- D A N I S H A G I N G R E S E A R C H C E N T E R - Why do we age so differently? Tinna Stevnsner for Christina Poulsen Hvitby (on maternity

- D A N I S H A G I N G R E S E A R C H C E N T E R -www.sdu.dk/darc

Why do we age so differently?

Tinna Stevnsner

for

Christina Poulsen Hvitby (on maternity leave)Enrolled in Ph.D.-programme October 2008

VELUX FONDEN

Funded by:

The role of genome maintenance in age-related fatigue

- and importance of synaptic mitochondrial maintenance in healthy aging


OutlineOutline Background

Mitochondria and oxidative damage

Project aims

Methods

Preliminary results Mitochondrial membrane potential Protein oxidation

Future plans


Mitochondria

2H+

H+

H+H+

H+

H+

NADH

NAD+H+

H+

H+

H+

H+H+

H+

ADP+ P

ATP

Complex II

Complex III

Complex I V

ATP Synthethase

O2 +H2O

e-

e-

ROS

O2

O2 2O2*-

+2H+H2O2

SOD2H2O

Gpx

2GSH GSSG

OH2- OH-

mtDNA BER

Complex I

Outer membrane

Inner membrane

Matrix


Mitochondrial free radical theory of agingMitochondrial free radical theory of aging(Harman, 1973)(Harman, 1973)

ROS Mitochondrial oxidative stress

Oxidative damage Proteins

Nucleic AcidsLipids

Carbohydrates

Repair mechanimsDNA repair

Protein Degradation

Accumulation of Lesions -DNA mutations

-Protein aggregation

AntioxidantsmnSOD

Gluthathione system

Aging

DiseaseDysfunction

al mitochondri

a

Frailty ?


The role of genome maintenance in age-related fatigue

- and importance of synaptic mitochondrial maintenance

in healthy aging The aims are

A. to investigate mitochondrial function as a biomarker for age-related fatigue

B. to investigate synaptic mitochondrial function and DNA repair capacity in premature (WRN) and age-related disease (Parkinsons, Alzheimers)

The research material includesA. 8 ml fresh blood samples from selected individuals from the 1953

Metropolit study with extreme phenotype regarding fatigue

B. WRN knockdown cell lines and fresh tissues from young and old mice - with and without neurodegenerative diseases such as Alzheimers and Parkinsons


Parameters to be measured Membrane potential ATP level ROS level Level of oxidative damages

The methods for investigating mitochondrial function as a biomarker for age related fatigue should cover a broad spectrum of mitochondrial function be adaptable for high throughput analysis require low sample volume


Mitochondrial functional Mitochondrial functional assaysassays

Membrane potential Flow cytometry Fluorescence spectrometry

ROS levels Flow cytometry Fluorescence Spectrometry

Seahorse Bioscience´s Extracellular flux (FX) Assay Oxygen consumption Fatty acid oxidation ATP turnover

- all the assays require fresh blood samples!


-Mitochondrial membrane potentialMitochondrial membrane potentialmeasured by flow cytometrymeasured by flow cytometry

(TMRE accumulation)(TMRE accumulation)100 101 10 2 103 104

FL2-H: FL2-Height

0

20

40

60

80

100

% o

f M

ax

WT_fccp.002 100

WT_norm.001 100

100 10 1 102 103 104

FL2-H: FL2-Height

0

20

40

60

80

100

% o

f M

ax

XPC.001 100

XPC-fccp.001 100

100 101 102 10 3 104

FL2-H: FL2-Height

0

20

40

60

80

100

% o

f M

ax

WT_fccp.002 100

WT_norm.001 100

XPC.001 100

XPC-fccp.001 100

FSC-H, SSC-H subset

100 101 102 103 104

FL2-H: FL2-Height

0

20

40

60

80

100

% o

f M

ax

XPC.001 100

WT_norm.001 100

FSC-H, SSC-H subset

100 101 10 2 103 104

FL2-H: FL2-Height

0

20

40

60

80

100%

of M

ax

WT_fccp.002 100

WT_norm.001 100

100 10 1 102 103 104

FL2-H: FL2-Height

0

20

40

60

80

100

% o

f M

ax

XPC.001 100

XPC-fccp.001 100

100 101 102 10 3 104

FL2-H: FL2-Height

0

20

40

60

80

100

% o

f M

ax

WT_fccp.002 100

WT_norm.001 100

XPC.001 100

XPC-fccp.001 100

100 101 102 103 104

FL2-H: FL2-Height

0

20

40

60

80

100

% o

f M

ax

XPC.001 100

WT_norm.001 100

Control:

Uncoupling by FCCP treatment

Test:

Difference between two cell lines (WT and XPC)


Carbonylation (oxidized Carbonylation (oxidized proteins)proteins)

in WRN whole cell extractsin WRN whole cell extractsW

RN

WT

WT

+ 1

25u

M H

2O

2

WT

+ 2

50u

M H

2O

2

WR

N +

250

uM

H2O

2

WR

N +

125

uM

H2O

2

WR

N +

500

uM

H2O

2

WT

+ 5

00u

M H

2O

2

WR

NW

T

WT

+ 1

25u

M H

2O

2

WT

+ 2

50u

M H

2O

2

WR

N +

250

uM

H2O

2

WR

N +

125

uM

H2O

2

WR

N +

500

uM

H2O

2

WT

+ 5

00u

M H

2O

2

set bset a

WR

NW

T

WT

125

WT

250

WR

N25

0

WR

N12

5

WR

N50

0

WT

500

WR

NW

T

WT

125

WT

250

WR

N25

0

WR

N12

5

WR

N50

0

WT

500

set cset a

WR

NW

T

WT

125

WT

250

WR

N25

0

WR

N12

5

WR

N50

0

WT

500

WR

NW

T

WT

125

WT

250

WR

N25

0

WR

N12

5

WR

N50

0

WT

500

set cset b

Preliminary data suggest increased carbonyl load in WRN knock-down cells compared to wild-type cells


Detection of oxidized proteinsDetection of oxidized proteinsin mitochondriain mitochondria

21 3 4 5 6 7 8 9 10 11

Preliminary data indicates an increased carbonyl load in mitochondria from premature aging cells (CSB) compared to wild-type cells


Methods for investigation of the importance of synaptic mitochondrial maintenance in healthy aging will include

Isolation of synaptosomal and soma mitochondria from mouse brains Incubation of isolated mitochondria with DNA substrates containing specific DNA lesions Quantitation of mitochondrial repair enzymes by Western blotting

Synapses


Future plansFuture plans Measure mitochondrial functions in lymphocytes isolated from

fresh blood samples from fatigue and non-fatigue individuals

Look for potential correlations between specific mitochondrial functions and fatigue – and correlate with results from telomere studies

Confirm prliminary data on mitochondrial carbonylation in premature aging syndrome cells

Characterize DNA repair in synaptosomal mitochondria vs. soma mitochondria in mice suffering from premature aging or neurodegeneration

Documents

- D A N I S H A G I N G R E S E A R C H C E N T E R - Why do we age so differently? Tinna Stevnsner for Christina Poulsen Hvitby (on maternity