Lab Update: Jan 08

Jonathon M. Sullivan MD, PhDAssociate Director for Basic ResearchDepartment of Emergency Medicine

Wayne State University

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.

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.

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.

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

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.

How Global Brain Ischemia Happens to Nice People:

TRANSIENT Global Brain Ischemia PERMANENT Global Brain Ischemia

(We focus on this one.)

From a Permanent Resident of Ratship Manor

How Does This Happen?

• Ischemia cocks the hammer.

• Reperfusion pulls the trigger.

Bad Things Happen During Ischemia

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!

Free Radical Damage

…And his trusty mount, Firestorm the Wonder Horse

Totally Rad

Free Radicals: The Horror Continues

Free Rads: Son of Superoxide

O2- + NO → ONO2-

Nitrosylates everything

O, NOO!

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.

Calpain…

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

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

How Calpain Gets His Freak On

Kalpain’s Krazy Killing Karnival

Protein Synthesis Inhibition…

…training his PERKy bow on your hapless ribosomes.

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

Let’s Review, Shall We?

The Protein Factory(Recently Moved to China)

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.

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!

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.

What is “The Long March?”

• 500 MORE Trivia points!

• Keenan can’t play.

??

Dear Leader

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.

Ribo Robots Rock

The Initiation Complex: Overview

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”

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).

So…who’s the kinase?

• The usual suspects: – GCN2

• Nope

– PRK• Nope

– HRK• Nope

– PERK• Bingo

PERKy isn’t always cute

More Geek-o-Vision

Apoptosis…

…and his unbalanced suicidal depression.

A Little Cell-Death Humor

How Caspases Can Screw Up Your Day

Extrinsic Apoptosis—Somebody Talks You Into Killing Yourself

Intrinsic Apoptosis—You Do It All By Yourself.

Head Crash

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

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:

SINGLE-DRUG THERAPY WILL NEVER WORK

…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.

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.

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.

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

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

» ?

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

Insulin Blocks Cytochrome c Release

SHAM (Non-Isch) 8 min Isch + 240 min Rep

240 min Rep + Insulin 2U/kg

Sullivan, Sanderson, Kumar 2003

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.

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.

MTA-BRAIN: Economy Class

Anybody Know a Good RecipeFor Chilled Rat?

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.

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

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!

The Refrige-a-RAT-or

The Refrige-a-RAT-or

Come on over and play!

Questions?

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