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Unraveling Survival Strategies

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. Two types of cell death?! (apoptosis or necrosis).

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Unraveling Survival Strategies

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  1. Unraveling Survival Strategies Valina L. Dawson, Ph.D. Johns Hopkins Institute for Cell Engineering

  2. Too Many Cell Types in the Nervous System? Cell Type Estimates: Outside the Brain ~300 Nervous System ~1,500-5,000 Two types of cell death?! (apoptosis or necrosis) C.F. Stevens Curr. Biol. 8:708

  3. Strategies Ordering the Cell Death Pathway Cause Trigger Cell Death Consequence Contributor Identification of Novel Neuronal Survival Pathways

  4. Preconditioning 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.” TRENDS in Neurosciences 26(5): 248-254, 2003

  5. Preconditioning and Tolerance Preconditioning 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

  6. Attenuated stroke severity after prodromal TIA: a role for ischemic tolerance in the brain? Stroke 1999 Sep;30(9):1851-4 CONCLUSIONS: Assuming that a TIA represents an adequate stimulus to elicit ischemic tolerance, the results suggest that ischemic tolerance might occur in the human brain. Neurology 2000 Jun 13;54(11):2089-94. 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. Stroke 2004 Mar;35(3):616-21.2004 CONCLUSIONS: The beneficial effect of TIAs on lesion size in ADC and T2 suggests the existence of endogenous neuroprotection in the human brain.

  7. Nitric Oxide Mediates NMDA Receptor-Induced Activation of p21ras & Preconditioning. NMDA-R Proc. Natl. Acad. Sci, USA 95: 5773-5778 (1998) Proc. Natl. Acad. Sci., USA., 97: 436-441 (2000). New Protein Synthesis Survival Proteins Expressed Ca2+ Calmodulin Ras-GDP NO NOS Ras-GTP Raf Mek Erk Nucleus Transcription Factors ?

  8. Functional Cloning Strategy Primary Cortical Neurons - Preconditioned with 10 min OGD Harvest mRNA cDNA Library Insert Library into a Retrovirus Infect Fibroblast Cultures Tx Menadione Amplify viable cell clones Tx Menadione Recover Genes PCR Sequence Bioinformatics Subclone into an expression vector Fibroblasts Neurons Assay Survival

  9. Functional Cloning of NeuroProtective Genes Menadione Control SC2 C139 Dong Liang

  10. Differential Analysis of Primary Library Expression (DAzLE) Proc. Natl. Acad. Sci. U.S.A., 101: 647-652, 2004 Proc. Natl. Acad. Sci. U.S.A., 101:2145-2150, 2004

  11. Screening for Survival Genes/Proteins Functional Cloning DAzLE Genes are functional Know that genes mediate survival Will identify pathways of survival in non-neuronal cells Genes are induced by PC stress Will identify gene fragments, full length can be cloned later Can be done in excitable cells (neurons) Strengths Cannot distinguish endogenous vs. preconditioning survival genes Cannot conduct in excitable cells (neurons) Genes must be FL-ORF Need additional experimental design to identify survival genes Requires a lot of sequencing Additional cloning required Weakness

  12. Protection Against Toxic Menadione

  13. OGD Neuroprotection NMDA

  14. +MK801 12 24 12 24 DAzLE PC Gene Set in Maximal Electroconvulsive Shock (MECS) MECS ± MK801 12hr-24hr after MECS

  15. DAzLE: Functional Categories of NMDA-induced Genes

  16. How to find PC Genes? NMDA Activated Genes Stress Genes Preconditioning Genes

  17. NMDA-induced Preconditioning 50 mM NMDA, 5 min 1hr-3hr-6hr-12hr-24hr MEK/NO inhibitor NMDA NMDA-R N-nitro-arginine nNOS null vs WT NOS p21 Ras Raf PD98059 Mek Erk New Transcription / Translation Preconditioning Mediated Neuroprotection

  18. Venn Diagram of Differentially Regulated Genes NMDA-induced Genes 658 Genes 129 108 Genes Genes 31 Genes MEK Pathway NO Pathway 246 Genes 218 Genes 12 Genes

  19. Antisense Antisense Knock-down of Selected Genes Abolishes Preconditioning 90% ** ** ** P < 0.001 ** 80% ** 70% ** 60% Cell Death 50% 40% 30% 20% 10% 0% Precond. 435 609 707 725 932 1012 Excito Sense (red: dead neurons)

  20. Iduna: Norse Goddess of Immortality and Healing

  21. Iduna protects cortical neurons from NMDA excitotoxicity

  22. Iduna sequence *** PAR Binding ***

  23. Localization of Iduna 1˚ Cortical Cultures Suk Jin Hong

  24. Regional Expression of Iduna Northern Blot Western Blot

  25. Iduna is Induced by Preconditioning Cortical Cultures Mouse Cortex Suk Jin Hong JHMI Sunghee Cho & Constantino Iadecola Weill College of Medicine at Cornell

  26. CA1 Control CA1 BCCAO 24h bilateral common carotid occlusion CA1/CA2 BCCAO 24h CA1 Control

  27. Cortex Outer Layer Naïve Control Cortex Outer Layer BCCAO 24h Cortex Inner Layer Naïve Control Cortex Inner Layer BCCAO 24h

  28. Cortex Naïve Control Cortex BCCAO 24h

  29. Mutant Constructs

  30. Iduna is a PAR binding protein Iduna-EGFP Iduna-EGFP control GFP GFP 101 56 36 29 Blot: a-GFP 32P-pADPr Overlay

  31. Mutation of the PAR Binding Site Prevents Neuroprotection by Iduna Iduna RING-finger WWE YR PAR 500 µM NMDA MNNG 120 120 *p < 0.001, Student’s t-test *p < 0.001, Student’s t-test 100 100 80 80 % Control Cell Death % Control Cell Death 60 60 * * * 40 40 20 20 0 0 Control Iduna DRF DWWE YR-AA Control Iduna DWWE YR-AA

  32. 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.

  33. Acknowledgments Ted M. Dawson Suk Jin Hong Xiujing Gu Huiwu Li Dong Liang Cheng Dai Mirella Gonzalez-Zulueta Hye-Young Yun Sika Zheng Kevin Becker Laura J. Klesse Robert G. Kalb Luis F. Parada Guy Poirer Jeff Haince Constantino Iadecola Sunghee Cho NIH-NINDS, NIH-NIDA AHA, McKnight Foundation, Abramson Foundation

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