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

3. 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.

4. 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.

5. 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.

6. 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

7. 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.

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

9. From a Permanent Resident of Ratship Manor

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

11. Bad Things Happen During Ischemia

12. 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!

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

14. Totally Rad

15. Free Radicals: The Horror Continues

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

17. A Free Radical Bestiary O2-, superoxide anionFrom mitochondria. Not very reactive, but initiates chain rxs H2O2, hydrogen peroxideFrom dismutation of O2-. Can diffuse across membranes. Bastard. OH, hydroxyl radicalMean SOB. Like Cheney, will attack almost anything. ROOH, organic hydroperoxideZombie molecules. RO, alkoxy and ROO, peroxy radicals More zombie molecules. Lipid forms are particularly ghoulish. OONO-, peroxynitriteEvil. Just evil.

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

19. 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

20. How Calpain Gets His Freak On

21. Kalpain’s Krazy Killing Karnival

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

23. Post-ischemic Suppression of Hippocampal Protein Synthesis Thilman et al., 1986 Nonischemic controls5 min I/ 30 min R 5 min I/ 90 min R5 min I/ 12 hr R

24. Let’s Review, Shall We?

25. The Protein Factory (Recently Moved to China)

26. 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.

27. 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 undamaged during 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!

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

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

30. Dear Leader

31. 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.

32. Ribo Robots Rock

33. The Initiation Complex: Overview

34. 60s 80s Initiation Complex eIF-5, eIF-4D eIF-2GDP eIF-2 Ternary Complex eIF-2B GTP GDP eIF-3 eIF-4C 40s 43s 40s Met-tRNA ATP ADP n m7Gm7G AUG (A) messenger RNA 40s eIF-4E eIF-4A, eIF-4G eIF-4B n m7Gm7G AUG (A) n m7Gm7G AUG (A) n m7Gm7G AUG (A) eIF-2  (P) blocks this step TRANSLATION INITIATION: GEEKVIEW “The 4 Side” “The 2 Side”

35. 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).

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

37. PERKy isn’t always cute

38. More Geek-o-Vision

39. Apoptosis… …and his unbalanced suicidal depression.

40. A Little Cell-Death Humor

41. How Caspases Can Screw Up Your Day

42. Extrinsic Apoptosis—Somebody Talks You Into Killing Yourself

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

44. Head Crash

45. ISCHEMIA ATP Ca 2+ influx Depolarization Excess Glutamate Lipolysis Free fatty acids Membrane damage REPERFUSION Reactive Oxygen Species (T) Mitochondrial stressCalpain proteolysis O2O2 Cytochrome c release from Mitochondria (T) APOPTOSIS NEURONAL DEATH (T) DNA damage Protein Synthesis, Altered mRNA selection

46. 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:

47. SINGLE-DRUG THERAPY WILL NEVER WORK

48. …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.

49. 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 19 th century.

50. 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.

51. 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

52. Hypothermia is Now the Foundation Let’s build on that. Pick our targets carefully. Anybody? –You already know: Excitotoxicity AND The Four Horsemen! –Apoptosis »Growth factors, caspase inhibitors –Free Radicals »PBN and other “spin traps,” NAC –Calpain »Calpain inhibitors. (Duh!) –Protein Synthesis Inhibition »?»?

53. Galocyanin-stained Autoradiographs Immunostained eIF2  (P) Control 10I- 90R 10I- 90R + Insulin 20 U/kg 25  m 50  m Insulin Rescues Post-ischemic Protein Synthesis Sullivan et al., 1999

54. Insulin Blocks Cytochrome c Release SHAM (Non-Isch) 8 min Isch + 240 min Rep 240 min Rep + Insulin 2U/kg Sullivan, Sanderson, Kumar 2003

55. MTA-BRAIN Multiply-Targeted Approach to Brain Resuscitation and Ischemic Neuroprotection Hypothermia 33-34 deg C – Apoptosis, ROS, Excitotoxicity, Calpain IGF-1 or Insulin – Apoptosis, Protein Synthesis Inhibition PBN or NAC – Reactive Oxygen Species Tat-NRB29c – Glutamate toxicity, ROS, ? Apoptosis Submitted as R21 proposal to NIH-NINDS. Cross your fingers.

56. MTA-BRAIN-Economy Class Submitted to WSU for Clinical Translational Science Award Hypothermia + IGF-1 Unlike entire MTA-BRAIN, positive animal results could be quickly translated to clinical study.

57. MTA-BRAIN: Economy Class

58. Anybody Know a Good Recipe For Chilled Rat?

59. Current State-of-the-Art Colbourne and Auer—Canadian Dudes, eh. Maintains hypothermia in awake, freely-moving rodents. Brain thermocouple telemetry (no longer manufactured) monitored by computer. Computer drives a system misters, fans, and heat lamps to maintain brain temperature. System cost: >$150,000 plus maintenance.

60. The Refrige-a-RAT-or Sullivan, Lagina, Freeman If it works, this system will induce selective brain hypothermia in the awake, freely moving rodent Estimated cost (excluding development costs): approx $3000 plus maintenance. Based on Peltier Technology

61. Peltier Effect Described by a French Dude. But it’s cool. Two metals of different thermopower. Drive a DC current across the interface. Creates a thermal gradient: hot side and a cold side. Used for cooling CPUs. Our idea: cool the carotids!

62. The Refrige-a-RAT-or

65. Come on over and play!

66. Questions? ? ?