1. - 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

2. - 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 Outline Background Mitochondria and oxidative damage Project aims Methods Preliminary results Mitochondrial membrane potential Protein oxidation Future plans

3. - 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+H+ H+H+ H+H+ NADH NAD + H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ ADP+ P ATP Complex II Complex III Complex I V ATP Synthethase O2O2 + H2OH2O e-e- e-e- ROS O2O2 O2O2 2O 2 *- +2H + H2O2H2O2 SOD 2H 2 O Gpx 2GSH GSSG OH 2 - OH - mtDNA BER Complex I Outer membrane Inner membrane Matrix

4. - 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 aging (Harman, 1973) ROS Mitochondrial oxidative stress Oxidative damage Proteins Nucleic Acids Lipids Carbohydrates Repair mechanims DNA repair Protein Degradation Accumulation of Lesions -DNA mutations -Protein aggregation Antioxidants mnSOD Gluthathione system Aging Disease Dysfunctional mitochondria Frailty ?

5. - 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 includes A. 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

6. - 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

7. - 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 assays 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!

8. - 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 potential measured by flow cytometry (TMRE accumulation) Control: Uncoupling by FCCP treatment Test: Difference between two cell lines (WT and XPC)

9. - 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 proteins) in WRN whole cell extracts Preliminary data suggest increased carbonyl load in WRN knock-down cells compared to wild-type cells

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

11. - 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

12. - 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 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