mitochondrial function decline and oxidative stress in human aging (...

Mitochondrial Function Decline and Oxidative Stress in Human Aging

(人體老化過程中的粒線體能量代謝功能衰退與氧化壓力 )

Yau-Huei Wei ( 魏耀揮 )Department of Biochemistry and Mol

ecular BiologyNational Yang-Ming University

Taipei, Taiwan 112October 31, 2007

Biochemical basis of aging

• Accumulation of oxidative damage during aging

• During aging, oxidative damage to biological macromolecules are accumulated. These include:– Peroxidation of lipids– Formation of protein carbonyls– Various forms of damage to DNA

• Macromolecular damage can cause malfunction of organelles, particularly the mitochondria

• It can also lead to necrosis and apoptosis of cells

The metabolic rate of a species ultimately determines its life

expectancy

Metabolism Aging

This relationship was described about a century ago

Relationship between body weight (W) and maximum life

span (MLSP) in mammals and birds

Relationship between MLSP and liver catalase activity

Relationship between MLSP and liver GPx activity

Relationship between MLSP and brain selenium-dependent

GSH-peroxidase in vertebrate species including birds

Relationship between MLSP and lung GSH-reductase activity in vertebrate species

including birds

Relationship between H2O2 production by liver mitochondria and MLSP in mammalian species

Overview of mitochondria

100 to 1000 mitochondri

a

2-10 copies of mtDNA

Electron transport chain

Mitochondrial DNA

Cell Mitochondrion

生

III

IIIIV

V

ROS

e-

H+c

Apoptosis

ATP

Q

c

Mitochondrial theory of agingOxidative phosphorylation 老

病 死Mitochondrial disease

Parkinson’s diseaseAlzheimer’s disease

Diabetes and metabolic syndrome

Cancer

Beta-oxidation of fatty acids

Heme synthesisSteroid hormone synthesis

Urea cycle

ROSOxidative damage

Dysfunctional macromolecules

Vicious cycle

生

III

IIIIV

V

ROS

e-

H+c

Apoptosis

ATP

Q

c

Mitochondrial theory of agingOxidative phosphorylation 老

病 死Mitochondrial disease

Parkinson’s diseaseAlzheimer’s disease

Diabetes and metabolic syndrome

Cancer

Beta-oxidation of fatty acids

Heme synthesisSteroid hormone synthesis

Urea cycle

ROSOxidative damage

Dysfunctional macromolecules

Vicious cycle

Scientific American 1996

The majority of intracellular ROS is derived from the mitochondrial

respiratory chain

Free-radical theory of aging

• First proposed in the mid-1950s

• Proposed by Dr. Denham Harman

• Endogenous oxygen radicals are generated in cells and result in cumulative damage to tissue cells

• Identification of superoxide dismutase (SOD) by Dr. Irwin Fridovich

Metabolism AgingROS

The targets of ROS and free radicals are random, indiscriminative and

cumulative

The common deletionthat is related to aging

Naked and compact Maternally inherited

Vulnerable to damage & mutation

Mitochondria play a role in aging and age-related diseases

The 4977 bp deletion of mtDNA in aging human liver

Yen TC et al. (1994) Free Radic Biol Med 16:207-214

A 13-bp direct repeat flanking the 4977 bp deletion of mtDNA

The strategy for the determination of multiple mtDNA deletions by long-range PCR techniques

Kao SH et al. (1998) Mol. Human Reprod. 7:657-666

Long-range PCR products amplified from mtDNA with large-scale deletions

Kao SH et al. (1998) Mol. Human Reprod. 7:657-666

Primer-shift PCR and corresponding products amplified from mtDNA with 4977 bp deletion

Kao SH et al. (1998) Mol. Human Reprod. 7:657-666

Large-scale deletions of mtDNA are accumulated in old human brain

Yang JH (1996) Arch. Dermatol. Res. 287:641-648

1

Yang JH (1996) Arch. Dermatol. Res. 287:641-648

Fahn HJ et al. (1996) Am. J. Respir. Crit. Care Med. 154:1141-1145

Large-scale deletion and oxidative damage of mtDNA increase exponentially in the human heart during aging

Age (year)

Experimental evidence supporting the occurrence and accumulation of different

mtDNA mutations in human skeletal muscle during ageing

Somatic mtDNA mutations in human aging

• Point mutations, deletions and tandem duplications of mitochondrial DNA (mtDNA) accumulate in a variety of tissues during aging in humans, monkeys and rodents.

• These mutations are unevenly distributed and can accumulate clonally in certain cells, causing a mosaic pattern of respiratory chain deficiency in tissues such as heart, skeletal muscle, liver and brain.

• These finding have provided great support of the “mitochondrial theory of aging”.

Wei YH et al. (1998) Ann. N.Y. Acad. Sci. 854:155-170

Science (1999) 286,774-779

Science (1999) 286:774-779

Science (1999)286,774-779

Science (1999) 286:774-779

Nature Genet. (2006) 385:515-517.

Respiratory chain deficiency and analysis of mtDNA deletions in substantia nigra neurons

Characterization and quantification of mtDNA deletion in substantia nigra neurons from individuals with Parkinso

n disease and from age-matched controls

Somatic mtDNA mutations

Reactive oxygen species

Oxidative DNA damage

Aging phenotypes

?

Overproduction of ROS results in DNA damage and aging

Nature (2004) 429:417-423

Hair loss and curvature of the spine in mutant mice

Kyphosis (curvature of the spine)

Reduced subcutaneous fat in mutant mice

Nature (2004) 429:417-423

Deletion and depletion of mtDNA in the POLG mutant mice

Somatic mtDNA mutations

Aging phenotype

sOxidative stress

Oxidative damageMitochondrial dysfunction

Apoptosis

?

Mitochondrial role in apoptosis

Science (2005) 309:481-484

Science (2005) 309:481-484

Hair loss and grayingkyphosis

Caspase 3 activation in aging

Science (2005) 309:481-484

Caspase 3 activation in D275A mice

Science (2005) 309: 481-484

Increased apoptosis in mutant mice

Science (2005) 309:481-484

Somatic mtDNA mutations

Reactive oxygen species

Oxidative DNA damage

Aging phenotypes

Apoptosis

Apoptosis induced by ROS may also play a role in aging

Mitochondrial roles in human aging

Apoptotic features in the mutant cybrid 51-10 harboring 4977 bp-deleted mtDNA after treating with STS or UV

irradiation

Ann. N.Y. Acad. Sci. (2004) 1011:133-145

Cell viability was decreased in the cybrids with mtDNA deletion compared to the cybrids

harboring wild-type mtDNA after exposure to UV irradiation

Cybrid lines

Lin 0 Lin 2

Viab

ility (%

)

0

10

20

30

40

50

60

70 (A)

Cybrid lines

1-3-16 51-10

Viab

ility (%

)

0

10

20

30

40

50 (B)

Cybrid lines

Lin 0 Lin 2

Viab

ility (%

)

0

10

20

30

40

50

60

70 (A)

Cybrid lines

1-3-16 51-10

Viab

ility (%

)

0

10

20

30

40

50 (B)

Lee CF et al. (2005) Ann. N.Y. Acad. Sci. 1042:429-438Mutant cybrids: 51-10 & Lin 2

Cybrid clones

1-3-16 51-10

Cas

pase

3 a

ctiv

ity (U

x10

-2/

g pr

otei

n)

0

50

100

150

200

250

ControlQ10

UVQ10+UV *

*

Cybrid clones

Lin 0 Lin 2

Casp

ase

3 ac

tivity

(Ux1

0-2

/g

prot

ein)

0

50

100

150

200

250

300

350

ControlQ10

UVQ10+UV

*

(A) (B)

Coenzyme Q10 attenuates UV-induced apoptosis in cybrids harboring 4977 bp- and 4366 bp-deleted mtDNA

n=3, *p < 0.05

Mutant cybrids: 51-10 & Lin 2 Lee CF et al. (2005) Ann. N.Y. Acad. Sci. 1042:429-438

Nature (2000) 408:239-247

ROS damages various cell components and triggers the activation of specific signaling

pathways

Nature (2000) 408:239-247

A number of signaling pathways are tightly regulated by ROS

cDNA microarray for aging study

Science (1999) 285:1390-1393

Science (1999) 285:1390-1393

Nature Genet (2000) 25:294-297

Nature Genet (2000) 25:294-297

Two interventions to reduce the rate of the aging process

• Caloric restriction– Restriction of nutrient intake to 60-70%

that of voluntary levels slows down aging in many organisms

• Alleviation of oxidative stress– Slows down aging by decreasing

oxidative stress

CCD-966SKSkin fibroblasts

H2O2 treatment for 90 min

incubated for 7 days

Senescence phenotype

Control Senescence

Increase in the proportion of -galactosidase positive skin fibroblasts after H2O2 treatment

A good cell model for molecular biological studies of aging in vitro

C 24hr 48hr 72hr

GAPDH

p-p53

-actin

p53

p21

MDM2

Rb

MDM2p53

p21

cyclin E

CDK2

Rb

P

PP

P

Rb E2F

H2O2 treatment

Increase in the p53 and p21 protein expression in H2O2-induced cellular senescence-like

phenotype

Rb

Cyclin A

GAPDH

-actin

p53

p21

MDM2C S

control

24hr

48hr

72hr

　 Control 24 hr 48 hr 72 hr

Sub G0 1.55 1.37 1.01 0.63

G1 73.13 73.68 75.53 81.95

S 4.55 4.30 5.15 4.11

G2/M 19.10 18.59 16.37 11.75

H2O2-treatment

H2O2-induced cellular senescence resulting in cell cycle arrest at G1 phase

Oxidative stress effect on mitochondria and nucleus

H2O2

mitochondrion

nucleus

• mitochondrial membrane potential• cytochome c oxidase activity

ROS

p53-dependent cycle arrest at G1 phase

Senescence

ROS

C 24 hr 48 hr 72 hr

H2O2 treatment

P-PKBThr308

GAPDH

Akt

FOXO3aP

FOXO1P

Catalase

P

MnSOD

p53

FOXO family, the downstream of Akt, regulates the antioxidant enzymes under oxidative stress

ROS Senescence

0

20

40

60

80

100

120

140

160

Control 24hr 48hr 72hr

250 M H2O2 treatment

DC

F r

elat

ive

inte

nsity

n=3

0

20

40

60

80

100

120

140

160

Control 24hr 48hr 72hr

250 M H2O2 treatment

DC

F r

elat

ive

inte

nsity

n=3

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Control Senescence

The mRNA level of catalase

Rel

ativ

e ra

tio (

%)

C S O A C E

C : controlS : senescenceO : oligomycinA : antimycin AC : CCCPE : EtBr

Down-regulation of catalase in cells with mitochondrial dysfunction

GAPDH

Treatment for 3 Days

catalase

＊

n=2 , ＊： P < 0.05

Why the expression level of catalase, a crucial

detoxification enzyme is decreased by H2O2?

Catalase promoter assay

Akt-mutated Foxo1-mutated

Down-regulation of catalase by ROS via PKB signaling in mesanglia cells

J. Cell. Physiol. (2007) 211(2):457-467.

Akt

FOXO1P

catalaseexpression

GAPDH

MnSOD

Catalase

N1 N2 N3 N4 N5 N6

1.0 1.5 1.7 1.3 0.9 0.8

1.0 1.4 1.2 1.1 1.5 1.7

GAPDH

MnSOD

Catalase

N1 M1 M2 M3 M4 M5

1.0 1.2 0.7 0.7 0.9 0.7

1.0 1.7 2.0 2.2 2.5 2.3

Differential expression of antioxidant enzymes in skin fibroblast of different ages

Normal subjects MERRF patients

Age Age

MnSOD was up-regulated but catalase was not changed in such a manner

When MnSOD is over-expressed without concurrent increase in catalase (CAT) or glutathione peroxidase (GPx), the accumulated H2O2 can be converted to the far more reactive .OH radicals via Fenton reaction in the affected cells.

·OH

NADH .O2- H2O2 2 H2O

2GSH GSSG

MnSOD

CAT

Defective ETC

O2

GPx

H2O + ½ O2

Over-expression of Mn-SOD leads to accumulation of H2O2

C 24hr 48hr 72hr

GAPDH

C S

PDH

GAPDH

PDH

H2O2 treatment

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Control Senescence

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Control Senescence

The RNA level of PDH

Rel

ativ

e ra

tio (

%)

Rel

ativ

e ra

tio (

%)

Changes in the PDH and PDK expression during cellular senescence

The RNA level of PDK

PDK

The protein level

Current Opinion in Cell Biology (2006) 18:598-608

Regulation of glucose metabolism by glycolysis and oxidative phosphorylation in human cells

2 ATP / glucose

36 ATP / glucose

Glycolysis

OXPHOS

Plasma membrane

Mitochondria

Down-regulation of pyruvate dehydrogenase in aging

Acta Biochim. Pol. (2005) 52:759-764

Active

Inactive

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Control Senescence

The mRNA level of PDH

Rel

ativ

e ra

tio (

%)

Cancer Res. (2006) 66: 8927-8930

Signaling molecules regulating glucose metabolism in cells with mitochondrial dysfunction or mtDNA

mutations

Metabolic alterations in cells of aging tissues

Mitochondrion

ROS

Nucleus

PI3K/Akt

FOXO1

Catalase

PDH

PDK

Pyruvate HIF

？

Repair system

+ SenescenceLDH

Lactate

Oxidative damage

Extension of life span by caloric restriction in animals

Scientific American 1996

Reduction of food intake decelerates aging

Scientific American 1996