MAMMALS ON ICE : MAMMALS ON ICE : HIBERNATIONHIBERNATION

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Myotis lucifugus, little brown bat

Spermophilus tridecemlineatus,13-lined ground squirrel

Spermophilus richardsonii,Richardson’s ground squirrel

• Seasonal phenomenon

• Pre-hibernation hyperphagia

• Gain up to 40% of body mass

• Need polyunsaturated fats

• Find hibernaculum: dark, near 0°C

• drop in body temperature• reduced heart rate• apnoic breathing• some muscle atrophy • periods of torpor lasting weeks• non-REM sleep• oleamide increases in brain

• suppression of carbohydrate oxidation

• RQ of 0.7 = lipid oxidation

Stewart JM, Boudreau NM, Blakely JA &Storey KB. 2002. J. Thermal Biol. 27, 309-315.

Oleamide in groundsquirrel brain

(ng/g)

Euthermic Hibernating0

200

400

600

800

Month

Body temperature

MR falls to fraction of normal

• Metabolism inhibited causing Tb to fall

• Metabolic rate falls to <5% of normal

• Smaller animals cool down faster

• Q10 values up to 15

• Reversible in arousal

• Torpor bout duration 4 days to 2 weeks

Estivation

METABOLIC RATE METABOLIC RATE DEPRESSIONDEPRESSION

Diapause

Freezing

Anoxia

Hibernation

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

Inhibitionand

Activation

Control at level ofControl at level ofenzyme functionenzyme function

METABOLISM IN METABOLISM IN HIBERNATIONHIBERNATION

• mRNA synthesis • Protein synthesis • Ion Pumping • Fuel use (esp. CHO)• O2 consumed

ATP turnover to <5% of normal

PRINCIPLES OF PRINCIPLES OF HIBERNATIONHIBERNATION

1. Metabolic rate reduction

2. Control by protein kinases(SAPKs, 2nd messenger PKs)

3. Selective gene activation

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

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

HIBERNATION INDUCED HIBERNATION 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%)

PROTEIN KINASESPROTEIN KINASES

PROTEIN

nATP nADP

PROTEIN-(P)n

• Covalent modification by phosphorylation

• Families of protein kinases: PKA (cAMP), PKG (cGMP), CaM (Ca2+), PKC (Ca2+, PL,DG)

• SAPKs : daisy chain phosphorylations

• Regulation via interconversion of active vs subactive forms of protein substrates

• p38, ERK (1/2), JNK, AMPK, AKT (mTOR)

PP

PP PP

PP

PiPiProtein

Phosphatase

ATPATP ADPADPProtein Kinase

P & deP enzymesseparate on ion

exchange columns

ReversibleReversiblephosphorylationphosphorylation

control of enzymescontrol of enzymes

PATHWAY CONTROL IN PATHWAY CONTROL IN HIBERNATIONHIBERNATION

• Glycolysis (GP, GS, PFK, PK)• Fat synthesis (ATP-CL, ACC)• CHO fuel use (PDH)• Translation (eIF2α, eEF2)• Ion pumps (NaK, Ca-ATPase)

• the usual suspects, TextBook

Phospho / de-Phospho

1. Novel PhosphoEnzymes: BioInformatics + Phospho-analyses 2. 32P-ATP labeling studies3. Purification / Properties 4. Structure / Function5. Phospho-sites

Post-translational Modifications:Post-translational Modifications: The Next Generation The Next Generation

Novel Phosphorylation ControlCK, GDH, Hexokinase, G6PDH,

LDH, NADP-IDH, α-GPDH, AMPD, GAPDH, FBPase, Antioxidant

enzymes

PTM: Acetylation, Methylation, SUMOylation

HIBERNATION INDUCED HIBERNATION 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%)

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 e.g. acetylation, phosphorylation - Regulatory non-coding RNAs

Transcription Transcription Suppression in Hibernator Suppression in Hibernator

MuscleMuscle

• Phospho-Histone H3 (Ser10) levels reduced

• Acetyl-Histone H3 (Lys23) levels reduced * Both inhibit Transcription ** Both inhibit Transcription *

• Histone Deacetylase activity increased 80%

• HDAC 1 & 4 protein levels increased

• RNA Polymerase II activity Decreased

Regulatory non-coding RNAs Regulatory non-coding RNAs

• Small RNAs ~22 nucleotides in length

• Highly conserved across species

• Bind to 3’ UTR of mRNAs

• Could be 1000, affect 60 % of genes

• Disease involvement

• Act to :

- Block translation of mRNA - Target mRNA for degradation

microRNAmicroRNA

Cuellar TL, McManus MT. J Endocrinol. 187(3):327-332, 2005.

Are miRNAs differentially regulated Are miRNAs differentially regulated in hibernators?in hibernators?

• Yes! Selected miRNAs were regulated in heart, muscle & kidney of hibernating 13-lined ground squirrels

(Morin, Dubuc & Storey, 2008, Biochim Biophys Acta 1779:628-633)

miRNA Fold change Process in higher vertebrates

Mir-1 2.0 Myogenesis

Mir-133a 2.4 Myogenesis

Mir-206 2.6 Myogenesis

Let-7 2.0 Cell cycle

Mir-26 2.4 Hypoxia

Mir-451 2.6 Erythropoiesis

Turning it all Turning it all offoff

Estivation

METABOLIC RATE METABOLIC RATE DEPRESSIONDEPRESSION

Diapause

Freezing

Anoxia

Hibernation

HIBERNATION INDUCED HIBERNATION 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%)

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

Sequenced genome(s) as of 2011 Sequenced genome(s) as of 2011

cDNA ARRAY SCREENINGcDNA ARRAY SCREENING

Euthermic Hibernating

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

GENE CHANGES IN GENE CHANGES IN HIBERNATIONHIBERNATION

• cDNA Library screen - Mitochondrial Genes - AOE - FABP, CPT, etc. - Shock proteins (GRP, HSP) - Transcription factors • DNA Chip ~1-2%

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

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 (**), NRF-1

• PPAR, PGC, RXR, chREBP, CREB-P

• STAT, SMAD, p53-P, HNF, AP (1,2)

• Methods: EMSA, CHiP

Nrf2/ARE pathwayNrf2/ARE pathway

Antioxidant proteins(e.g. GSTs, HO1)

ARE

Nucleus

Cytoplasm

Small Maf

Nrf2

Keap1

Actin

Reactive Oxygen Species (ROS)

Activation

Dissociation

Nrf2 P

Nrf2 P

Small Maf Nrf2 P

Nrf-2Nrf-2• Increased Nrf-2 protein & P

• Increased Nrf-2 in the Nucleus

• Increased levels of co-Tf: MafG

• Downstream gene activation:

• GST, HO-1, HO-2, Peroxiredoxin

• Thioredoxin, SOD (Cu/Zn & Mn)

Nrf2/ARE pathwayNrf2/ARE pathway

Antioxidant proteins(e.g. GSTs, HO1)

ARE

Nucleus

Cytoplasm

Small Maf

Nrf2

Keap1

Actin

Reactive Oxygen Species (ROS)

Activation

Dissociation

Nrf2 P

Nrf2 P

Small Maf Nrf2 P

Protein Regulation of Nrf2Protein Regulation of Nrf2

100 kDa

57 kDa

E H

Nrf2

Actin

Nrf2

Euthermicvs

Hibernating

Squirrel Nrf2 57kDa and 100kDa protein expression(hibernating vs.euthermic)

00.5

11.5

22.5

33.5

44.5

B.A.T. Brain Heart Kidney Liver Lung Muscle W.A.T.rati

o:

hib

ern

ati

ng

vs

. eu

the

rmic

57KDa

100KDa

* *

* *

* *

**

*

RatioHib:Euth

Nrf2 distribution between nuclearNrf2 distribution between nuclearand cytoplasmic fractionsand cytoplasmic fractions

E H E H Nucleus Cytoplasm

57 kDa

100 kDa

Nucleus CytoplasmE H E H

57 kDa

100 kDa

Moved to nucleus

Nrf2 (57 KDa and 100 KDa) Protein Expression inMuscle Nuclear and Cytoplasmic Fractions

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Muscle Nuclear Fraction Muscle Cytoplasmic Fraction

Hib

ern

atin

g v

s. E

uth

erm

ic

57KDa

100KDa

**

*

Muscle

Hib

: E

uth

Nrf2 (57 KDa and 100 KDa) Protein Expression in LiverNuclear and Cytoplasmic Fractions

00.20.40.60.8

11.21.41.6

Liver Nuclear Fraction Liver Cytoplasmic Fraction

Hib

ern

atin

g v

s. E

uth

erm

ic

57KDa

100KDa

*

*

Liver

Hib

: E

uth

Nrf2 Timecourse in HeartNrf2 Timecourse in Heart

• Nrf2 protein in early and late hibernation

Up-regulation “cascade”.

Active incold room

(37°C)

Entrance

(T-drop)

Early Hibern.

(~5-7°C)

Late Hibern.

(~5-7°C)

Early Arousal(T-gain)

FullyAroused(37°C)

Nrf2

CuZnSOD

AFAR1

HO-1

PeroxiredoxinsPeroxiredoxins

• Detoxify / reduce hydroperoxides

• Expressed at high levels

• ARE in promoter region of Prdx genes

•Nrf2 activated

0

2

4

6

8

10

12

14

16

18

Brown adipose tissue Heart

scan

ned

in

ten

sit

y o

f h

ibern

ati

ng

versu

s

eu

therm

ic

peroxiredoxin-1

peroxiredoxin-2

peroxiredoxin-3

BAT

Heart

Euth Hib

Prdx1 Prdx3Prdx2Peroxiredoxin protein levels

Euth HibEuth HibR

ati

o b

and inte

nsi

tyH

ib :

Euth

Peroxiredoxin ActivityPeroxiredoxin Activity

• Protein level correlates with increased activity

• Assays in BAT and heart with thioredoxin, thioredoxin reductase and NADPH:

Kim et al., 2005

0

0.5

1

1.5

2

2.5

3

3.5

4

Brown adipose tisuue Heart

Rat

io o

f hib

erna

ting

vers

us e

uthe

rmic

act

ivity

Relative activity: Hibernating: euthermic

A

ctiv

ity

rati

o

Hib

: E

uth

BAT Heart

Nrf ConclusionNrf Conclusion Activation of the Nrf2 pathway:Activation of the Nrf2 pathway:

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

Increased PRDX, HO & TRX protein and activity

Result:Result:

Detoxification of ROS, intracellular signaling control

GENE CHIPS TRANSCRIPTION FACTORPROFILING

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

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

• Applications of MRD research • Novel phosphorylations• Atrophy, hypertrophy -- autophagy for survival • Turning it all off -- microRNA• Epigenetics & adaptation • Life span extension• Antioxidant Defense• Cell cycle suppression• Unity through evolution

NEW DIRECTIONSNEW DIRECTIONS

PRIMATE HIBERNATION!!GREY MOUSE LEMUR

Hibernation and medicineHibernation and medicine

Primates !!

Novel phosphorylations Novel phosphorylations

PP

PP PP

PP

PiPiProtein

Phosphatase

ATPATP ADPADPProtein Kinase

Atrophy – HypertrophyAtrophy – Hypertrophy

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 Evolution Unity through Evolution

WWCeDWWCeD

HIBERNATIONHIBERNATION• J. STOREY• S. EDDY• D. HITTEL• J. MacDONALD• A. FAHLMAN• P. MORIN• C. HOLDEN• H. MEHRANI• J. NI

• M. HAPSATOU• S. TESSIER• M. WU• S. BROOKS• C. FRANK• J. HALLENBECK• D. THOMAS• A. RUBTSOV• J. STEWART

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Funded by NSERC CanadaFunded by NSERC Canada