www.carleton.ca/~kbstorey

FREEZE TOLERANT FREEZE TOLERANT ANIMALS ANIMALS

• Insects (many)Insects (many)

• Intertidal molluscs & Intertidal molluscs & barnacles barnacles

• Amphibians & Amphibians & Reptiles:Reptiles: - Frogs (6 species) - Frogs (6 species) - Hatchling turtles - Hatchling turtles - Garter snakes - Garter snakes - Lizards (some) - Lizards (some)

VERTEBRATE VERTEBRATE FREEZE FREEZE

TOLERANCETOLERANCE

WOOD FROGRana sylvatica

WOOD FROGRana sylvatica

Wood frog, Rana sylvatica

SURVIVING FREEZINGSURVIVING FREEZING

• Extracellular freezing only

• 65-70% of body water frozen

• Acclimationrequired

• Protectants - high glucose, glycerol, sorbitol - ice nucleators

WOOD FROG WOOD FROG CRYOPROTECTANTSCRYOPROTECTANTS

• 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

GLYCOGEN GLYCOGEN PHOSPHORYLASEPHOSPHORYLASE

Glycogen + Pi

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

LiverPhosphorylase a

Activity, U/g

PROTEIN KINASESPROTEIN KINASES

PROTEIN

nATP nADP

PROTEIN-(P)n

• 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

PROTEIN KINASESPROTEIN KINASES

PROTEIN

nATP nADP

PROTEIN-(P)n

• Covalent modification by phosphorylation

• 2-3% of genome, over 700 kinases

• Reversal via protein phosphatases

• Regulation involves activation cycles

Nucleus

GENESON/OFF

mRNAs

[ i + e Factors]

PROTEINS

Ca+2

KINASES (2nd)

PATHWAYS

SMW

CHO

AA

ATP

?SAPK

ATP

ADP

MITOGENES

FAT

[Trans.F] Na

K

ETC

P PROT

FREEZE INDUCED FREEZE INDUCED CHANGESCHANGES

• 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 )

CELL PROCESSESCELL PROCESSES

• DNA/RNA synthesis• Protein synthesis • Fuel metabolism• Ion pumping • Work done

ATP turnover to <5% of normal

FREEZE INDUCED FREEZE INDUCED CHANGESCHANGES

• 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

FREEZE INDUCED GENE FREEZE INDUCED GENE CHANGESCHANGES

• Most Genes ‘inactivated’• mRNA decreases• Epigenetic changes ! • Few Genes activated - transcription factors - shock proteins - antioxidants

FREEZE INDUCED GENE FREEZE INDUCED GENE CHANGESCHANGES

• Most Genes ‘inactivated’• mRNA decreases• Epigenetic changes ! • Few Genes activated - transcription factors - shock proteins - antioxidants

TURNING OFF GENES:TURNING OFF GENES:Role of EpigeneticsRole of Epigenetics

Epigenetics:Epigenetics: - Stable changes in gene activity that do not involve changes in DNA sequence

Common mechanisms:Common mechanisms: - DNA methylation

- Histone modification / histone variants

- Regulatory non-coding RNAs

microRNA (miRNA)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

miRNA processing pathwaymiRNA processing pathway

Steps of interest in our lab:

Dicer ProteinDicer Protein ExpressionExpression

miRNAmiRNAExpressionExpression

Source: www.ambion.com

Storey Lab approach to Storey Lab approach to miRNAmiRNA

Image: www.exiqon.com

1) miRNA array comparing samples from control vs frozen/hibernating

2) Lead Identification

3) Q-PCR Validation

Image: www.promega.com

Are miRNAs differentially regulated in Are miRNAs differentially regulated in our animal models?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:

miRNA Fold change Process in higher mammals

Mir-1 5.0 Myogenesis

Mir-133a 5.4 Myogenesis

Mir-206 4.6 Myogenesis

Let-7 4.0 Cell cycle

Mir-26 3.4 Hypoxia

Mir-451 7.6 Erythropoiesis

FREEZE INDUCED FREEZE INDUCED CHANGESCHANGES

• 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

GENE ACTIVATION GENE ACTIVATION (TRANSCRIPTION)(TRANSCRIPTION)

• Global rate of mRNA synthesis depressed. Method: nuclear run-on

• Are selected genes up-regulated ?

• TO ASSESS GENE UP-REGULATION:TO ASSESS GENE UP-REGULATION:

What new mRNAs are created - cDNA library, Gene Chip

Result: only 1 %1 % of Genes are UPUP

FREEZE INDUCED GENE FREEZE INDUCED GENE CHANGESCHANGES

• Most Genes ‘inactivated’• mRNA decreases• Epigenetic changes ! • Few Genes activated

- transcription factors - shock proteins

- antioxidant enzymes

cDNA ArrayscDNA Arrays- Methods- Methods- MaterialsMaterials- SourcesSources- Publications- Publications

Transcription Factors UP : Transcription Factors UP : GENES Respond GENES Respond

Epigenetics = OFF) : Epigenetics = OFF) : • microRNAmicroRNA

• phospho-RNA Polymerasephospho-RNA Polymerase• Histones modified Histones modified

• HDAC / HAT changesHDAC / HAT changes

TRANSCRIPTION FACTORS 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

TRANSCRIPTION FACTORS TRANSCRIPTION FACTORS

• ATF (Glucose Regulated Proteins)

• HIF (O2), HSF (Hsp)

• NFkB (IkB-P), Nrf-2 (GST)Nrf-2 (GST),, NRF-1

• PPAR, PGC, RXR, chREBP, CREB-P

• STAT, SMAD, p53-P, HNF, AP (1,2)

• Role : MR, Diabetes, Organ Preservation

FREEZE-INDUCED FREEZE-INDUCED GENES: WOOD FROGSGENES: WOOD FROGS

cDNA Library / Gene Chip

• Transcription Factors NRF -2 • Antioxidant enzymes

Nrf2 / ARE pathwayNrf2 / ARE pathway

Antioxidant proteinsAntioxidant proteins(e.g. GSTs, HO1)(e.g. GSTs, HO1)

AREARE

NucleusNucleus

CytoplasmCytoplasmSmall Maf

Nrf2

Keap1

Actin

Reactive Oxygen Species (ROS)Reactive Oxygen Species (ROS)

Activation

Dissociation

Nrf2 P

Nrf2 P

Small Maf Nrf2 P

NRF-2NRF-2

• Increased Nrf-2 protein & P-protein

• Increased Nrf-2 in the Nucleus

• Increased levels of co-Tf: MafG

• Downstream gene activation: Downstream gene activation:

• mRNA …. Protein Synthesis…..

• GST, HO-1, HO-2, Peroxiredoxin

ANTIOXIDANT ENZYMESANTIOXIDANT ENZYMES

Conclusions: FreezingConclusions: Freezing

Activation of the Nrf2 pathway: Activation of the Nrf2 pathway:

Tissue-specific activation, along with downstream gene protein products

Increased GST protein and activity

Result:Result: Detoxification of ROS,

intracellular signaling control

TRANSCRIPTION FACTORS TRANSCRIPTION FACTORS

• ATFATF (Glucose Regulated Proteins)

• HIFHIF (O2), HSFHSF (Hsp)

• NFkB (IkB-P), NFkB (IkB-P), Nrf-2 (GST), NRF-1NRF-1

• PPAR, PGC, RXR, chREBP, CREB-PPPAR, PGC, RXR, chREBP, CREB-P

• STAT, SMAD, p53-P, HNF, AP (1,2)STAT, SMAD, p53-P, HNF, AP (1,2)

• Role : MR, Diabetes, Organ Preservation

In: Hypometabolism in Animals: Hibernation, Torpor and Cryobiology (Lovegrove, B.G., and McKechnie, A.E., eds.) University of KwaZulu-Natal, Pietermaritzburg, pp. 101-108.

Upstream TATAA mRNA Coding Sequence

TBP

Transcription Factors

AUGRNA Pol II

AAAAA...*Transcription

Inducible Factors

Upstream Factors

Basal Machinery

Downstream Factors

Regulation of Gene TranscriptionRegulation of Gene Transcription

GENE CHIPS TRANSCRIPTION FACTORPROFILING

Data LeadsData Leads

ELISAs in platesELISAs in plates

Confirm by EMSAConfirm by EMSA

Confirm byConfirm byRT-PCR,RT-PCR,Northern blotsNorthern blots

TfDownstream genesDownstream genes

Protein levels - enzyme assay - antibodies : protein - functional analysis e.g. HIF EPO

Where do we go from Where do we go from here?here?

• Novel proteins• Novel phosphorylations• Turning it all off -- microRNA• Epigenetics & adaptation • Life span extension• Antioxidant defense• Cell cycle suppression• Climate Change & Winter

NEW DIRECTIONSNEW DIRECTIONS

Freeze Tolerance & Medicine:Freeze Tolerance & Medicine:Unique Animal Stress ModelUnique Animal Stress Model

Diabetes model: resistance to damage by

high glucose

Organ Cryopreservation

NOVEL PROTEINS:NOVEL PROTEINS: FR10, FR47, Li16FR10, FR47, Li16

Novel Phosphorylations Novel Phosphorylations

PP

PP PP

PP

PiPiProtein

Phosphatase

ATPATP ADPADPProtein Kinase

Turning It All Turning It All OffOff

Epigenetics in AdaptationEpigenetics in Adaptation

Life Span Life Span ExtensionExtension

Unavoidable Metabolic Costs Unavoidable Metabolic Costs

Unity through Unity through Evolution Evolution

WWCeDWWCeD

Molecular Adaptation toMolecular Adaptation to Climate Change:Climate Change:

Challenges for Amphibians & ReptilesChallenges for Amphibians & Reptiles

Freezing survival

Estivation

Anoxia tolerance

GENE CHIPSTRANSCRIPTION FACTOR

PROFILING

Data LeadsData Leads

ELISAs in platesELISAs in plates

Confirm by EMSAConfirm by EMSA

Confirm byConfirm byRT-PCR,RT-PCR,Northern blotsNorthern blots

Tf

Downstream genesDownstream genes

ROLE & CONTROL OF SYSTEM

Transgenics

Cell AssayRNAi Knock out Epigenetics

FUNCTIONAL ASSAYS

Protein levels - enzyme assay - antibodies : protein - functional analysis e.g. HIF EPO

FREEZE TOLERANCEFREEZE TOLERANCE

• 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

www.carleton.ca/~kbstoreywww.carleton.ca/~kbstoreyFunded by NSERC CanadaFunded by NSERC Canada