LIVING WITHOUT OXYGEN

www.carleton.ca/~kbstorey

FREEZE TOLERANTANIMALSFREEZE TOLERANTANIMALS

• Insects (many)• Insects (many)

• Intertidal molluscs & barnacles• Intertidal molluscs & barnacles

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

VERTEBRATEFREEZE TOLERANCEVERTEBRATEFREEZE TOLERANCE

WOOD FROGRana sylvatica

WOOD FROGRana sylvatica

Wood frog, Rana sylvatica

SURVIVING FREEZINGSURVIVING FREEZING

• Extracellularfreezing only

• 65-70% of bodywater frozen

• Acclimationrequired

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

WOOD FROGCRYOPROTECTANTSWOOD FROGCRYOPROTECTANTS

•Blood glucose rises from ~5 mM to 200-400 mM

•Glucose triggered by ice formation

•Made from liverglycogen (180 mg/g)

•Liver is ~12% of body mass

•Glucose distribution via Blood: Liver > Core organs > Periphery

Blood

Liver

Heart

Kidney

Muscle

GLYCOGENPHOSPHORYLASEGLYCOGENPHOSPHORYLASE

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

Liver

Phosphorylase 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

GENES

ON/OFF

mRNAs

[ i + e Factors]

PROTEINS

Ca+2

KINASES (2nd)

PATHWAYS

SMW

CHO

ATP

SAPK

ATP

ADP

MITO

GENES

FAT

[Trans.F]

ETC

PROT

FREEZE INDUCEDCHANGESFREEZE INDUCEDCHANGES

•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 INDUCEDCHANGESFREEZE INDUCEDCHANGES

•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 INDUCEDGENE CHANGESFREEZE INDUCEDGENE CHANGES

•Most Genes ‘inactivated’

•mRNA decreases

•Epigenetic changes !

•Few Genes activated

- transcription factors

- shock proteins

- antioxidants

FREEZE INDUCEDGENE CHANGESFREEZE INDUCEDGENE CHANGES

•Most Genes ‘inactivated’

•mRNA decreases

•Epigenetic changes !

•Few Genes activated

- transcription factors

- shock proteins

- antioxidants

TURNING OFF GENES:Role of EpigeneticsTURNING OFF GENES:Role 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 bedefined, 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

Expression Expression

miRNAmiRNA

ExpressionExpression

Source: www.ambion.com

Storey Lab approach tomiRNAStorey Lab approach tomiRNA

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 regulatedin our animal models?Are miRNAs differentially regulatedin our animal models?

•Yes! Selected miRNAs were up-regulated inhibernating 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 highermammals

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 INDUCEDCHANGESFREEZE INDUCEDCHANGES

•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(TRANSCRIPTION)GENE ACTIVATION(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

1 %UP Result: only 1 % of Genes are UP

FREEZE INDUCEDGENE CHANGESFREEZE INDUCEDGENE CHANGES

•Most Genes ‘inactivated’

•mRNA decreases

•Epigenetic changes !

•Few Genes activated

- transcription factors

- shock proteins

- antioxidant enzymes

cDNA Arrays- MethodscDNA Arrays- Methods

- Materials- Materials

- Sources- Publications- Sources- Publications

Transcription Factors UP :GENES RespondTranscription Factors UP :GENES Respond

Epigenetics = OFF) :Epigenetics = OFF) :

• microRNA• microRNA

• phospho-RNA Polymerase• phospho-RNA Polymerase

• Histones modified• Histones modified

• HDAC / HAT changes• HDAC / HAT changes

TRANSCRIPTIONFACTORSTRANSCRIPTIONFACTORS

•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

TRANSCRIPTIONFACTORSTRANSCRIPTIONFACTORS

•ATF (Glucose Regulated Proteins)

•HIF (O2), HSF (Hsp)

Nrf-2 (GST),•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

FREEZE-INDUCEDGENES: WOOD FROGSFREEZE-INDUCEDGENES: 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

CytoplasmCytoplasm

Small

Maf

Nrf2

Keap1

Actin

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

Activation

Dissociation

Nrf2

Small

Maf

Nrf2

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

TRANSCRIPTIONFACTORSTRANSCRIPTIONFACTORS

•ATF•ATF (Glucose Regulated Proteins)

•HIF HSF •HIF (O2), HSF (Hsp)

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

•PPAR, PGC, RXR, chREBP, CREB-P•PPAR, 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 andCryobiology (Lovegrove, B.G., and McKechnie, A.E., eds.)University of KwaZulu-Natal, Pietermaritzburg, pp. 101-108.

Upstream

TATAA

mRNA Coding Sequence

TBP

Transcription Factors

AUG

RNAPol II

AAAAA...

Transcription

InducibleFactors

UpstreamFactors

BasalMachinery

DownstreamFactors

Regulation of Gene TranscriptionRegulation of Gene Transcription

GENE CHIPS

TRANSCRIPTION FACTOR

PROFILING

Data LeadsData Leads

ELISAs in platesELISAs in plates

Confirm by EMSAConfirm by EMSA

Confirm byConfirm by

RT-PCR,RT-PCR,

Northern blotsNorthern blots

Downstream genesDownstream genes

Protein levels

- enzyme assay

- antibodies : protein

- functional analysis

e.g. HIF  EPO 

$F:\ALL my pictures unprocessed\2009_04_07\IMG_3876 copy.jpg$

Where do we go from here?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 DIRECTIONSNEW DIRECTIONS

Freeze Tolerance & Medicine:Freeze Tolerance & Medicine:

Unique Animal Stress ModelUnique Animal Stress Model

Diabetes model: resistanceto damage by high glucose

OrganCryopreservation

NOVEL PROTEINS:NOVEL PROTEINS:

FR10, FR47, Li16 FR10, FR47, Li16

Novel PhosphorylationsNovel Phosphorylations

PiPi

Protein

Phosphatase

ATPATP

ADPADP

Protein

Kinase

$H:\All-Photos-Most\ANIMALS\Red-eared turtle 2008\IMG_0536sm.jpg$

Turning It All OffTurning It All Off

Epigenetics in AdaptationEpigenetics in Adaptation

$H:\All-Photos-Most\ANIMALS\Wood Frogs\Wood Frog Oct 07-5.jpg$

Life Span ExtensionLife Span Extension

Unavoidable Metabolic CostsUnavoidable Metabolic Costs

Unity through EvolutionUnity through Evolution

WWCeDWWCeD

$H:\All-Photos-Most\ANIMALS\Hyla versicolor\Spring 2004\DSCN0899-sm.jpg$

$H:\All-Photos-Most\ANIMALS\Eurosta-Epiblema\Gall photos fall 2005\DSCN3054-sm.jpg$

Molecular Adaptation toMolecular Adaptation to

Climate Change: Climate Change:

Challenges for Amphibians & ReptilesChallenges for Amphibians & Reptiles

Freezing

survival

Estivation

Anoxia

tolerance

$H:\All-Photos-Most\ANIMALS\Painted turtles\Painted Turtles - Hatchlings\Hatchling turtles - fake nests Mar 2006\DSCN3331-flip.JPG$

GENE CHIPS

TRANSCRIPTION FACTOR

PROFILING

Data LeadsData Leads

ELISAs in platesELISAs in plates

Confirm by EMSAConfirm by EMSA

Confirm byConfirm by

RT-PCR,RT-PCR,

Northern blotsNorthern blots

Downstream genesDownstream 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 

$F:\ALL my pictures unprocessed\2009_01_19\IMG_3329.JPG$

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/~kbstorey

Funded by NSERC CanadaFunded by NSERC Canada