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.

8-oxodG/10 dG in mtDNA

Oxidative stress mt-DNA damage mt-DNA deletions Oxidized proteins Lipid peroxidation Lipid-adduct formation Decrease in repair systems
H2O2 Production (nmoles/min/mg protein)
Dityrosine/Tyrosine (mmol/mol)
1.5
0.075

5 4 3 2 1 0 12-AL 26-AL 26-CR

**

Vastus Lateralis Ad Libitum Caloric Restriction

H2O2 Production (nmol/min/mg protein)

0.5 0.4 0.3 0.2 0.1 0.0

Isolated Mitochondria

1.0

0.025

8-oxodG/10 dG in nDNA

0.050
1.5

Age (Months)

1.0

0.5

0.000 4 14 30

0.5

**

0.0 AD CR

0.0 12-AL 26-AL 26-CR

12AD

26AD

26CR

Age (Months)

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

25 20

TNF-

TNF- (pg/mL)

15 10 5

6AD 26AD 26CR

TNF receptor-associated death domain (TRADD) TNF-R-associated factor 2 (TRAF2) Receptor interacting protein (RIP) Inhibitor of B kinase (IKK) complex

0 6AD 26AD 26CR

*P = 0.0001 vs 6AD

P = 0.0001 vs 26AD

0.035

NFB Activation (OD450nm)

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Phillips and Leeuwenburgh (unpublished)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?
Humans
Fibers Through Midbelly
20000

Animals
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.

Density of Striated M uscle Cells (%)

Muscle for control of urination 5-weeks

100

75

50

56-years

25

25

50

75

100

15

Age (Years)

Apoptotic Index (%)

10

81-years

25

50 Age (Years)

75

100

Myocyte Cell loss During Aging and CR

Fibers Through Midbelly
750

Epitrochlearis

** p <0.05 = ad lib< CR and Young

Type II Fiber

500

**

250

5-AL

30-AL

30-CR

Fibers Through Midbelly

20000

Vastus Lateralis

Fibers Through Midbelly

1000

Soleus

10000

**

500

5-AL

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

8.0

EDL

+
Specific Force (N/cm2)

30

EDL

6.0

Force/BW (N/kg)

20

4.0

Type II Fiber

10

2.0

0.0 Young Old Old-CR

0 Young Old Old-CR

5.0 4.0

30

Soleus
Specific Force (N/cm2)

Soleus

Force/BW (N/kg)

*
3.0 2.0 1.0 0.0 Young Old Old-CR

20

10

Type I Fiber
0 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
A EDL, Young B EDL, Old C EDL, Old-CR
Extracellular Space (%)
10 8 6

EDL

*
+

4 2 0 Young Old Old-CR

D Soleus, Young

E Soleus, Old

F Soleus, Old-CR
10

Soleus

Extracellular Space (%)

8 6

*
+

4 2 0

Scale bar, 100 m

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
200

Cleaved Caspase-3 (% of control)

300

Procaspase-3 (% of control)

150 100 50 0 12AD 26AD 26CR

200

**

100

**

0 12AD 26AD 26CR

30

Caspase-3 Activity (OD/mg protein)

C
XIAP Content (% of control)

150

* **

20

100

10

50

0 12AD 26AD 26CR

0 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
200

Cytosolic ARC (% of control)

**

150 100 50 0 AD12

200

AD26

CR26

Mitochondrial ARC (% of control)

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

400

Procaspase-12 (% control)

300 200 100 0 12AD 26AD 26CR

**
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.

Cleaved Caspase-12 (% of control)

150

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.

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 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

Calorie Restriction

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 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

Calorie Restriction

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

3000

Meters Run Per Day

2000

1000

0 2 4 6 8 10 12 14 16 18 20 22 24

Months
500 450

Sed-restricted Sedentary Exercise

Body weight (grams)

400 350 300 250 200 2 4 6 8 10 12 14 16 18 20 22 24

Age (months)

ACKNOWLEDGEMENTS

Biochemistry of Aging Lab:

Barry Drew, PhD (USA)

Collaborators:
Gustavo Barja, PhD (Spain)

Funding Provided by:

National Institute of Health

Amie Dirks, PhD (USA)

Sharon Phaneuf (USA) Suma K. PhD (India) Rajani Shelke, PhD (India) Colin Selman, PhD (Scotland) Tracey Philips, (Scotland) Mina Hiona (Greece) Young Yang (Korea)

Ricardo Gredilla (Spain)

Steve Dodd, PhD (USA) Scott Powers, PhD (USA) Angela Lezza, PhD (Italy) Nicola Gadaleta, PhD (Italy) David Julian, PhD (USA) Tilman Grune, PhD (Germany)

National Institute on Aging

Society of Geriatric Cardiology

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 Minerals Calcium Phosphorus Sodium Chlorine Potassium Magnesium Iron Maganese Zinc Copper Iodine Cobalt Selenium 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 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/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/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 1.06 0.39 0.41 1.61 0.95 0.24 0.96 0.28 0.90 0.71 0.92 0.70 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 -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 ---

% % % % % % % % % % %

% % % % % % mg/Kg mg/Kg mg/Kg mg/Kg mg/Kg mg/Kg mg/Kg

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