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Lab Update: Jan 08
Jonathon M. Sullivan MD, PhDAssociate Director for Basic ResearchDepartment of Emergency Medicine
Wayne State University
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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.
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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.
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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.
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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
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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.
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How Global Brain Ischemia Happens to Nice People:
TRANSIENT Global Brain Ischemia PERMANENT Global Brain Ischemia
(We focus on this one.)
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From a Permanent Resident of Ratship Manor
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How Does This Happen?
• Ischemia cocks the hammer.
• Reperfusion pulls the trigger.
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Bad Things Happen During Ischemia
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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!
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Free Radical Damage
…And his trusty mount, Firestorm the Wonder Horse
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Totally Rad
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Free Radicals: The Horror Continues
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Free Rads: Son of Superoxide
O2- + NO → ONO2-
Nitrosylates everything
O, NOO!
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A Free Radical Bestiary•O2-, superoxide anion From mitochondria. Not very
reactive, but initiates chain rxs
H2O2, hydrogen peroxide From dismutation of •O2-. Can diffuse across membranes. Bastard.
•OH, hydroxyl radical Mean SOB. Like Cheney, will attack almost anything.
ROOH, organic hydroperoxide Zombie molecules.
RO•, alkoxy and ROO•, peroxy radicals
More zombie molecules. Lipid forms are particularly ghoulish.
OONO-, peroxynitrite Evil. Just evil.
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Calpain…
…and his trusty blade, Fubaring, sword of mayhem.
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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
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How Calpain Gets His Freak On
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Kalpain’s Krazy Killing Karnival
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Protein Synthesis Inhibition…
…training his PERKy bow on your hapless ribosomes.
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Post-ischemic Suppression ofHippocampal Protein Synthesis
Thilman et al., 1986
Nonischemic controls 5 min I/ 30 min R
5 min I/ 90 min R 5 min I/ 12 hr R
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Let’s Review, Shall We?
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The Protein Factory(Recently Moved to China)
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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.
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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!
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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.
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What is “The Long March?”
• 500 MORE Trivia points!
• Keenan can’t play.
??
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Dear Leader
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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.
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Ribo Robots Rock
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The Initiation Complex: Overview
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60s
80s Initiation Complex
eIF-5, eIF-4D
eIF-2•GDP
eIF-2
Ternary Complex
eIF-2BGTP
GDP
eIF-3
eIF-4C
40s
43s
40s
Met-tRNA
ATP
ADP
nm7G AUG (A)
messenger RNA
40s
eIF-4E
eIF-4A, eIF-4G eIF-4Bnm7G AUG (A)
nm7G AUG (A)
nm7G AUG (A)
eIF-2(P)blocksthis step
TRANSLATION INITIATION:GEEKVIEW
“The 4 Side” “The 2 Side”
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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).
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So…who’s the kinase?
• The usual suspects: – GCN2
• Nope
– PRK• Nope
– HRK• Nope
– PERK• Bingo
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PERKy isn’t always cute
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More Geek-o-Vision
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Apoptosis…
…and his unbalanced suicidal depression.
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A Little Cell-Death Humor
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How Caspases Can Screw Up Your Day
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Extrinsic Apoptosis—Somebody Talks You Into Killing Yourself
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Intrinsic Apoptosis—You Do It All By Yourself.
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Head Crash
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ISCHEMIA
ATP
Ca2+ influx
DepolarizationExcessGlutamate
Lipolysis
Free fatty acids Membrane damage
REPERFUSION
Reactive Oxygen Species (T)
Mitochondrial stress Calpain proteolysis
O2
Cytochrome crelease from
Mitochondria (T)
APOPTOSIS
NEURONAL DEATH (T)
DNAdamage
ProteinSynthesis,
AlteredmRNA selection
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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:
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SINGLE-DRUG THERAPY WILL NEVER WORK
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…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.
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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.
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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.
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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
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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
» ?
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Galocyanin-stainedAutoradiographs
ImmunostainedeIF2(P)
Control
10I- 90R
10I- 90R +Insulin20 U/kg
25 m 50 m
Insulin Rescues Post-ischemic Protein Synthesis
Sullivan et al., 1999
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Insulin Blocks Cytochrome c Release
SHAM (Non-Isch) 8 min Isch + 240 min Rep
240 min Rep + Insulin 2U/kg
Sullivan, Sanderson, Kumar 2003
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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.
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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.
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MTA-BRAIN: Economy Class
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Anybody Know a Good RecipeFor Chilled Rat?
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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.
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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
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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!
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The Refrige-a-RAT-or
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The Refrige-a-RAT-or
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Come on over and play!
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Questions?
??