Sullivan, Patrick


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Sullivan, Patrick

  1. 1. MITOCHONDRIAL DYSFUNCTION IN ACUTE CNS INJURY <br />Patrick Sullivan, Ph.D.<br />Associate Professor of Anatomy & Neurobiology<br />Endowed Chair of the Spinal Cord and Brain Injury Research Center<br />University of Kentucky College of Medicine<br />
  2. 2. Mitochondrial Dysfunction following TBI/SCI<br />How does it manifest?<br />Time course of dysfunction.<br />How does it contribute to cell death?<br />
  3. 3. “Powerhouse of the cell”<br /><br />
  4. 4. Mitochondria: Death Switch of the Cell?<br />Ca2+<br />Glutamate<br />Death Triggers<br />Bax<br />Oxidants<br />Caspases<br />Cyto C<br />Cyto C<br />Caspase 9<br />Apaf-1<br />dATP<br />ATP<br /><br />Downstream<br />Caspases<br />ROS<br />NECROSIS<br />APOPTOSIS<br />MPTP<br />Smac/Diablo<br />AIF<br />
  5. 5. Electron Transport System (ETS)<br />Creates membrane potential (ΔΨ) across the inner membrane<br /> ATP synthesis<br />Ca2+ cycling<br />
  6. 6. Mitochondrial Calcium Transport<br />Ca2+<br />2H+<br />Ca2+<br />Driven by <br />2Na+<br />Ca2+<br />
  7. 7. Ca2+<br />PTP<br />2H+<br />Ca2+<br />2Na+<br />Mitochondrial Calcium Transport and mPT<br />Driven by <br />
  8. 8. Mitochondrial Respiration<br />RCR = State III/State IV<br />Healthy >5<br />
  9. 9. Loss of Mitochondrial Bioenergetics <br />following TBI<br />
  10. 10. Acute loss of Mitochondrial Function depends on Injury Severity<br />Mean ± SD<br />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.<br />
  11. 11. Overview of Peroxynitrite Formation/Reactions in Mitochondria<br />Radi et al., 2002<br />
  12. 12. Mitochondrial Oxidative Damage following TBI<br />
  13. 13. Loss of Mitochondrial Enzyme Activities 3 hrs post-injury<br />
  14. 14. Mitochondria Dysfunction can be Targeted up to 24 hrs post-injury<br />
  15. 15. Mitochondrial Ca2+ Load Following TBI<br />
  16. 16. Post-traumatic Time Course Changes in Cortical Mitochondrial Calcium Buffering Capacity<br />Isolated then challenged<br />Snapshot of in vivo levels<br />
  17. 17. Motor Ctx<br />Motor Ctx<br />SC Ventral horn<br />In Situ ROS Formation<br /> Basal level of ROS<br /> Motor Neurons<br />- Dihidroethidium (DHET)<br /> - 3hr prior<br /> - converted to ethidium by O2-•<br />140%↑<br />J Comp Neurol (2004) 474:524-534<br />
  18. 18. Mitochondrial Lipid Peroxidation<br /> - non synaptic mitochondria<br /> - 4 hydroxynonenal (HNE)<br />40%↑<br />J Comp Neurol (2004) 474:524-534<br />
  19. 19. mtDNA Oxidation<br />Non synaptic mitochondria<br /><ul><li> Nucleotide base oxidation
  20. 20. chromatography/mass spec
  21. 21. levels of oxidized bases higher in SC</li></ul>Oxidation of mitochondrial DNA<br />Immunoreactivity for8-hydroxyguanosine<br /> - specific marker for oxidative damage to DNA<br /> - serial confocal micrographs<br />*cyclophilin D mRNA was significantly increased in the SC (1.58-fold; p=0.014)<br />J Comp Neurol (2004) 474:524-534<br />
  22. 22. Loss of Mitochondrial Homeostasis following SCI: Role of ROS<br />
  23. 23. Oxidative Damage following SCI<br />Mitochondrial<br />Fraction<br />Cytosolic<br />Fraction<br />
  24. 24. Mitochondrial Oxidative Damage to ETS and loss of <br />Enzyme Activities following SCI<br />
  25. 25. Mitochondrial Bioenergetics after<br /> with and without Post-injury Treatments<br />
  26. 26. Synaptic Mitochondrial <br />Respiratory Control Ratio (RCR)<br />State III<br />State IV<br />RCR > 5.0<br /> RCR decreased significantly <br /> following SCI.<br />15 and 30 min post-SCI treatment with <br /> DNP preserved the mitochondrial <br /> integrity. However tempol was <br /> ineffective. <br />1 hr post-injury treatment had no <br /> effect on RCR.<br />RCR =<br />Vehicle<br />DNP<br />TEMPOL<br />*p < 0.05compared to sham <br />#p < 0.05 compared to vehicle <br />
  27. 27. Sham<br />Vehicle<br />DNP<br />TEMPOL<br />Synaptic Mitochondrial oxidative markers:<br /> Post-SCI Treatment <br />15 min <br />1 hr <br />30 min <br />*<br />*<br />*<br />#<br />#<br />#<br />#<br />*p<0.05 compared to sham<br />#p<0.05 compared to Vehicle<br />Protein Carbonyls increased significantly following contusion SCI.<br /> Treatment with DNP and TEMPOL (15 and 30 min post-SCI) reduced the <br /> protein carbonyls to normality.<br />
  28. 28. Summary<br />Mitochondrial dysfunction occurs rapidly and is progressive over 24 hrs post-injury.<br />Mitochondrial oxidative damage coincides with loss of bioenergetics.<br />Mitochondrial Calcium overload plays a pivotal role leading to mPT and cell death.<br />Mitochondrial Dysfunction may be amendable to treatment in first 24 hrs<br />
  29. 29. Acknowledgments<br />UK (Team MITO)<br />Andrea Sebastain<br />Dr. Laurie Davis<br />Kristen Day<br />Dr. Jignesh Pandya<br />Dr. Ryan Readnower<br />Dr. Andrew Sauerbeck<br />Rabchevsky Lab<br />Dr. Sasha Rabchevsky<br />Dr. Samir Patel<br />Travis Lyttle<br />Scheff Lab<br />Dr. Stephen Scheff<br />Dr. Leslie Gilmer<br />Springer Lab<br />Dr. Joe Springer<br />Dr. Melanie McEwen<br />Travis Lyttle<br />Sponsored by:<br />NIH/NINDS<br />KSCHIRT<br />