Why do we age so differently?

1. Tinna Stevnsner for Christina Poulsen Hvitby (on maternity leave) Enrolled in Ph.D.-programme October 2008 VELUX FONDEN Why do we age so differently? The role of genome maintenance in age-related fatigue - and importance of synaptic mitochondrial maintenance in healthy aging Funded by:

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

3. Mitochondria H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ ROS Complex I V mtDNA Outer membrane BER Complex III ATP Synthethase Complex II O2 SOD Gpx 2O2*- Complex I H2O2 2H2O NADH O2 +2H+ O2 + 2GSH GSSG H2O NAD+ Inner membrane OH- OH2- 2H+ ADP+ P ATP e- e- Matrix

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

5. The role of genome maintenance in age-related fatigue- and importance of synaptic mitochondrial maintenancein healthy aging The aims are to investigate mitochondrial function as a biomarker for age-related fatigue to investigate synaptic mitochondrial function and DNA repair capacity in premature (WRN) and age-related disease (Parkinsons, Alzheimers) • The research material includes • 8 ml fresh blood samples from selected individuals from the 1953 Metropolit study with extreme phenotype regarding fatigue • WRN knockdown cell lines and fresh tissues from young and old mice - with and without neurodegenerative diseases such as Alzheimers and Parkinsons

6. 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. Mitochondrial functional assays Membrane potential Flow cytometry Fluorescence spectrometry • Seahorse Bioscience´s Extracellular flux (FX) Assay • Oxygen consumption • Fatty acid oxidation • ATP turnover • ROS levels • Flow cytometry • Fluorescence Spectrometry - all the assays require fresh blood samples!

8. Mitochondrial membrane potentialmeasured by flow cytometry(TMRE accumulation) Control: Uncoupling by FCCP treatment Test: Difference between two cell lines (WT and XPC)

9. 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. Detection of oxidized proteinsin mitochondria Preliminary data indicates an increased carbonyl load in mitochondria from premature aging cells (CSB) compared to wild-type cells

11. 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. 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