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www.carleton.ca/~kbstorey

LIFE IN THE COLD - An Ice Stor-e-y . www.carleton.ca/~kbstorey. FREEZE TOLERANT ANIMALS. Insects (many) Intertidal molluscs & barnacles Amphibians & Reptiles: - Frogs (6 species) - Hatchling turtles - Garter snakes - Lizards (some).

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  1. LIFE IN THE COLD - An Ice Stor-e-y www.carleton.ca/~kbstorey

  2. FREEZE TOLERANT ANIMALS • Insects (many) • Intertidal molluscs & barnacles • Amphibians & Reptiles: - Frogs (6 species) - Hatchling turtles - Garter snakes - Lizards (some)

  3. VERTEBRATE FREEZE TOLERANCE

  4. WOOD FROGRana sylvatica

  5. WOOD FROGRana sylvatica

  6. Wood frog, Rana sylvatica

  7. SURVIVING FREEZING • Extracellular freezing only • 65-70% of body water frozen • Acclimationrequired • Protectants - high glucose, glycerol, sorbitol - ice nucleators

  8. Blood Liver Heart Kidney Muscle WOOD FROG CRYOPROTECTANTS • Blood glucose rises from ~5 mM to 200-400 mM • Glucose triggered by ice formation • Made from liver glycogen (180 mg/g) • Liver is ~12% of body mass • Glucose distribution via Blood: Liver > Core organs > Periphery

  9. Liver Phosphorylase a Activity, U/g 0 2 5 30 60 2 3 1 2 3 1 3 1 3 4 min hours days min hours TIME OF FREEZING TIME OF THAW GLYCOGEN PHOSPHORYLASE Glycogen + Pi kinase Phos a Phos b phosphatase Glucose-1-P + glycogen (n-1)

  10. PROTEIN PROTEIN-(P)n nATP nADP PROTEIN KINASES • Covalent modification by phosphorylation • Families of protein kinases: PKA (cAMP), PKG (cGMP), CaMK (Ca2+), PKC (Ca2+,PL,DG) • SAPKs : daisy chain phosphorylations • Regulation is via interconversion of active vs subactive forms of protein substrates

  11. PROTEIN PROTEIN-(P)n nATP nADP PROTEIN KINASES • Covalent modification by phosphorylation • 2-3% of genome, over 700 kinases • Reversal via protein phosphatases • Regulation involves activation cycles

  12. [ i + e Factors] Nucleus mRNAs GENES ON/OFF CHO PROTEINS [Trans.F] Na ATP K PATHWAYS AA SAPK P PROT ? Ca+2 SMW FAT GENES ADP ATP KINASES (2nd) MITO ETC

  13. FREEZE INDUCED CHANGES • Protein Synthesis slows to 1% • Pumps & channels closed • Energy Production slows to 5% • Energy Utilization slows to 2% • Few ‘SAP’ kinases activated • Results: P38, AMPK, JUNK, ERK, PKA, PKC, PERK, Fuel Pathways • Protein Phosphatases ( 1, 2A, 2C )

  14. CELL PROCESSES • DNA/RNA synthesis • Protein synthesis • Fuel metabolism • Ion pumping • Work done ATP turnover to <5% of normal

  15. FREEZE INDUCED CHANGES • Protein Synthesis slows to 1% • Pumps & channels closed • Energy Production slows to 5% • Energy Utilization slows to 2% • Few ‘SAP’ kinases activated • Gene ‘inactivation’ ( mRNA) • Few Genes activated

  16. FREEZE INDUCED GENE CHANGES • Most Genes ‘inactivated’ • mRNA decreases • Epigenetic changes ! • Few Genes activated - transcription factors - shock proteins - antioxidants

  17. FREEZE INDUCED GENE CHANGES • Most Genes ‘inactivated’ • mRNA decreases • Epigenetic changes ! • Few Genes activated - transcription factors - shock proteins - antioxidants

  18. TURNING OFF GENES:Role of Epigenetics Epigenetics: - Stable changes ingene activity that do not involve changes in DNA sequence Common mechanisms: - DNA methylation - Histone modification / histone variants - Regulatory non-coding RNAs

  19. microRNA (miRNA) • Small RNAs ~22 nucleotides in length • Highly conserved across species • Bind to 3’ UTR of mRNAs • All repression mechanism(s) yet to be defined, but include: - Block translation of mRNA - Help bind mRNA into stress granules - Target mRNA for degradation

  20. miRNA processing pathway Steps of interest in our lab: Dicer Protein Expression miRNA Expression Source: www.ambion.com

  21. Storey Lab approach to miRNA 3) Q-PCR Validation • miRNA array comparing • samples from control • vs frozen/hibernating 2) Lead Identification Image: www.exiqon.com Image: www.promega.com

  22. Are miRNAs differentially regulated in our animal models? • Yes! Selected miRNAs were up-regulated in hibernating 13-lined ground squirrels (Morin, Dubuc & Storey, 2008, Biochim Biophys Acta 1779:628-633) • How about frogs? New data for muscle of frozen wood frogs:

  23. FREEZE INDUCED CHANGES • Protein Synthesis slows to 1% • Pumps & channels closed • Energy Production slows to 5% • Energy Utilization slows to 2% • Few ‘SAP’ kinases activated • Gene ‘inactivation’ ( mRNA) • Few Genes activated

  24. GENE ACTIVATION (TRANSCRIPTION) • Global rate of mRNA synthesis depressed. Method: nuclear run-on • Are selected genes up-regulated ? • TO ASSESS GENE UP-REGULATION: What new mRNAs are created - cDNA library, Gene Chip Result: only 1 % of Genes are UP

  25. FREEZE INDUCED GENE CHANGES • Most Genes ‘inactivated’ • mRNA decreases • Epigenetic changes ! • Few Genes activated - transcription factors - shock proteins - antioxidant enzymes

  26. cDNA Arrays- Methods • Materials • Sources- Publications

  27. Transcription Factors UP : GENES Respond • Epigenetics = OFF) : • microRNA • phospho-RNA Polymerase • Histones modified • HDAC / HAT changes

  28. TRANSCRIPTION FACTORS • ATF (Glucose Regulated Proteins) • HIF (O2), HSF (Hsp) • NFkB (IkB-P), Nrf-2 (GST), NRF-1 • PPAR, PGC, RXR, chREBP, CREB-P • STAT, SMAD, p53-P, HNF, AP (1,2) • Methods: EMSA, PCR

  29. TRANSCRIPTION FACTORS • ATF (Glucose Regulated Proteins) • HIF (O2), HSF (Hsp) • NFkB (IkB-P), Nrf-2 (GST), NRF-1 • PPAR, PGC, RXR, chREBP, CREB-P • STAT, SMAD, p53-P, HNF, AP (1,2) • Role : MR, Diabetes, Organ Preservation

  30. FREEZE-INDUCED GENES: WOOD FROGS cDNA Library / Gene Chip • Transcription Factors • NRF -2 • Antioxidant enzymes

  31. Reactive Oxygen Species (ROS) Actin Keap1 P P P Nrf2 Dissociation Nrf2 Cytoplasm Nucleus Activation Antioxidant proteins (e.g. GSTs, HO1) Nrf2 Nrf2 Small Maf Small Maf ARE Nrf2 / ARE pathway

  32. NRF-2 • Increased Nrf-2 protein & P-protein • Increased Nrf-2 in the Nucleus • Increased levels of co-Tf: MafG • Downstream gene activation: • mRNA …. Protein Synthesis….. • GST, HO-1, HO-2, Peroxiredoxin

  33. ANTIOXIDANT ENZYMES

  34. Conclusions: Freezing • Activation of the Nrf2 pathway: • Tissue-specific activation, along with downstream gene protein products • Increased GST protein and activity • Result: •  Detoxification of ROS, intracellular signaling control

  35. TRANSCRIPTION FACTORS • ATF(Glucose Regulated Proteins) • HIF(O2), HSF(Hsp) • NFkB (IkB-P), Nrf-2 (GST), NRF-1 • PPAR, PGC, RXR, chREBP, CREB-P • STAT, SMAD, p53-P, HNF, AP (1,2) • Role : MR, Diabetes, Organ Preservation

  36. Regulation of Gene Transcription Transcription Factors Transcription * AAAAA... In: Hypometabolism in Animals: Hibernation, Torpor and Cryobiology (Lovegrove, B.G., and McKechnie, A.E., eds.) University of KwaZulu-Natal, Pietermaritzburg, pp. 101-108. TBP RNA Pol II AUG Upstream TATAA mRNA Coding Sequence Inducible Factors Upstream Factors Basal Machinery Downstream Factors

  37. TRANSCRIPTION FACTOR PROFILING GENE CHIPS Data Leads Confirm by RT-PCR, Northern blots ELISAs in plates Confirm by EMSA Downstream genes Tf Protein levels - enzyme assay - antibodies : protein - functional analysis e.g. HIF  EPO 

  38. Where do we go from here? Novel proteins Novel phosphorylations Turning it all off -- microRNA Epigenetics & adaptation Life span extension Antioxidant defense Cell cycle suppression Climate Change & Winter NEW DIRECTIONS

  39. Freeze Tolerance & Medicine: Unique Animal Stress Model Organ Cryopreservation Diabetes model: resistance to damage by high glucose

  40. NOVEL PROTEINS: FR10, FR47, Li16

  41. Protein Kinase ATP ADP P P P P Protein Phosphatase Pi Novel Phosphorylations

  42. Turning It All Off

  43. Epigenetics in Adaptation

  44. Life Span Extension

  45. Unavoidable Metabolic Costs

  46. Unity through Evolution WWCeD

  47. Molecular Adaptation to Climate Change: Challenges for Amphibians & Reptiles Freezing survival Estivation Anoxia tolerance

  48. TRANSCRIPTION FACTOR PROFILING GENE CHIPS Data Leads ELISAs in plates Confirm by EMSA Confirm by RT-PCR, Northern blots Downstream genes Tf ROLE & CONTROL OF SYSTEM Protein levels - enzyme assay - antibodies : protein - functional analysis e.g. HIF  EPO  Transgenics Cell Assay RNAi Knock out Epigenetics FUNCTIONAL ASSAYS

  49. J. STOREY D. McNALLY J. MacDONALD T. CHURCHILL S. GREENWAY C. HOLDEN S. WU J. NILES J. DU A. DeCROOS K. BIGGAR O. AGUILAR R. ROUFAYEL C. BROOKS L. ZHENHONG Q. CAI F. SCHUELER S. BROOKS B. RUBINSKY R. BROOKS FREEZE TOLERANCE Funded by NSERC Canada www.carleton.ca/~kbstorey

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