VERTEBRATE FREEZE VERTEBRATE FREEZE TOLERANCETOLERANCE

ADAPTATIONS TO COLDADAPTATIONS TO COLD

Below 0°C

Freeze Freeze AvoidanceAvoidance

Freeze Freeze ToleranceTolerance HibernationHibernation

Invertebrates

Some reptiles& amphibians

MigrationMigration

Mammals

Above 0°C

Others

Stay warmStay warm

Supercool

WOOD FROGRana sylvatica

TO SURVIVE FREEZINGTO SURVIVE FREEZING• Alter metabolism to synthesize cryoprotectants (polyols, sugars)

• Defend against intracellular desiccation

• Suppress metabolic rate

ACCOMPLISHED BY:ACCOMPLISHED BY:

• Activate signaling enzymes in every cell

- ‘SAP’ kinases

- Role: reversible controls on cell processes

Up-regulate selected genesUp-regulate selected genes

SURVIVING FREEZINGSURVIVING FREEZING

• Extracellular freezing only

• Up to 70% ofbody water frozen

• High ‘polyols’

• Acclimation required

• Glucose

• Glycerol

• Sorbitol

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

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

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

• Gene ‘inactivation’ ( mRNA )• Few Genes activated (1-2%)

p38 Pathway Signalingp38 Pathway Signaling

Path activated by ??

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

ROLE OF ROLE OF TRANSCRIPTIONTRANSCRIPTION

• 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

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

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

FREEZE-INDUCED FREEZE-INDUCED GENES: WOOD FROGSGENES: WOOD FROGScDNA Library / Gene Chip

• Transcription Factors• Mito ETC; Transporters• AOE & Shock proteins • The Unknowns: Fr10, Li16, FR47

Storey KB 2004. Cryobiology 48, 134-145

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

CONTROL REGION OF A CONTROL REGION OF A TYPICAL EUKARYOTIC GENETYPICAL EUKARYOTIC GENE

Epigenetics: Epigenetics: • microRNAmicroRNA

• RNA Polymerase-PRNA Polymerase-P• Histones modified Histones modified

• HDAC / HAT changesHDAC / HAT changes

FREEZE-INDUCED FREEZE-INDUCED GENES: WOOD FROGSGENES: WOOD FROGScDNA Library / Gene Chip

• Transcription Factors NRF -2 • AOE

Storey KB 2004. Cryobiology 48, 134-145

NRF-2NRF-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

Nrf2/ARE pathwayNrf2/ARE pathway

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

AREARE

NucleusNucleus

CytoplasmCytoplasm

Small Maf

Nrf2

Keap1

Actin

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

Activation

Dissociation

Nrf2 P

Nrf2 P

Small Maf Nrf2 P

Regulation of Nrf2 ProteinRegulation 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 Nrf2Nrf2

Actin

Nrf2Nrf2

Liver

Regulation of MafGRegulation of MafG

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

AREARE

Small Maf Nrf2 P

mafG protein expression

0

0.5

1

1.5

2

2.5

brain muscle gut liver skin

Re

lati

ve

pro

tein

lev

el

control

frozen

recovery

MafG protein

a

a

a

a

a

a

Glutathione S-Transferase Pi isozyme in Wood Frogs

mRNA increasesmRNA increases- transcriptional control- transcriptional control

Protein increasesProtein increases- translational control- translational control

aa

a

a

a

a

a

a

GST Pi protein

GST Pi mRNA expression

0

0.5

1

1.5

2

2.5

3

brain gut liver kidney heart muscle

Re

lati

ve

mR

NA

le

ve

ls control

frozen

recovery

*a

a

GST Pi mRNA

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

Activated in early-late torpor, along with downstream gene protein products

Increased GST protein and activity

Result:Result:

Detoxification of H2O2, intracellular signaling control

ANTIOXIDANT ENZYMESANTIOXIDANT ENZYMES

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

GENE CHIPSTRANSCRIPTION FACTOR

PROFILING

Data LeadsData Leads

ELISAs in plates

Confirm by EMSA

Confirm byRT-PCR,Northern blots

TfDownstream 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

Unique Animal Stress ModelUnique Animal Stress Model

Vertebrate whole-body freeze tolerance

Tissue cryopreservation

Tolerance of extreme ischemia and hyperglycemia

GENESGENES

Transcription

RNAsRNAs

Control byControl bytranscriptional regulationtranscriptional regulation

Control byControl bytranslational regulationtranslational regulationTranslation

PROTEINSPROTEINS(ENZYMES)(ENZYMES)

Control byControl byproteasesproteases INACTIVEINACTIVE

ENZYMEENZYME

NoModification

FUNCTIONALFUNCTIONALENZYMESENZYMES

Covalentmodification

Degradation

Control by post-Control by post-translationaltranslationalmodificationmodification

ACTIVEACTIVEENZYMESENZYMES

InhibitionandActivation

Control at level ofControl at level ofenzyme functionenzyme function

www.carleton.ca/~kbstoreywww.carleton.ca/~kbstorey