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Sullivan, Patrick

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  • 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
  • 19. mtDNA Oxidation
    Non synaptic mitochondria
    • Nucleotide base oxidation
    • 20. chromatography/mass spec
    • 21. levels of oxidized bases higher in SC
    Oxidation 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