Fiskum, Gary

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Fiskum, Gary

  1. 1. MITOCHONDRIAL MECHANISMS OF BRAIN INJURY AND TARGETS FOR NEUROPROTECTION Gary Fiskum University of Maryland School of Medicine Department of Anesthesiology and the Center for Shock, Trauma, and Anesthesiology Research (STAR)
  2. 2. ACKNOWLEDGMENTS <ul><li>Robert E. Rosenthal, M.D. </li></ul><ul><li>Mary McKenna, Ph.D. </li></ul><ul><li>Courtney Robertson, M.D. </li></ul><ul><li>Susanna Scafidi, M.D. </li></ul><ul><li>Andy Saladino, M.D. </li></ul><ul><li>Brian Polster, Ph.D. </li></ul><ul><li>Julie Hazelton, M.S. </li></ul><ul><li>Irina Balan, Ph.D. </li></ul><ul><li>Deborah Stein, M.D. </li></ul><ul><li>Bizhan Aarabi, M.D. </li></ul><ul><li>Tiffany Greco, Ph.D. Student </li></ul><ul><li>NINDS, NICHD, US Army </li></ul>
  3. 3. DISCLOSURE <ul><li>Previously supported by Sigma Tau, SpA, a primary manufacturer and distributor of carnitines. </li></ul><ul><li>Co-PI on a NICHD grant that studies the ability of acetyl-L-carnitine to protect against brain injury, using a rat model of neonatal hypoxic ischemia. </li></ul>
  4. 4. OBJECTIVES <ul><li>Mitochondria roles in metabolic failure, oxidative stress, and apoptosis. </li></ul><ul><li>Neuroprotection via mitochondria-targeted interventions. </li></ul><ul><li>Influence of age, gender and brain cell type on mitochondrial response to injury and to interventions (for discussion). </li></ul>
  5. 5. Elevated Intracellular Ca 2+ Impaired Oxidative Phosphorylation Generation of Reactive Oxygen Species Release of Mitochondrial Apoptogens Mitochondrial Ca 2+ Overload Trauma / Ischemia / Seizures Necrosis Apoptosis Abnormal Signal Transduction Inflammation Inflammation
  6. 6. AIF AIF ATP Pro-Casp3
  7. 7. MITOCHONDRIAL MECHANISMS AND TARGETS FOR INTERVENTION <ul><li>Bioenergetic dysfunction / metabolic failure </li></ul><ul><li>Oxidative stress </li></ul><ul><li>Apoptosis </li></ul>
  8. 8. BIOENERGETIC DYSFUNCTION / METABOLIC FAILURE <ul><li>Permeability transition pore opening </li></ul><ul><ul><li>Direct pore inhibitors (Cyclosporin A, etc.) </li></ul></ul><ul><ul><li>Redox power (pyruvate, Nrf2 activators, etc.) </li></ul></ul><ul><li>Impaired metabolic flux (glycolysis, pyruvate dehydrogenase, NADH catabolism) </li></ul><ul><ul><li>Alternative fuels (ketone bodies, pyruvate, acetyl-L-carnitine) </li></ul></ul><ul><ul><li>Mitochondrial biogenesis (pioglitazone) </li></ul></ul><ul><ul><li>PARP inhibition, NADH precursors (nicotinamide) </li></ul></ul><ul><ul><li>Protect against oxidative stress </li></ul></ul>
  9. 9. REDOX REGULATION OF THE PTP E T C PTP NADH NAD + O 2 H 2 O O 2 - O 2 SH (closed) S-SR (open) H 2 O 2 X X + - ONOO - NO . ATP ADP + P i OH . Fe 2+ H 2 O GPX GSH GSSG TH NADPH NADP NADH + NADP NAD + NADPH + - SOD GR U Malic Enzyme, Isocitrate DH, Glutamate DH Ca 2+ Ca 2+ CyD CsA Trx-SH Trx-S-S NADPH NADP TrxR
  10. 10. KEAP1 Nrf2 SH Sulforaphane S KEAP1 Nrf2 ARE Cytoprotective Genes mRNA Proteins <ul><li>Antixoxidant (NQO1) </li></ul><ul><li>Heme oxygenase 1 </li></ul><ul><li>Glutathione synthesis </li></ul><ul><li>NADPH reduction </li></ul>P ATP ADP PKC/PI3K ? Inhibition of Mitochondrial Permeability Transition ? Protection against oxidative stress and inflammation Nrf2-MEDIATED GENOMIC POST-CONDITIONING AGAINST ISCHEMIA/REPERFUSION INJURY Sulforaphane
  11. 11. Nrf2-MEDIATED GENOMIC POST-CONDITIONING AGAINST BRAIN INJURY Enhancing expression of Nrf2-driven genes protects the blood brain barrier after brain injury. Zhao et al., J Neurosci. 2007 27:10240-8. Role of the Nrf2-ARE pathway in early brain injury after experimental subarachnoid hemorrhage. Chen et al., J Neurosci Res. 2011 89:515-23 The role of Nrf2 signaling in the regulation of antioxidants and detoxifying enzymes after traumatic brain injury in rats and mice. Hong et al., Acta Pharmacol Sin. 2010 31:1421-30. Sulforaphane improves cognitive function administered following traumatic brain injury. Dash et al., Neurosci Lett. 2009 460:103-7.
  12. 12. HYPOTHESIS Sulforaphane post-treatment protects against short-term neuronal death and neurologic impairment in a clinically translational, canine cardiac arrest and resuscitation model.
  13. 13. SULFORAPHANE SIGNIFICANTLY REDUCES NEUROLOGIC INJURY AND HIPPOCAMPAL NEURONAL DEATH AFTER CARDIAC ARREST - SFP +SFP - SFP +SFP
  14. 14. HYPOTHESIS Nrf2 activation increases mitochondrial antioxidant related proteins, thereby increasing resistance to permeability transition pore opening.
  15. 15. SULFORAPANE TREATMENT INCREASES MITOCHONDRIAL GLUTATHIONE AND ANTIOXIDANT PROTEINS
  16. 16. SULFORAPHANE TREATMENT INCREASES MITOCHONDRIAL RESISTANCE TO PEROXIDE-INDUCED PTP OPENING AND NAD(P)H OXIDATION
  17. 17. BIOENERGETIC DYSFUNCTION / METABOLIC FAILURE <ul><li>Permeability transition pore opening </li></ul><ul><ul><li>Direct pore inhibitors (Cyclosporin A, etc.) </li></ul></ul><ul><ul><li>Redox power (pyruvate, Nrf2 activators, etc.) </li></ul></ul><ul><li>Impaired metabolic flux (glycolysis, pyruvate dehydrogenase, NADH catabolism) </li></ul><ul><ul><li>Alternative fuels (ketone bodies, pyruvate, acetyl-L-carnitine) </li></ul></ul><ul><ul><li>Mitochondrial biogenesis (resveratrol, </li></ul></ul><ul><ul><li>PARP inhibition, NADH precursors (nicotinamide) </li></ul></ul><ul><ul><li>Protect against oxidative stress </li></ul></ul>
  18. 18. ACETYL-L-CARNITINE Present at high mid-micromolar concentrations in tissue and at low micromolar concentrations in serum. Can enter mitochondria through carnitine translocase and react with CoA to form acetyl-CoA plus free carnitine.
  19. 19. DIRECT METABOLIC PROTECTION BY ALCAR AS AN ALTERNATIVE FUEL ALCAR Ketone bodies
  20. 20. ALCAR NEUROPROTECTION <ul><li>Neuroprotection in canine cardiac arrest (CA) model </li></ul><ul><li>Neuroprotection in rat MCAO stroke models </li></ul><ul><li>Reduces lactate/pyruvate </li></ul><ul><li>Reduces oxidative protein modifications </li></ul><ul><li>May act as an alternative fuel to bypass impairment of oxidative energy metabolism at pyruvate dehydrogenase </li></ul><ul><li>May be particularly effective in pediatric brain injury </li></ul>
  21. 21. ALCAR PROTECTION AGAINST SCI Patel et al., J Neurochem, 2010
  22. 22. HYPOTHESIS ALCAR post-treatment protects against cell death and neurologic impairment in a rat model of pediatric brain injury.
  23. 23. ALCAR IMPROVES BEAM WALKING AND NOVEL OBJECT RECOGNITION AT 3-7 DAYS AFTER CCI IN PND 21 RATS Scafidi et al., Develop Neurosci, 2010 ALCAR 100 mg/kg ip at 1, 4, 12, and 24 hr after injury
  24. 24. ALCAR REDUCES LESION VOLUME AT 7 DAYS AFTER CCI IN PND 21 RATS Scafidi et al., Develop Neurosci, 2010
  25. 25. ALCAR ACETATE IS METABOLISED BY PND 21 RAT BRAIN TO OXIDATIVE ENERGY METABOLITES AND GABA Scafidi et al., J Neurochem, 2010 2- 13 C ALCAR ip at 100 mg/kg then NMR of frozen brain extracts at 15, 60, and 120 min
  26. 26. ALCAR, MITOCHONDRIAL BIOGENESIS AND Nrf2 Acetyl-L-carnitine feeding to unloaded rats triggers in soleus muscle the coordinated expression of genes involved in mitochondrial biogenesis. Cassano et al., Biochim Biophys Acta. 2006 1757:1421-8. Combined R-alpha-lipoic acid and acetyl-L-carnitine exerts efficient preventative effects in a cellular model of Parkinson's disease. Zhang et al., J Cell Mol Med. 2010 14:215-25. Acetyl-L-carnitine-mediated neuroprotection during hypoxia is attributed to ERK1/2-Nrf2-regulated mitochondrial biosynthesis Hota et al., Hippocampus. 2011 epub May 3. .
  27. 27. OXIDATIVE STRESS <ul><li>Mitochondrial Generation and Targets of Action </li></ul><ul><ul><li>Excessive mitochondrial production of ROS </li></ul></ul><ul><ul><li>Impaired detoxification of ROS </li></ul></ul><ul><ul><li>Oxidation of critical proteins (e.g., PDHC), lipids (e.g., cardiolipin), and DNA/RNA </li></ul></ul><ul><ul><li>Mitochondrial fission and promotion of apoptosis </li></ul></ul><ul><li>Mitochondria-Associated Interventions </li></ul><ul><ul><li>Avoid excessive oxygen beyond what is needed for oxidative energy metabolism </li></ul></ul><ul><ul><li>Mild uncoupling (proton ionophores, ATP-sensitive K channel openers) </li></ul></ul><ul><ul><li>Mitochondria-targeted antioxidants (MitoQ, triphenylphosphonium- and hemi-gramicidin S-moieties ) </li></ul></ul><ul><ul><li>Other antioxidants (glutathione ester, Nrf2 activators, etc.) </li></ul></ul><ul><ul><li>Suppress inflammation </li></ul></ul><ul><ul><li>Genomic post-conditioning against oxidative stress and inflammation </li></ul></ul>
  28. 28. Circulation 2010;122;S768-S786 Kronick Silvers, Arno L. Zaritsky, Raina Merchant, Terry L. Vanden Hoek and Steven L. Geocadin, Janice L. Zimmerman, Michael Donnino, Andrea Gabrielli, Scott M. Mary Ann Peberdy, Clifton W. Callaway, Robert W. Neumar, Romergryko G. for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Part 9: PostCardiac Arrest Care: 2010 American Heart Association Guidelines Animal data suggests that ventilations with 100% oxygen (generating PaO2 >350 mm Hg at 15 to 60 minutes after ROSC) increase brain lipid peroxidation, increase metabolic dysfunctions, increase neurological degeneration, and worsen short-term functional outcome when compared with ventilation with room air or an inspired oxygen fraction titrated to a pulse oximeter reading between 94% and 96%. 82–87 Provided appropriate equipment is available, once ROSC is achieved, adjust the FIO2 to the minimum concentration needed to achieve arterial oxyhemoglobin saturation >94%, with the goal of avoiding hyperoxia while ensuring adequate oxygen delivery.
  29. 29. OXIDATIVE STRESS <ul><li>Mitochondrial Generation and Targets of Action </li></ul><ul><ul><li>Excessive mitochondrial production of ROS </li></ul></ul><ul><ul><li>Impaired detoxification of ROS </li></ul></ul><ul><ul><li>Oxidation of critical proteins (e.g., PDHC), lipids (e.g., cardiolipin), and DNA/RNA </li></ul></ul><ul><ul><li>Mitochondrial fission and promotion of apoptosis </li></ul></ul><ul><li>Mitochondria-Associated Interventions </li></ul><ul><ul><li>Avoid excessive oxygen beyond what is needed for oxidative energy metabolism </li></ul></ul><ul><ul><li>Mild uncoupling (proton ionophores, ATP-sensitive K channel openers) </li></ul></ul><ul><ul><li>Mitochondria-targeted antioxidants (MitoQ, triphenylphosphonium- and hemi-gramicidin S-moieties ) </li></ul></ul><ul><ul><li>Other antioxidants (glutathione ester, Nrf2 activators, etc.) </li></ul></ul><ul><ul><li>Suppress inflammation </li></ul></ul><ul><ul><li>Genomic post-conditioning against oxidative stress and inflammation </li></ul></ul>
  30. 30. HYPOTHESIS In vitro experiments help elucidate mechanisms by which oxidative stress and other factors impair or improve mitochondrial bioenergetics.
  31. 31. 50 75 100 125 150 0 20 40 60 80 120 140 100 O 2 consumption (% baseline) Time (min) 175 100 Glu + DETA-NO Control DETA-NO (200  M) Glutamate (100  M) NITRIC OXIDE AND GLUTAMATE SYNERGISTICALLY IMPAIR NEURONAL RESPIRATION pyruvate DETA-NO ± Glut FCCP MK801/ CNQX
  32. 32. -50 -25 0 25 50 0 20 40 60 80 120 140 100 O 2 consumption (%  baseline) Time (min) 75 100 PEROXYNITRITE DECOMPOSITION CATALYST FeTMPyP PROTECTS AGAINST RESPIRATORY INHIBITION control glutamate control or glu FCCP pyruvate MK801+ CNQX+Nif control glutamate control or glu or glu+NO FCCP pyruvate MK801+ CNQX+Nif glutamate + DETA-NO control glutamate control or glu or glu+NO FCCP pyruvate MK801+ CNQX+Nif glutamate + DETA-NO glutamate + DETA-NO + FeTM-PyP
  33. 33. OXIDATIVE STRESS <ul><li>Mitochondrial Generation and Targets of Action </li></ul><ul><ul><li>Excessive mitochondrial production of ROS </li></ul></ul><ul><ul><li>Impaired detoxification of ROS </li></ul></ul><ul><ul><li>Oxidation of critical proteins (e.g., PDHC), lipids (e.g., cardiolipin), and DNA/RNA </li></ul></ul><ul><ul><li>Mitochondrial fission and promotion of apoptosis </li></ul></ul><ul><li>Mitochondria-Associated Interventions </li></ul><ul><ul><li>Avoid excessive oxygen beyond what is needed for oxidative energy metabolism </li></ul></ul><ul><ul><li>Mild uncoupling (proton ionophores, ATP-sensitive K channel openers) </li></ul></ul><ul><ul><li>Mitochondria-targeted antioxidants (MitoQ, triphenylphosphonium- and hemi-gramicidin S-moieties ) </li></ul></ul><ul><ul><li>Other antioxidants (glutathione ester, Nrf2 activators, etc.) </li></ul></ul><ul><ul><li>Suppress inflammation </li></ul></ul><ul><ul><li>Genomic post-conditioning against oxidative stress and inflammation </li></ul></ul>
  34. 34. TRANSLATIONAL RESEARCH ON MITOCHONDRIA AND TBI <ul><li>Combined human patient data </li></ul><ul><ul><li>Microdialysis </li></ul></ul><ul><ul><li>MRI/MRS </li></ul></ul><ul><ul><li>CSF and serum biomarkers </li></ul></ul><ul><li>Human brain mitochondrial pathology </li></ul><ul><ul><li>Histology and electron microscopy </li></ul></ul><ul><ul><li>Neurochemistry </li></ul></ul><ul><li>Human clinical trials targeting mitochondria </li></ul><ul><ul><li>Cyclosporin A </li></ul></ul><ul><ul><li>ALCAR </li></ul></ul>

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