- 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
- 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
OutlineOutline Background
Mitochondria and oxidative damage
Project aims
Methods
Preliminary results Mitochondrial membrane potential Protein oxidation
Future plans
- 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
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
- 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
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 ?
- 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
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
- 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
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
- 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
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!
- 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
-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)
- 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
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
- 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
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
- 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
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
- 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
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
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