U.S. patent application number 17/148614 was filed with the patent office on 2021-05-13 for agents and methods using thereof for the prevention and treatment of stem cell muscle disorders.
The applicant listed for this patent is ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE (EPFL). Invention is credited to JOHAN AUWERX, KEIR MENZIES, DONGRYEOL RYU, HONGBO ZHANG.
Application Number | 20210137959 17/148614 |
Document ID | / |
Family ID | 1000005345582 |
Filed Date | 2021-05-13 |
United States Patent
Application |
20210137959 |
Kind Code |
A1 |
ZHANG; HONGBO ; et
al. |
May 13, 2021 |
AGENTS AND METHODS USING THEREOF FOR THE PREVENTION AND TREATMENT
OF STEM CELL MUSCLE DISORDERS
Abstract
The present invention relates to agents that induce
mitochondrial unfolded protein response (UPR.sup.mt) in muscle stem
cells and prevents or reverse process of muscle stem cell
senescence. Further, the invention relates to methods and
compositions useful in the prevention and/or treatment of muscle
stem senescence.
Inventors: |
ZHANG; HONGBO; (GUANGZHOU,
CN) ; MENZIES; KEIR; (LUSKVILLE, CA) ; AUWERX;
JOHAN; (BUCHILLON, CH) ; RYU; DONGRYEOL;
(BUSAN, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE (EPFL) |
LAUSANNE |
|
CH |
|
|
Family ID: |
1000005345582 |
Appl. No.: |
17/148614 |
Filed: |
January 14, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15758365 |
Mar 8, 2018 |
10905704 |
|
|
PCT/EP2016/071044 |
Sep 7, 2016 |
|
|
|
17148614 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 21/00 20180101;
C12N 2501/999 20130101; C12N 2501/10 20130101; A61K 38/1825
20130101; A61K 31/65 20130101; A61K 31/165 20130101; A61K 35/34
20130101; A23L 33/10 20160801; A23L 33/13 20160801; A61K 31/706
20130101; C12N 5/0658 20130101; A61K 45/06 20130101 |
International
Class: |
A61K 31/706 20060101
A61K031/706; A61K 35/34 20060101 A61K035/34; A23L 33/10 20060101
A23L033/10; A61K 31/165 20060101 A61K031/165; A61K 45/06 20060101
A61K045/06; A61K 31/65 20060101 A61K031/65; A61K 38/18 20060101
A61K038/18; A23L 33/13 20060101 A23L033/13; A61P 21/00 20060101
A61P021/00; C12N 5/077 20060101 C12N005/077 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 8, 2015 |
EP |
15184341.4 |
Claims
1. A pharmaceutical composition comprising at least one UPR.sup.mt
inducing agent and further comprising an agent useful for the
prevention and/or treatment of diseases or disorders associated
with MuSCs senescence and/or decreased MuSCs number and/or for
promoting muscle tissue growth and/or repair, wherein said diseases
or disorders are selected from Duchenne's muscular dystrophy (DMD),
Becker's muscular dystrophy, Congenital muscular dystrophy, Distal
muscular dystrophy, Emery-Dreifuss' muscular dystrophy,
Facio-scapulo-humeral muscular dystrophy, Limb-girdle muscular
dystrophy, myotonic muscular dystrophy, oculopharyngeal muscular
dystrophy, non-mitochondrial myopathies, myotonia, congenital
myopathies selected from nemaline myopathy, multi/minicore myopathy
and centronuclear myopathy, metabolic myopathies, inflammatory
myopathies, muscle stem cell senescence caused by nutritional
deficiencies, chronic obstructive pulmonary disease, cachexia of
cancer, diseases resulting from clinical treatments with
anthracyclines and/or for promoting muscle tissue growth and/or
repair after graft, and wherein said mitochondrial UPR.sup.mt
inducing agent selected from nicotinamide riboside (NR),
thiamphenicol (TAP) and analogues thereof.
2. A food supplement comprising at least one agent selected from
one UPR.sup.mt inducing agent and further comprising an agent
useful for the prevention and/or treatment of diseases or disorders
associated with MuSCs senescence and/or decreased MuSCs number
and/or for promoting muscle tissue growth and/or repair, wherein
said diseases or disorders are selected from Duchenne's muscular
dystrophy (DMD), Becker's muscular dystrophy, Congenital muscular
dystrophy, Distal muscular dystrophy, Emery-Dreifuss' muscular
dystrophy, Facio-scapulo-humeral muscular dystrophy, Limb-girdle
muscular dystrophy, myotonic muscular dystrophy, oculopharyngeal
muscular dystrophy, non-mitochondrial myopathies, myotonia,
congenital myopathies selected from nemaline myopathy,
multi/minicore myopathy and centronuclear myopathy, metabolic
myopathies, inflammatory myopathies, muscle stem cell senescence
caused by nutritional deficiencies, chronic obstructive pulmonary
disease, cachexia of cancer, diseases resulting from clinical
treatments with anthracyclines and/or for promoting muscle tissue
growth and/or repair after graft, and wherein said mitochondrial
UPR.sup.mt inducing agent selected from nicotinamide riboside (NR),
thiamphenicol (TAP) and analogues thereof.
3. A method of preventing and/or treating of diseases or disorders
associated with skeletal MuSCs senescence and/or decreased MuSCs
number, wherein said diseases or disorders are selected from
Duchenne's muscular dystrophy (DMD), Becker's muscular dystrophy,
Congenital muscular dystrophy, Distal muscular dystrophy,
Emery-Dreifuss' muscular dystrophy, Facio-scapulo-humeral muscular
dystrophy, Limb-girdle muscular dystrophy, myotonic muscular
dystrophy, oculopharyngeal muscular dystrophy, non-mitochondrial
myopathies, myotonia, congenital myopathies selected from nemaline
myopathy, multi/minicore myopathy and centronuclear myopathy,
metabolic myopathies, inflammatory myopathies, muscle stem cell
senescence caused by nutritional deficiencies, chronic obstructive
pulmonary disease, cachexia of cancer, diseases resulting from
clinical treatments with anthracyclines and/or for promoting muscle
tissue growth and/or repair after graft in a subject, said method
comprising administering an effective amount of a mitochondrial
UPR.sup.mt inducing agent selected from nicotinamide riboside (NR),
thiamphenicol (TAP) and analogues thereof or a pharmaceutical
composition thereof to a subject.
4. The method according to claim 3, wherein the subject is
suffering from a non-mitochondrial myopathy.
5. The method according to claim 3, wherein the MuSCs senescence is
a muscle dystrophy or muscle wasting.
6. The method according to claim 3, wherein the MuSCs senescence is
muscle frailty and sarcopenia in aging.
7. The method according to claim 3, wherein said mitochondrial
UPR.sup.mt inducing agent is to be administered orally.
8. The method according to claim 3, wherein said mitochondrial
UPR.sup.mt inducing agent is to be administered parenterally.
9. The method according to claim 3, wherein said mitochondrial
UPR.sup.mt inducing agent is nicotinamide riboside (NR).
10. The method according to claim 3, wherein said mitochondrial
UPR.sup.mt inducing agent is thiamphenicol (TAP) or analogues
thereof.
11. The method according to claim 3, wherein said mitochondrial
UPR.sup.mt inducing agent is to be administered in combination with
a co-agent useful for preventing or treating a disease or disorder
associated with MuSCs senescence and/or decreased MuSCs number.
12. The method according to claim 11, wherein said co-agent is
selected from an inhibitor of transforming growth factor .beta.
(TGF.beta.) family protein/receptor, a myostatin inhibitor, a
follistatin-derived peptide, FS I-I, P38, a JAK-STAT signalling
pathway inhibitor, SB203580, SB202190, BIRB796, AG490,
5,15-Diphenylporphyrin, a muscle stem cell activator, a Notch
signalling activator, an anabolic stimulator of the muscle, and
IGF-1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/758,365, filed Mar. 8, 2018, which is the U.S. national
stage application of International Patent Application No.
PCT/EP2016/071044, filed Sep. 7, 2016.
[0002] The Sequence Listing for this application is labeled
"Seq-List.txt" which was created on Feb. 9, 2018 and is 9 KB. The
entire content of the sequence listing is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the field of
muscle stem cell medicine and in particular regenerative therapies
and muscle transplantation. In particular, the invention relates to
methods and compositions useful in the regeneration of damage human
tissue, ex vivo propagation of stem/progenitor cells and in the
treatment of muscle diseases.
BACKGROUND OF THE INVENTION
[0004] In adults, tissue homeostasis is highly dependent on adult
stem cells (SCs) function in multiple tissues. These adult SCs are
not only essential in continuously-proliferating tissues, such as
hematopoietic-, intestinal- and skin-systems, but also in normally
quiescent tissues, such as skeletal muscle and brain that require
regeneration following damage or with disease (Wagers and Weissman,
2004, Cell, 116: 639). Adult stem cells (SCs) are essential for
tissue maintenance and regeneration yet are susceptible to SC
senescence during aging that is a decline in adult SC quantity and
function. SC senescence is at least partly responsible for the loss
of tissue homeostasis and regenerative capacity (Kuilman et al.,
2010, Genes & Development, 24: 2463; Lopez-Otin et al., 2013,
Cell 153: 1194).
[0005] With respect to skeletal muscle, homeostasis and
regeneration depends on the normally quiescent muscle stem cells
(MuSCs), which are activated upon muscle damage to expand and give
rise to differentiated progeny that regenerate damaged muscle
fibers (Yin et al., 2013, Physiological Reviews, 93: 23;
Tabebordbar et al., 2013, Annual Review of Pathology, 8: 441).
These responses are blunted in aged muscle due to a quantitative
and qualitative decline in MuSCs (Tang et al., 2011, Cold spring
Harbor symposia on quantitative biology 76: 1001; Price et al.,
2014, Nature Med., 20: 1094). In aging, MuSC dysfunction may be
attributed to both extrinsic signals (Conboy et al., 2005, Nature
433: 760; Chakkalakal et al., 2012, Nature, 490: 335) and/or
intrinsic cellular senescence signalling pathways (Sousa-Victor et
al., 2014, Nature, 506: 316). One general regulator of cellular
senescence, cyclin-dependent kinase inhibitor 2A (CDKN2A,
p16.sup.INK4A), is increasingly expressed in geriatric MuSCs (Burd
et al., 2013, Cell, 152: 316), eliciting permanent cell cycle
withdrawal and senescence of MuSCs in very old mice (Sousa-Victor
et al., supra). However, before this stage, reductions in MuSC
number and function can already be observed (Tang et al., supra;
Sousa-Victor et al., supra) indicating that MuSC senescence may be
initiated at an earlier time point. Several recent reports support
the idea that pre-geriatric mice, approximately two-years-old, can
exhibit features of MuSC senescence (Price et al., supra; Bernet et
al., 2014, Nature Med., 20: 265; Cosgrove et al., 2014, Nature
Med., 20: 255; Tierney et al., 2014, Nature Med., 20: 1182; Liu et
al., 2013, Cell Rep., 4: 189). However, the early mechanisms that
instigate MuSC senescence are still largely unknown.
[0006] One of the hallmarks of organismal aging is the appearance
of mitochondrial dysfunction (Kuilman et al., supra; Lopez-Otin et
al., supra). Recent evidence has shown that mitochondrial
dysfunction, induced by calorie-dense diets or aging, can result
from oxidized nicotinamide adenine dinucleotide (NAD.sup.+)
depletion, while NAD.sup.+ repletion, using precursors such as
nicotinamide riboside (NR), can reverse this process (Canto et al.,
2012, Cell Metabolism, 15: 1034; Pirinen et al., 2014, Cell
Metabolism, 19: 1034; Mouchiroud et al., 2013, Cell, 154: 430;
Yoshino et al., 2011, Cell Metabolism 14: 528; Gomes et al., 2013,
Cell, 155:1624). It is generally assumed that stem cells rely
predominantly on glycolysis for energy, a process that would reduce
cellular NAD.sup.+ (Folmes et al., 2012, Cell Stem Cell, 11: 596).
However, mitochondrial function was linked to muscle and neural
stem cell maintenance and activation (Cerletti et al., 2012, Cell
Stem Cell, 10: 525; Ryall et al., 2015, STEM 16: 171, Stein et al.,
2014, EMBO J., 33: 1321), yet its role in SC senescence is
unknown.
[0007] Disorders that are related to muscle stem cell senescence
include muscle dystrophy diseases, such as Duchenne's muscular
dystrophy (DMD), Becker's muscular dystrophy (BMD), Congenital
muscular dystrophy, Distal muscular dystrophy, Emery-Dreifuss'
muscular dystrophy, Facio-scapulo-humeral muscular dystrophy,
Limb-girdle muscular dystrophy, Myotonic muscular dystrophy and
Oculopharyngeal muscular dystrophy. It further includes other
inherited myopathies, such as myotonia, congenital myopathies
(includes nemaline myopathy, multi/minicore myopathy, centronuclear
myopathy), metabolic myopathies (includes glycogen storage diseases
and lipid storage disorder), inflammatory myopathies, such as
dermatomyositis, polymyositis, inclusion body myositis and
auto-immune myositis. These diseases further include muscle frailty
and sarcopenia in aging (Sousa-Victor et al., supra) and other
acquired myopathies, such as drug/toxic agents-induced myopathy,
alcoholic myopathy, myositis ossificans, rhabdomyolysis and
myoglobinurias. Other diseases linked to muscle stem cell
senescence include muscle wasting induced by nutritional
deficiencies. Diseases linked to muscle stem cell senescence may be
developed in the context of other diseases, such as chronic
obstructive pulmonary disease (COPD), chronic inflammatory
syndromes, and cachexia of cancer. Further, diseases linked to
muscle stem cell senescence may be developed as a result of
clinical treatments that use compounds such as anthracyclines (i.e.
doxorubicin) that can cause severe skeletal muscle and cardiac
muscle toxicity leading to heart failure (Piegari et al., 2013,
Basic Res Cardiol. 108(2): 334).
[0008] In order to prevent some muscle stem senescence and related
muscular dystrophies, some dietary interventions such as creatine
supplements, resveratrol, protein--rich diets, and exercise
regimens are recommended and the use mesenchymal stem cells
transplantation or MuSC transplantation are being investigated.
[0009] Therefore, there is a significant need for the development
of strategies to prevent or delay MuSCs senescence in order to
facilitate muscle regeneration after injury or be used in diseases
related to impair MuSCs function and in aging.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to the unexpected findings
that the induction of the mitochondrial unfolded protein response
(UPR.sup.mt) and of prohibitin proteins can directly impact the
regulation of muscle stem cell senescence. The induction of the
mitochondrial unfolded protein response (UPR.sup.mt) and
specifically of prohibitin proteins by a mitochondrial UPR.sup.mt
inducing agent such as nicotinamide riboside (NR) or thiamphenicol
rejuvenates MuSCs in aged mice. Strategies that induce
mitochondrial unfolded protein response (UPR.sup.mt) and of
prohibitin proteins could therefore be utilized to reprogram
dysfunctional SCs in aging and disease to improve healthspan in
mammals.
[0011] One aspect of the invention provides a mitochondrial
UPR.sup.mt inducing agent for use in the prevention and/or
treatment of diseases or disorders associated with skeletal MuSCs
senescence and/or for promoting muscle tissue growth and/or
repair.
[0012] Another aspect of the invention provides a use of a
mitochondrial UPR.sup.mt inducing agent for the preparation of a
composition for prevention and/or treatment of diseases or
disorders associated with skeletal MuSCs senescence and/or for
promoting muscle tissue growth and/or repair.
[0013] Next, aspect of the invention provides a composition
comprising a mitochondrial UPR.sup.mt inducing agent and an agent
useful in the prevention and/or treatment of diseases or disorders
associated with skeletal MuSCs senescence and/or useful for
promoting muscle tissue growth and/or repair.
[0014] Another aspect of the invention provides a muscle stem cell
culture medium or a composition for preservation of muscle cells,
muscle grafts and muscle tissues comprising a mitochondrial
UPR.sup.mt inducing agent.
[0015] Another aspect of the invention provides a method of
preventing and/or treating of diseases or disorders associated with
skeletal MuSCs senescence and/or promoting muscle tissue growth
and/or repair in a subject, said method comprising administering an
effective amount of a mitochondrial UPR.sup.mt inducing agent or a
pharmaceutical composition thereof in a subject.
[0016] Another aspect of the invention provides a method of in vivo
maintaining and/or extending stemness of skeletal muscle stem cell
population comprising contacting a skeletal muscle stem cell
population or a muscle stem cell containing sample with a
composition of the invention.
[0017] Another aspect of the invention provides a method for
promoting muscle tissue growth and/or repair, in particular for
improving muscle cell/tissue survival, comprising using a
composition or a method of the invention.
[0018] Another aspect of the invention provides an ex-vivo method
for preparing a muscle graft sample in view of promoting muscle
tissue growth and/or repair, in particular for improving
cell/tissue survival after grafting said graft sample.
[0019] A further aspect of the invention provides a kit for
skeletal muscle stem cell culture or for preservation of muscle
cells, samples or tissues comprising at least one mitochondrial
UPR.sup.mt inducing agent or a composition of the invention with
instructions of use.
[0020] Another aspect of the invention provides a method of
cell-based therapy, said method comprising administering, grafting
a skeletal muscle stem cell composition of the invention. Said
skeletal muscle stem cells could be prepared according to a method
of the invention.
DESCRIPTION OF THE FIGURES
[0021] FIG. 1 shows reduction of mitochondrial content and
oxidative respiration in MuSCs during aging. A: GSEA demonstrates
up- and downregulated signaling pathways in MuSCs from two-year-old
mice, compared to four-month-old mice. Signaling pathways are
ranked on the basis of normalized enrichment scores (NESs);
positive and negative NESs indicate down- or upregulation in aged
MuSCs, respectively. Specific pathways related to MuSC function are
marked in black. B: Top 10 ranked downregulated pathways in MuSCs
from aged animals (GSE47177), based on gene ontology (GO)
enrichment. Pathways are ranked by family wise error rate (FWER) p
values. The top 5 significant down-regulated pathways are marked in
grey. C: Area-proportional Venn diagram representing 113 common
genes between the significantly downregulated genes (p<0.05) in
MuSC transcriptomes originating from aged mice (GSE47177 and
GSE47401), and genes from the human mitochondrial transcriptome. D:
Pie chart illustrating the percent composition of the common 113
mitochondrial genes found in C. TXN, transcription, TLN,
translation. E, Custom gene-set analysis showing enrichment of
OXPHOS, TCA cycle and UPR'' related transcripts from MuSCs of young
(Y) and aged (A) mice obtained from three independent data sets
(GSE47177, GSE47401 and GSE47104). Roman numerals indicate
corresponding OXPHOS complexes.
[0022] FIG. 2 shows reduction of mitochondrial content and
oxidative respiration in MuSCs during aging. A-D: MuSCs were
isolated from young (3 months old) and aged (22-24 months old)
C57BL/6J mice either freshly (A, C and D) or under in vitro cell
culture for three generations (B); A: qPCR validation of
transcriptional changes in mitochondrial genes of freshly sorted
MuSCs. B: OCR in isolated primary MuSCs, cultured in vitro for
three generations. C-D: Relative gene expression for UPR.sup.mt
genes (C) and cell senescence markers (D) in freshly sorted MuSCs.
Data are normalized to 36b4 mRNA transcript levels. All data are
shown as mean.+-.s.e.m. A-D, n=6 mice per group. *P<0.05,
**P<0.01. All statistical significance was calculated by
Student's t test.
[0023] FIG. 3 shows improved muscle stem cell numbers and muscle
function in NR-treated aged mice. Young (3 months old) and aged
(22-26 months old) C57BL/6J mice received a dietary supplement with
NR (400 mg/kg/day) for 6 weeks. All results are compared to
age-matched mice given a control diet. A-C: FACS contour plots of
Sca-1, Lin- (CD11b- CD23-, CD45-) cells isolated from muscle
tissue. Percentage of the marker CD34+/integrin
.alpha.7+/Lin-/Sca-1-cells, MuSC are noted in contour plots (A),
and quantified relative to the total Lin-/Sca-1-cell population (B)
or to the entire live cell population (C) for all treatment groups.
D: Representative images of PAX7 immunostained (arrows) tibialis
anterior (TA) muscle cross-sections from control and NR-treated
aged mice. Arrows point to PAX7 positive SCs. 20.times.20 .mu.m
insets shows single MuSCs. Scale bar=50 .mu.m. E-G: Comparison of
maximal running distance (E), running period (F) and grip strength
(G) between control and NR-treated aged mice. H: TA muscle
structure in tissue-sections from NR-treated aged mice with 7 and
14 days of regeneration CTX induced muscle damage. Images show
representative H/E staining of muscle cross sections. Scale bar=100
.mu.m. I-J: Representative images (I) and quantification (J) of
immunostained TA muscle cross-sections taken from control and
NR-treated mice 7 days after CTX-induced muscle damage. Arrows
point to PAX7 positive MuSCs. 20.times.20 .mu.m insets show single
MuSCs. Scale bar=50 .mu.m. K: Quantification of the signal
intensity ratio between MYOD1 and PAX7 in PAX7 positive muscle
MuSCs, performed on sections isolated 7 days after muscle damage.
L: Representative images of newly regenerated muscle fibers,
indicated by eMyHC immunostaining (arrows), 7 days after muscle
damage. Scale bar=50 M: The schema of MuSCs transplantation
experiments. MuSCs were double sorted from control and NR diet
treated B6 mice and transplanted into the different hind limbs of
the same Mdx mouse. N: Dystrophin immunostaining (arrows) of TA
muscle sections in Mdx mice 4-weeks after receiving
transplantations of MuSCs isolated from control or NR-treated aged
C56BL/6J donors. Scale bar=100 .mu.m. All data are represented as
mean.+-.s.e.m. *p<0.05, **p<0.01. ***p<0.001. A-E and I-M,
n=3-5 mice per group; F-H, n=10 control diet; n=7 NR-treated mice;
N-O, n=12 donor mice, n=3 recipient mice for each treatment.
[0024] FIG. 4 shows that NR treatment prevents MuSC senescence by
increasing mitochondrial respiration. A-B: Immunostaining (A,
.gamma.H2AX indicated by arrows) and quantification (B) of
.gamma.H2AX staining in freshly sorted MuSCs from aged mice.
20.times.20 .mu.m insets show single MuSCs. C: .beta.-galactosidase
staining of freshly sorted MuSCs from aged mice. D-E,
Quantification (D) of .gamma.H2AX and cleaved CASP3 immunostained
(E) primary MuSCs, isolated from control or NR-treated aged mice
and cultured in vitro for three generations. Scale bar=10 .mu.m. F:
Western blots showing the expression of .gamma.H2AX, cleaved
caspase3, and .beta.-actin in C2C12 myoblasts upon NR treatment at
the indicated time points. G: Colony formation ability assay in
freshly FACS sorted MuSCs from aged mice control or treated with
NR. H-I: Quantification of transcript expression for cell
senescence markers (H) or mitochondrial OXPHOS and TCA genes (I) in
aged MuSCs isolated from mice treated with NR. J: Basal and
uncoupled oxidative respiration and glycolysis, based on OCR and
extracellular acidification rate (ECAR), in C2C12 myoblasts that
were challenged with PBS or NR for 6 hours (control--white bars;
NR-black bars). All data are represented as mean.+-.s.e.m.
*p<0.05, **p<0.01. A-E, n=3 mice per group; G, n=24 in each
group; H and I, n=6 mice per group.
[0025] FIG. 5 shows that effects of NR on MuSC senescence are
mediated by prohibitin activation of UPR.sup.mt. A: Expression of
HSP60, CLPP and prohibitins in C2C12 myoblasts upon NR treatment at
the indicated time points. B: Quantification of transcript
expression for UPR.sup.mt and prohibitin genes in MuSCs from aged
(22-24 months old) C57BL/6J mice following 6 weeks of chow or NR
supplemented (400 mg/kg/day) diets. C: Expression of prohibitins
and cell cycle related genes in C2C12 myoblasts after a combined
Phb1 and Phb2 shRNA knockdown in combination with a 6-hour NR
treatment. D: Expression of prohibitins and cell cycle related
genes in C2C12 myoblasts with the combined overexpression of Phb1
and Phb2. All data are represented as mean.+-.s.e.m. *p<0.05,
**p<0.01. B, n=6 mice per group.
[0026] FIG. 6 shows increased stem cell number and stemness in
NR-treated Mdx mice. Mdx mice (one-months-old) received a dietary
supplement with NR (400 mg/kg/day) for 10 weeks. All results are
compared to Mdx mice given a control diet. A: .beta.-galactosidase
staining of MuSCs isolated from C57BL/10 SnJ or Mdx mice and
cultured in vitro for three generations. Scale bar=10 .mu.m. B-D:
FACS contour plots of Sca-1.sup.-, Lin.sup.- (CD11b.sup.-
CD23.sup.-, CD45.sup.-) cells isolated from muscle tissue.
Percentage of the CD34.sup.+/integrin
.alpha.7.sup.+/Lin.sup.-/Sca-1.sup.- MuSC populations are noted in
in contour plots (B), and quantified relative to the total
Lin.sup.-/Sca-1.sup.- cell population (C) or to the entire live
cell population (D), control--white bars, NR-black bars. E-F:
Immunostaining of muscle stem cells (PAX7) (E) and newly
regenerated muscle fibers (eMyHC) (F) in tissue-sections of
NR-treated Mdx mice 7 days after CTX-induced muscle damage. Arrows
point to PAX7.sup.+ MuSCs (E) and eMyHC (F). 20.times.20 .mu.m
insets show single MuSCs. Scale bar=50 .mu.m. G-I: FACS contour
plots (G), quantification (H) and distribution (I) of MuSC
autofluorescence as a measure of the relative NAD(P)H concentration
upon UV light excitation. Autofluorescence emission was detected
using 405/450 nm. Arrow in (I) points to the highly autofluorescent
stem cell population. J: .beta.-galactosidase staining of
FACS-sorted MuSCs from C57BL/6 (B6), untreated (Mdx) or NR-treated
Mdx (Mdx with NR) mice challenged with PBS or NR for 6 hours. K:
Immunostaining showing .gamma.H2AX and cleaved caspase-3 in MuSCs
cultured in vitro for three generations. Arrow points to a
.gamma.H2AX-positive nucleus. Scale bar=10 .mu.m. L: Muscle
structure in tissue-sections from NR-treated Mdx mice with 7 days
of recovery following CTX induced muscle damage. Images show
representative H/E staining of muscle cross sections. Scale bar=100
.mu.m. All data are represented as mean.+-.s.e.m. *p<0.05,
**p<0.01. A-I and K-L, n=3-5 per treatment group; J, n=3 mice
and n=6 in vitro treatments.
[0027] FIG. 7 shows expression of prohibitins and cell cycle
related genes in C2C12 myoblasts following different treatment
periods with 50 .mu.g/ml TAP, which induces a mitonuclear imbalance
and UPR.sup.mt.
DETAILED DESCRIPTION
[0028] As used herein "adult stem cells" or "SCs" or "somatic stem
cells" refers to undifferentiated cells, found throughout the body
after development, capable of self-renewal (ability of multiply by
cell division while still maintaining cell's undifferentiated
state). The function of said cells is to replenish dying cells and
regenerate damaged tissues of the organ from which they originate,
potentially regenerating the entire organ from a few cells.
[0029] As used herein "muscle stem cells" or "MuSCs" or "satellite
cells" that refers to adult stem cells of muscle, having the
capacity to self-renew and to differentiate into myocytes, which
fuse amongst each other or with the existing myofibers to compose
the muscle fiber units. The known markers of MuSCs include, but are
not limited to several transcription factors PAX7, MYF5 and cell
surface antigens CD34, Integrin .alpha.7 and M-Cadherin (Yin et
al., 2013, Physiological Reviews, 93: 23; Tabebordbar et al., 2013,
Annual Review of Pathology, 8: 441).
[0030] As used herein "SC senescence" refers to a stable and
irreversible loss of proliferative capacity (stable cell cycle
arrest), despite continued viability and metabolic activity.
Specific markers of MuSCs senescence include, among others,
.beta.-galactosidase activation, H2.gamma.AX phosphorylation,
downregulation of cell cycle regulators (Mki67, Cdk4, Ccndl,
Cdknla), and induction of inflammatory factors (IL6 and IL18)
(Kuilman et al., supra; Lopez-Otin et al., supra). Cdkn1a and
Cdkn2a are among the most important general regulators of cellular
senescence that are increased in senescent muscle SCs (Burd et al.,
supra; Lopez-Otin et al., supra).
[0031] The expression "skeletal muscle stem cell senescence"
includes muscle dystrophy diseases, includes Duchenne's muscular
dystrophy (DMD), Becker's muscular dystrophy (BMD), Congenital
muscular dystrophy, Distal muscular dystrophy, Emery-Dreifuss'
muscular dystrophy, Facio-scapulo-humeral muscular dystrophy,
Limb-girdle muscular dystrophy, Myotonic muscular dystrophy and
Oculopharyngeal muscular dystrophy. It further includes other
inherited myopathies, such as myotonia, congenital myopathies
(includes nemaline myopathy, multi/minicore myopathy, centronuclear
myopathy), metabolic myopathies (includes glycogen storage diseases
and lipid storage disorder), inflammatory myopathies, such as
dermatomyositis, polymyositis, inclusion body myositis and
auto-immune myositis. Other diseases linked to muscle stem cell
senescence include muscle wasting induced by nutritional
deficiencies. Diseases linked to muscle stem cell senescence may be
developed in the context of other diseases, such as chronic
obstructive pulmonary disease (COPD), chronic inflammatory
syndromes, and cachexia of cancer. Further, diseases linked to
muscle stem cell senescence may be developed as a result of
clinical treatments that use compounds such as anthracyclines (i.e.
doxorubicin) that can cause severe skeletal muscle and cardiac
muscle toxicity leading to heart failure (Piegari et al., 2013,
Basic Res Cardiol. 108(2): 334).
[0032] According to a particular aspect, muscle stem cells and
muscle stem cell-containing samples for graft purposes are
allogeneic and autologous.
[0033] The term "cell-based therapy" or "cell-based tissue
regeneration" include cell replacement therapies making use of
allogenic or autologous muscle stem cells, or in the direct
induction of tissue regeneration by in situ stimulation of resident
muscle stem cells (e.g. inducing resident stem cells mobilization
and differentiation for repair), as alternatives to surgical
interventions and muscle organ/tissue transplantation. Methods and
compositions according to invention can be advantageously used in
methods of "cell-based therapy" or "cell-based tissue regeneration"
methods used to produce differentiated muscle tissue from
progenitor cells or stem cells.
[0034] As used herein the term "skeletal muscle stem cell (MuSC)
sample" or "muscle stem cell (MuSC) containing sample" comprises
any ex-vivo sample comprising muscle stem cell isolated from a
source of said cells (e.g. human or mouse skeletal muscle
tissue).
[0035] As used herein, the term "muscle stem cell culture medium"
refers to any standard cell stem cell culture medium, optionally
comprising appropriate differentiation factors, the nature of which
may be adapted to the nature of the cell, in particular culture
medium suitable for stem cell expansion such as for example culture
media described in the following examples or described in Boitano
et al., 2010, Science 329, 1345-8.
[0036] According to a particular aspect, the medium for isolation
and maintenance of muscle cells or muscle tissues according to the
invention may further comprise of fetal bovine serum (FBS),
recombinant human basic fibroblast growth factor (rhFGF), or
chicken embryo extract, penicillin and streptomycin.
[0037] As used herein, the term "mitochondrial UPR.sup.mt inducing
agent" is an agent which is able to induce UPR.sup.mt such as
measured by several UPR.sup.mt markers including HSP60, CLPP,
HSP70/Mortalin and prohibitins.
[0038] Mitochondrial UPR.sup.mt inducing agent can be easily
identified by known techniques, such via monitoring the induction
of CLPP, HSP60, HSP70/Mortalin, or prohibitins proteins, the
imbalance between proteins encoded in mtDNA or nDNA, the induction
of cellular NAD.sup.+ contents, and the reduction of cellular
PARylation status. Examples of said agent include, but are not
limited nicotinamide riboside (NR), thiamphenicol, or thiamphenicol
analogs thereof such as amphenicols. According to another aspect,
mitochondrial UPR.sup.mt inducing agents are tetracyclines or
analogues thereof. Identification of any further agents to able to
induce UPR.sup.mt can be identified by standard methods known to
the skilled person.
[0039] As used herein, the term "thiamphenicol analogs" includes
amphenicols such as chloramphenicol. Amphenicols are antibiotics
with a phenylpropanoid structure such as chloramphenicol,
azidamfenicol and florfenicol.
[0040] As used therein, the term "tetracyclines" includes
doxocycline and minocycline.
[0041] As used herein, the term "MuSCs cell depleted subjects" mean
subjects presenting a significant reduction in the quantity and
quality of muscle tissue specific adult muscle stem cells and more
specifically aged human subjects, MuSCs are reduced.
[0042] As used herein, "treatment" and "treating" and the like
generally mean obtaining a desired pharmacological and
physiological effect. The effect may be prophylactic in terms of
preventing or partially preventing a disease, symptom or condition
thereof and/or may be therapeutic in terms of a partial or complete
cure of a disease, condition, symptom or adverse effect attributed
to the disease. The term "treatment" as used herein covers any
treatment of a disease in a mammal, particularly a human, and
includes: (a) preventing the disease from occurring in a subject
which may be predisposed to the disease but has not yet been
diagnosed as having it for example based on familial history or
age; (b) inhibiting the disease, i.e., arresting its development;
or relieving the disease, i.e., causing regression of the disease
and/or its symptoms or conditions such as improvement or
remediation of damage. In particular, a method according to the
invention is useful in the maintenance and/or extension of stemness
of stem cell population; prevention of cell senescence/apoptosis of
stem cell population; maintenance and/or prevention of the
reduction of stem cell proliferation/cell cycle process;
maintenance and or prevention of the reduction of differentiation
potential of stem cell population.
[0043] The term "subject" as used herein refers to mammals. For
example, mammals contemplated by the present invention include
human, primates, domesticated animals such as dogs, cats, cattle,
sheep, pigs, horses, laboratory rodents and the like.
[0044] The term "efficacy" of a treatment or method according to
the invention can be measured based on changes in the course of
disease or condition in response to a use or a method according to
the invention. For example, the efficacy of a treatment or method
according to the invention can be measured through the measurement
of through the measurement of muscle damage parameters from blood
biochemical measurements of creatine kinase, aspartate
aminotransferase and total protein levels. The efficacy of a
treatment or method according to the invention can be measured
through the measurement of muscle force, as well as immunostaining
of MuSCs number and the analysis of regeneration of damaged
muscle.
[0045] The terms "effective amount", "therapeutic effective
amount", and "prophylactic effective amount" refer to a dosage of a
compound or composition effective for eliciting a desired effect,
commensurate with a reasonable benefit/risk ratio and will vary
from subject to subject, depending, for example, on species, age,
and general condition of a subject, severity of the side effects or
disorder, identity of the particular compound(s), mode of
administration, and the like. In certain embodiments, the desired
dosage can be delivered using multiple administrations. Those terms
as used herein may also refer to an amount effective at bringing
about a desired in vivo effect in an animal, preferably, a human,
such as induction of proliferation of tissue specific stem cells
and the acceleration of tissue regeneration.
[0046] The efficacy of a treatment or method according to the
invention can be measured by determining the level of cell
maturation or of cell differentiation in the cell culture medium
using standard methods in the art, including visual observation by
microscopy, detection of markers which are specific for the
targeted differentiated tissue by immunological staining or
blotting and by molecular assays of mRNA, chromatin, nuclear DNA,
mtDNA, or microRNA.
[0047] Use According to the Invention
[0048] According to an embodiment, the invention provides a
mitochondrial UPR.sup.mt inducing agent for use in the prevention
and/or treatment of a disease or disorder associated with muscle SC
senescence and/or decreased muscle SCs number and/or for promoting
muscle tissue growth and/or repair.
[0049] According to another embodiment, the invention provides a
use of a mitochondrial UPR.sup.mt inducing agent for the
preparation of a composition or a food supplement for the
prevention and/or treatment of a disease or disorder associated
with associated with muscle SC senescence and/or decreased muscle
SCs number and/or for promoting muscle tissue growth and/or
repair.
[0050] According to another embodiment, the invention provides a
mitochondrial UPR.sup.mt inducing agent or composition thereof for
use in the treatment of an injured muscle tissue notably after an
injury or trauma.
[0051] According to another embodiment, the invention provides a
method for promoting tissue growth and/or repair, in particular for
improving cell/tissue survival, said method comprising contacting
or administering to a muscle stem cell or to an isolated muscle
tissue in culture before transplantation/grafting to a mammal in
need thereof (ex-vivo), a mitochondrial UPR.sup.mt inducing agent
or composition thereof in an amount effective to stimulate
differentiation, maturation, proliferation, survival of cells and
tissues and/or maintain and/or extend stemness of stem cell
population.
[0052] A method of preparation of a cell composition for cell-based
therapy comprising a step of contacting with or administering to a
muscle stem cell a mitochondrial UPR.sup.mt inducing agent or
composition thereof.
[0053] According to a further embodiment of the invention, is
provided a kit for muscle stem cell culture or muscle tissue graft
preparation or preservation comprising at least one mitochondrial
UPR.sup.mt inducing agent or mixtures of formulations thereof
together with instructions for use.
[0054] A method of preparation of a graft organ, cell or tissue
comprising a step of contacting said graft organ, cell or tissue
with a mitochondrial UPR.sup.mt inducing agent or composition
thereof.
[0055] According to a further embodiment, the invention provides a
method of prevention and/or treatment of diseases or disorders
associated with muscle SC senescence, said method comprising
grafting of a cell composition or graft sample prepared according
to methods described herein.
[0056] According to a particular embodiment, the invention provides
a method for promoting muscle tissue growth and/or repair in a
subject in need thereof, said method comprising administering an
effective amount of a mitochondrial UPR.sup.mt inducing agent or
composition thereof in said subject.
[0057] According to a particular aspect, a disease or disorder
associated with muscle SC senescence and/or decreased muscle SCs
number is selected from muscle dystrophies, myopathies and muscle
frailty and sarcopenia of the aged.
[0058] According to a particular aspect, a method of the invention
is an ex-vivo method useful for maintaining and/or extending
stemness of a muscle stem cell population.
[0059] According to another particular embodiment of the invention,
is provided a method for ex-vivo preparing a graft sample
comprising the steps of: [0060] a) providing a MuSC-containing
sample in a stem cell culture medium; [0061] b) contacting said
MuSC-containing sample with at least one mitochondrial UPR.sup.mt
inducing agent or a mixture thereof in an amount effective to
stimulate the survival and the maintenance of the stemness of the
stem cells within the sample increased as compared to a sample in
absence of said mitochondrial UPR.sup.mt inducing agent.
[0062] According to a further aspect, said MuSC-containing sample
is further combined with a muscle tissue or organ to be grafted,
before grafting. Isolated muscle stem cells can be treated with at
least one UPR.sup.mt inducing agent or a mixture thereof, or in
combination with another agent useful to proliferation and
maintenance of stemness.
[0063] According to a particular aspect, the method of muscle graft
sample preparation of the invention is useful for promoting muscle
tissue growth and/or repair following graft sample grafting.
[0064] According to another particular embodiment, is provided an
ex-vivo method of the invention wherein stemness (e.g.
self-renewing capacity of SCs) is assessed by quantifying stemness
markers such as transcription factors PAX7, MYF5 and cell surface
antigens CD34, Integrin .alpha.7 and M-Cadherin of the cell
preparation obtained after step b).
[0065] According to another embodiment, is provided a muscle stem
cell culture medium comprising at least one mitochondrial
UPR.sup.mt inducing agent, optionally further comprising a cocktail
of cytokines and growth factors useful for stem cell expansion.
[0066] According to a further embodiment, the agent mitochondrial
UPR.sup.mt inducing agent is selected from NR, thiamphenicol or
analogues thereof, such as amphenicols.
[0067] According to another further embodiment, the mitochondrial
UPR.sup.mt inducing agent is NR.
[0068] According to another further embodiment, the mitochondrial
UPR.sup.mt inducing agent is thiamphenicol or an analogue
thereof.
[0069] According to another further embodiment, the mitochondrial
UPR.sup.mt inducing agent is a tetracycline or an analogue
thereof.
[0070] In particular, treatment of diseases or disorders associated
with MuSC senescence comprises promoting tissue homeostasis, tissue
regeneration, and the ability of stem cells to infiltrate tissues
upon transplantation. This includes maintenance and/or extension of
stemness of stem cell population; maintenance and/or prevention of
the reduction of stem cell proliferation/cell cycle process;
maintenance and/or prevention of the reduction of differentiation
potential of stem cell population.
[0071] Muscle cells and muscle graft samples obtained by a method
according to the invention can be formulated for clinical stem cell
or graft or tissue transplantation, or for augmentation stem
function or for cell-based therapy in a subject in need
thereof.
[0072] Compositions According to the Invention
[0073] Mitochondrial UPR.sup.mt inducing agent or formulations
thereof may be administered as a pharmaceutical formulation or a
food supplement or may be formulated as stem cell culture or organ
preservation media, which can contain one or more agents according
to the invention in any form described herein. The compositions
according to the invention, together with a conventionally employed
adjuvant, carrier, diluent or excipient may be placed into the form
of pharmaceutical compositions and unit dosages thereof, and in
such form may be employed as solids, such as tablets or filled
capsules, or liquids such as solutions, suspensions, emulsions,
elixirs, or capsules filled with the same, all for oral use, or in
the form of sterile injectable solutions for parenteral (including
subcutaneous) use by injection or continuous infusion. Injectable
compositions are typically based upon injectable sterile saline or
phosphate-buffered saline or other injectable carriers known in the
art. Such pharmaceutical compositions and unit dosage forms thereof
may comprise ingredients in conventional proportions, with or
without additional active compounds or principles, and such unit
dosage forms may contain any suitable effective amount of the
active ingredient commensurate with the intended daily dosage range
to be employed.
[0074] Compositions of this invention may be liquid formulations
including, but not limited to, aqueous or oily suspensions,
solutions, emulsions, syrups, and elixirs. The compositions may
also be formulated as a dry product for reconstitution with water
or other suitable vehicle before use. Such liquid preparations may
contain additives including, but not limited to, suspending agents,
emulsifying agents, non-aqueous vehicles and preservatives.
Suspending agents include, but are not limited to, sorbitol syrup,
methylcellulose, glucose/sugar syrup, gelatin, hydroxyethyl
cellulose, carboxymethyl cellulose, aluminum stearate gel, and
hydrogenated edible fats. Emulsifying agents include, but are not
limited to, lecithin, sorbitan monooleate, and acacia.
Preservatives include, but are not limited to, methyl or propyl
p-hydroxybenzoate and sorbic acid. Dispersing or wetting agents
include but are not limited to poly(ethylene glycol), glycerol,
bovine serum albumin, Tween.RTM., Span.RTM..
[0075] Compositions of this invention may also be formulated as a
depot preparation, which may be administered by implantation or by
intramuscular injection.
[0076] Solid compositions of this invention may be in the form of
tablets or lozenges formulated in a conventional manner. For
example, tablets and capsules for oral administration may contain
conventional excipients including, but not limited to, binding
agents, fillers, lubricants, disintegrants and wetting agents.
Binding agents include, but are not limited to, syrup, accacia,
gelatin, sorbitol, tragacanth, mucilage of starch and
polyvinylpyrrolidone. Fillers include, but are not limited to,
lactose, sugar, microcrystalline cellulose, maize starch, calcium
phosphate, and sorbitol. Lubricants include, but are not limited
to, magnesium stearate, stearic acid, talc, polyethylene glycol,
and silica. Disintegrants include, but are not limited to, potato
starch and sodium starch glycollate. Wetting agents include, but
are not limited to, sodium lauryl sulfate. Tablets may be coated
according to methods well known in the art.
[0077] The compounds of this invention can also be administered in
sustained release forms or from sustained release drug delivery
systems.
[0078] According to a particular embodiment, compositions according
to the invention are for intravenous use.
[0079] According to a particular aspect, the formulations of the
invention are oral formulations.
[0080] According to a particular embodiment, compositions according
to the invention are food supplement.
[0081] In another particular aspect, the compositions according to
the invention are adapted for delivery by repeated
administration.
[0082] In another particular aspect, the compositions according to
the invention are adapted for the stem cell culture or graft
preparation or transplantation.
[0083] According to a particular embodiment, compositions of the
invention are veterinary compositions.
[0084] According to a particular embodiment, compositions of the
invention are adapted for topical delivery.
[0085] Further materials as well as formulation processing
techniques and the like are set out in Part 5 of Remington's "The
Science and Practice of Pharmacy", 22.sup.nd Edition, 2012,
University of the Sciences in Philadelphia, Lippincott Williams
& Wilkins, which is incorporated herein by reference.
[0086] Mode of Administration
[0087] Mitochondrial UPR.sup.mt inducing agents or formulations
thereof may be administered in any manner including orally,
parenterally, intravenously, rectally, or combinations thereof.
Parenteral administration includes, but is not limited to,
intravenous, intra-arterial, intra-peritoneal, subcutaneous and
intramuscular. The compositions of this invention may also be
administered in the form of an implant, which allows slow release
of the compositions as well as a slow controlled iv infusion.
[0088] According to a particular aspect, mitochondrial UPR.sup.mt
inducing agents or formulations thereof are to be administered by
injection.
[0089] According to a particular aspect, the mitochondrial
UPR.sup.mt inducing agent or formulation thereof are to be
administered orally.
[0090] Typically, a dosage rate of NR can be at a dosage rate
ranging from about 1 mg/kg/day to about 400 mg/kg/day.
[0091] Typically, a dosage rate of thiamphenicol can be at a dosage
rate ranging from about 5 to about 15 mg/kg/day.
[0092] The dosage administered, as single or multiple doses, to an
individual will vary depending upon a variety of factors, including
pharmacokinetic properties, patient conditions and characteristics
(sex, age, body weight, health, size), extent of symptoms,
concurrent treatments, frequency of treatment and the effect
desired.
[0093] Combination
[0094] According to the invention, mitochondrial UPR.sup.mt
inducing agents or formulations thereof, including pharmaceutical
formulations thereof can be administered alone or in combination
with a co-agent (e.g. multiple drug regimens) useful for preventing
or treating a disease or disorder associated with muscle SC
senescence and/or decreased muscle SCs number.
[0095] According to the invention, mitochondrial UPR.sup.mt
inducing agents or formulations thereof, including pharmaceutical
formulations thereof can be administered alone or in combination
with a co-agent (e.g. multiple drug regimens) useful for graft
muscle tissue improvement, in particular for promoting muscle
tissue growth and/or repair, in particular for improving
cell/tissue survival.
[0096] According to the invention, mitochondrial UPR.sup.mt
inducing agents or formulations thereof, can be administered to a
subject prior to, simultaneously or sequentially with other
therapeutic regimens or co-agents useful for preventing or treating
a disease or disorder associated with muscle SC senescence and/or
decreased muscle SCs number or useful for promoting muscle tissue
growth and/or repair.
[0097] A compound of the invention or a formulation thereof
according to the invention that is administered simultaneously with
said co-agents can be administered in the same or different
composition(s) and by the same or different route(s) of
administration.
[0098] According to a particular embodiment, is provided a
formulation (such as a food supplement or a pharmaceutical
composition) comprising a mitochondrial UPR.sup.mt inducing agent,
combined with at least one co-agent useful for preventing or
treating a disease or disorder associated with muscle SC senescence
and/or decreased SCs number or useful for promoting muscle tissue
growth and/or repair. These co-agents include but are not limited
to transforming growth factor .beta. (TGF.beta.) family
protein/receptor inhibitors, such as the myostatin inhibitor or
follistatin-derived peptide FS I-I (Tsuchida, 2008, Acta Myol,
27(1):14-18), P38 and JAK-STAT signalling pathway inhibitors,
including compounds such as SB203580, SB202190, BIRB796, AG490,
5,15-Diphenylporphyrin (Bernet et al., supra; Cosgrove et al.,
supra; Price et al., supra); muscle stem cell activators, such as
Notch signalling activators (Conboy et al., 2005, Nature, 433:760)
and anabolic stimulators of the muscle, such as IGF-1 (Musaro, et
al., 2004, Basic Appl Myol, 14(1):29-32).
[0099] Patients
[0100] According to an embodiment, subjects according to the
invention are subjects suffering from disease or disorders
associated with muscle stem cells senescence, in particular stem
cell related muscular dystrophy, such as Duchenne's muscular
dystrophy (DMD), Becker's muscular dystrophy (BMD), Congenital
muscular dystrophy, Distal muscular dystrophy, Emery-Dreifuss'
muscular dystrophy, Facio-scapulo-humeral muscular dystrophy,
Limb-girdle muscular dystrophy, Myotonic muscular dystrophy and
[0101] Oculopharyngeal muscular dystrophy.
[0102] In another particular embodiment, subjects according to the
invention are subjects suffering from disease or disorders
associated with muscle stem cells senescence, in particular
inherited myopathies that includes diseases such as myotonia,
congenital myopathies such as nemaline myopathy, multi/minicore
myopathy and centronuclear myopathy, metabolic myopathies such as
glycogen storage diseases and lipid storage disorder, inflammatory
myopathies, such as dermatomyositis, polymyositis, inclusion body
myositis and auto-immune myositis.
[0103] In another particular embodiment, subjects according to the
invention are subjects suffering from non-mitochondrial
myopathies.
[0104] In a particular embodiment, subjects according to the
invention are subjects suffering from disease or disorders
associated with muscle stem cells senescence resulting from a
traumatic injury.
[0105] In a particular embodiment, subjects according to the
invention are subjects suffering from disease or disorders
associated with muscle stem cells senescence in frailty and
sarcopenia, resulting from aging.
[0106] In another particular embodiment, subjects according to the
invention are muscle stem cells depleted subjects, in particular
aged subjects.
[0107] In another particular embodiment, subjects according to the
invention are subjects suffering from disease or disorders
associated with muscle stem cells senescence, in particular muscle
wasting induced by nutritional deficiencies.
[0108] In another particular embodiment, subjects according to the
invention are subjects suffering from disease or disorders
associated with muscle stem cells senescence, in particular
developed in the context of diseases, such as chronic obstructive
pulmonary disease (COPD), chronic inflammatory syndromes, and
cachexia of cancer.
[0109] In another particular embodiment, subjects according to the
invention are subjects suffering from disease or disorders
associated with muscle stem cells senescence, in particular
acquired myopathies, such as drug/toxic agents-induced myopathy,
alcoholic myopathy, myositis ossificans, rhabdomyolysis and
myoglobinurias.
[0110] In an embodiment, subjects according to the invention are
subjects undergoing graft transplantation.
[0111] References cited herein are hereby incorporated by reference
in their entirety. The present invention is not to be limited in
scope by the specific embodiments and drawings described herein,
which are intended as single illustrations of individual aspects of
the invention, and functionally equivalent methods and components
are within the scope of the invention. The examples illustrating
the invention are not intended to limit the scope of the invention
in any way.
EXAMPLES
[0112] CASP3 (caspase-3), Cdknla (cyclin-dependent kinase inhibitor
1A or p21), CTX (cardiotoxin), DAPI
(4',6-diamidino-2-phenylindole), eMyHC (embryonic myosin heavy
chain), GO (gene ontology), GSEA (gene set enrichment analysis),
OCR (oxygen consumption rate), OXPHOS (oxidative phosphorylation),
UPR.sup.mt (mitochondrial unfolded protein response), NES
(normalized enrichment scores), NR (nicotinamide riboside), TCA
(tricarboxylic acid cycle), TA (tibialis anterior).
Example 1: Identification of Mitochondrial Dysfunction as a
Biomarker of MuSCs Senescence
[0113] To identify the role of mitochondrial function in muscle SCs
senescence, MuSCs from young and aged mice were compared. To
identify the principal mechanisms initiating MuSC senescence,
publically available MuSC gene expression datasets from young
(.about.3 months) and aged (.about.24 months) mice were compared
with the use of gene set enrichment analysis. (GSEA; GEO dataset
IDs: GSE47177, GSE47401 and GSE47104) as described below.
[0114] Bioinformatic analysis. Quadriceps microarray data from
young and aged mice MuSCs (Price et al., supra; Bernet et al.,
supra; Liu et al., supra) were analyzed for transcript expression
using the Kyoto encyclopedia of genes and genomes (KEGG), gene
ontology (GO) or gene set enrichment analysis (GSEA) analysis. Raw
microarray data are also publicly available on Gene Expression
Omnibus (GEO) database under the accession numbers GSE47177,
GSE47401 and GSE47104. All gene expression heat maps were draw
using GENE-E software.
[0115] Gene expression analyses. Total RNA was extracted from MuSCs
by sorting cells directly into TriPure RNA isolation reagent
(Roche) or from cultured C2C12 myoblasts using TriPure reagent
according to the product manual. Total RNA was transcribed to cDNA
using QuantiTect Reverse Transcription Kit (Qiagen). Expression of
selected genes was analyzed using the LightCycler480 system (Roche)
and LightCycler.RTM. 480 SYBR Green I Master reagent (Roche). The
acidic ribosomal protein 36b4 gene (ribosomal protein, large, P0,
Rplp0) was used as housekeeping reference. Primer sets for
quantitative real-time PCR analyses are shown in Table 1 below.
TABLE-US-00001 TABLE 1 Gene name Forward primer Reverse Primer 36b4
AGATTCGGGATATGCTGTTGG AAAGCCTGGAAGAAGGAGGTC SEQ ID NO: 1 SEQ ID NO:
2 Ndufb5 CTTCGAACTTCCTGCTCCTT GGCCCTGAAAAGAACTACG SEQ ID NO: 3 SEQ
ID NO: 4 Sdha GGAACACTCCAAAAACAGAC CCACCACTGGGTATTGAGTAG CT SEQ ID
NO: 5 AA SEQ ID NO: 6 Sdhc GCTGCGTTCTTGCTGAGACA
ATCTCCTCCTTAGCTGTGGTT SEQ ID NO: 7 SEQ ID NO: 8 Cox5b
AAGTGCATCTGCTTGTCTCG GTCTTCCTTGGTGCCTGAAG SEQ ID NO: 9 SEQ ID NO:
10 Atp5b GGTTCATCCTGCCAGAGACTA AATCCCTCATCGAACTGGACG SEQ ID NO: 11
SEQ ID NO: 12 Mdh2 TTGGGCAACCCCTTTCACTC GCCTTTCACATTTGCTCTGG SEQ ID
NO: 13 TC SEQ ID NO: 14 Idh2 GGAGAAGCCGGTAGTGGAGAT
GGTCTGGTCACGGTTTGGAA SEQ ID NO: 15 SEQ ID NO: 16 Idh3a
CCCATCCCAGTTTGATGTTC ACCGATTCAAAGATGGCAAC SEQ ID NO: 17 SEQ ID NO:
18 Cdkn1a GTGGGTCTGACTCCAGCCC CCTTCTCGTGAGACGCTTAC SEQ ID NO: 19
SEQ ID NO: 20 Mki67 TTGGAAAGGAACCATCAAGG TTTCTGCCAGTGTGCTGTTC SEQ
ID NO: 21 SEQ ID NO: 22 Cdk4 CCGGTTGAGACCATTAAGGA
CACGGGTGTTGCGTATGTAG SEQ ID NO: 23 SEQ ID NO: 24 Ccna2
AAGAGAATGTCAACCCCGAAA ACCCGTCGAGTCTTGAGCTT SEQ ID NO: 25 SEQ ID NO:
26 Ccnd1 GAGCGTGGTGGCTGCGATGC GGCTTGACTCCAGAAGGGCTT AA SEQ ID NO:
27 CAAT SEQ ID NO: 28 Ccne1 CAAAGCCCAAGCAAAGAAAG
CCACTGTCTTTGGAGGCAAT SEQ ID NO: 29 SEQ ID NO: 30 Cdc6
GACACAAGCTACCATGGTTT CAGGCTGGACGTTTCTAAGTT SEQ ID NO: 31 SEQ ID NO:
32 IL6 GGTGACAACCACGGCCTTCCC AAGCCTCCGACTTGTGAAGTG SEQ ID NO: 33 GT
SEQ ID NO: 34 IL18 GTGAACCCCAGACCAGACTG CCTGGAACACGTTTCTGAAA SEQ ID
NO: 35 GA SEQ ID NO: 36 Hsp60 ACAGTCCTTCGCCAGATGAG
TGGATTAGCCCCTTTGCTGA AC SEQ ID NO: 37 SEQ ID NO: 38 Hsp10
CTGACAGGTTCAATCTCTCC AGGTGGCATTATGCTTCCAG AC SEQ ID NO: 39 SEQ ID
NO: 40 Clpp CACACCAAGCAGAGCCTACA TCCAAGATGCCAAACTCTTG SEQ ID NO: 41
SEQ ID NO: 42 Phb TCGGGAAGGAGTTCACAGAG CAGCCTTTTCCACCACAAAT SEQ ID
NO: 43 SEQ ID NO: 44 Phb2 CAAGGACTTCAGCCTCATCC GCCACTTGCTTGGCTTCTAC
SEQ ID NO: 45 SEQ ID NO: 46
[0116] Animals. Young (1 month old) and aged (20-24 months old)
C57BL/6JRj mice, purchased from Janvier Labs, and five weeks old
male C57BL/10SnJ mice or C57BL/10ScSn-Dmdmdx/J, purchased from The
Jackson Laboratory, were fed with pellets containing vehicle or NR
(400 mg/kg/day) for 6-8 weeks. The pellets were prepared by mixing
powdered chow diet (D12450B, Research Diets Inc.) with water or
with NR dissolved in water. Pellets were dried under a laminar flow
hood for 48 hours. All mice were housed in micro-isolator cages in
a room illuminated from 7:00 am-7:00 pm with ad libitum access to
diet and water.
[0117] FACS based muscle stem cell isolation. Gastrocnemius,
soleus, quadriceps, and tibialis anterior muscles from both limbs
were excised and transferred into PBS on ice. All muscles were
trimmed, minced and digested with 0.1 mg/ml of type II collagenase
(Sigma) in PBS for 15 min at 37.degree. C. Samples were then
centrifuged at 750 g for 5 min and further digested in 1 mg/ml of
collagenase/dispase (Roche) for 30 mins at 37.degree. C. Muscle
slurries were sequentially filtered through 100, 70 and 40 pm cell
strainers. The isolated cells were then washed in washing buffer
(PBS+2.5% FBS) then resuspended in 200 .mu.l of washing buffer and
immediately stained with antibodies, including the MuSC markers
CD31 (1:800, eBioscience, eFluor450 conjugated); CD34 (1:200,
eBioscience, eFluor660 conjugated); CD45 (1:200, eBioscience,
eFluor450 conjugated); CD11b (1:400, eBioscience, eFluor450
conjugated); Sca-1 (1:1000, eBioscience, PE-Cy7 conjugated); and
.alpha.7 integrin (1:300, MBL) for 30 min at 4.degree. C. Secondary
staining was performed with a mixture of goat anti-mouse antibody
(1:800, Life technologies, Alexa Fluor 488 conjugated) and
propidium Iodide (PI, Sigma) for 15 min at 4.degree. C. in the
dark. Stained cells were analysed and sorted using the FACSAria II
instrument (BD Biosciences). Debris and dead cells were excluded by
forward scatter, side scatter and PI gating. Cells were sorted
either directly on slides for immunostaining and into TriPure
(Roche) reagent for RNA extraction.
[0118] Respirometry on MuSCs. Basal and uncoupled oxygen
consumption rates (OCRs) were measured using the Seahorse
extracellular flux bioanalyzer (XF96, Seahorse Bioscience Inc.). To
uncouple mitochondria, 5 uM of FCCP was injected after a basal
respiration measurement. All measurements were performed in
triplicates and results were normalized to total cell number seeded
(primary MuSCs) assessed using a Bradford kit (Bio-Rad).
[0119] Enrichment scores of young versus aged datasets demonstrate
the upregulation of senescence pathways and downregulation of cell
cycle pathways with age (FIG. 1A) that is consistent with the
paradigm that irreversible cell cycle arrest is a primary marker of
cellular senescence (Kuilman et al., supra; Lopez-Otin et al.,
supra). In all three datasets, citric acid cycle (TCA, also known
as the tricarboxylic acid cycle or the Krebs cycle) and oxidative
phosphorylation (OXPHOS) pathways were amongst the most
downregulated pathways in aged MuSCs, despite the general
assumption that MuSCs predominantly rely on glycolysis (FIG. 1A).
Gene ontology (GO) term analysis, of genes significantly
(p<0.05) downregulated in aged MuSCs, further demonstrated that
many of these pathways were related to mitochondrial function (FIG.
1B). Common downregulated genes during aging indicated a
substantial overlap (113 genes; 11.59%) with mitochondrial genes
(mitochondrial genes as in Mercer et al., 2011, Cell, 146: 645)
(FIG. 1C) in contrast to the minimal (11 genes; 1.92%) overlap
amongst common upregulated genes. Among the 113 downregulated
mitochondrial genes in aged MuSCs, 41.6% were related to the TCA
cycle and OXPHOS (FIG. 1D), which is significantly higher than
their percent composition of the whole mitochondrial proteome
(.about.14%) (Sickmann et al., 2003, PNAS, 100: 13207; Pagliarini
et al., 2008, Cell, 134: 112). This indicates a dominant decline of
mitochondrial respiratory genes in aged MuSCs. The reduction in
mitochondrial OXPHOS and TCA cycle genes is consistent for all
independent datasets (FIG. 1E).
[0120] Confirming dysfunctional mitochondrial respiration, isolated
primary aged and young MuSCs were isolated. Reductions in OXPHOS
and TCA cycle transcripts were found (FIG. 2A), matched by a
reduction in oxidative respiration rates (FIG. 2B). Interestingly,
several important markers and regulators of the mitochondrial
unfolded protein response (UPR.sup.mt), a stress response pathway
that mediates adaptations in mitochondrial content and function,
were significantly downregulated in aged MuSCs (FIGS. 1E and 2C,
D). Notably, despite the absence of consistent changes in CDKN2A or
MAPK14 (p38) pathways, previously reported to regulate MuSC
senescence, there was a downregulation of cell cycle-related gene
expression (FIG. 2D). The reduction in cell cycle signalling was
accompanied by an upregulation of the cyclin-dependent kinase
inhibitor 1A (CDKN1A)-mediated pathway (FIG. 2D), suggesting that
early senescence in MuSCs may involve CDKN1A.
[0121] This data show that mitochondrial oxidative respiration is
important for the functional maintenance of adult MuSCs during
aging as a dominant decline mitochondrial OXPHOS and TCA cycle
genes can be observed in aged MuSCs.
Example 2: NR Treatment Improves MuSCs Function in Aging Mice
[0122] The effect of a mitochondrial UPR.sup.mt inducing agent in
the treatment of loss of MuSC numbers during aging was studied as
follows:
[0123] Animals and FACS based muscle stem cell isolation as
described in Example 1.
[0124] Endurance running test. Mice were fasted 2 hours before
running on a treadmill. The exercise regimen commenced at a speed
of 9 cm/s with an inclination of 5 degrees. The speed was gradually
increased 3 cm/s every 12 minutes. Mice were considered to be
exhausted, and removed from the treadmill, following the
accumulation of 5 or more shocks (0.1 mA) per minute for two
consecutive minutes. The distance traveled and time before
exhaustion is registered as maximal running distance and
period.
[0125] Grip strength test. Muscle strength was assessed by a grip
strength behavior task. The grasp strength of each mouse for all
four limbs was measured on a pull-grid assembly connected to a grip
strength meter (Columbus Instruments). The mouse was drawn along a
straight line parallel to the grid until the grip is broken,
providing the peak force in grams. This was repeated 4 times with 5
minute intervals between measurements.
[0126] Cardiotoxin-induced muscle damage. Animals were anesthetized
using Isoflurane in oxygen from a precision vaporizer. 50 .mu.l of
20 .mu.M Naje Mossambica mossambica cardiotoxin (Sigma) was
injected intramuscularly cross the skin and directly into the
tibialis anterior (TA) muscle. Mice were sacrificed at 7 and 14
days after injury. TA muscles were immediately embedded in Thermo
Scientific.TM. Shandon.TM. Cryomatrix.TM. and frozen in isopentane,
cooled in liquid nitrogen, for 2 mins before being transferred to
dry ice and stored at -80.degree. C.
[0127] MuSCs transplantation. 5,000-8,000 double-sorted MuSCs
isolated from NR or normal chow diet C57B/6J mice were resuspended
in 10 .mu.l of F10 media with 20% FBS and injected directly into
cardiotoxin (CTX) pre-injured tibialis anterior (TA) muscle of Mdx
mice 24 hrs after the injury. The CTX pre-injury was performed as
described above. Recipient mice were sacrificed 4 weeks after
transplantation, TA muscle were harvested and prepared for
cryosection.
[0128] Histology. TA muscles were harvested from anaesthetized mice
and immediately frozen in Tissue-TEK.RTM. OCT compound (PST).
8-.mu.m cryosections were collected and fixed with 4%
paraformaldehyde, which are either stained with haematoxylin/eosin
(HE) or antibodies. For immunostainings, heat activated antigen
retrieval was performed in pH 6.0 citrate buffer for 10 min at
65.degree. C. After washing with PBS-0.1% tween 20 (PBST), the
sections were blocked with 10% affinipure Fab goat anti mouse IgG
(Jackson Immunoresearch) in PBST for 60 min and PBST containing 2%
BSA and 5% goat serum for 30 min at room temperature. Primary
antibodies were then applied over night at 4.degree. C. The
following antibodies were used: anti-eMHC (Developmental Studies
Hybridoma Bank, DSHB, University of Iowa), anti-Pax7 (DSHB,
University of Iowa), anti-Laminin (Sigma). Subsequently, the slides
were washed in PBST and incubated with appropriate secondary
antibodies and labeling dyes. For immunofluorescence, secondary
antibodies were coupled to Alexa-488 or Alexa-568 fluorochromes
(Life technology), and nuclei were stained with DAPI (Invitrogen).
After washing in PBST, tissue sections were mounted with Dako
mounting medium (Dako).
[0129] Compared to young, aged mouse muscle contained fewer MuSCs
(FIG. 2A-C). However, nicotinamide riboside (NR) treatment
attenuated the loss of MuSC numbers during aging, while also
evoking gains in younger mice (FIG. 3 A-C). The increase in aged
MuSC numbers was confirmed with PAX7 staining, a known MuSC marker
(Yin et al., supra) (FIG. 3D). The effect of NR in young or aged
mice was not due to changes in muscle mass or body weight, as they
were comparable amongst all groups over this short treatment
period. Consistent with the increase of MuSC numbers, NR treatment
significantly enhanced muscle function as indicated by improvements
in maximal running times and distances, along with limb grip
strength (FIG. 3E-G). Impairments in muscle regeneration efficiency
have been linked to the decline in aged MuSC function Yang et al.,
supra). The action of NR on muscle regeneration with cardiotoxin
(CTX)-induced muscle damage was tested (Yin et al., supra). Indeed,
NR treatment accelerated muscle regeneration in aged and young mice
(FIG. 311). NR-induced improvements in regeneration were paralleled
by increases in PAX7-positive MuSCs in aged mice (FIG. 3I-J), with
a trend to increase in young mice. NR treatment also improved the
stemness of the aged MuSCs, as demonstrated by a reduction in
MYOD1-positive PAX7 immunostained cells, e.g. MuSCs started to
differentiation (FIG. 3K). Complementing the improvements in MuSC
function, 7 days after CTX-induced damage, NR-treated aged mice
exhibited improvements in embryonic myosin heavy chain staining
(eMyHC), a protein expressed in fetal and newly regenerating adult
muscle fibers (Sartore et al., 1982, Nature, 298: 294) (FIG. 3L).
Finally, compared to controls, MuSCs transplanted from NR-treated
aged mice into Mdx mice (FIG. 3M), a mouse model of Duchenne
muscular dystrophy, more effectively replenished the MuSC
compartment and stimulated myogenesis of dystrophin-positive
myofibers, demonstrating an improved engraftment potential for
NR-treated MuSCs (FIG. 3N).
[0130] This data demonstrate that NR can attenuate the loss of MuSC
numbers during aging, enhanced muscle function, improve muscle
regeneration after induced muscle damage, improved the stemness of
the aged MuSCs and MuSC transplantation efficiency.
Example 3: NR Prevents MuSCs Senescence
[0131] The effect of a mitochondrial UPR.sup.mt inducing agent of
the invention in the prevention of MuSC senescence during aging was
studied as follows:
[0132] Animals and FACS based muscle stem cell isolation as
described in Example 1.
[0133] Histology. TA muscles were harvested and the immunestaining
was performed as described in Example 2. The following antibodies
were used: anti-.gamma.H2AX Ser 139 (Millipore),
anti-activated-caspase3 (Cell signaling). Secondary antibodies were
coupled to Alexa-488 or Alexa-568 fluorochromes (Life technology),
and nuclei were stained with DAPI (Invitrogen).
[0134] .beta.-galactosidase assay. MuSCs were sorted directly and
cultured primary MuSCs were grown on 8 chamber slides (Thermo
Scientific). Senescence-associated .beta.-galactosidase activity
was detected using the senescence .beta.-galactosidase staining kit
(Cell signaling), according to manufacturer's instructions.
[0135] Myogenesis assay. Five MuSCs were sorted directly into wells
of a Matrigel-coated 96-well cell culture plate, containing MuSC
growth medium (F10, 20% FBS, 2.5 ng/ml bFGF, lx pen/strep), using
the automated cell deposition unit (ACDU) of the FACSAria II
instrument (BD Biosciences). Cells were cultured at 37.degree. C.
for 5 days. Cell colony formations were counted using the DM IL LED
Inverted Microscope (Leica) after fixation in freshly made or
defrosted 4% paraformaldehyde (PFA) for 10 min.
[0136] Cell culture and treatments. FACS sorted MuSCs were grown on
a 10% Matrigel (Corning)-coated dish and flasks with Fams F-10
media (Gibco), supplemented with 20% fetal bovine serum (FBS,
Gibco), 2.5 ng/ml basic fibroblast growth factor (bFGF, Sigma) and
penicillin/streptomycin (lx, Gibco). Dishes were coated with 10%
growth factor-free Matrigel solution on ice for 7 min then
transferred to a 37.degree. C. cell culture incubator overnight
before use. Cells were grown for three generations in vitro before
experiments with cells plated and passaged at 10.sup.3 cells/ml and
50% confluencies, respectively. C2C12 mouse myoblasts were grown in
DMEM (4.5 g/1 glucose, Gibco) supplemented with 10% FBS and
penicillin/streptomycin (lx, Gibco).
[0137] Western blotting. C2C12 cells were lysed in a buffer
composed of 50 mM Tris, 150 mM KCl, EDTA 1 mM, NP40 1%,
nicotinamide 5 mM, sodium butyrate 1 mM and protease inhibitors
cocktail (Roche) at pH 7.4. Proteins were separated by SDS-PAGE and
transferred onto nitrocellulose membranes. Blocking and antibody
incubations were performed in 3% BSA. The following primary
antibodies were used: anti-cleavage caspase 3 (Cell Signalling);
Anti-.gamma.H2AX (Millipore); anti-.beta.-actin (Sigma). All
secondary antibodies were from Jackson Immunoresearch. Antibody
detection reactions were developed by enhanced chemiluminescence
(Advansta, CA, USA) using x-ray films or imaged using the c300
imaging system (Azure Biosystems).
[0138] To explain the improvements in aged MuSCs following NR
treatment, the ability of NR to prevent MuSC senescence was
examined. Freshly isolated MuSCs from NR-treated young and aged
mice were immunostained with .gamma.H2AX, a marker of DNA damage
(Kuilman et al., supra). .gamma.H2AX positive nuclei were more
abundant in aged MuSCs, yet staining was reduced with NR treatment
(FIG. 4A, B). The reduction of the nuclear damage response was
confirmed by .beta.-galactosidase staining, a classical senescence
marker (Kuilman et al., supra) (FIG. 3C). To evaluate whether the
effect of NR on MuSC senescence depends on the in vivo environment
(MuSC niche), isolated MuSCs from untreated or NR-treated mice were
cultured them ex vivo for three generations. Again, reductions in
.gamma.H2AX positive nuclei and cleaved caspase-3 (CASP3) (a marker
for apoptosis or cell death) immunostaining was found, in MuSCs
isolated from NR-treated mice (FIG. 3D, E). Moreover, a 6-hour NR
treatment in late passage C2C12 myoblasts reduced the expression of
cell senescence and apoptosis markers (Hara et al., 1996, Molecular
and Cellular Biol., 16: 859) (FIG. 3F). This is further supported
by the enhanced proliferation ability of MuSCs isolated from
NR-treated aged mice, as indicated by their enhanced potential to
form myogenic colonies (FIG. 3G).
[0139] As the in vitro culture conditions do not change the
stemness under in vivo treatments, it supports that NR would exert
a protective effect against MuSC senescence that is not dependent
on extrinsically mediated factors.
Example 4: Rejuvenating MuSCs by Activating the UPR.sup.mt and
Prohibitin Pathways
[0140] The effect of a mitochondrial UPR.sup.mt inducing agent of
the invention in the rejuvenation of MuSCs was studied as
follows:
[0141] Animals, FACS based muscle stem cell isolation and gene
expression analyses as described in Example 1.
[0142] Western blotting performed as described in Example 3. The
following primary antibodies were used: anti-HSP60 (Enzo Life
Science); anti-.beta.-actin (Sigma); anti-PHB (Biolegend);
anti-PHB2 (Santa Cruz); anti-CKD4 (Novus biologicals); anti-CCND1
(Santa Cruz); anti-CCND3 (Santa Cruz); anti-HSP90 (BD Biosciences);
HSP70 (Abcam); and anti-CLPP (Sigma).
[0143] Cell culture and treatments were prepared according to
description of Example 3. C2C12 mouse myoblasts were grown in DMEM
(4.5 g/1 glucose, Gibco) supplemented with 10% FBS and
penicillin/streptomycin (lx, Gibco). Cell transformation with Phb
(Santa Cruz) and Phb2 shRNA (Santa Cruz) were performed using
jetPEI DNA transfection kit (Polyplus), according to manufacturer's
instructions. Cells were treated with 1 mM NR or PBS for 6 hours
before cell harvesting or fixation.
[0144] Respirometry on C2C12 myoblasts. Basal and uncoupled oxygen
consumption rates (OCRs) and the extracellular acidification rate
(ECAR) was measured using the Seahorse extracellular flux
bioanalyzer (XF96, Seahorse Bioscience Inc.). To uncouple
mitochondria, 5 uM of FCCP was injected after a basal respiration
measurement. All measurements were performed in triplicates and
results were normalized to total protein amount (C2C12 cells),
assessed using a Bradford kit (Bio-Rad).
[0145] In contrast to the up-regulated CDKN1A senescence pathway
seen in aged MuSCs (FIG. 2D), NR significantly reduced mRNA levels
of CDKN1A, and related senescence indicators, while increasing the
expression of cell cycle related genes, in freshly isolated MuSCs
(FIG. 411). This protective effect of NR on MuSC senescence relies
on changes in mitochondrial function as NR largely rescued
mitochondrial TCA and OXPHOS gene expression in aged MuSCs (FIG.
4I). This is consistent with increases in oxidative respiration and
reductions in glycolysis in NR-treated C2C12 cells (FIG. 4J). The
trend in oxidative respiration was replicated in primary MuSCs
isolated from NR-treated aged mice. As the mitochondrial unfolded
protein response (UPR.sup.mt) is known to be induced by NR
(Mouchiroud et al., supra), several UPR.sup.mt markers were
similarly induced in NR-treated C2C12 mouse myoblasts (FIG. 5A).
The mechanism of how UPR.sup.mt regulates senescence was tested
examining its effect on prohibitins, a family of stress response
proteins. Prohibitins are known to sense mitochondria stress and
modulate senescence in fibroblasts in mammals (Coates et al., 2001,
Exp. Cell Res., 265: 268). Intriguingly, the expression of
prohibitins, Phb1 and Phb2, is significantly reduced in the
bioinformatics analysis (FIG. 1E), and in freshly isolated aged
MuSCs. However, NR treatment induced Phb1 and Phb2 expression in
both young and aged MuSCs (FIG. 5B) and in C2C12 myoblasts (FIG.
5A), consistent with the upregulation of UPR.sup.mt markers and
cell cycle genes. The effects of NR on cell senescence were
furthermore PHB-dependent, as knockdown and overexpression of
prohibitins inhibits and stimulates cell cycle gene expression,
respectively (FIG. 5C, D).
[0146] These results indicate that NR activates UPR.sup.mt and the
prohibitin signaling pathway, thereby reversing MuSC
senescence.
Example 5: NR Reprograms Senescence Prone MuSCs in Mdx Mice
[0147] The effect of a mitochondrial UPR.sup.mt inducing agent of
the invention in reprogramming of senescence of MuSCs was studied
as follows:
[0148] Animals, FACS based muscle stem cell isolation as described
in Example 1.
[0149] .beta.-galactosidase assay as described in Example 3.
[0150] Cardiotoxin-induced muscle damage and Histology as described
in Example 2. The following antibodies were used: anti-eMHC
(Developmental Studies Hybridoma Bank, DSHB, University of Iowa),
anti-Pax7 (DSHB, University of Iowa), anti-.gamma.H2AX Ser 139
(Millipore) and anti-activated-caspase3 (Cell signaling).
[0151] Determining cellular redox ratio. NAD+ and NADH
quantification and ratio were measured using a kit from Biovision
(Milpitas, Calif.), following providers' instructions.
[0152] With continuous muscle regeneration, MuSCs in Mdx mice are
abnormally active at a young age, leading to MuSC depletion and
dysfunction later in life. As a result, primary MuSCs isolated from
14-week-old Mdx mice were significantly more senescent compared to
control mice (FIG. 6A). Similar to the effect in aged animals, NR
treatment of Mdx mice increased MuSC numbers by .about.1.8 fold in
vivo (FIG. 6B-D), as also confirmed by PAX7 immunostaining (FIG.
6E). Along with the increase in MuSCs, there was an increase in
regenerated muscle fibers following NR treatment (FIG. 6F). Thus
the self-renewal capacity of Mdx mouse MuSCs was tested. The
cellular redox ratio decreases as MuSCs differentiate (Fulco et
al., 2003, Molecular Cell, 12: 51), which can be detected by the
increase in 405/450 autofluorescence (Quinn et al., 2013,
Scientific Reports, 3: 3432). In line with NR increasing Mdx mouse
MuSC numbers, we found a significant reduction in autofluorescence
from MuSCs isolated from these animals (FIG. 6G-I). We then
performed .beta.-galactosidase staining on primary MuSCs isolated
from Mdx mice, with or without NR treatment, and cultured these
cells with NR or vehicle in vitro. This demonstrated that MuSCs
isolated from NR-treated mice were less prone to senescence (FIG.
6J). In addition, when these MuSCs were treated with NR in vitro
there was a further reduction in senescence (FIG. 6J). The
prevention of MuSCs senescence in NR-treated Mdx mice was confirmed
by the attenuation of .gamma.H2AX and cleaved caspase-3
immunostaining (FIG. 6K). To evaluate MuSC function, CTX-induced
muscle regeneration was examined in NR-treated mice. Consistent
with the prevention of MuSC senescence, muscle regeneration was
improved with NR (FIG. 6L).
[0153] These results show that NR treatment can increase MuSCs
numbers in mouse model of Duchenne muscular dystrophy and further
prevent MuSCs senescence.
Example 6: Thiamphenicol Induces UPR.sup.mt in Myoblasts
[0154] The activity of TAP as another a mitochondrial UPR.sup.mt
inducing agent of the invention in was assayed in myoblast as
follows:
[0155] Western blotting. C2C12 cells were lysed in a buffer
composed of 50 mM Tris, 150 mM KCl, EDTA 1 mM, NP40 1%,
nicotinamide 5 mM, sodium butyrate 1 mM and protease inhibitors
cocktail (Roche) at pH 7.4. Proteins were separated by SDS-PAGE and
transferred onto nitrocellulose membranes. Blocking and antibody
incubations were performed in 3% BSA. The following primary
antibodies were used: anti-cleavage caspase 3 (Cell Signalling);
Anti-.gamma.H2AX (Millipore); anti-.beta.-actin (Sigma). All
secondary antibodies were from Jackson Immunoresearch. Antibody
detection reactions were developed by enhanced chemiluminescence
(Advansta, CA, USA) using x-ray films or imaged using the c300
imaging system (Azure Biosystems).
[0156] The following primary antibodies were used: anti-HSP60 (Enzo
Life Science); anti-PHB (Biolegend); anti-PHB2 (Santa Cruz);
anti-CKD4 (Novus biologicals); anti-CCND1 (Santa Cruz); anti-CCND3
(Santa Cruz); anti-HSP90 (BD Biosciences); HSP70 (Abcam);
anti-MT-CO1 (Biolegend); anti-ATP5A (Biolegend); anti-Grp78
(Abcam); and anti-CLPP (Sigma).
[0157] UPR.sup.mt induction by thiamphenicol (TAP) which also
induced prohibitins and cell cycle gene expression in C2C12 cells
(FIG. 7) is supporting the ability of this agent in attenuating the
senescence-signaling cascade in those cells.
[0158] These results support that TAP can be used as another
mitochondrial UPR.sup.mt inducing agent and would be able to MuSC
senescence.
TABLE-US-00002 Sequence listing Nucleic acid sequence of 36b4
forward primer SEQ ID NO: 1: AGATTCGGGATATGCTGTTGG Nucleic acid
sequence of 36b4 reverse primer SEQ ID NO: 2: AAAGCCTGGAAGAAGGAGGTC
Nucleic acid sequence of Ndufb5 forward primer SEQ ID NO: 3:
CTTCGAACTTCCTGCTCCTT Nucleic acid sequence of Ndufb5 reverse primer
SEQ ID NO: 4: GGCCCTGAAAAGAACTACG Nucleic acid sequence of Sdha
forward primer SEQ ID NO: 5: GGAACACTCCAAAAACAGACCT Nucleic acid
sequence of Sdha reverse primer SEQ ID NO: 6:
CCACCACTGGGTATTGAGTAGAA Nucleic acid sequence of Sdhc forward
primer SEQ ID NO: 7: GCTGCGTTCTTGCTGAGACA Nucleic acid sequence of
Sdhc reverse primer SEQ ID NO: 8: ATCTCCTCCTTAGCTGTGGTT Nucleic
acid sequence of Cox5b forward primer SEQ ID NO: 9:
AAGTGCATCTGCTTGTCTCG Nucleic acid sequence of Cox5b reverse primer
SEQ ID NO: 10: GTCTTCCTTGGTGCCTGAAG Nucleic acid sequence of Atp5b
forward primer SEQ ID NO: 11: GGTTCATCCTGCCAGAGACTA Nucleic acid
sequence of Atp5b reverse primer SEQ ID NO: 12:
AATCCCTCATCGAACTGGACG Nucleic acid sequence of Mdh2 forward primer
SEQ ID NO: 13: TTGGGCAACCCCTTTCACTC Nucleic acid sequence of Mdh2
reverse primer SEQ ID NO: 14: GCCTTTCACATTTGCTCTGGTC Nucleic acid
sequence of Idh2 forward primer SEQ ID NO: 15:
GGAGAAGCCGGTAGTGGAGAT Nucleic acid sequence of Idh2 reverse primer
SEQ ID NO: 16: GGTCTGGTCACGGTTTGGAA Nucleic acid sequence of Idh3a
forward primer SEQ ID NO: 17: CCCATCCCAGTTTGATGTTC Nucleic acid
sequence of Idh3a reverse primer SEQ ID NO: 18:
ACCGATTCAAAGATGGCAAC Nucleic acid sequence of Cdkn1a forward primer
SEQ ID NO: 19: GTGGGTCTGACTCCAGCCC Nucleic acid sequence of Cdkn1a
reverse primer SEQ ID NO: 20: CCTTCTCGTGAGACGCTTAC Nucleic acid
sequence of Mki67 forward primer SEQ ID NO: 21:
TTGGAAAGGAACCATCAAGG Nucleic acid sequence of Mki67 reverse primer
SEQ ID NO: 22: TTTCTGCCAGTGTGCTGTTC Nucleic acid sequence of Cdk4
forward primer SEQ ID NO: 23: CCGGTTGAGACCATTAAGGA Nucleic acid
sequence of Cdk4 reverse primer SEQ ID NO: 24: CACGGGTGTTGCGTATGTAG
Nucleic acid sequence of Ccna2 forward primer SEQ ID NO: 25:
AAGAGAATGTCAACCCCGAAA Nucleic acid sequence of Ccna2 reverse primer
SEQ ID NO: 26: ACCCGTCGAGTCTTGAGCTT Nucleic acid sequence of Ccnd1
forward primer SEQ ID NO: 27: GAGCGTGGTGGCTGCGATGCAA Nucleic acid
sequence of Ccnd1 reverse primer SEQ ID NO: 28:
GGCTTGACTCCAGAAGGGCTTCAAT Nucleic acid sequence of Ccne1 forward
primer SEQ ID NO: 29: CAAAGCCCAAGCAAAGAAAG Nucleic acid sequence of
Ccne1 reverse primer SEQ ID NO: 30: CCACTGTCTTTGGAGGCAAT Nucleic
acid sequence of Cdc6 forward primer SEQ ID NO: 31:
GACACAAGCTACCATGGTTT Nucleic acid sequence of Cdc6 reverse primer
SEQ ID NO: 32: CAGGCTGGACGTTTCTAAGTT Nucleic acid sequence of IL6
forward primer SEQ ID NO: 33: GGTGACAACCACGGCCTTCCC Nucleic acid
sequence of IL6 reverse primer SEQ ID NO: 34:
AAGCCTCCGACTTGTGAAGTGGT Nucleic acid sequence of IL18 forward
primer SEQ ID NO: 35: GTGAACCCCAGACCAGACTG Nucleic acid sequence of
IL18 reverse primer SEQ ID NO: 36: CCTGGAACACGTTTCTGAAAGA Nucleic
acid sequence of Hsp60 forward primer SEQ ID NO: 37:
ACAGTCCTTCGCCAGATGAGAC Nucleic acid sequence of Hsp60 reverse
primer SEQ ID NO: 38: TGGATTAGCCCCTTTGCTGA Nucleic acid sequence of
Hsp10 forward primer SEQ ID NO: 39: CTGACAGGTTCAATCTCTCCAC Nucleic
acid sequence of Hsp10 reverse primer SEQ ID NO: 40:
AGGTGGCATTATGCTTCCAG Nucleic acid sequence of Clpp forward primer
SEQ ID NO: 41: CACACCAAGCAGAGCCTACA Nucleic acid sequence of Clpp
reverse primer SEQ ID NO: 42: TCCAAGATGCCAAACTCTTG Nucleic acid
sequence of Phb forward primer SEQ ID NO: 43: TCGGGAAGGAGTTCACAGAG
Nucleic acid sequence of Phb reverse primer SEQ ID NO: 44:
CAGCCTTTTCCACCACAAAT Nucleic acid sequence of Phb2 forward primer
SEQ ID NO: 45: CAAGGACTTCAGCCTCATCC Nucleic acid sequence of Phb2
reverse primer SEQ ID NO: 46: GCCACTTGCTTGGCTTCTAC
Sequence CWU 1
1
46121DNAArtificial Sequence36b4 forward primer 1agattcggga
tatgctgttg g 21221DNAArtificial Sequence36b4 reverse primer
2aaagcctgga agaaggaggt c 21320DNAArtificial SequenceNdufb5 forward
primer 3cttcgaactt cctgctcctt 20419DNAArtificial SequenceNdufb5
reverse primer 4ggccctgaaa agaactacg 19522DNAArtificial
SequenceSdha forward primer 5ggaacactcc aaaaacagac ct
22623DNAArtificial SequenceSdha reverse primer 6ccaccactgg
gtattgagta gaa 23720DNAArtificial SequenceSdhc forward primer
7gctgcgttct tgctgagaca 20821DNAArtificial SequenceSdhc reverse
primer 8atctcctcct tagctgtggt t 21920DNAArtificial SequenceCox5b
forward primer 9aagtgcatct gcttgtctcg 201020DNAArtificial
SequenceCox5b reverse primer 10gtcttccttg gtgcctgaag
201121DNAArtificial SequenceAtp5b forward primer 11ggttcatcct
gccagagact a 211221DNAArtificial SequenceAtp5b reverse primer
12aatccctcat cgaactggac g 211320DNAArtificial SequenceMdh2 forward
primer 13ttgggcaacc cctttcactc 201422DNAArtificial SequenceMdh2
reverse primer 14gcctttcaca tttgctctgg tc 221521DNAArtificial
SequenceIdh2 forward primer 15ggagaagccg gtagtggaga t
211620DNAArtificial SequenceIdh2 reverse primer 16ggtctggtca
cggtttggaa 201720DNAArtificial SequenceIdh3a forward primer
17cccatcccag tttgatgttc 201820DNAArtificial SequenceIdh3a reverse
primer 18accgattcaa agatggcaac 201919DNAArtificial SequenceCdkn1a
forward primer 19gtgggtctga ctccagccc 192020DNAArtificial
SequenceCdkn1a reverse primer 20ccttctcgtg agacgcttac
202120DNAArtificial SequenceMki67 forward primer 21ttggaaagga
accatcaagg 202220DNAArtificial SequenceMki67 reverse primer
22tttctgccag tgtgctgttc 202320DNAArtificial SequenceCdk4 forward
primer 23ccggttgaga ccattaagga 202420DNAArtificial SequenceCdk4
reverse primer 24cacgggtgtt gcgtatgtag 202521DNAArtificial
SequenceCcna2 forward primer 25aagagaatgt caaccccgaa a
212620DNAArtificial SequenceCcna2 reverse primer 26acccgtcgag
tcttgagctt 202722DNAArtificial SequenceCcnd1 forward primer
27gagcgtggtg gctgcgatgc aa 222825DNAArtificial SequenceCcnd1
reverse primer 28ggcttgactc cagaagggct tcaat 252920DNAArtificial
SequenceCcne1 forward primer 29caaagcccaa gcaaagaaag
203020DNAArtificial SequenceCcne1 reverse primer 30ccactgtctt
tggaggcaat 203120DNAArtificial SequenceCdc6 forward primer
31gacacaagct accatggttt 203221DNAArtificial SequenceCdc6 reverse
primer 32caggctggac gtttctaagt t 213321DNAArtificial SequenceIL6
forward primer 33ggtgacaacc acggccttcc c 213423DNAArtificial
SequenceIL6 reverse primer 34aagcctccga cttgtgaagt ggt
233520DNAArtificial SequenceIL18 forward primer 35gtgaacccca
gaccagactg 203622DNAArtificial SequenceIL18 reverse primer
36cctggaacac gtttctgaaa ga 223722DNAArtificial SequenceHsp60
forward primer 37acagtccttc gccagatgag ac 223820DNAArtificial
SequenceHsp60 reverse primer 38tggattagcc cctttgctga
203922DNAArtificial SequenceHsp10 forward primer 39ctgacaggtt
caatctctcc ac 224020DNAArtificial SequenceHsp10 reverse primer
40aggtggcatt atgcttccag 204120DNAArtificial SequenceClpp forward
primer 41cacaccaagc agagcctaca 204220DNAArtificial SequenceClpp
reverse primer 42tccaagatgc caaactcttg 204320DNAArtificial
SequencePhb forward primer 43tcgggaagga gttcacagag
204420DNAArtificial SequencePhb reverse primer 44cagccttttc
caccacaaat 204520DNAArtificial SequencePhb2 forward primer
45caaggacttc agcctcatcc 204620DNAArtificial SequencePhb2 reverse
primer 46gccacttgct tggcttctac 20
* * * * *