Nature 506, 316321 (20 February 2014)

Go J. Yoshida M.D.,Ph.D. (E-mail:goscm@tmd.ac.jp)Dept. of Stem Cell Biology, Medical Research Institute

Tokyo Medical and Dental University, Tokyo, Japan

Senescence regulation in stem cellsSenescence is a state in which a cell no longer has

the ability to proliferate.

Senescence accompanies changes in nuclear

morphology and formation of a distinct chromatin

structure, called senescence-associated

heterochromatic foci (SAHF).

Cell. 2003;113:703-716.

p16Ink4a promotes SAHF formation.

Epigenetic regulation of the p16INK4a locus in

senescent cells

In senescent cells, polycomb proteins are lost or displaced from the INK4A locus by a

number of established mechanisms, including (d) EZH2 downregulation resulting in the

loss of H3K27 methylation, (e) the histone demethylase JMJD3, which actively removes

the H3K27me3 mark and (f) the SWI/SNF chromatin remodelling complex, which is

proposed to displace polycombs from the INK4A locus. We also propose a novel

mechanism (g) by which polycomb proteins may be displaced by transcriptional

activators.

Oncogene. 2011;30:2901-11.

The pRb pathway and SAHF formation in senescent cells

There is a inversed relation between p16INK4a and p-Rb.

pRb protein functions to recruit repressive enzymatic activities to E2F-regulated target gene promoters.

The remarkably stable state of cellular senescence is thought to be maintained by compacted heterochromatic or closed chromatin, called SAHF, which accumulates around genes encoding cell cycle and proliferation proteins.

Oncogene. 2011;30:2901-11.

Aging Cell. 2002;1:132-9.

The arrows indicate satellite cells stained

positively for N-CAM (peroxidase, brown)

whereas the arrowheads give examples of

myonuclei (blue).

Cold Spring Harb.2011;76:101111.

Age-associated decline in muscle regeneration

Aged muscle (right, 24 months of

age) shows a significant deficit in

the number of regenerating fibers

and an increase in necrotic fibers

compared with young muscle.

Prevention of age-related increase in fibrosis during

muscle regeneration by heterochronic parabiosis

established for 1month

muscle tissue damage (freeze injuries)

3 days later, BrdU was administered.

2 days later, muscle tissue was fixed. Science. 2007;317:807-10.

6month 24month

The effects of the aged environment on myogenic

progenitor cell fate and muscle regeneration are

mediated by the Wnt signaling pathway.

Science. 2007;317:807-10.

Satellite CellsSkeletal Muscle Stem Cells

to exist close proximity to skeletal muscle fibers

to exhibit regenerative activity of skeletal muscle

to be required for early post-natal muscle growth

and for repair of catastrophic muscle injury

SC markers: Pax7, Myf5, MyoD, Cav1, N-CAM etc.

Physiol Rev. 2013;93:23-67.

Stem Cells Int. 2013;2013:420164.

Regulatory switch between

quiescent and activated states

of satellite cells

Notch and Wnt signaling antagonize

each other to define the state of

satellite cells.

Stem Cells Int. 2013;2013:420164.Notch-predominant Wnt-predominant

Functional dysregulation of stem cells during

aging: a focus on skeletal muscle stem cells

FEBS J. 2013;280:40514062.

Dev Biol. 2006;294:50-66.

: sarcopeniaLoss of muscle mass: sarcopenia

Loss of self-renewal: senescence

geroconversion

Bmi1 plays a crucial role in the maintenance of the stem cell pool in postnatal skeletal muscle and is

essential for efficient muscle regeneration after injury, possibly through its actions on p16ink4a and p19arf

Regenerating fibers express fetal myosin heavy chain (MyHC) initially and then switch to adult isoforms while they mature.

PLoS One. 2011;6(11):e27116.

Bmi-1-/- MEF underwent premature aging.

Nature. 1999;397:164-8.

SAMP8 pre-mature aging model

sarcopenia in senescence-accelerated mice (SAMP8)

Exp Gerontol. 2005;40:562-72.

type I fiber type IIA fiber

SAMP8 strains exhibit

accelerated muscle aging

as compared with SAMR1.

senescence-prone inbred

strains (SAMP)

senescence-resistant inbred

strains (SAMR)

Type-I fiber size was not

different between SAM

strains.

Major Hypothesis

The geriatric satellite stem cells show the irreversible

switch of quiescence-to-senescence (geroconversion) and

lose self-renewal potential via PRC1/p16INK4a/Rb/E2F axis.

young: 23 months of age

adult: 5-6 months of age

old: 2024 months of age

geriatric: 28-32 months of age

young: 23 months of age

adult: 5-6 months of age

old: 2024 months of age

geriatric: 28-32 months of age

A reduced number of satellite cells in aged mice is not

responsible for the significant regenerative decline.

Injection of cardiotoxin (CTX

1X10-5M) to tibialis anterior

muscle

After 21days

eMHC (embryonic myosin heavy chain) is a differentiation marker of skeletal muscle.

Pax7-positive satellite

cells in resting phase

Satellite-cell-intrinsic potential of regeneration 1week

after transplantation into young recipient mice

Equal numbers of sorted satellite cells labelled with

GFP were transplanted into muscles of young mice.

Pax7+ quiescent satellite cells from geriatric mice

showed a reduced activation rate early after injury

compared to adult/old cells.

Pax7/MyoD double-positive satellite cells

are defined as activated satellite cells.

Quiescent geriatric satellite cells exhibited a

defective activation capacity 24hr after

transplantation into a young host

The only significantly up-regulated senescence

gene in cluster G1 was the master regulator of

senescence p16INK4a (Cdkn2a)

group of genes with increased

expression in geriatric satellite cells

H2Aub mark in the INK4a locus was significantly

reduced in quiescent satellite cells from geriatric mice

Polycomb repressive complex 1

(PRC1): E3 ubiquitin ligase complex

that is specific for histone H2A.

PRC1 comple possess H2A-K119

ubiquitin E3 ligase activities

Canonical PRC1

complex

Nature 2004;431:873-878.

Mol Cell. 2005;20:845-54.

H2Aub: histone H2A mono-ubiquitination at

lysine 119 (PRC1 repressive marker)

RD domain is the main binding site for Bmi1 of

p16INK4a locus.

p16INK4a expression changed the satellite cell activation

ectopic p16INK4a overexpression in

quiescent young satellite cells

prevented their activation

p16INK4a silencing rescued the permanent

cell-cycle arrest and allowed geriatric

satellite cell activation

p16INK4a silencing in geriatric satellite cells

before engraftment into young mice

p16INK4a silencing in geriatric satellite cells

recovered the self-renewal potential

Self-renewing satellite cells; Pax7(+)/ Ki-67(-)

p16INK4a overexpression reduced the

capacity of young reserve satellite cells

to reactivate.

Activation capacity of satellite cells were

reduced after successive myogenesis rounds

(repeated injuries).

Geriatric satellite cells underwent an accelerated

entry into senescence under proliferative pressure

Transplanted geriatric satellite cells also displayed

signs of deep senescence at times of maximal

proliferative expansion after injury

Geriatric satellite cells exposed to proliferative

conditions presented high p16INK4a expression levels

correlating with reduced phosphorylated Rb protein

p16INK4a interference reduced geroconversion in

geriatric satellite cells

Ex vivo culture of muscle satellite cells

J Stem Cell Res Ther 2012;S11:003.

adult: 30 years of age

old: 65 years of age

geriatric: 96 years of age

Ex vivo analysis

Active p16INK4a/Rb axis in human geriatric stem cells

Conclusion

Geriatric age induces intrinsic alterations in

muscle stem-cell regenerative functions, which

cannot be recovered by a young host environment.

p16INK4a expression is specifically induced in

geriatric satellite cells and drives geroconversion

at the expense of proliferation.

p16INK4a seems to be positively associated with

reduced myogenic potential and increased cellular

senescence in human satellite cells from geriatric

individuals with sarcopenia.