1. VERTEBRATE FREEZE TOLERANCE

2. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians Migration Mammals Above 0°C Others Stay warm Supercool

3. WOOD FROG Rana sylvatica

4. TO SURVIVE FREEZING Alter metabolism to synthesize cryoprotectants (polyols, sugars) Defend against intracellular desiccation Suppress metabolic rate ACCOMPLISHED BY: Activate signaling enzymes in every cell - ‘SAP’ kinases - Role: reversible controls on cell processes Up-regulate selected genes Up-regulate selected genes

5. SURVIVING FREEZING Extracellular freezing only Up to 70% of body water frozen High ‘polyols’ Acclimation required Glucose Glycerol Sorbitol

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

7. 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 Blood Liver Heart Kidney Muscle

8. GLYCOGEN PHOSPHORYLASE Glycogen + P i kinase Phos a Phos b phosphatase Glucose-1-P + glycogen (n-1) 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 Liver Phosphorylase a Activity, U/g

9. PROTEIN KINASES PROTEIN nATPnADP PROTEIN-(P)n Covalent modification by phosphorylation Families of protein kinases: PKA (cAMP), PKG (cGMP), CaMK (Ca 2+ ), PKC (Ca 2+,PL,DG) SAPKs : daisy chain phosphorylations Regulation is via interconversion of active vs subactive forms of protein substrates

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

11. 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 (1-2%)

12. p38 Pathway Signaling  Path activated by ??

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 Gene ‘inactivation’ ( mRNA) Few Genes activated

14. ROLE OF TRANSCRIPTION Global rate of mRNA synthesis depressed. Method: nuclear run-on Are selected genes up-regulated ? TO ASSESS GENE UPREGULATION:TO ASSESS GENE UPREGULATION: What new mRNAs are created - cDNA library, Gene Chip

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

16. cDNA Arrays - Methods - Materials - Sources - Publications

17. FREEZE-INDUCED GENES: WOOD FROGS cDNA Library / Gene Chip Transcription Factors Mito ETC; Transporters AOE & Shock proteins The Unknowns: Fr10, Li16, FR47 Storey KB 2004. Cryobiology 48, 134-145

18. TRANSCRIPTION FACTORS ATF (Glucose Regulated Proteins) HIF (O 2 ), 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

19. CONTROL REGION OF A TYPICAL EUKARYOTIC GENE Epigenetics: microRNA microRNA RNA Polymerase-P RNA Polymerase-P Histones modified Histones modified HDAC / HAT changes HDAC / HAT changes

20. FREEZE-INDUCED GENES: WOOD FROGS cDNA Library / Gene Chip Transcription Factors NRF -2 AOE Storey KB 2004. Cryobiology 48, 134-145

21. NRF-2 Increased NFR-2 protein Increased NFR-2 in the Nucleus Increased levels of co-Tf: MafG Downstream gene activation: GST, HO-1, HO-2, Peroxiredoxin

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

23. Regulation of Nrf2 Protein Nrf2 protein, 40 kDa Nrf2 protein, 82 kDa a a a a a a a a a 82 kDa 40 kDa C C F F Nrf2 Actin Nrf2 Liver

24. Regulation of MafG Antioxidant proteins (e.g. GSTs, HO1) ARE Small Maf Nrf2 P MafG protein a a a a a a

25. Glutathione S-Transferase Pi isozyme in Wood Frogs mRNA increases - transcriptional control Protein increases - translational control a a a a a a a a GST Pi protein a a GST Pi mRNA

26. Conclusions Activation of the Nrf2 pathway:  Activated in early-late torpor, along with downstream gene protein products  Increased GST protein and activityResult:  Detoxification of H 2 O 2, intracellular signaling control

27. ANTIOXIDANT ENZYMES

28. TRANSCRIPTION FACTORS ATF (Glucose Regulated Proteins) HIF (O 2 ), 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. GENE CHIPS TRANSCRIPTION FACTOR PROFILING Data Leads ELISAs in plates Confirm by EMSA Confirm by RT-PCR, Northern blots Tf Downstream genes ROLE & CONTROL OF SYSTEM Transgenics Cell Assay RNAi Knock out Epigenetics FUNCTIONAL ASSAYS Protein levels - enzyme assay - antibodies : protein - functional analysis e.g. HIF  EPO 

30. Unique Animal Stress Model Vertebrate whole-body freeze tolerance Tissue cryopreservation Tolerance of extreme ischemia and hyperglycemia

31. GENES Transcription RNAs Control by transcriptional regulation Control by translational regulation Translation PROTEINS(ENZYMES) Control by proteases INACTIVEENZYME No Modification FUNCTIONALENZYMES Covalent modification Degradation Control by post- translationalmodification ACTIVEENZYMES Inhibition and Activation Control at level of enzyme function www.carleton.ca/~kbstorey