Unraveling Survival Strategies

Valina L. Dawson, Ph.D.Johns Hopkins Institute for Cell

Engineering

Too Many Cell Types in the Nervous System?

Cell Type Estimates:

Outside the Brain ~300

Nervous System ~1,500-5,000

C.F. Stevens Curr. Biol. 8:708

Two types of cell death?!

(apoptosis or necrosis)

Ordering the Cell Death PathwayCauseTrigger

Contributor Consequence

Cell Death

Identification of Novel Neuronal Survival Pathways

Strategies

Preconditioning

TRENDS in Neurosciences 26(5): 248-254, 2003

Preconditioning is a phenomenon in which “any stimulus capable of causing injury to a tissue or organ can, when applied close to (but below) the threshold of damage, activate endogenous protective mechanisms, thus potentially lessening the impact of subsequent, more severe stimuli.”

Preconditioning and TolerancePreconditioning Stimuli: ischemia, oxygen glucose deprivation, excitotoxins, reactive oxygen species, cytokines/ceramide, activation of adenosine receptors or KATP channels, spreading depression, inhibition of oxidative phosphorylation.

Acute Tolerance: Occurs immediately following preconditioning, is sustained for several hours and most often associated with post-translational protein modifications.

Sustained Tolerance: Maintained for several days and requires new protein synthesis

Attenuated stroke severity after prodromal TIA: a role for ischemic tolerance in the brain?

CONCLUSIONS: The beneficial effect of TIAs on lesion size in ADC and T2 suggests the existence of endogenous neuroprotection in the human brain.

Stroke 2004 Mar;35(3):616-21.2004

CONCLUSIONS: This study suggests that ischemic tolerance may play a role in patients with ipsilateral TIAs before cerebral ischemia, allowing better recovery from a subsequent ischemic stroke.

Neurology 2000 Jun 13;54(11):2089-94.

Stroke 1999 Sep;30(9):1851-4CONCLUSIONS: Assuming that a TIA represents an adequate stimulus to elicit ischemic tolerance, the results suggest that ischemic tolerance might occur in the human brain.

Ca2+

NMDA-R

Calmodulin

NOS NO

TranscriptionFactors

Nucleus

Ras-GDP

Ras-GTPRafMekErk

?

Proc. Natl. Acad. Sci, USA 95: 5773-5778 (1998)Proc. Natl. Acad. Sci., USA., 97: 436-441 (2000).

Nitric Oxide Mediates NMDA Receptor-Induced Activation of p21ras & Preconditioning.

New Protein SynthesisSurvival ProteinsExpressed

Functional Cloning StrategyPrimary Cortical Neurons - Preconditioned with 10 min OGD

Harvest mRNA

cDNA Library

Insert Library into a Retrovirus

Infect Fibroblast Cultures

Amplify viable cell clones

Recover Genes

Subclone into an expression vector

Assay Survival

Tx Menadione

Neurons Fibroblasts

SequenceBioinformatics

Tx MenadionePCR

Functional Cloning of NeuroProtective Genes

Control Menadione

C139SC2

Don

g Li

ang

Differential Analysis of Primary Library

Expression (DAzLE)

Proc. Natl. Acad. Sci. U.S.A., 101: 647-652, 2004Proc. Natl. Acad. Sci. U.S.A., 101:2145-2150, 2004

Screening for Survival Genes/Proteins

Functional Cloning

DAzLE

Strengths

WeaknessCannot distinguish endogenous vs. preconditioning survival genesCannot conduct in excitable cells (neurons)Genes must be FL-ORF

Genes are functionalKnow that genes mediate survivalWill identify pathways of survival in non-neuronal cells

Genes are induced by PC stressWill identify gene fragments, full length can be cloned laterCan be done in excitable cells (neurons)Need additional experimental design to identify survival genesRequires a lot of sequencingAdditional cloning required

Protection Against Toxic Menadione

Neu

ropr

otec

tion

OGD

NMDA

DAzLE PC Gene Set inMaximal Electroconvulsive Shock (MECS)

MECS ± MK801 12hr-24hr after MECS

2424+MK801+MK801

1212 24241212

DAzLE: Functional Categories of NMDA-induced Genes

NMDA Activated GenesStress Genes

PreconditioningGenes

How to find PC Genes?

NMDA-induced PreconditioningNMDA-induced Preconditioning

NMDANMDA

NMDA-R

NOS

p21 Ras

Raf

Mek

Erk

New Transcription / Translation

Preconditioning Mediated Neuroprotection

N-nitro-argininenNOS null vs WT

PD98059

50 50 M NMDA, 5 minM NMDA, 5 min

1hr-3hr-6hr-12hr-24hr1hr-3hr-6hr-12hr-24hrMEK/NO inhibitorMEK/NO inhibitor

NMDA-inducedGenes

658 Genes

MEKPathway

246 Genes

129Genes

NOPathway

218 Genes

31Genes

108Genes

12Genes

Venn Diagram of Differentially Regulated GenesVenn Diagram of Differentially Regulated Genes

Antisense Knock-down of Selected Genes Antisense Knock-down of Selected Genes Abolishes PreconditioningAbolishes Preconditioning

Cel

l D

eath

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Precond. 435 609 707 725 932 1012 Excito

****

** ****

** P < 0.001

Sense Antisense

(red: dead neurons)

Iduna: Norse Goddess of Immortality and Healing

Iduna protects cortical neurons from NMDA excitotoxicity

Iduna sequence

***

***PAR Binding

Localization of Iduna1˚ Cortical Cultures

Suk Jin Hong

Regional Expression of IdunaN

orth

ern

Blo

tW

este

rn B

lot

Iduna is Induced by PreconditioningCortical Cultures

Mouse Cortex

Suk Jin Hong JHMISunghee Cho & Constantino IadecolaWeill College of Medicine at Cornell

CA1 Control CA1 BCCAO 24hbilateral common carotid occlusion

CA1 Control CA1/CA2 BCCAO 24h

Cortex Outer Layer BCCAO 24h

Cortex Inner Layer BCCAO 24h

Cortex Outer Layer Naïve Control

Cortex Inner Layer Naïve Control

Cortex BCCAO 24hCortex Naïve Control

Mutant Constructs

32P-pADPr Overlay Blot: -GFP

56

36

29

101

GFPId

una-EGFP

GFPId

una-EGFP

contro

l

Iduna is a PAR binding protein

RING-finger WWEYR

Iduna

PAR

0

20

40

60

80

100

120

Control WWE YR-AA

% C

on

trol C

ell

Death

Iduna

*

*p < 0.001, Student’s t-test

500 µM NMDA

0

20

40

60

80

100

120

Control WWE YR-AA

% C

on

trol C

ell

Death

Iduna

*

*p < 0.001, Student’s t-test

MNNG

Mutation of the PAR Binding Site Prevents Neuroprotection by Iduna

RF

*

Thoughts for the Future……There are novel survival proteins and

pathways in the brain that need to be uncovered and understood.These survival proteins/pathways have tremendous promise as future therapeutic targets.With appropriate experimental design, gene screening is a powerful tool to reveal new survival pathways.Hopefully, understanding novel survival pathways may illuminate novel death pathways in the nervous system.

AcknowledgmentsAcknowledgmentsTed M. DawsonTed M. DawsonSuk Jin HongSuk Jin HongXiujing GuXiujing GuHuiwu LiHuiwu LiDong LiangDong LiangCheng DaiCheng DaiMirella Gonzalez-Mirella Gonzalez-ZuluetaZuluetaHye-Young YunHye-Young YunSika ZhengSika Zheng

NIH-NINDS, NIH-NIDAAHA, McKnight Foundation, Abramson Foundation

Kevin BeckerKevin BeckerLaura J. KlesseLaura J. KlesseRobert G. KalbRobert G. KalbLuis F. ParadaLuis F. ParadaGuy PoirerGuy PoirerJeff HainceJeff HainceConstantino Constantino

IadecolaIadecolaSunghee ChoSunghee Cho