vertical bars the adventitia). c DAPI staining of heart aorta, showing a decreased number of nuclei in the HGPS mice, rescued by Remodelin treatment
(dotted white lines delineate the aorta edges). d Smooth muscle actin (SMA) staining (green) of heart aorta sections, showing loss of integrity of the artery
wall in HGPS mice, improved by Remodelin treatment (dotted white lines delineate the aorta). e, f Representative western blotting analysis of the
representative heart (e) and lung (f) tissues from end-point mice, showing that Remodelin decreased γH2AX levels in LmnaG609G/G609G tissues (see
quantification below each Western blot, relative to total H2AX levels). Western blots were performed more than once on n ≥ 4/group
NATURE COMMUNICATIONS | (2018)9:1700
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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03770-3
E14.5
Expected
10
*
5
Number of mice
Mrpl23-ps1
Dnajb2
60
Angptl4
40
Eps8l1
20
Arrdc2
*
0.50
0.25
+/
–
+/
–
0.75
0.50
0.25
0.00
0.00
WT
c
1.00
Nat10 +/–
Nat10 +/–
WT
17
72
β-actin
WT
Nat10 +/–
–4
Cyp26b1
Gm10709
Pdk4
Klf15
Fosl2
Otud1
Plin1
Bhlhe40
Nat10
Gm15501
Dnajb1
Irs2
130
γH2AX
Lamin A
Progerin
Lamin C
–2
Nrn1
Lung (4 week old)
NAT10
Gene expression changes (log fold)
0.75
0
Map3k6
***
1.25
2
Fam107a
N
at
10
T
W
RNA expression (RQ)
Lung - 12 weeks old
1.00
N
at
10
+/
–
+/
–
N
at
10
T
W
N
at
10
****
1.25
4
Fstl4
0
0
40
KDa
Relative NAT10
levels (A.U.)
Number of mice
15
Gm4202
Trim9
80
20
RNA expression (RQ)
Heart - 12 weeks old
Observed
Expected
Observed
25
b
d
3 weeks old
log fold change
a
Fos
1
Nr4a1
Hspa1b
0.5
Atf3
Hp
0
LmnaG609G/G609G
WT
Nat10 +/–
LmnaG609G/G609G
Hspa1a
Gm12966
C330021F23Rik
Fig. 3 Engineering and characterization of a Nat10+/− mouse model. a Number of observed embryos (E14.5) and mice (21 days), compared to the expected
numbers (Mendelian frequencies) (*p < 0.01; Chi-square analysis). b, c Nat10+/− mice display ~50% reduction in Nat10 transcript level (b each bar
indicates individual mice ± s.d. of n = 5 technical replicates/mouse) or protein expression in the indicated tissues (c representative western blot; blots have
been performed more than once on n ≥ 3 mice), and the quantification is presented on the right panel, relative to actin levels. d Heatmap of genes
differentially expressed in the heart of Nat10+/− mice, compared to the wild-type, from RNAseq analysis (n = 2/genotype)
treatment or reduced Nat10 gene dosage decreased acetyl-αtubulin K40 levels in extracts from cells and mouse tissues
(Fig. 5a, b; Supplementary Fig. 9a, b), in cultured cells (Fig. 5c),
and as detected by in situ mouse-tissue staining (Fig. 5d;
Supplementary Fig. 9cd). These results thus indicated that αtubulin K40 acetylation is increased in both human HGPS cells
and in the mouse HGPS model, in a manner counteracted by
Remodelin treatment, thereby suggesting this acetylation mark as
a potential readout for NAT10 inhibition in vivo.
Discussion
HGPS, a debilitating premature aging disease whose features
strikingly resemble the accelerated aspects of normal aging,
represents a paradigm for translational medicine in the arena of
aging research33. The complex nature of this segmental syndrome
makes it difficult to target therapeutically, with standard therapeutic approaches mainly aiming at preventing the expression or
the accumulation of progerin on the nuclear envelope. Thus,
strategies that have so far received most attention largely involve
the targeting enzymes participating in the progerin pathway10,
including HMG-CoA reductase, farnesyl-pyrophosphate synthase, farnesyl transferase21, isoprenylcysteine carboxyl methyltransferase, as well as modulating Lmna pre-mRNA splicing by
morpholino compounds24. Notably, there is encouraging evidence that HGPS children treated with the FTI lonafarnib display
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NATURE COMMUNICATIONS | (2018)9:1700
improved vascular stiffness and bone structure21, as well as 33%
increased survival, based on Kaplan–Meier area under the curve
estimations10. Because FTI treatment is now essentially standardof-care and the HGPS patient population is small (~1 in 18
million)34, further clinical trials will likely be arranged as combined therapies with lonafarnib22.
Our results show that NAT10 genetic depletion or its chemical
inhibition by the compound Remodelin lead to healthspan
enhancements— as indicated by effects on age-dependent BW
loss, cardiac function, back curvature, fitness, and genomic
instability—in both homozygous and heterozygous HGPS mice,
through a mechanism that appears to be independent of progerin.
Reinforcing our conclusions, we have recently carried out further
studies with a Remodelin derivative, Remodelin-fluor (Supplementary Fig. 10a), that in HGPS cells showed the same effect as
Remodelin at half the Remodelin dose (Supplementary Fig. 10b).
Using this compound in mice, we observed hair graying (Supplementary Fig. 10c) and no drug-induced weight loss (Supplementary Fig. 10d). Moreover, Remodelin-fluor treatment led to a
significant decrease in the timeframe of body-weight loss along
with 30% increase in Kaplan–Meier area under the curve for
treated mice, as compared to vehicle-treated mice (Supplementary Fig. 10e; Supplementary Table 3). While we did not see
Remodelin-induced effects on low mineral density and bone
mineral content in HGPS mice (see Supplementary Data 3), we
observed significant disease amelioration by Remodelin treatment
| DOI: 10.1038/s41467-018-03770-3 | www.nature.com/naturecommunications
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a
% Survival to
20% body weight loss
(end-point)
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03770-3
b
100
**p<0.0052
WT (n=7)
50
LmnaG609G/G609G
Nat10 +/+ (n=34)
Nat10 +/– (n=16)
0
0
25
50
75
Age (days)
100
WT
125
LmnaG609G/G609G
d
LmnaG609G/G609G
Nat10 +/+
800
LmnaG609G/G609G
Nat10 +/–
650
g
9
12
Age (weeks)
p21 RNA expression
(RQ)
f
n.s.
2.5
* *
2.0
1.5
WT (n=5)
LmnaG609G/G609G
Nat10 +/+ (n=6)
Nat10 +/– (n=6)
1.0
0.5
0.0
1.0
0.5
0.0
LmnaG609G/G609G
Nat10 +/+ (n=7;n=5)
Nat10 +/– (n=4; n=8)
LmnaG609G/G609G
Nat10 +/+ (n=5)
Nat10 +/– (n=8)
700
**
WT (n=3)
WT (n=4)
750
1.5
9
12
Age (weeks)
***
100
% Survival to 20% body
weight loss (end-point)
Heart rate (bmp)
e
Relative kyphotic index
(A.U.)
c
WT
Nat10 +/– Nat10 +/+
Lmna+/G609G
Nat10 +/+ (n=30)
Nat10 +/– (n=13)
50
*p =0.025
0
0
200
250
300 350
Age (days)
400
450
Genotype
Fig. 4 Genetic depletion of Nat10 enhances the health of LmnaG609G mice. a LmnaG609G/G609GNat10+/− mice show a 21% increased median age at the endpoint, compared to LmnaG609G/G609G (103 days vs. 85 days respective median age at end-point; based on the mice being terminated upon reaching 20%
body weight loss); (**Log-rank, Mantel-Cox test; Chi-square 32.61; also see Supplementary 3). b–d Appearance (b) of back curvature (c) in LmnaG609G/
G609G mice is delayed by Nat10 depletion, as observed by images of terminal mice and X-rays from 9 week-old females, and quantified (kyphotic index)
over time (d) (mean ± s.e.m.; individual data points represented; mixed model analysis shows a significant difference between LmnaG609G/G609GNat10+/+
and LmnaG609G/G609GNat10+/− genotypes **p = 0.01; raw data and extended conclusions and statistics are presented in Supplementary Data 3). e
Progressive heart function failure observed in LmnaG609G/G609G mice over time is delayed by Nat10 depletion, as observed by the heart rate measurements
at indicated times (mean ± s.e.m.; individual data points represented; mixed model analysis shows a significant difference between LmnaG609G/G609GNat10
+/+ and LmnaG609G/G609GNat10+/− genotypes ***p = 0.004; raw data, extended conclusions, and statistics are presented in Supplementary Data 3). f
RNA expression from heart tissues shows decreased p21 expression in LmnaG609G/G609GNat10+/−, compared to controls (mean ± s.e.m.; n.s. not
significant, *p < 0.05 two-tailed Student’s t-test; individual data points represented). g Lmna+/G609GNat10+/− mice show a 17% increased median age at
end-point, compared to Lmna+/G609G (333 days vs. 285 days respective median age at end-point; based on mice being terminated upon reaching a 20%
body weight loss); (*Log-rank, Mantel-Cox test; Chi-square 4.98; also see Supplementary Table 3) and more than 90 days between the longest lived (20%
body weight loss) Lmna+/G609GNat10+/− and the longest lived Lmna+/G609G mouse
and Nat10 genetic depletion in critical organs, such as the heart.
Notably, these cardiovascular effects were not only restricted to
the aorta, but also included impacts on other large vessels of the
heart, such as the coronary arteries. These effects are of potential
clinical relevance because advanced coronary disease is prevalent
in HGPS patients, even in the absence of high blood pressure29,35.
We also note that, in contrast to FTI36, Remodelin decreases
markers of genome instability in HGPS cells and organs. We
showed in our previous work that combining FTI and Remodelin
NATURE COMMUNICATIONS | (2018)9:1700
in HGPS patient cells does not improve the cellular phenotypes
further, compared to Remodelin alone (see Supplementary Fig. 8
of Larrieu et al., Science 201425). However, as they act in different
pathways, it is possible that the phenotypes in vivo would benefit
from the drug combination, which will likely be the scope of
ensuing studies.
Collectively, our data thus highlights the potential for NAT10
inhibitors in treating HGPS in combination with lonafarnib,
where additive therapeutic effects might be anticipated. While we
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a
c
Remodelin:
NAT10
130
Lamin A
95
Lamin C
72
Ac-α-Tubulin
(K40)
55
Ac-α-Tubulin
(K40)
Merged
HGPS
+ Vehicle
Progerin
Tubulin
55
KDa
HGPS
+ Remodelin
1.2
0.8
0.4
0.0
1
2
3
d
4
+ Vehicle
130
NAT10
*
Lamin A
Progerin
Lamin C
Ac-α-Tubulin
(K40)
95
72
55
+ Vehicle
LmnaG609G/G609G
b
Wild type
Relative
Ac-α-Tubulin (K40)
levels (A.U.)
Lamin A/C
HGPS
– +
Healthy
+ Vehicle
Healthy
–
+
55
KDa
+ Remodelin
2.5
0
1. Wild type
2. LmnaG609G/G609G + Vehicle
3. Lmna
G609G/G609G
+ Remodelin
4. LmnaG609G/G609GNat10 +/–
DAPI ; Ac-α-Tubulin (K40)
Aorta
% Cells with ac-α-Tubulin (K40)
in the Aorta wall
5
LmnaG609G/G609G
Relative
Ac-α-Tubulin (K40)
levels (A.U.)
Tubulin
50
n.s.
**
*
40
30
20
10
0
Fig. 5 Identification of potential readouts for Nat10 inhibition in cells and tissues. a,b Representative images of western blots showing that 1 µM Remodelin
treatment for 7 days decreases the high alpha-Tubulin (α-tubulin) K40 acetylation in HGPS-patient derived cells (a) and mouse tissues (b). In panel b
NAT10 chemical (lane 3) or genetic (lane 4) inhibition reverses the high α-tubulin K40 acetylation levels in heart tissues from indicated mice; *indicates a
cross-reacting band. We note that the ratio between Lamin A and C appears to vary between tissues. All western blotting experiments were performed
independently at least three times (n ≥ 3/genotype). c Representative immunofluorescence images of acetyl-α-tubulin K40 in HGPS-patient derived cells,
as compared to matching healthy fibroblasts. Scale bar = 20 µm. The K40 α-tubulin acetylation (magenta) is increased in the HGPS-patient derived cells
and decreased upon Remodelin treatment. d Representative immunofluorescence images (left) and quantification (right) of acetyl-α-tubulin K40 in aortas
of terminal mice of the indicated genotypes and treatments. Scale bar = 10 µm. The K40 α-tubulin acetylation (green; white arrowheads point to example
of cells that show increased K40 acetylation) is increased in LmnaG609G/G609G mice and significantly decreased in such mice, upon Remodelin treatment (n
= 3; mean ± s.d.; individual data points represented; n.s. not significant, *p < 0.05; **p < 0.01, ***p < 0.001; two-tailed Student’s t-test). For better
visualization, these are higher magnification snapshots (red dotted squares) from images in Supplementary Fig. 9d. Quantification was performed on full
size aorta images from n = 3 independent mice
found that complete Nat10 deletion resulted in early embryonic
lethality in mice, Nat10 haploinsufficiency or chemical inhibition
via Remodelin treatment did not confer any profound side effects.
Because NAT10 is a 115 kDa protein with multiple functional
domains, it could be that the lethality associated with its total loss
is linked to another aspect of the NAT10 protein, other than its
N-acetyltransferase function. In this regard, we note that in
Caenorhabditis elegans, a null allele of NAT10, nath-10(tm2624),
causes fully penetrant embryonic lethality in the homozygous
state. By contrast, the nath-10(N2) hypomorphic allele,
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containing a polymorphism in the N-acetyltransferase domain,
did not cause pathology in a homozygous setting, but instead
conferred increased fitness and a strong competitive advantage
over WT animals37. While these and our data are encouraging
from the perspective of considering NAT10 inhibition as a
therapeutic approach for HGPS, the fact that our understanding
of NAT10 biology is still in its infancy means that any clinical
studies should be explored with caution.
The small number of HGPS patients and the diverse nature of
their pathologies provide challenges for the evaluation of
| DOI: 10.1038/s41467-018-03770-3 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03770-3
potentially new HGPS therapies in the clinic22. If NAT10 inhibitors are explored in clinical settings, it will thus be extremely
valuable to assess target engagement. In this regard, it will be of
interest to investigate the potential of the gene-expression signatures that we have found to be associated with HGPS cells, and
which are rebalanced toward WT profiles by either Remodelin
treatment or NAT10 depletion. Furthermore, our data suggests
that acetylated α-tubulin lysine 40 (K40), a known NAT10 target31,32, might also be used as a readout for NAT10 inhibition in
cells and in vivo. These findings also correlated with our previous
data, indicating that NAT10 inhibition ameliorates HGPS cellular
phenotypes, at least in part, by mediating microtubule destabilization25. As α-tubulin acetylation at K40 is elevated in HGPS
tissues and the cells are compared to controls, it will be of interest
to explore whether this could be used to monitor disease progression and also enhance our understanding of disease pathobiology. Finally, we speculate that because the hallmarks of HGPS
are present at lower levels in the vasculature and other tissues of
aged-normal individuals33, NAT10 targeting might offer therapeutic opportunities in broader settings. In accord with such a
possibility, we have recently reported the effects of NAT10
inhibition in normally aged smooth muscle cells26, that might
suggest the potential for NAT10 inhibition in the context of
normal ageing.
Methods
Synthesis of Remodelin and derivatives. All solvents and reagents were purified
using standard techniques, or used as supplies from commercial sources (SigmaAldrich). NMR spectra were acquired on a Bruker 500 MHz instrument, with
deuterated solvents at 300 K. Notation for the 1H NMR spectral splitting patterns
includes: singlet (s), doublet (d), triplet (t), broad (br), and multiplet/overlapping
peaks (m). Signals are quoted as values in ppm and coupling constants (J) are
quoted in Hertz. Mass spectra were recorded on a Micromass® Q-Tof (ESI)
spectrometer. General procedure: The appropriate ketone or aldehyde was dissolved in isopropanol at a final concentration of 0.5 M and refluxed for 24 h in the
presence of an equimolar amount of thiosemicarbazide. The corresponding thiosemicarbazones were isolated by filtration and recrystallized from hot ethanol.
Equimolar amounts of thiosemicarbazones and the desired haloketones were
stirred at room temperature in isopropanol overnight at a final concentration of
0.2 M. The resulting products were recrystallized from hot ethanol several times to
yield pure products and were used without further purification.
4-(4-cyanophenyl)-2-(2-cyclopentylidenehydrazinyl)thiazole (Remodelin): 2cyclopentylidenehydrazine-1-carbothioamide (1 g, 4.46 mmol) and 2-bromo-4’cyanoacetophenone (700 mg, 4.45 mmol) were stirred overnight in 12 ml of
isopropanol at room temperature. The precipitate was filtered and recrystallized
from hot ethanol to yield the hydrobromide salt of the desired compound (559 mg,
1.98 mmol, 45%) as light-yellow needles.1H NMR (500 MHz, CDCl3): δ 12.11 (br
s), 7.84 (d, J = 9.0 Hz, 2H), 7.81 (d, J = 9.0 Hz, 2H), 6.84 (s, 1H), 2.61 (t, J = 9.0 Hz,
2H), 2.51 (t, J = 9.0 Hz, 2H), 1.94–1.80 (m, 4H); 13C NMR (125 MHz, CDCl3): δ
173.8, 169.5, 138.8, 133.5, 131.3, 126.3, 118.0, 114.1, 103.8, 33.7, 31.2, 25.2, 25.0;
HRMS (m/z): [M]+ calcd. for C15H15N4S, 283.1009; found, 283.1017. Molecule 4
was resuspended in DMSO at 10 mg/ml.
4-(4-trifluoromethylphenyl)-2-(2-cyclopentylidenehydrazinyl)thiazole
(Remodelin Fluor): 2-cyclopentylidenehydrazine-1-carbothioamide (3.0 g, 18.7
mmol) and 2-bromo-4’-(trifluoromethyl)acetophenone (5.0 g, 18.7 mmol) were
stirred in isopropanol (150 mL) at r.t. for 24 h. The precipitate was filtered and
recrystallized three times from hot ethanol to yield the hydrobromide salt of
Remodelin Fluor as bright yellow needles (1.5 g, 3.7 mmol, 20%). 1H NMR (300
MHz, CDCl3) δ 11.12 (br s, 2H), 7.80 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H),
6.87 (s, 1 H), 2.53 (t, J = 7.0 Hz, 2 H), 2.47 (t, J = 7.0 Hz, 2 H), 1.93–1.75 (m, 4 H).
13 C NMR (75 MHz, CDCl3) δ 171.9, 169.4, 140.3, 131.8 (q, J = 32.0 Hz), 131.6,
126.5 (br q, J = 3.5 Hz, 2 C), 126.1 (2 C), 123.7 (q, J = 274.0 Hz), 103.5, 33.5, 30.6,
25.1, 24.9. HRMS (m/z): [M + H] + calculated for C15H15F3N3S, 326.0933;
found, 326.0950. See Fig. S2A for the synthesis reaction.
Animals—ethical information. For the studies at the Wellcome Trust Sanger
Institute (WTSI) the care and use of all mice used to generate data for this protocol
was carried out in accordance with UK Home Office regulations, UK Animals
(Scientific Procedures) Act of 2013 under UK Home Office licenses which
approved this, and were reviewed regularly by the WTSI Animal Welfare and
Ethical Review Board. For the studies at Crown Biosciences the protocols and any
amendment(s) or procedures involving the care and use of animals in this study
were reviewed and approved by the Institutional Animal Care and Use Committee
(IACUC) of Crown Biosciences. During the study, the care and use of animals were
NATURE COMMUNICATIONS | (2018)9:1700
conducted in accordance with the regulations of the Association for Assessment
and Accreditation of Laboratory Animal Care (AAALAC AN-1308-017-66).
Animals—housing and husbandry. At WTSI, mice are maintained in a specific
pathogen-free unit on a 12-h light and 12-h dark cycle with lights off at 19:30 and
no twilight period. The ambient temperature is 21 ± 2 °C, and the humidity is 55 ±
10%. Mice are housed using a stocking density of 3–5 mice per cage (overall
dimensions of caging: 365 × 207 × 140 mm (length × width × height), floor area
530 cm2) in individually ventilated caging (Tecniplast, Sealsafe 1284 L) receiving 60
air changes per hour. In addition to Aspen bedding substrate, standard environmental enrichment of two Nestlets, a cardboard fun tunnel and three wooden chew
blocks are provided. Mice were given water and diet (Teklad Global 18% Protein
Rodent Diet/Envigo) ad libitum. At Crown Biosciences, mice were housed at an
average temperature of 23.5 °C with a 7:00 am–19:00 pm light and 19:00 pm – 7:00
am (next day) darkness cycle in polysulfone IVC cages (3 mice/cage; 325 mm ×
210 mm × 180 mm). Mice were fed Co60 irradiation sterilized dry granule mouse
diet. Animals had free access to food and water during the entire study period.
Mice had no drug or test naïve prior to treatment.
Engineering of the Nat10tm1a(KOMP)Wtsi (Nat10+/−) mice. Mice carrying the
knockout-first conditional-ready allele Nat10tm1a(KOMP)Wtsi (abbreviated to
Nat10tm1a) were generated on a C57BL/6 N background as part of the Sanger
Mouse Genetics Project (MGP). Detailed description of the Sanger Mouse Genetics
Project methodology has been reported38. Briefly, a promoter-containing cassette
(L1L2_ Bact_P) was introduced upstream of the critical Nat10 exon 4 at position
103754720 of Chromosome 2, Build GRCm38. The vector containing Nat10tm1a
was electroporated into C57BL/6 N derived JM8A3.N1 ES cells. Correct ES cell
gene targeting was confirmed by long-range PCR and quantitative PCR. Targeted
ES cells were microinjected into blastocysts and used to generate chimeras. Germline transmission was confirmed by genotyping PCR analyzes (http://www.
knockoutmouse.org/kb/25/). Mice obtained from heterozygous intercross were
genotyped for the Nat10tm1a allele by PCR. Mice were killed by CO2 inhalation
followed by cervical dislocation.
Use of the LmnaG609G and LmnaG609G Nat10+/−mice. LmnaG609G (C57BL/6N)
mice were imported from the laboratory of Carlos-Lopez Otin39 and re-derived on
the line C57BL/6NTac at the Wellcome Trust Sanger Institute (WTSI). Mouse
genotyping was performed from tail biopsies39. Nat10 KO mice were generated in
the C57BL/6NTac background as part of the Mouse Genetics Project at the
WTSI40. The double mutant combinations were maintained on the same C57BL/
6NTac background. Experimental animals were maintained under close supervision according to the following protocol. Upon weaning LmnaG609G/G609G
homozygous animals and littermate Lmna+/+ wild-type (WT) controls were set up
in experimental cohorts and weighed weekly. WT and mutant experimental animals were housed together randomly in multiple cages to reduce cage bias.
Treatment and weight measurements were carried out by the mouse facility
technicians blind of the scientific background or goals of the experiment. When
animals reached 10% BW loss they were weighed every other day and wet pellets
provided on the floor daily. Upon 15% BW loss animals were weighed and
monitored daily and culled when they passed over 19% BW loss. A number of male
mice of the LmnaG609G/G609G genotype have presented with penile prolapse and
upon detection they have been culled and indicated as incidence of penile prolapse
(Supplementary Fig. 5a). Over the course of the study three mice of the LmnaG609G/
G609G genotype have been found dead. No drug or naïve test was performed prior
to treatment or testing. Animals have been killed by CO2 inhalation followed by
cervical dislocation. WTSI facility runs periodic health reports that indicate that the
mice were free of known viral, bacterial and parasitic pathogens. For analysis of
embryonic development of Nat10 KO mice, timed matings were performed at noon
and the day of vaginal plug detection was defined as embryonic day E0.5. Movies
and pictures were made using a Sony Cyber-shot DSC-HX10V GPS camera. Mouse
health evaluation was performed by trained technicians using established
protocols41.
Small-molecule dosing. Remodelin and Remodelin Fluor were dissolved in a
solution of 20% DMSO, 65% (45% 2-Hydroxypropyl-b-cyclodextrin solution,
H5784 Sigma Aldrich) and 15% Tween 80 (P8074 Sigma Aldrich). The vehicletreated animals were given this solution alone, without Remodelin. Remodelin and
Remodelin Fluor were administered daily by oral gavage at 100 mg per kg per day
and 50 mg per kg per day respectively (defined as non-toxic doses in toxicity
studies), from day 21 and until culled. Dosing needle—Instech FTP-20–30 Plastic
feeding tubes, 20ga × 30 mm 1 ml syringes and 20ga dosing needles were used as
they are appropriate for the volume to be administered and for the size of the mice.
During each dosing session, the vehicle only was administered to each animal in
the control group before administering Remodelin to each animal in the treated
group to avoid cross-contamination. BWs were recorded for each mouse before
dosing and the dose volume was calculated according to the BW. The mice were
restrained by scruffing the back of the neck, the dosing needle was presented
through the mouth down into the esophagus in a smooth motion. Once in situ, the
dose (calculated to a 100–200 µl volume) was administered to the mouse.
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Additional food was moistened and added to the floor of each cage to facilitate food
consumption following the dosing procedure. The end-point criteria were represented by 20% BW loss, mice that were found dead or moribund or mice that
presented with penile prolapse, a distinctive clinical phenotype for the progeric
male mice. All animals were used to represent the survival curves; n = 1
Remodelin-treated LmnaG609G/G609G mouse presented with haemangioma at
79 days of age and was censored into the survival analysis. Treatment and weight
measurements were carried out by the mouse facility technicians blind of the
scientific background or goals of the experiment. The comparison between
LmnaG609G/G609G vehicle and LmnaG609G/G609G no treatment showed no significant difference (p = 0.56). We utilized an informal method of randomizing
within each batch to assign mice to treatment using mouse database ID. Mice were
used from multiple cages and litters.
Assessment of remodelin toxicity in vivo and dosing. Remodelin toxicity was
assessed by the Crown Biosciences on 12 6-week-old C57BL/6N female mice, with
a BW of 20 g on average (see Table S2. Animal supplier: Shanghai Laboratory
Animal Center SLAC, Shanghai, China). At WTSI, Remodelin and Remodelin
Fluor were dissolved in a solution of 20% DMSO, 65% (45% 2-Hydroxypropyl-bcyclodextrin solution, H5784 Sigma Aldrich) and 15% Tween 80 (P8074 Sigma
Aldrich). The vehicle-treated animals were given this solution alone, without
Remodelin. Remodelin and Remodelin Fluor were administered daily by oral
gavage at 100 mg per kg per day and 50 mg per kg per day respectively (defined as
non-toxic doses in toxicity studies), from day 21 and until culled. Animals have
been killed by CO2 inhalation followed by cervical dislocation.
Pharmacokinetics evaluation of Remodelin. The pharmacokinetics of Remodelin
was assessed by the Crown Bioscience (Supplementary Tables 1 and 2) and the
Xenogesis Ltd., Nottingham, UK (Supplementary Fig. 1c). The administration of
Remodelin and sample collection in each WT study group are shown in the following experimental design tables Tables S1 and S2 (IV = intravenous; PO = per os
(by mouth)). The LmnaG609G mice were treated with 100 mg/kg Remodelin for
6 weeks and tissues and plasma collected 1 h after the last dosing. Animals were
randomly assigned to groups. Before grouping and treatment, all animals were
weighed, and assigned into groups using randomized block based on their BW.
Within each block, experimental animals were randomly assigned to the different
groups. Randomized block design was used to assign experimental animals to
ensure that each animal has the same probability of being assigned to any given
treatment groups and therefore minimizing systematic error. Animals showing
obvious signs of severe distress and/or pain were humanely killed. Animal that had
lost significant body mass (>20%, emaciated) were killed. Animals found to have
other severe health problems, i.e., prolonged diarrhea, persistent anorexia, lethargy
or failure to respond to gentle stimuli, labored respiration, or that could not get to
adequate food or water, etc., were removed from the study and killed (n = 3; subcutaneous administration; data not shown). LC/MS/MS method development was
used to assess the test compound in plasma. A minimum of 6 standards with
LLOQ < 5 ng/mL and a minimum of five standards back were calculated to within
±20% of their nominal concentrations. A total of six QC samples at three concentrations (Low, Mid, and High) were included in sample runs with a minimum
of four QC back were calculated to within ±20% of their nominal concentrations.
Plasma samples were analyzed following the above criteria.
Cell lines. Normal skin primary fibroblasts GM03440 and Hutchinson Gilford
Progeria Syndrome (HGPS) skin primary fibroblasts AG11513 and A11498 were
purchased from Coriell Cell Repositories and used between passage number 9–17.
Cells were grown in Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich)
supplemented with 10% fetal bovine serum (BioSera), 2mM L-glutamine, 100 U/ml
penicillin, 100 μg/ml streptomycin. All cell lines were tested for mycoplasma
contamination using the Charles River Mycoplasma Testing Services.
Antibodies. Antibodies used in this study are: Lamin A/C (sc-6215 Santa-Cruz
1:200 for Western Blotting and 1:100 for Immunofluorescence), NAT10 (13365-1AP ProteinTech Europe 1:400 for Western Blotting), γH2AX (05-636 Millipore,
1:200 for Western Blotting and 1:100 for Immunofluorescence), H2AX (ab11175
Abcam, 1:500 for Western Blotting), β Actin (ab8226, Abcam, 1:1000 for Western
Blotting), Acetyl α Tubulin (K40) (5335 Cell Signaling, 1:500 for Western Blotting
and 1:100 for Immunofluorescence), α Tubulin (T9026 Sigma-Aldrich, 1:1000 for
Western Blotting and 1:400 for Immunofluorescence).
Immunoblotting of mouse tissues. Mice were killed by CO2 inhalation followed
by cervical dislocation. Mouse tissues were snap-frozen in liquid N2 and immediately stored at −80 °C. 40 µl/g of protein extraction buffer (50 mM Tris pH 7.5,
150 mM NaCl, 0.1% NP-40, 0.5% CHAPS, 5 mM MgCl2, 10% glycerol, MilliQ
distilled water) or Laemmli Buffer (S3401 SIGMA). Mini-protease and miniphosphatase tablets were added to each sample in conjunction with a stainless-steel
bead (7 mm; QIAGEN) and physically disrupted using the TissueLyser LT (QIAGEN), in 2 × 5 min cycles with 5 min rest on ice in between. After removing the
stainless-steel bead, the lysates were sonicated using the BioruptorTM Next Gen
(Diagenode), 2 × 20 min cycles (30 s on, 30 s off), and a 20 min rest period on ice in
10
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between. The tissue debris was separated from the protein content by centrifugation using a bench Eppendorf centrifuge 5417R (Eppendorf) for 30 min, at 4 °C,
and 16400 rpm. The quantity of protein was measured using PierceTM BCA protein
assay kit (Thermo Scientific) and Multiskan GO (Thermo Scientific) at an absorbance of 562 nm.
Immunoblotting of cell extracts. Total cell extracts were prepared by scraping
cells in SDS lysis buffer (4% SDS, 20% glycerol, and 120 mM Tris-HCl, pH 6.8),
boiling for 5 min at 95 °C, followed by ten strokes through a 25-gauge needle.
Before loading, cell or tissue lysates were diluted with a solution of 0.01% bromophenol blue and 200 mM DTT and boiled for 10 min at 95 °C. Proteins from
individual mice/cell lines were resolved by SDS-PAGE on 4%–12% gradient gels
(NUPAGE, Life Sciences) and transferred onto nitrocellulose membranes (Protran;
Whatman). Secondary antibodies conjugated to IRDye were from LI-COR Biosciences. Western blots shown are representative of three repeats. Detection and
quantification was performed with an imager (Odyssey; LI-COR Biosciences).
Uncropped Western blots are presented in Supplementary Figure 11.
Immunofluorescence. Isolation and culture of adult mouse fibroblasts from skin
and lungs were performed using an established protocol42. Cells were washed with
PBS and fixed for 10 min with 4% PFA in PBS. Cells were permeabilised for 5 min
with PBS/0.2% Triton X-100 and blocked with PBS/0.2% Tween 20 (PBS-T)
containing 5% BSA. Coverslips were incubated for 1 h with primary antibodies and
for 30 min with appropriate secondary antibodies coupled to Alexa Fluor 488 or
594 fluorophores (Life Technologies), before being incubated with 2 μg/ml DAPI.
Pictures were acquired with a FluoView 1000 confocal microscope (Olympus) and
images were quantified using ImageJ. All the immunofluorescence experiments
were performed independently at least three times and the pictures shown in the
figures are representative images of at least three experiments.
Histology and immunohistochemistry. All major organs were isolated following
killing and then fixed in 10% formalin overnight. On the second day the fixed
organs were transferred to 70% ethanol, were placed in cassettes, embedded in
paraffin and serial 5 μm sections were collected on Superfrost Plus slides (Fisher)
using a Leica microdissection system (LMD7000). Hematoxilin and eosin (HE) and
immunohistochemistry staining were performed using standard protocols43. The
organs were examined for abnormalities by a Board Certified Veterinary Pathologist (MJ). Sections of heart containing aortic outflow, pulmonary artery and
myocardium were stained using a primary antibody against alpha smooth muscle
actin (SMA-Sigma Aldrich Cat No: A2547 1:1000) and a secondary antibody
(Alexa 488 Life Technologies at 1:100). The heart sections were also stained using
DAPI alone and with an antibody against Acetyl α Tubulin (K40) (5335 Cell
Signaling). Representative histology images were obtained from whole slide images
scanned on a Hamamatsu NanoZoomer in brightfield and fluorescence modes. The
thickness of the subcutaneous fat layer in the skin and nuclei number in the aorta
were measured using whole slide images and the Hamamatsu NDP view software
at a magnification of ×200 (resolution of 0.45 μm per pixel). The skin (always the
same region/the flanks) and aorta regions for analysis were identified by a
pathologist (MJ) and manually annotated using the HALO image analysis software
(Indica Labs). The cells inside these annotated regions were identified and counted
using the HALO software CytoNuclear v1.5 algorithm, the output of cell density
(cells/mm2) was used to differentiate between the treatment groups. SMA intensity
quantification was performed using ImageJ.
Oocyte culture and immunofluorescence. Oocytes were isolated from ovaries of
8-week old C57BL/6 N female mice and cultured in M2 medium covered by
mineral oil at 37 °C. Isolated oocytes were maintained in prophase arrest by
addition of 250 µM dbcAMP (Sigma; D0627). To induce resumption of meiosis,
oocytes were released into dbcAMP-free medium. For immunofluorescence,
oocytes were fixed for 30 min at 37 °C in 100 mM HEPES (pH 7; titrated with
KOH), 50 mM EGTA (pH 7; titrated with KOH), 2% formaldehyde (methanol
free) and 0.2% Triton X-100. Fixed oocytes were incubated in PBS with 0.1% Triton
X-100 overnight at 4 °C. Antibody incubations were performed in PBS, 3% BSA,
and 0.1% Triton X-100. Primary antibodies used were rat anti-Nup98 (Abcam,
ab50610; 1:100) and rat anti-tyrosinated-α-tubulin (YOL1/34, AbD Serotec;
1:3000). Secondary antibodies used were Alexa-Fluor-488-labeled anti-rat (Molecular Probes; 1:400). DNA was stained with 5 mg/ml Hoechst 33342 (Molecular
Probes). Images were acquired with a Zeiss LSM710 microscope equipped with a
63 × C-Apochromat 1.2 NA water-immersion objective.
Generation of oocytes and in vitro fertilization. Three 4- to 5-week-old C57BL/
6NTac females per sperm sample were super-ovulated by intraperitoneal (IP)
injection of 5IU of pregnant mare’s serum at 17:00 h (on a 12 h light/dark cycle, on
at 07:00/off at 19:00) followed 48 h later by an IP injection of 5IU human chorionic
gonadotrophin. Oviducts were dissected at ~07:50 am on the day of the in vitro
fertilization (IVF), and cumulus-oocyte complexes were transferred into the IVF
fertilization dish containing human tubal fluid (HTF) + glutathione (GSH). An
aliquot of 20 µl of sperm from the pre-incubation dish was then added to the
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fertilization dish. After allowing 3–4 h for fertilization to occur the embryos were
washed and cultured overnight in HTF at 37 °C, 5% CO2 in air.
High-throughput phenotypic screen. The high-throughput phenotyping screen,
is a series of standardized tests performed on all mice that enter the screen and
conducted according to standard operating procedures (SOPs). The tests cover a
broad range of biological areas, including metabolism, cardiovascular, neurological
and behavioral, bone, sensory and hematological systems and plasma chemistry.
The data were obtained following the SOPs available at IMPReSS (www.
mousephenotype.org/impress)40. Factors predicted to affect the variables were
standardized where possible. If this was not possible, measures were taken to
reduce potential bias. For example, the impact of different people performing the
test was minimized (known as the “minimized operator”) as defined in the Mouse
Experimental Design Ontology (MEDO)44. The data captured with the MEDO
ontology can be found at http://www.mousephenotype.org/about-impc/arriveguidelines. In addition, pre-established reasons were defined for QC failures (e.g.,
insufficient sample, error with equipment during test) and detailed using IMPRESS.
This provides standardized options and criteria as agreed by area experts as to
when data can be discarded. All discarded data is retained and tracked in a
database to allow QC-failed data to be audited. Phenotyping data are collected at
regular intervals on age, sex, and strain-matched wildtype (control) mice. In total,
at least seven homozygote mice of each sex per knockout line were generated for
phenotyping. If no homozygotes were obtained from ≥28 offspring from heterozygote intercrosses at P14, the line was declared homozygous lethal. Similarly, if
less than 13% of the pups resulting from intercrossing were homozygous survive to
P14, the line was judged as being homozygous subviable. In this event, heterozygote
mice were examined in the phenotyping screen. The random allocation of mice to
experimental group (wildtype vs. knockout) was driven by Mendelian Inheritance.
Because of the high-throughput nature of the phenotyping screen, blinding the
operators to the identity of knockout lines (both which line to be studied and the
zygosity of the individual mouse) during phenotyping was not employed as the
cage cards used to identify the mice includes genotype information. However, in a
high throughput environment without a defined hypothesis, the potential bias is
minimized. In all cases, the individual mouse was considered the experimental unit.
Further experimental design strategies (e.g., exact definition of a control animal) is
defined using a standardized ontology as detailed in Karp et al.44 and is available
from the IMPC portal (http://www.mousephenotype.org/about-impc/arriveguidelines). For each line, n ≥ 5 mice/genotype were studied.
Comparison between mouse genotypes. Mutant mice at 9 or 12 weeks of age
were analyzed in separate groups (n ≥ 3 for each sex/genotype). Prior to entering
this workflow, due to the progeria phenotype, mice were assessed for adverse health
and welfare to ensure that mice were in a suitable condition for analysis. No mice
were excluded from study on this basis. Mice were anaesthetized with 110 mg/kg
BW ketamine and 11 mg/kg BW xylazine given intraperitoneally. Mice were then
imaged sequentially with three modalities, high resolution X-Ray imaging (MX-20,
Faxitron, Tucson, AZ), Dual-energy X-ray Absorptiometry for body composition
(Piximus II, GE Healthcare, Hatfield, UK), and with light photography for imaging
of dysmorphology. A whole-body lateral image using the MX-20 was collected for
the analysis of spinal curvature by trained persons using a standard defined
position to minimize inconsistencies between mice. Following this, and while still
under anesthesia, blood collection was performed to obtain samples for plasma
chemistry and hematological analysis via the retro-orbital route using capillary
tubes (cat. no. 078042; Scientific Laboratory Supplies). Mice were then culled by
cervical dislocation and heart removal, followed by removal of other organs for
analysis. During this series of procedures, the experimenters collecting images and
blood samples were not blinded from the genotypes of the mice. However, the
analysis of the blood parameters was performed blind and uploaded onto a database. The kyphosis index (KY) was calculated as the ratio between a line drawn
between the caudal margin of the last cervical vertebra to the caudal margin of the
sixth lumbar vertebra and a line perpendicular to this from the dorsal edge of the
vertebra at the point of greatest curvature45. For the data collected on the back
curvature, a multilevel regression model was performed using R (package:nlme
version 3.1). A model (Eq. 1), treating genotype, sex and age as fixed effects whilst
the repeat measure nature of the dataset was accounted for by treating each mouse
as a random effect, was fitted to the data. The genotype effect was tested and
contrasts used to directly compare LmnaG609G/G609G and LmnaG609G/G609GNat10
+/− mice if the genotype effect was significant. For the hypothesis test of primary
interest, the impact of genotype, p-values were adjusted to account for the multiple
comparisons completed to control the false discovery rate to 0.05. Visual inspection
of the data was used to assess whether variance was equal and no outliers were
present thus ensuring the assumptions of the model were met.
Y Genotype þ Sex þ Ageþð1jMouseÞ
ð1Þ
For the survival analysis, the end-point criteria were represented by 20% BW
loss, mice that were found dead or moribund or mice that presented with penile
prolapse, a distinctive clinical phenotype for the progeric male mice. All animals
were used to represent the survival curves; one LmnaG609G/G609GNat10+/− was
reported by the technician staff with a swollen abdomen at 32 days of age and
NATURE COMMUNICATIONS | (2018)9:1700
requested to be culled. The mouse was active and within the weight range for the
age. No abnormality was found at necropsy but because it was culled for other
reasons than end-point criteria it was censored from the analysis.
Statistical analysis. For all analyzes, the individual mouse was considered the
experimental unit within the studies. Survival distributions of the different cohorts
were plotted using the Kaplan–Meier estimator and statistical analysis was performed using log-rank (Mantel–Cox) test. For survival analysis, we have completed
power calculations for a large size effect and with an n of 10/group. We could
detect a 0% to 60% change in survival after treatment, 91% of the time (power of
Fisher Exact test, 0.91). To meet the assumption of this statistical method, censoring of an individual mouse could only occur when the culling of a mouse was
not related to the genotype/assessed-phenotype (e.g., fight wound leading to overt
clinical presentation).
Nat10+/− and LmnaG609G high-throughput phenotyping data. Knockout data
collected across multiple batches were compared to a year’s worth of control data
collected on mice from the same genetic background. For the continuous data, an
iterative top down mixed modeling strategy fitting Eq. 1 was performed using
PhenStat46, an R package version 2.6.047 freely available from Bioconductor48. The
package’s mixed model framework was used as default except the argument
equationType was set to withoutWeight and dataPointsThreshold was set to 2. The
model optimization implemented will adjust for unequal variances. The genotype
contribution test p value was adjusted for multiple testing to control the false
discovery rate to 5%. This statistical method has been studied through simulations
and resampling studies49 and found to be robust and reliable with a multi-batch
workflow, where the knockout mice are split into multiple phenotyping batches.
Y Genotype þ Sex þ Genotype Sexþð1jBatchÞ
ð2Þ
For the categorical data, a Fisher Exact Test was fitted comparing the
proportions seen between wildtype and knockout mice for each sex independently
using PhenStat FE Framework with the default settings. This simple method is
appropriate for categorical phenotyping data as discussed in Karp et al.46. The
minimum p value returned from the two tests for a variable was adjusted for
multiple testing to control the false discovery rate to 5%. The number of caudal
vertebrae were recoded to a categorical variable by classifying mice with less than
28 vertebrae as “low,” those with greater than 29 as “high” and all others as
“normal.” For ABR data, the knockout dataset was smaller with only four data
points for each variable, therefore to meet the assumption of the test, the data was
analyzed using a reference range plus methodology which calls a significant
phenotype when the majority of animals lie outside the natural variation seen in
the control animals47. The implementation within PhenStat RR framework is based
on classifying the analyzable variable values as high, normal or low based on the
natural variation seen within the control data and comparing the proportions seen
with a Fisher Exact Test. The minimum p value returned from the two tests ((1)
increase in high classification and (2) increase in low classification) for a variable
was adjusted for multiple testing to control the false discovery rate to 5%. As a high
throughput program with many variables and multiple analysis tools, a single
power calculation would not help; instead, the pipeline has been developed through
empirically selecting a workflow which has historically given hits at a rate that
would be cost effective for the program.
Data analysis for genotype comparison. Mixed model data analysis was performed using R (package: nlme version 3.1). An iterative top down modeling
strategy was implemented starting with the fully loaded model (Eq. 2). For the
Origins of Bone and Cartilage Disease (OBCD) screen50, where only one sex was
collected, Eq. 3 details the starting model. The final model was selected by first
selecting a structure for the random effects, then a covariance structure for the
residual, and then the model reduced by removing non-significant fixed effects.
Then the genotype effect was tested, model diagnostics assessed and contrasts used
to directly compare LmnaG609G/G609G and LmnaG609G/G609GNat10+/− mice if the
genotype effect was significant. During the model building stage, the hypotheses
were tested with a threshold of p < 0.05. For the hypothesis test of primary interest,
the impact of genotype, p-values were adjusted to account for the multiple comparisons completed to control the false discovery rate to 0.05. The difficulty
associated with the breeding and viability challenged the production of these mice
for the array of phenotyping used in this paper. 136 mating pairs were set up over
more than 3 years that generated 181 LmnaG609G homozygous (single or Nat10
double-mutant) mice that were used at specific ages together with littermate
controls. The n was thus limited by breeding constraint and we have used n > 5.
Y Genotype þ Age þ ð1jBatchÞ
ð3Þ
Heart rate comparison. Heart measurements were performed using the ecgTUNNEL (emka TECHNOLOGIES), a noninvasive ECG system, using the manufacturer’s
recommendations51. All ECG recording sessions were performed during daytime and
the data analyzed using the iox2, data acquisition, and analysis software (emka
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TECHNOLOGIES). Each animal was put inside the tunnel, which was then closed,
ensuring the animal was properly restrained. To minimize the effects of stress, animals were allowed to stay in the restraining system for 1 min before starting ECG
recordings. Indeed, direct observation of the animals and ECG traces proved that they
were calm and that the heart rate was stable. For data acquisition, a series of repeated
measurements were done on the same animal at each time-point and data for the
same animal was collected over the different week intervals. For the data collected on
the heart rate screen, a multilevel regression model was performed using R (package:
nlme version 3.1). A model [Eq. 1], treating genotype, sex, and age as fixed effects
whilst the repeat measure nature of the dataset was accounted for by treating each
mouse as a random effect, was fitted to the data. The genotype effect was tested and
contrasts used to directly compare LmnaG609G/G609G and LmnaG609G/G609GNat10+/−
mice if the genotype effect was significant. For the hypothesis test of primary interest,
the impact of genotype, p-values were adjusted to account for the multiple comparisons completed to control the false discovery rate to 0.05. Visual inspection of the
data was used to assess whether variance was equal and no outliers were present thus
ensuring the assumptions of the model were met.
RNA extraction and qPCR analysis. RNA was extracted from tissues from n > 5
independent mice/group using the RNeasy fibrous tissue mini kit (50; cat. No.7404;
Qiagen) and quantified using the NanoDrop 1000 Spectrophotometer (Thermo
Fisher Scientific). 2 µg RNA/sample was used to produce cDNA using the HighCapacity RNA-to-cDNA kit (cat. No. 4387406; Applied Biosystems/Thermo Fisher
Scientific). qPCR was carried out using the TaqMan system (Universal Master Mix
II, with UNG, 4440038; Applied Biosystems/Thermo Fisher Scientific) on an
Applied Biosystems Quant Studio 3 machine. n ≥ 3 mice were used for each genotype, with 50 ng cDNA for each sample run in triplicate or quadruplicate. Thermo
Fisher Scientific Nat10 (Mm00462302_m1) and Cdkn1a/p21 (Mm00432448_m1)
primers were used as experimental probes while Gapdh (Mm99999915_g1), Actb
(Mm00607939_s1) and/or Rn18s (Mm03928990_g1) were used as endogenous
controls. The Relative Standard Curve pre-set program was used throughout and
data analysis was performed using the Relative Quantification application, powered
by the Thermo Fisher Scientific cloud platform. To account for technical replicates
the mean value for a mouse was calculated and used for the statistical assessment.
For each sample, data was normalized to the endogenous controls and represented
as relative to the wildtype control. Data was evaluated by visual inspection and an F
test of the variance was calculated in order to estimate normality and equal variance. All graphs and part of the statistical analysis in the manuscript (Student’s ttests; Kaplan–Meier estimator and statistical analysis; Fishers exact test) were
generated and calculated using GraphPad Prism version 7.0a for Mac OS X,
GraphPad Software, La Jolla, California, USA, www.graphpad.com.
RNAseq data analysis. RNA was extracted as described above and quality control
assessed using the 2100 Bioanalyzer (Agilent Technologies). Because of financial
constrains we used n = 2 mice/group. Transcriptome data was obtained using
paired end sequencing, with read lengths of 150 bp, on a NextSeq 500 machine.
Trimmed reads were aligned using STAR aligner (version 2.4.2a) to the mouse
genome assembly GRCm38. Normalization of the read counts and differential
expression analysis was performed using three commonly used software programs:
DeSeq252, edgeR53, and Cuffdiff 254. Our conservative approach defined genes
differentially expressed as those found to be in common between the results of at
least two of the three software programs mentioned above. The log-2 fold change in
gene expression presented in Fig. 2d and Fig. 4e correspond to the log-2 fold
change returned by DeSeq2. DeSeq2 and edgeR used as an input raw read counts
produced by featureCounts from the Bioconductor (version 3.3) Rsubread package
(14) in R version 3.3.1. For the DeSeq2 and edgeR analyzes, we filtered out genes
that had 0 or 1 read support across all samples. In the DeSeq2 differential
expression analysis, we selected genes that were up or down regulated at a FDR
lower than 0.1. In edgeR differential expression analysis, we selected genes up or
down regulated with a p-value less than 0.05. Gene ontology analysis was performed using the mouse genome informatics visual annotation display55. For the
analysis of the biological term fold enrichment, a ratio between the frequency of
genes in our set to frequency of genes in the whole genome was calculated56.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Data availability. All data presented in the manuscript are available from the
corresponding authors upon reasonable request. The mouse phenotypic data from
the present manuscript are available in the supplementary Data. Mouse phenotypic
data are available from IMPC and Zenodo. RNAseq data are available from
ArrayExpress under accession code E-MTAB-6578.
27.
28.
Received: 25 September 2017 Accepted: 12 March 2018
29.
30.
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Cancer Research UK (CRUK) program grant C6/ A18796 and a Wellcome Trust
Investigator Award (206388/Z/17/Z). Institute core funding is provided by CRUK
(C6946/A24843) and the Wellcome Trust (WT203144). D.L was funded by a Project
Grant from the Medical Research Council, UK MR/L019116/1. Research in the D.J.A.
laboratory is supported by CRUK and the Wellcome Trust. Research in the R. R.
laboratory is supported by the European Research Council (Grant N° 647973), and the
Emergence Ville de Paris Program. M.D. was supported by the European Research
Council grant DDREAM. Some data in this publication form part of the subject matter of
patent WO 2015/150824. The funders had no role in study design, data collection, and
analysis, decision to publish or in preparation of the manuscript.
Author contributions
G.B., D.L., and S.P.J. coordinated and conceptualized the study and wrote the manuscript. D.L. performed the human and mouse derived cell culture, prepared the smallmolecule doses for animal gavage, generated immunofluorescence and western blots with
help from MD and analyzed the related data. G.B. analyzed the survival and fertility data,
performed the heart rate measurements, kyphotic index measurements, and sperm
counts as well as mouse primary cell line derivations. G.B. helped with tissue collection
throughout the study. A.B. genotyped all the mice in this study, did all the mouse protein
extractions and analyzed the weighting data. C.C. did the Remodelin and Remodelin
fluor gavages and mouse weighting with covering help from other mouse facility staff and
supervised daily by A.K. A.K. helped throughout the study with end-point criteria
assessment making sure consistency was achieved. M.A. performed the RNAseq analysis
and pathway analysis with help from G.B. N.G. performed the RNA extractions and did
the qPCR analysis with help from G.B. C.J.L. and J.K.W. supervised the phenotypic
analysis; C.J.L. assembled all the phenotypic pipeline raw data produced by Sanger Mouse
Genetics Project that performed all the pipeline phenotypic measurements in this study
as well as the IVF analysis. N.K. performed the statistical analysis on the phenotypic
pipeline data and helped throughout with all the statistical analysis in the paper. N.K.
made an important contribution to manuscript material and methods writing. M.J. and J.
A. performed the pathologic staining and assessments. D.C. performed the meiotic
spreads analysis. R.R. designed and synthesized the small molecules Remodelin and
Remodelin Fluor used in the study. D.J.A. helped supervise all the mouse work. S.P.J. and
D.L. supervised the work. All the authors commented and edited the manuscript and
figures.
Additional information
Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467018-03770-3.
Competing interests: D.L., S.P.J., and R.R. are named inventors on a patent describing
compounds that include Remodelin. The remaining authors declare no competing
interests.
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Acknowledgements
We thank all members of the Steve Jackson laboratory for help and support—particularly
Kate Dry for editing the manuscript—and Dr Carlos Lopez-Otin for sharing his
LmnaG609G mouse model with us. Research in the Jackson laboratory is funded by
© The Author(s) 2018
Sanger Mouse Genetics Project
Carl Shannon, Mark Sanderson, Amy Gates, Joshua Dench, Valerie Vancollie, Catherine McCarthy,
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Yvette E. Hooks, Catherine L. Tudor, Angela L. Green, Fiona L. Kussy, Elizabeth J. Tuck, Emma J. Siragher,
NATURE COMMUNICATIONS | (2018)9:1700
| DOI: 10.1038/s41467-018-03770-3 | www.nature.com/naturecommunications
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| DOI: 10.1038/s41467-018-03770-3 | www.nature.com/naturecommunications