Maternal care and Gene - Environment Interactions Defining

Maternal care and Gene -

Environment Interactions Defining Development

Michael J Meaney

PhD

James McGill ProfessorDept. Psychiatry

McGill UniversityDouglas Hospital Research Centre

The development of an individual is an active process of adaptation that occurs within a social and economic context:

•

To resource (food, shelter, safety) availability.

•

To social interactions.

•

To independence from the parent.

Early experience

AbuseFamily strifeEmotional neglectHarsh discipline

Health Risks

DepressionDrug abuseAnxietyDiabetesHeart diseaseObesity

Mechanism?

Developmental Origins of Adult Disease

Early experience


Health Risks


Individual differencesin neural and endocrine responses to stress (defensive responses)

Stress Diathesis Models

Early experience


Health Risks


Individual differencesin neural and endocrine responses to stress

Poverty

Early experience


DepressionDrug AbuseAnxietyObesityDiabetesHeart Disease

Poverty

Effects of poverty on emotional and cognitive development are mediated by parental factors (Conger, McLloyd, Eisenberg).

Poverty

Maternalemotionaldistress

Parenting

Childbehaviouralproblems

Linver, Brooks-Gunn & Kohen

Dev Psychol

2002; same model predicts child cortisol

levels (Lupien, McEwen, Meaney

Biol

Psychiatry 2000)

Poverty

Homeenvironment

Childcognitive

development

Environmentaladversity

Parenting/Home

resources

Childdevelopment

Nutrient Supply

Mate Quality

Violence

Infection

Population density

Parentalinvestment

Defensive Strategies

Foraging/Metabolism

Reproductive Strategies

Environmental Parental Developmentalsignal mediation outcome

Robert Hinde: Evolution has shaped the young to use parental signals as a ‘forecast’

of the quality of the environment

into which they have been born. For most species, there isno single, optimal phenotype.

Evolutionary biology -

Maternal effects

Environmentaladversity -mother

Mother -

offspringinteractions

Programming of defensive responses in offspring

Over her lifespan?

• Seed

•

Propulagate

• Yolk

• Uterine

•

Maternal care

Evolutionary Biology: Maternal Effects

Defense to snake predation in skink lizards

Most frequent prey

• smaller•

shorter tails

•

less reactive to snake cues

If mother has been exposed to the scent of apredatory snake then offspring are larger, withlonger tails and ….

Control Perfume Snake0

1

2

3

4

Response to snake odours

Tong

ue-F

lick

Offspringare significantlylarger and withlonger tails

Scanning electron micrograph showing typical and predator-induced morphs of Daphnia cucullata of the same clone.

Predatorexposed

Control

Inducible defenses

25

15

20

10

30

K C

C C

N A K+

Agrawal

et al. Nature 1999

Rel

ativ

e he

lmet

size

Rel

ativ

e he

lmet

size

Nutrient Supply

Mate Quality

Violence

Infection

Population density

Parentalinvestment

Defensive Strategies

Foraging/Metabolism

Reproductive Strategies

Environmental Parental Developmentalsignal mediation outcome

Robert Hinde: Evolution has shaped the young to use parental signals as a ‘forecast’

of the quality of the environment

into which they have been born. For most species, there isno single, optimal phenotype.

Evolutionary biology -

Maternal effects

Cultu

reSocial

Econ

omics

Family

Violence

Parent-Infant

Summary

•

Parental care affects the activity ofgenes in the brain that regulate stressresponses, neural development and reproduction.

•

This parental effect involves a forma “plasticity”

at the level of the DNA.

Epigenetics: Any functional change in the genome thatdoes not involve an alteration of DNA sequence.

Creating diversity in phenotype from a common genome

Multiple phenotypes from a common genotype

C T A C G T A C T C G G A A T C T C GCH3CH3CH3

C T A C G T A C T C G G A A T C T C G

Genetic code is defined by the sequence of four nucleotides that produce proteins and other molecules that serve cell function.

Protein

Epigenetic effects refer to modifications of the chemistry of the DNA, but not

to a change of sequence. Epigenetics alters the activity of the

gene, but not its function.

Epigenetics

Any functional change in the genome thatdoes not involve an alteration of DNA sequence.




Protein




Warning!!!Incomprehensible scientific jargon

will follow…


•

Epigenetic effects refers to modifications of thechemistry of the DNA, but not

to a change of sequence.

•

Epigenetics alters the activity of the gene, but not its function.


•



•


•

DNA methylation: The addition of a methyl group onto a cytosine.


•



•


•


•

DNA methylation is chemically very stable (potentially lasting

forthe life of the organism).


•



•


•


•

DNA methylation is chemically very stable (potentially lasting

forthe life of the organism).

•

DNA methylation silences gene expression.

Nucleosome core particle: ribbon traces for the 146-bp DNA phosphodiester backbones (brown and turquoise) and eight histone protein chains (Luger et

al. Nature 1997).

+ -

Prevents TFbinding to DNA

TF binding involvesalteration of

chromatin structure

Acetyl group

B. Turner. Chromatin structure and gene regulation. 2001.




RNAs, proteins




SIN

SIN

CH3

CH3

CH3

CH3 MECP2

HDACCH3

CH3 MECP2

DNA Methylation can inhibit gene expressionby blocking transcription factors binding

HDAC HDAC: Histone

deacetylase

Methylated DNAbinding protein

HDAC

B. Turner. Chromatin structure and gene regulation. 2001.

Parental care Epigenetic mark PhenotypeGene expression

Naturally-occurringvariations in maternal care

Expression of specific genesin brain regions

HPA function

Stable individual differencesin stress reactivity

Maternal licking/grooming

Source of tactile stimulation/nurturance: Enhances Source of tactile stimulation/nurturance: Enhances activity of endocrine systems (e.g., GH/IGF) that activity of endocrine systems (e.g., GH/IGF) that promote somatic growth, suppresses those promote somatic growth, suppresses those (glucocorticoids) that inhibit growth(glucocorticoids) that inhibit growth

Variations in maternal careVariations in maternal care

Freq

uenc

y co

unt

% Licking/grooming

Variations in maternal careVariations in maternal care

Freq

uenc

y co

unt

% Licking/grooming

Low LGMean -

1SDHigh LG

Mean + 1SD

HighLow

Days of Age

Lick

ing/

groo

min

g

Variations in maternal care X DaysVariations in maternal care X Days

* No differencesin time with pups

Are these naturallyAre these naturally--occurring variationsoccurring variationsin maternal behaviour associated within maternal behaviour associated with

the development of individual differencesthe development of individual differencesin endocrine and behavioural responses in endocrine and behavioural responses

to stress?to stress?

* Effects hold for both males and females

fetal postnatal peripubertal adulthood

Maternal care Outcomes

autonomicautonomicoutputoutput

(Noradrenaline)(Noradrenaline)HPA outputHPA output

(glucocorticoids)(glucocorticoids)

(NA)

Baso-lateral

complex

Centraln.

mPFC

Locus coer.PB nNTSP

VN

Sensoryinput

(Stressor)

Hippocampus BNST

autonomicautonomicoutputoutputHPA outputHPA output

(NA)

Baso-lateral

complex

Centraln.

mPFC

Locus coer.PB nNTSP

VN

Sensoryinput

(Stressor)

Hippocampus BNSTGCrec

GlucocorticoidReceptor

Hypothalamus

Pituitary

Adrenals

Hippocampus

Glucocorticoids

Stress

(-)

CRF

ACTH

CRF: corticotropin releasing factor. ACTH: adrenocorticotropin

Hypothalamus

Pituitary

Adrenals

Hippocampus

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Hippocampus

Glucocorticoids

(-)

CRF

ACTH

(-)

Individual differences in glucocorticoid receptorlevels lead to altered pituitary-adrenal responses

to stress

Hypothalamus

Pituitary

Adrenals

High LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Low LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)


to stress

Adult offspring of High LG mothers show more modest HPA responses to stress

Pre S10 S20 P20 P40 P60 P1200

200

400

600

800

1000High LGLow LG

Plas

ma

AC

TH (p

g/m

l)

Pre 10 20 40 60 80 1000

10

20

30

40

50

60

70

80High LGLow LG

Cor

ticos

tero

ne (µ

g/dl

)

Intra-hippocampal infusion of a GR antagonist completely eliminates the maternal effect on HPA responses to stress

Hypothalamus

Pituitary

Adrenals

High LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Low LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)


to stress

Adaptive advantages of increased stress reactivity(Central CRF systems, HPA axis, Catechols)

• Increased resistance to sepsis (infection).• Increased resistance to famine.• Decreased mortality due to aggressive conflict.

Poverty: Pathogens, nutritional deprivation and violence

Hypothalamus

Pituitary

Adrenals

High LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Low LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)


to stress

High LG Low LG

Hypothalamus

Pituitary

Adrenals

High LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Hypothalamus

Pituitary

Adrenals

Low LG offspring

Glucocorticoids

(-)

CRF

ACTH

(-)

Cross-fostering reveals evidence for direct, postnataleffects of maternal care

Hypothalamus

Pituitary

Adrenals

Hippocampus

Glucocorticoids

(-)

CRF

ACTH

(-)

?

How might maternal licking/groomingregulate hippocampal glucocorticoid

receptor gene activity and HPA function?

And how do these effects persist over the lifespan

of the offspring?

SignalReceptor

Extracellular signalMaternalMaternalCareCare

DNA

Transcription

Factors

Intracellular signal

gene

Relevant gene -

environment interaction

cAMP

NGFI-A

5-HT7

rec

5-HTTactile stimulationTactile stimulation(maternal LG)(maternal LG)

GR gene

PKA

(T3)

(H-P-T)

Transcription

Factor

Summary of in vivo and in vitro studies

CTL5-HT

7 15 30 500

50100150200250

Days in CultureG

R le

velscAMP

NGFI-A

5-HT7

rec

5-HT

GR gene

PKA

Represents period of 5-HT exposure

*

1.5

1.0

0.5

0.0

NGFI-A mRNA levels

cAMP

NGFI-A

5-HT7

rec

5-HT

GR gene

PKA

Day 6 pups

Low LG High LG

*0.9

0.6

0.3

0.0

NGFI-A mRNA (stroking)

R/S R/C HC

Ctl 5-HT 5-HT0

1

2

3

4

+ NGFI-A antisense

GR

ir (R

OD

)

*

Weaver et al., 2006

In vitro (primary hippocampal neuronal cultures) studies

cAMP

NGFI-A

5-HT7

rec

5-HT

GR gene

PKA

What are the relevantgenomic targets?

Clone the 5’ untranslatedregion of the rat hippocampal glucocorticoid receptor gene

cAMP

NGFI-A

5-HT7

rec

5-HT

GR gene

PKA

Gene organization

Non-coding, regulatoryregion (contains enhancers,

repressors, etc.).

Coding regionresponsible for protein

synthesis.

(+ or -)

Clone the 5’ untranslated region of the rat hippocampal glucocorticoid receptor gene

Glucocorticoid receptor gene

(~110 kb)

1 2 3 4 5 6 7 8 9 101112 2 3 4

5 6 78 9

Variable exon 1 region Constant region

5’ 3’

(McCormick et al. Mol Endo 2000)

Transfection studies with promoter-reporter constructs reveal exon 17

sequence has considerable transactivational capacity.

Critical forHippocampal

GR

1 2 3 4 5 6 7 8 9 101112 2 3 4

5 6 78 9


5’ 3’

1681 ccc1741 ctctgctagt gtgacacact t1cg2cgcaact c3cgcagttgg 4cggg5cg6cgga ccacccctg7c1801 ggctctgc8cg gctggctgtc accct9cgggg gctctggctg c10cgaccca11cg ggg12cgggct1861 c13cgag14cggtt ccaagcct15cg gagtggg16cg gggg17cgggag ggagcctggg agaa

DNA sites that regulate glucocorticoid receptor gene

NGFI-A

GR Promoter 17 Sequence

1 2 3 4 5 6 7 8 9 101112 2 3 4

5 6 78 9

5’ 3’

NGFI-A binding to the GR(17

) promoterin neonates

**

00.10.20.30.40.50.60.70.80.9

Ant

ibod

y/in

put (

RO

D) 1.0

NGFI-A

cAMP

NGFI-A

5-HT7

rec

5-HT

GR gene

PKA

…..

Exon 1 (Noncoding region) Exons 2- 9 Coding region

5’ 3’

(+)

Glucocorticoid receptor mRNA

Glucocorticoid receptor protein

Offspring of High LG mothers

1177

NGFI-A

…..


5’ 3’

(+)




1177

NGFI-A

But…..

*

1.5

1.0

0.5

0.0

1.5

1.0

0.5

0.0

Pup Adult

NGFI-A levels

So, while increased levels of NGFI-A can explain the increased activity of the glucocorticoid receptor genein the pup, it does not explain why the difference is still observed in adult animals?

…..


5’ 3’

(+)




1177

NGFI-A

Stable modificationof the DNA????

GR expression & HPA function

C T A C G T A C T C G G A A T C T C GCH

3CH

3CH

3

•

DNA methylation occurs at cytosines.•

DNA methylation is chemically very stable.


CH3

CH3

CH3

CH3 MECP2

HDACCH3

CH3 MECP2

DNA Methylation can inhibit gene expressionby blocking transcription factors binding

HDAC HDAC: Histone

deacetylase

Methylated DNAbinding protein

X

X

DNA methylation silences gene expression

1681 ccc1741 ctctgctagt gtgacacact t1cg2cgcaact c3cgcagttgg 4cggg5cg6cgga ccacccctg7c1801 ggctctgc8cg gctggctgtc accct9cgggg gctctggctg c10cgaccca11cg ggg12cgggct1861 c13cgag14cggtt ccaagcct15cg gagtggg16cg gggg17cgggag ggagcctggg agaa

DNA sites that regulate glucocorticoid receptor gene

NGFI-A

GR Promoter 17 Sequence

1 2 3 4 5 6 7 8 9 101112 2 3 4

5 6 78 9

5’ 3’

High LG Low LG

5’…T G C G G G G G C G G G G ……

3’

5’…T G C G G G G G C G G G G …….3’CH3

CH3

5’…T G C G G G G G C G G G G …….3’

CH3

CH3

5’…T G C G G G G G C G G G G ……

3’

(no methylation)

(methylated only at 3’; High LG)

(methylated only at 5’)

(methylated at both 5’

and 3’; Low LG)

NGFI-A binding to ….

(High LG)

(Low LG)

*

1.5

1.0

0.5

0.0

1.5

1.0

0.5

0.0

Pup Adult

NGFI-A levels

So while levels of NGFI-A are similar in animals rearedby High or Low licking/grooming mothers, the NGFI-A

site in the adult offspring of Low LG mothers is methylated and therefore cannot interact with NGFI-A.

But….

Maternal Care

Glucocorticoid receptorgene expression

CRF expression

Individual differences in stress reactivity

GC feedbacksensitivity

Differential methylation of GR promoter… differences in NGFI-A

binding.

cAMP

NGFI-A

5-HT7

rec


GR gene

PKA

(T3)

(H-P-T)

Do these ‘maternal’

signals alter the methylationof the exon 17 GR promoter?

Do comparable processes occur in humans?

•

Post-mortem studies of hippocampus.

•

Samples from suicide victims/controls.

•

QSBB (Gustavo Turecki) -

forensicphenotyping.

•

Human exon 1F promoter (Turner & Muller, J Molec Endo, 2005)

Hippocampal samples from humans

•

Human brain bank (suicide victims vs controls).

•

All suicide victims (and none of the controls)experienced verified abuse in childhood.

Human glucocorticoid receptor gene

A B C D E F G H I J

2 3 4 5 6 7 8 9


5’ 3’

Human glucocorticoid receptor gene

A B C D E F G H I J

2 3 4 5 6 7 8 9


5’ 3’

Control Suicide0.0

0.5

1.0

1.5GRtotal

GR

mR

NA

Control Suicide0.0

0.3

0.6

0.9

1.2 GR1F

GR

1-F

var

iant

s

McGowan et al. PlosOne 2008, Nature Neuroscience 2009

Suicide vs abuse -

GR expression

GRtotal

GR

mR

NA

/GA

PDH

(log

con

c.)

Control Suicide Suicide-

Abuse + Abuse

0.0

0.4

0.8

1.2

*

GR

-1F

mR

NA

/GA

PDH

(log

con

c.)


Abuse + Abuse

0.0

0.5

1.0

1.5

*


GR

1-F

CpG

Met

hyla

tion

(%)


Abuse + Abuse

0

20

40

60 *

0

20

40

60

30 31 32CpG sites

Suicide vs abuse -

CpG methylation


Control NGFI-A expression

Co-transfection studies (NGFI-A vector w/human GR exon 1F-luciferase construct)

0

2000

4000

6000

8000

10000

12000

14000

Luci

fera

se a

ctiv

ity

mPlasmid nmPlasmid Antisense



DNA methylation serves as an interface between the dynamic environment and the fixed genome

DNA methylation serves to imprint social factors, such as maternal behavior, upon the offspring’s genome.

cAMP

NGFI-A

5-HT7

receptor


Synaptic plasticity

PKA CBP

Maternal Care

Expression of genes NMDArec

sub-units (Hipp)

BDNF/bFGF

Learning & Memory

Synaptogenesis

Documents

Maternal care and Gene - Environment Interactions Defining