1. MITOCHONDRIAL DYSFUNCTION IN ACUTE CNS INJURY Patrick Sullivan, Ph.D. Associate Professor of Anatomy & Neurobiology Endowed Chair of the Spinal Cord and Brain Injury Research Center University of Kentucky College of Medicine

2. Mitochondrial Dysfunction following TBI/SCI How does it manifest? Time course of dysfunction. How does it contribute to cell death?

3. “Powerhouse of the cell” http://www.puc.edu/Faculty/Gilbert_Muth/art0072.jpg

4. Mitochondria: Death Switch of the Cell? Ca2+ Glutamate Death Triggers Bax Oxidants Caspases Cyto C Cyto C Caspase 9 Apaf-1 dATP ATP  Downstream Caspases ROS NECROSIS APOPTOSIS MPTP Smac/Diablo AIF

5. Electron Transport System (ETS) Creates membrane potential (ΔΨ) across the inner membrane ATP synthesis Ca2+ cycling

6. Mitochondrial Calcium Transport Ca2+ 2H+ Ca2+ Driven by  2Na+ Ca2+

7. Ca2+ PTP 2H+ Ca2+ 2Na+ Mitochondrial Calcium Transport and mPT Driven by 

8. Mitochondrial Respiration RCR = State III/State IV Healthy >5

9. Loss of Mitochondrial Bioenergetics following TBI

10. Acute loss of Mitochondrial Function depends on Injury Severity Mean ± SD Gilmer, L.K., Roberts, K.N.,Joy, K.M, Sullivan, P.G., and Scheff, S.W. Early mitochondrial dysfunction following cortical contusion injury. J. Neurotrauma 2009, Aug; 26 (8): 1271-80.

11. Overview of Peroxynitrite Formation/Reactions in Mitochondria Radi et al., 2002

12. Mitochondrial Oxidative Damage following TBI

13. Loss of Mitochondrial Enzyme Activities 3 hrs post-injury

14. Mitochondria Dysfunction can be Targeted up to 24 hrs post-injury

15. Mitochondrial Ca2+ Load Following TBI

16. Post-traumatic Time Course Changes in Cortical Mitochondrial Calcium Buffering Capacity Isolated then challenged Snapshot of in vivo levels

17. Motor Ctx Motor Ctx SC Ventral horn In Situ ROS Formation Basal level of ROS Motor Neurons - Dihidroethidium (DHET) - 3hr prior - converted to ethidium by O2-• 140%↑ J Comp Neurol (2004) 474:524-534

18. Mitochondrial Lipid Peroxidation - non synaptic mitochondria - 4 hydroxynonenal (HNE) 40%↑ J Comp Neurol (2004) 474:524-534

20. chromatography/mass spec

21. levels of oxidized bases higher in SCOxidation of mitochondrial DNA Immunoreactivity for8-hydroxyguanosine - specific marker for oxidative damage to DNA - serial confocal micrographs *cyclophilin D mRNA was significantly increased in the SC (1.58-fold; p=0.014) J Comp Neurol (2004) 474:524-534

22. Loss of Mitochondrial Homeostasis following SCI: Role of ROS

23. Oxidative Damage following SCI Mitochondrial Fraction Cytosolic Fraction

24. Mitochondrial Oxidative Damage to ETS and loss of Enzyme Activities following SCI

25. Mitochondrial Bioenergetics after with and without Post-injury Treatments

26. Synaptic Mitochondrial Respiratory Control Ratio (RCR) State III State IV RCR > 5.0 RCR decreased significantly following SCI. 15 and 30 min post-SCI treatment with DNP preserved the mitochondrial integrity. However tempol was ineffective. 1 hr post-injury treatment had no effect on RCR. RCR = Vehicle DNP TEMPOL *p < 0.05compared to sham #p < 0.05 compared to vehicle

27. Sham Vehicle DNP TEMPOL Synaptic Mitochondrial oxidative markers: Post-SCI Treatment 15 min 1 hr 30 min * * * # # # # *p<0.05 compared to sham #p<0.05 compared to Vehicle Protein Carbonyls increased significantly following contusion SCI. Treatment with DNP and TEMPOL (15 and 30 min post-SCI) reduced the protein carbonyls to normality.

28. Summary Mitochondrial dysfunction occurs rapidly and is progressive over 24 hrs post-injury. Mitochondrial oxidative damage coincides with loss of bioenergetics. Mitochondrial Calcium overload plays a pivotal role leading to mPT and cell death. Mitochondrial Dysfunction may be amendable to treatment in first 24 hrs

29. Acknowledgments UK (Team MITO) Andrea Sebastain Dr. Laurie Davis Kristen Day Dr. Jignesh Pandya Dr. Ryan Readnower Dr. Andrew Sauerbeck Rabchevsky Lab Dr. Sasha Rabchevsky Dr. Samir Patel Travis Lyttle Scheff Lab Dr. Stephen Scheff Dr. Leslie Gilmer Springer Lab Dr. Joe Springer Dr. Melanie McEwen Travis Lyttle Sponsored by: NIH/NINDS KSCHIRT