Transcript Apoptosis and Aging in Post Mitotic Tissues. Christiaan

CELL SIGNALING AND APOPTOSIS IN
AGING
Christiaan Leeuwenburgh, Ph.D.
University of Florida
Biochemistry of Aging Laboratory
Web Page: http://grove.ufl.edu/~cleeuwen/
International Association of Biomedical Gerontology, 10th Congress
University of Cambridge, England
2003
Simplified Signaling
Pathways of Apoptosis
Pollack M, Leeuwenburgh C:
J Gerontol A Biol Sci Med Sci 2001;56:B475-482.
Caloric Restriction (CR)
• CR increases mean and maximum life-span improves
neuronal and muscle function as well as reduces
cardiomyopathy.
•
•
•
•
1) reduces oxidant production
2) improves calcium homeostasis
3) reduces inflammation
4) reduces DNA damage
Mitochondrial Dysfunction and Aging
Drew, B., Phaneuf, S., Dirks, A., Selman, C., Gredilla, R., Lezza, A., Barja, G., and Leeuwenburgh, C. (2003). Am J Physiol
Regul Integr Comp Physiol 284, R474-R480.
Leeuwenburgh, C., Wagner, P., Holloszy, J.O., Sohal, R.S., and Heinecke, J.W. 1997.. Arch Biochem Biophys 346:74-80.
 Oxidative stress
 mt-DNA damage
 mt-DNA deletions
 Oxidized proteins
 Lipid peroxidation
 Lipid-adduct
formation
•  Decrease in repair
systems
0.5
+
0.3
0.2
0.1
AD
CR
Ad Libitum
Caloric Restriction
26AD
26CR
**
3
2
1
0
12-AL
0.025
4
12AD
4
0.050
0.000
0.0
0.0
Vastus Lateralis
5
1.5
26-AL
26-CR
Age (Months)
5
0.4
0.075
8-oxodG/10 dG
in nDNA
*
1.0
0.5
Isolated
Mitochondria
Dityrosine/Tyrosine
(mmol/mol)
1.5
H2O2 Production
(nmol/min/mg protein)
H2O2 Production
(nmoles/min/mg protein)
5
8-oxodG/10 dG
in mtDNA
•
•
•
•
•
•
14
Age (Months)
30
1.0
**
0.5
0.0
12-AL
26-AL
26-CR
Age (Months)
Endoplasmic (Sarcoplasmic) Reticulum “Stress”


Damage due to
reactive nitrogen
and oxygen species
Squier, T.C., and D.J. Bigelow. 2000. Protein oxidation
and age-dependent alterations in calcium homeostasis.
Front Biosci. 5:D504-26.
The effects of age and calorie restriction on
TNF- / NF-B signaling in skeletal muscle
TNF- / NF-B signaling
TNF-
25
*
6AD
26AD
26CR
TNF-
(pg/mL)
20
15

10
5
TNF receptor-associated death domain (TRADD)
0
6AD
TNF-R-associated factor 2 (TRAF2)
Receptor interacting protein (RIP)
26AD
26CR
*P = 0.0001 vs 6AD

P = 0.0001 vs 26AD
Inhibitor of B kinase (IKK) complex
*
NFB Activation
(OD450nm)
0.035
0.030
0.025
#
0.020
0.015
0.010
0.005
0.000
6AD
26AD
26CR
Phillips and Leeuwenburgh (unpublished)
Myocardial Aging, Sarcopenia,
Neurodegeneration, Oxidative Stress and
Apoptosis
• in total number of skeletal and heart myocytes as well
as neurons with age
•May lead to accelerated decline in cardiac functional
capacity, sarcopenia, neurodegenerative diseases
•Oxidative Stress and Apoptosis may be one major factor
Neuron loss in the brain with
age and improved brain
function with CR
• Improved responses to enclosed alleys
• Previous studies on neuronal loss with aging reported disparate
results.
• Studies often measured neuron density in a given structure
instead of total neuron number.
• Some studies suggest that most neocortical areas and certain
hippocampal subfields lose 25 to 50% of their neurons with age in
humans.
• Specific regions of the brain appear to be effected (hippocampus,
hilus of the dentate gyrus and the subiculum)
Morrison, J. H., and Hof, P. R. (1997) Science 278, 412-419; Mattson, M. P. (2000). Nat Rev Mol Cell Biol 1,
120-129.
Ingram, D. K., Weindruch, R., Spangler, E. L., Freeman, J. R., and Walford, R. L. (1987) J Gerontol 42, 7881. Ingram, D. K., Chefer, S., Matochik, J., Moscrip, T. D., Weed, J., Roth, G. S., London, E. D., and Lane,
M. A. (2001) Ann N Y Acad Sci 928, 316-326.
Is there skeletal muscle fiber loss with age
in humans and animals?
Animals
Fibers Through
Midbelly
Humans
20000
Vastus Lateralis
10000
**
0
5-AL
18-AL
36-AL
Bua EA, McKiernan SH, Wanagat J,
McKenzie D, Aiken JM. Mitochondrial
abnormalities are more frequent in
muscles undergoing sarcopenia.
J Appl Physiol. 2002 Jun;92(6):2617-24.
Lexell J, Taylor CC, Sjostrom M. What is the cause of
the ageing atrophy? Total number, size and
proportion of different fiber types studied in whole
vastus lateralis muscle from 15- to 83-year-old men. J
Neurol Sci. 1988 Apr;84(2-3):275-94.
Pesce V, Cormio A, Fracasso F, Vecchiet J, Felzani G,
Lezza AM, Cantatore P, Gadaleta MN. Age-related
mitochondrial genotypic and phenotypic alterations
in human skeletal muscle Free Radic Biol
Med,1;30(11):1223-33, 2001.
56-years
Density of Striated M uscle
Cells (%)
•Muscle for control of urination
5-weeks
100
75
50
25
0
25
81-years
Apoptotic Index
(%)
15
50
75
100
Age (Years)
10
5
0
25
50
Age
(Years)
75
100
750
Epitrochlearis
500
** p <0.05 =
ad lib< CR and Young
**
250
0
5-AL
Fibers Through
Midbelly
Type II Fiber
1000
30-AL
30-CR
Soleus
Fibers Through
Midbelly
Fibers Through
Midbelly
Myocyte Cell loss During Aging and CR
20000
Vastus Lateralis
**
10000
0
5-AL
500
18-AL
36-AL
Deep portion of VL
muscle is a type 2
fiber
0
5-AL
30-AL
30-CR
Type I Fiber
Lee, C.M., Aspnes, L.E., Chung, S.S., Weindruch, R., and Aiken, J.M. 1998. Influences of caloric
restriction on age-associated skeletal muscle fiber characteristics and mitochondrial changes in rats
and mice. Ann N Y Acad Sci 854:182-191.
Muscle Function with Age and Calorie
Restriction
EDL
Force/BW
(N/kg)
6.0
30
+
*
Type II Fiber
4.0
Specific Force
(N/cm2)
8.0
EDL
+
*
20
10
2.0
0
0.0
Young
5.0
Old
Young
Old-CR
30
Soleus
Specific Force
(N/cm2)
Force/BW
(N/kg)
4.0
*
3.0
2.0
1.0
Old
Old-CR
Soleus
*
20
10
Type I Fiber
0
0.0
Young
Old
Old-CR
Young
Old
Old-CR
Anthony M. Payne, Stephen L. Dodd, and Christiaan Leeuwenburgh. Life-long calorie restriction in Fischer-344
rats attenuates age related loss in skeletal muscle specific force and extracellular space. J Appl Physiol (September
12, 2003). 10.1152/japplphysiol.00758.2003.
CR attennuated the age associated rise in
extracellular space in the fast extensor
digitorum longus (Type 2) muscle
B – EDL, Old
10
C – EDL, Old-CR
Extracellular Space
(%)
A – EDL, Young
EDL
8
*
6
+
4
2
0
Young
E – Soleus, Old
Old-CR
F – Soleus, Old-CR
10
Extracellular Space
(%)
D – Soleus, Young
Old
Soleus
8
*
6
+
4
2
0
Scale bar, 100 μm
Young
Old
Old-CR
Does caloric restriction attenuate age
related alterations in apoptosis and cell
signaling?
• Are caspases effected?
• Inhibitors of caspases?
• Are other key pro- and anti-apoptotic
proteins effected?
• And do they influence the apoptotic
potential (survival/death)?
•Scientific American
Caspase-3; cleaved caspase-3; and X-linked inhibitor-ofapoptosis (XIAP) protein content as well as the enzymatic
activity of caspase-3 in the gastrocnemius muscle
300
A
*
150
100
**
50
Cleaved Caspase-3
(% of control)
Procaspase-3
(% of control)
200
0
26AD
200
**
100
12AD
26CR
150
C
XIAP Content
(% of control)
Caspase-3 Activity
(OD/mg protein)
*
0
12AD
30
B
20
10
0
26AD
26CR
*
D
**
100
50
0
12AD
26AD
26CR
12AD
26AD
26CR
Oxidative damage, mtDNA deletions, and ETC
abnormalities are co-localized along a single muscle fiber
and exhibit atrophy
Wanagat et al. 2001. Mitochondrial DNA deletion mutations colocalize with segmental electron transport system
abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia. Faseb J. 15:322-32
Bua EA, McKiernan SH, Wanagat J, McKenzie D, Aiken JM. Mitochondrial abnormalities are more frequent in
muscles undergoing sarcopenia. J Appl Physiol. 2002 Jun;92(6):2617-24.
Fiber Splitting
COX-/SDH++
Wanagat et al. 2001. Mitochondrial DNA deletion mutations colocalize with segmental electron transport system
abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia. Faseb J. 15:322-32
Koseki, T., Inohara, N., Chen, S., and
Nunez, G. (1998) ARC, an inhibitor of
apoptosis expressed in skeletal
muscle and heart that interacts
selectively with caspases. Proc Natl
Acad Sci U S A 95, 5156-5160
Neuss, M., Monticone, R., Lundberg,
M. S., Chesley, A. T., Fleck, E., and
Crow, M. T. (2001) The apoptotic
regulatory protein ARC (apoptosis
repressor with caspase recruitment
domain) prevents oxidant stressmediated cell death by preserving
mitochondrial function. J Biol Chem
276, 33915-33922
Cytosolic (A) and mitochondrial (B) ARC
content in the gastrocnemius muscle
Cytosolic ARC
(% of control)
200
A
**
150
100
50
0
AD12
Mitochondrial ARC
(% of control)
200
AD26
CR26
B
*
150
**
100
50
0
12AD
26AD
26CR
Coupling endoplasmic reticulum
stress to the cell death program
Rao, R.V., Castro-Obregon, S.,
Frankowski, H., Schuler, M., Stoka, V.,
del Rio, G., Bredesen, D.E., and Ellerby,
H.M. 2002. Coupling endoplasmic
reticulum stress to the cell death
program: An Apaf-1-independent
intrinsic pathway. JBC
Nakagawa, T., and Yuan, J. (2000) Crosstalk between two cysteine protease
families. Activation of caspase-12 by
calpain in apoptosis. J Cell Biol 150,
887-894.
Nakagawa, T., Zhu, H., Morishima, N., Li,
E., Xu, J., Yankner, B. A., and Yuan, J.
(2000) Caspase-12 mediates
endoplasmic-reticulum-specific
apoptosis and cytotoxicity by amyloidbeta. Nature 403, 98-103.
Caspase-12 and cleaved caspase-12 in the gastrocnemius muscle
Procaspase-12
(% control)
400
*
A
300
**
200
Nakagawa, T., and Yuan, J. (2000) Cross-talk
between two cysteine protease families.
Activation of caspase-12 by calpain in
apoptosis. J Cell Biol 150, 887-894.
100
0
Cleaved Caspase-12
(% of control)
12AD
150
26AD
26CR
Nakagawa, T., Zhu, H., Morishima, N., Li, E.,
Xu, J., Yankner, B. A., and Yuan, J. (2000)
Caspase-12 mediates endoplasmic-reticulumspecific apoptosis and cytotoxicity by
amyloid-beta. Nature 403, 98-103.
B
100
**
50
0
12AD
26AD
26CR
Caspase-12
• Intracellular calcium levels increase with age.
Several studies have suggested that intracellular
calcium handling is drastically improved
following periods of caloric restriction
• A ~350% increase in the expression of caspase12 (caspase located at the sarcoplasmic
reticulum) with age, CR reduced this ageassociated rise
• These data suggest that the caspase-12-mediated
pathway of apoptosis may play a key role in
sarcopenia and is attenuated by CR
Table 1. Overview of changes ( increase,  decrease,  no change) in apoptosis and apoptotic
regulatory proteins in skeletal muscle with aging and calorie restriction (12AD v 26AD and 26CR v 26AD).
Aging
Calorie Restriction
Gastrocnemius Muscle
Markers for Apoptosis
Apoptosis
Caspase-3 (procaspase)1
Caspase-3 (cleaved caspase)1
Caspase-3 (activity)2








Mitochondrial
Cytochrome c2
Apaf-11
Caspase-9 (procaspase)1
Caspase-9 (cleaved caspase)1
Apoptosis inducing factor1
Apoptosis inducing factor4












Inhibitors
XIAP2
ARC1
ARC2
ARC3








Sarcoplasmic Reticulum
Caspase-12 (procaspase)1
Caspase-12 (cleaved caspase)1




1Total
tissue; 2Cytosolic; 3Mitochondrial; 4Nuclear.
Dirks, A., and Leeuwenburgh, C. (2004). FRBM. Dirks, A., and Leeuwenburgh, C. (2002). Am J Physiol Regul Integr Comp
Physiol 282, R519-527.
Dirks and Leeuwenburgh
Table 2. Overview of changes ( increase,  decrease,  no change) in apoptosis and apoptotic
regulatory proteins in cerebral cotices with age and calorie restriction (12AD v 26-27AD and 26-27CR v
27AD).
Aging
Calorie Restriction
Markers for Apoptosis
Apoptosis
Cleaved PARP
Caspase-3 (cleaved caspase)1
Caspase-3 (activity)2








Receptor Mediated
Caspase-2 (cleaved caspase)1
Caspase-2 (activity)2




Mitochondrial Mediated
Caspase-9 (cleaved caspase)1
Caspase-9 (activity)1
Cytochrome c2
Apaf-11








Inhibitors
XIAP2
ARC2




1Total
tissue; 2Cytosolic; 3Mitochondrial; 4Nuclear.
Hiona, A., and Leeuwenburgh, C (2004). Unpublished data.
Shelke, R. R. J., and Leeuwenburgh, C. (2003) Life-long calorie restriction (CR) increases expression of
apoptosis repressor with a caspase recruitment domain (ARC) in the brain. FASEB J., 02-0803fje.
Summary
• CR is able to attenuate the age-associated increase in
apoptosis in skeletal muscle and neurons by altering
several key apoptotic proteins towards cellular survival,
thereby reducing the potential for sarcopenia and
neurodegenerative diseases.
• A diminished activation of mitochondrial-mediated, ERmediated and death receptor-mediated pathways with lifelong caloric restriction could have a profound affect on
apoptosis and the susceptibility to apoptosis, cognition, as
well as on muscle function.
• The information obtained from these studies could
potentially permit the development of physiological or
genetic interventions that may attenuate the loss of skeletal
muscle myocytes (sarcopenia) and neurodegeneration
indicative of advancing age.
Life-long exercise by wheel running
Meters Run Per Day
3000
2000
1000
0
2
4
6
8
10 12 14 16 18 20 22 24
Months
500
Sed-restricted
Sedentary
Exercise
Body weight
(grams)
450
400
350
300
250
200
2
4
6
8
10
12
14
Age
(months)
16
18
20
22
24
ACKNOWLEDGEMENTS
Biochemistry of Aging Lab:
Collaborators:
Funding Provided by:
Barry Drew, PhD (USA)
Gustavo Barja, PhD (Spain)
National Institute of Health
Amie Dirks, PhD (USA)
Ricardo Gredilla (Spain)
National Institute on Aging
Sharon Phaneuf (USA)
Steve Dodd, PhD (USA)
Society of Geriatric Cardiology
Suma K. PhD (India)
Scott Powers, PhD (USA)
Rajani Shelke, PhD (India)
Angela Lezza, PhD (Italy)
Colin Selman, PhD (Scotland)
Nicola Gadaleta, PhD (Italy)
Tracey Philips, (Scotland)
David Julian, PhD (USA)
Mina Hiona (Greece)
Tilman Grune, PhD (Germany)
Young Yang (Korea)
The End
Complete Data
Web Page: http://grove.ufl.edu/~cleeuwen/
NIH-31/NIA Fortified Diet (CR)NIH31 Average Nutrient Composition (AL)
Average Nutrient Composition
NIH-31/NIA Fortified
NIH-31
Protein
%
18.74
18.42
Fat
%
4.41
4.47
Fiber
%
4.58
4.05
Ash
%
6.51
6.64
Nitrogen-Free Extract
%
55.04
55.91
Gross Energy
kcal/g
3.95
4.02
Digestible Energy
kcal.g
3.36
-Metabolizable Energy
kcal.g
3.07
-Linoleic Acid
%
1.79
-Moisture
%
-10.51
Amino Acids
Arginine
Methionine
Histidine
Leucine
Lysine
Tryptophan
Valine
Cystine
Isoleucine
Threonine
Pherylalanine
+Tyrosine
%
%
%
%
%
%
%
%
%
%
%
NIH-31/NIA Fortified
1.10
0.36
0.42
1.50
0.96
0.22
0.88
0.26
0.76
0.71
1.53 Pherylalanine
Tyrosine
NIH-31
1.06
0.39
0.41
1.61
0.95
0.24
0.96
0.28
0.90
0.71
0.92
0.70
Minerals
Calcium
Phosphorus
Sodium
Chlorine
Potassium
Magnesium
Iron
Maganese
Zinc
Copper
Iodine
Cobalt
Selenium
%
%
%
%
%
%
mg/Kg
mg/Kg
mg/Kg
mg/Kg
mg/Kg
mg/Kg
mg/Kg
NIH-31/NIA Fortified
1.03
0.93
0.30
0.48
0.59
0.20
336.41
156.01
48.41
13.28
2.01
0.53
0.30
NIH-31
1.06
0.92
0.26
0.42
0.59
0.20
300.20
152.80
50.40
13.20
1.94
0.53
--
Vitamins
Vitamin A
Vitamin A3
Vitamin D3
Vitamin E
Choline
Niacin
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Menadione
Folic Acid
Biotin
Vitamin B12
Vitamin C
Carotene
IU/g
IU/g
IU/g
mg/Kg
mg/g
mg/Kg
mg/Kg
mg/Kg
mg/Kg
mg/Kg
mg/Kg
mg/Kg
mg/Kg
mcg/Kg
mg/g
mg/Kg
NIH-31/NIA Fortified
40.49
-7.00
52.15
2.60
116.16
55.07
13.16
11.04
123.44
111.01
2.13
0.38
93.80
---
NIH-31
-30.73
4.19
38.30
1.96
92.20
39.50
10.20
7.80
77.30
22.00
1.70
0.13
53.00
---