Transcript Slide 1

2011 Brookdale Foundation Retreat
Senescent cell clearance in healthspan extension
Darren Baker
Assistant Professor of Biochemistry and Molecular Biology
Brookdale Leadership in Aging Fellow 2011
Tuesday June 7, 2011
Mayo
Clinic
Kogod
Center on
Aging
Cellular senescence
The Hayflick limit (1962)
Young tissue (fully functional)
Old tissue (dysfunctional)
Cell
damage
Normal (healthy) cell
Senescent cell
Dysfunctional cell
Methalloproteinases, Cytokines, etc…
Significance: Senescent cells accumulate with age at sites of age-related pathology
Hypothesis: Elimination of senescent cells has a rejuvenating affect on aged tissues
–/H
–/H
4%
H/H
+/–
10%
H/H
+/H
+/–
30%
+/H
60%
+/+
Impaired wound healing
Reduced adipose tissue
Reduced dermal thickness
100%
+/+
BubR1 hypomorphic mice- a progeroid mouse model
BubR1
Lordokyphosis
Gliosis
Arterial stiffening
Impaired respiration
Infertility
Bilateral
cataracts
Craniofacial
dysphorisms
4-5 fold reduced median and maximum lifespan
Sarcopenia (muscle atrophy)
Reduced stress tolerance
Pathways activated by BubR1 hypomorphism
Eye
BubR1+/+
Adipose tissue
BubR1H/H
BubR1+/+
BubR1H/H
p16
p19
p21
p53
Tubulin
INK-ATTAC
p16Ink4a
promoter
FKBP Caspase 8-Flag
Cell
membrane
IRES EGFP
-2617
M
M
M
M
M
M
+ AP20187
(dimerization)
FKBP
Caspase 8
Myristoylation
Inactive Caspase 8
Active Caspase 8
Apoptosis
Experimental design
BubR1H/H
x
p16Ink4a-FKBP-Casp8
Interbreeding
1-month-old mouse
1-month-old mouse
Healthy mouse tissue
Healthy mouse tissue
p16Ink4a-FKBP-Casp8
BubR1H/H
p16Ink4a-FKBP-Casp8
BubR1H/H
Untreated (–AP)
Treated (+AP)
Dysfunctional aged
mouse tissue
Aged mouse tissue
Dysfunctional cell
p16Ink4a-FKBP-Casp8
BubR1H/H
p16Ink4a-FKBP-Casp8
BubR1H/H
Methalloproteinases,
Cytokines, etc…
Senescent cell
Functional cell
p16Ink4a expression overlaps with the INK-ATTAC transgene
BubR1H/H;INK-ATTAC3
GFP intensity
p16
5-month-old IAT
Relative expression IAT
% of cells
BubR1H/H;
INK-ATTAC3
SkM
IAT
Eye
Liver
Heart
IL-6
BubR1H/H;
INK-ATTAC3 GFP–
BubR1H/H;
INK-ATTAC3 GFP+
INK-ATTAC3
p21
BubR1HH;
INK-ATTAC3
SkM
IAT
Eye
Liver
Heart
SkM
IAT
Eye
Liver
Heart
GFP
EGFP relative expression
FKBP-Casp8 rel. expression
INK-ATTAC3
Pai1
INK-ATTAC3
Experimental design
BubR1H/H
x
p16Ink4a-FKBP-Casp8
Interbreeding
1-month-old mouse
1-month-old mouse
Healthy mouse tissue
p16Ink4a-FKBP-Casp8
BubR1H/H
p16Ink4a-FKBP-Casp8
BubR1H/H
Untreated (–AP)
9-months-old
Healthy mouse tissue
Treated (+AP)
Dysfunctional aged
mouse tissue
9-months-old
Aged mouse tissue
Dysfunctional cell
p16Ink4a-FKBP-Casp8
BubR1H/H
p16Ink4a-FKBP-Casp8
BubR1H/H
Methalloproteinases,
Cytokines, etc…
Senescent cell
Functional cell
Senescent cell clearance delays age-related dysfunction
9-months-old females
H/H;ATTAC3
+AP
H/H;ATTAC3
–AP
*
40
30
20
10
45
*
300
**
250
200
150
100
50
0
35
*
**
*
30
H/H;ATTAC3–AP
25
H/H;ATTAC3 +AP
20
H/H;ATTAC5 –AP
15
H/H;ATTAC5 +AP
10
5
0
IAT
Inguinal
adipose
40
Fiber diameter (μm)
50
350
**
Thickness (μm)
Cell diameter (μm)
60
0
Dermis
Dermis
Adipose
Adipose
Gastro
Gastro
ABD
Abdominal
*
600
500
400
300
3,5
**
160
140
120
*
100
80
60
200
40
100
20
Running
time (s)
2,5
*
2
1,5
1
0,5
0
0
**
3
Energy (J)
800
700
180
**
Distance (m)
Running time (s)
900
Distance
(m)
0
Energy expended (J)
H/H;ATTAC3–AP
H/H;ATTAC3 +AP
H/H;ATTAC5 –AP
H/H;ATTAC5 +AP
INK-ATTAC clears senescent cells
INK-ATTAC3
+ Rosiglitazone
+ AP20187
SA-b-GAL
SA-b-GAL
INK-ATTAC3
+ Rosiglitazone
– AP20187
H/H;ATTAC3
+AP
H/H;ATTAC5
–AP
H/H;ATTAC5
+AP
SA-β-Gal
H/H;ATTAC3
–AP
There are fewer indicators of senescence in skeletal muscle and eye as well
Goals for my fellowship
1. Test whether removal of senescent cells from
aged tissues has rejuvenating potential
BubR1H/H
x
p16Ink4a-FKBP-Casp8
Interbreeding
Healthy mouse tissue
1-month-old
mouse p16Ink4a-FKBP-Casp8
Normal cell
BubR1H/H
Premature aging
Dysfunctional aged
mouse tissue
2 to 5-monthold mouse p16Ink4a-FKBP-Casp8
BubR1H/H
Dysfunctional cell
Senescent cell
Methalloproteinases,
Cytokines, etc…
AP20187 treatment
(clearance of senescent cells)
Rejuvenated mouse tissue
8-month-old
p16Ink4a-FKBP-Casp8
mouse
BubR1H/H
Functional cell
Goals for my fellowship
1. Test whether removal of senescent cells from
aged tissues improves function
1. Determine if elimination of senescent cellsdelays
or prevents tumorigenesis
INK-ATTAC transgene- targeting cancer cells?
Collado et. al., Nature, 2005
INK-ATTAC- targeting the cancer microenvironment
MMTV-rtTA;NeuNT
MTB-TAN
x
p16Ink4a-FKBP-Casp8
Interbreeding
Healthy mouse tissue
p16Ink4a-FKBP-Casp8
MTB-TAN
Normal cell
Aging
Dysfunctional aged
mouse tissue
p16Ink4a-FKBP-Casp8
MTB-TAN
Dysfunctional cell
Senescent cell
Methalloproteinases,
Cytokines, etc…
Clear senescent cells, then activate the
oncogene for 6 weeks with doxycycline
water to induce mammary tumors
Moody et. al., Cancer Cell, 2002
Acknowledgements
Jan van Deursen laboratory
Kogod Center on Aging
Tobias Wijshake
Tyler Mann
Jim Kirkland
Tamar Tchkonia
Tamar Pirtskhalava
Nathan LeBrasseur