Advancements in Cerebral Resuscitation Research: Insights from Wayne State University

1. Lab Update: Jan 08 Jonathon M. Sullivan MD, PhD Associate Director for Basic Research Department of Emergency Medicine Wayne State University

2. Hey! Where’s Everybody Going? • It won’t be that bad, I promise. • Broad brushstrokes, with a minimum of icky geekishness • No test. • We’re headed for translational stuff: bench to bedside. • If you’re not careful, you might learn something.

3. Your Basic Science Lab • For more than twenty years, this lab has been focused on the problem of cerebral resuscitation after cardiac arrest. • Dozens of papers. • Millions in extramural funding. • Produced more than a dozen PhDs, including 3 EM MD-PhDs. • Edward C. Thomas Endowed Chair.

4. Our Lab…is a Very, Very, Very Fine Lab • Gary Krause, MD, MS – Edward C. Thomas Endowed Chair, Associate Chair for Research, Director for Basic Science. • Jonathon Sullivan MD, PhD – Associate Director for Basic Science. • Rita Kumar, PhD – Assistant Professor • Anthony Lagina, MD – Assistant Professor • Thomas Sanderson, PhD – Post-doc. • Michael DeO’Gracia – Research Assistant • Jeffrey Groom – assistant, part-time scut dog.

5. Toys…we has them. • Fluorescence microscope • Two rat surgery/anesthesia stations • Downdraft table • Isoflurane vaporizers • Rat laryngoscope • Isothermal blankets • Art line, ekg, etc. • Electron microscope • Croystatic microtome • PCR • Cell culture facility • Spectrophotometer • Full range of electrophoresis equipment • Ultracentrifuge • i-Pod-compatible Boom Box

6. Our Problem: Brain Ischemia Sux • After > 5 min transient global brain ischemia, selectively vulnerable neurons go on to die. • Unfortunately, these happen to be the neurons you think and remember with.

7. How Global Brain Ischemia Happens to Nice People: TRANSIENT Global Brain Ischemia PERMANENT Global Brain Ischemia (We focus on this one.)

8. From a Permanent Resident of Ratship Manor

9. How Does This Happen? • Ischemia cocks the hammer. • Reperfusion pulls the trigger.

10. Bad Things Happen During Ischemia

11. Worse Things Happen During Reperfusion • The Four Horsemen of the Brainocalypse: • Free Radical Damage • Inhibition of Protein Synthesis • Calpain-Mediated Proteolysis • Apoptosis Who drew this? 500 trivia points!

12. Free Radical Damage …And his trusty mount, Firestorm the Wonder Horse

13. Totally Rad

14. Free Radicals: The Horror Continues

15. Free Rads: Son of Superoxide O, NOO! O2- + NO → ONO2- Nitrosylates everything

16. A Free Radical Bestiary

17. Calpain… …and his trusty blade, Fubaring, sword of mayhem.

18. Who is this Calpain Dude? • Usually a nice guy. • Neutral cysteine proteases • Two flavors: • Calpain I (μ-calpain): neurons • Calpain II (m-calpain): glia • Cytoskeletal remodeling • Synaptic plasticity • Neurite outgrowth • Activated by calcium flux

19. How Calpain Gets His Freak On

20. Kalpain’s Krazy Killing Karnival

21. Protein Synthesis Inhibition… …training his PERKy bow on your hapless ribosomes.

22. Post-ischemic Suppression ofHippocampal Protein Synthesis Nonischemic controls 5 min I/ 30 min R 5 min I/ 90 min R 5 min I/ 12 hr R Thilman et al., 1986

23. Let’s Review, Shall We?

24. The Protein Factory(Recently Moved to China)

25. A Target-Rich Environment • All tuckered out. • Not enough ATP during reperfusion • DNA badness. • Free Rads or nucleases go all all Braveheart on the DNA. • Denaturation or other physical/chemical changes • Transcriptional badness. • RNA polymerase is confused, damaged, drunk or dead. • mRNA badness. • mRNA can't be processed correctly, or • gets damaged, or • fails to get out of the nucleus. • Translational badness. Ribosome can't translate the mRNA into protein.

26. The Long March: Nucleus to Ribosome • Brain ATP levels return to near-normal levels during early reperfusion (early 80s). • Brain nuclear and mitochondrial DNA is undamagedduring early reperfusion. (Your lab, 1991 and 1992) • The transcriptional machinery is intact (early 90s). • mRNA makes it out of the brain intact (Your lab, early 90s). • “Washed" or purified ribosomes isolated from brains after an ischemic insult could still translate mRNA to protein in an in vitro system (Your lab, mid-90s). • And that’s it!

27. Clearly, this is Emergency Medicine Research He’s losing it. Bummer. @#$%&*!! I know he’s sick but I can’t find anything wrong! Maybe he just wants a work excuse, man.

28. What is “The Long March?” • 500 MORE Trivia points! • Keenan can’t play. ?

29. Dear Leader

30. Wait a Minute! • What about dirty ribosomes? • Experiments demonstrated that inhibition of translation during reperfusion was at the level of translation initiation. • Translation initiation is a complex process, involving the assembly of over 140 proteins, ribonucleotides, and ribonucleoproteins in a translation initation complex.

31. Ribo Robots Rock

32. The Initiation Complex: Overview

33. TRANSLATION INITIATION: GEEKVIEW 40s eIF-3 40s “The 4 Side” “The 2 Side” eIF-4C Met-tRNA messenger RNA Ternary Complex 40s (A) m7G AUG n eIF-4E 43s eIF-2 eIF-4B eIF-4A, eIF-4G (A) m7G AUG n GDP ATP ADP GTP (A) eIF-2B m7G AUG n eIF-2(P) blocks this step eIF-5, eIF-4D eIF-2•GDP 60s (A) m7G AUG n 80s Initiation Complex

34. Short answer: eIF2α (P) • eIF2α gets phosphorylated during early brain reperfusion (DeGracia, 1998) • eIF2α(P) maps to selectively vulnerable neurons (DeGracia and Sullivan, 1999) • Dephosphorylation of eIF2α during early reperfusion restores protein synthesis (Sullivan, 1999).

35. So…who’s the kinase? • The usual suspects: • GCN2 • Nope • PRK • Nope • HRK • Nope • PERK • Bingo

36. PERKy isn’t always cute

37. More Geek-o-Vision

38. Apoptosis… …and his unbalanced suicidal depression.

39. A Little Cell-Death Humor

40. How Caspases Can Screw Up Your Day

41. Extrinsic Apoptosis—Somebody Talks You Into Killing Yourself

42. Intrinsic Apoptosis—You Do It All By Yourself.

43. Head Crash

44. Excess Glutamate Depolarization Protein Synthesis, Altered mRNA selection ISCHEMIA ATP Ca2+ influx Mitochondrial stress Lipolysis Calpain proteolysis Free fatty acids Membrane damage REPERFUSION O2 Reactive Oxygen Species (T) DNA damage Cytochrome c release from Mitochondria (T) APOPTOSIS NEURONAL DEATH (T)

45. Several Conclusions Follow • The earliest events (during ischemia) are probably not preventable. • Even so, treatment must begin early to be effective (upon ROSC), to prevent propagation and intercalation of pathologic processes. • Target Rich Environment. • Most important:

46. SINGLE-DRUG THERAPY WILL NEVER WORK

47. …Never Has, Never Will • Calcium channel blockers: • Fail. • Glutamate receptor antagonists: • Bupkes. • Glutamate release inhibition • Loser. • Free radical scavengers: • Snake-eyes. • Any number of Also-Rans: • Also ran.

48. One Therapy Stands Alone • Only one intervention has been shown to improve neurologic outcome and survival after cardiac arrest. What is it? • Correctomundo! • Took us long enough: therapeutic hypothermia was first used in the 19th century.

49. Brain Freeze • First deployed for neuroprotection in the 1940s. • Eventually abandoned • Target temps much lower than today’s • V-fib • Coagulopathies • Sepsis • Technical issues • Eventually, people got tired of this crap.

50. Hypothermia is NOT Single Therapy • Hypothermia has been shown to: • Improve cell survival signaling processes (Akt, PKC, etc) • Inhibit cytochrome c release from mitochondria • Decrease free radical production and propagation • Decrease lipolysis • Effect salutary changes in glutamate receptor composition and signaling