dna methylation in alzheimer’s and down syndrome · 2011-04-25 · down syndrome (trisomy 21)...

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DNA Methylation in Alzheimer’s and Down syndrome B T k MD PhD Ben T yck o MD , PhD Taub Institute for Research on Alzheimer’s Disease and the Aging Brain and the Aging Brain Columbia University Medical Center

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Page 1: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

DNA Methylation in Alzheimer’s and Down syndrome

B T k MD PhDBen Tycko MD, PhDTaub Institute for Research on Alzheimer’s Disease

and the Aging Brainand the Aging BrainColumbia University Medical Center

Page 2: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Why study DNA methylation?Why study DNA methylation?

• Promoter CpG methylation silences gene expression;Promoter CpG methylation silences gene expression; methylation elsewhere can activate genes.

• Methylation is perturbed in disease – best studied inMethylation is perturbed in disease  best studied in cancer but now of interest (NIH Roadmap) in many other diseases – especially neurological ones.p y g

• Methylation can be manipulated by drugs and diet (5‐aza‐dC; folate); )

• Mapping CpG methylation can highlight genetic‐epigenetic interactions – help to find disease risk loci.ep ge et c te act o s e p to d d sease s oc

Page 3: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

DNA methylation: faithfully transmitted toDNA methylation: faithfully transmitted to daughter cells  in proliferating tissues

CpG

G C

me

CpG

G C

me

CpG

meGpC

me

GpC

DNMT1 re‐methylates daughterGpC

me me

DNMT1 re‐methylates daughter strands in S‐phase

CpG

GpC

CpG

GpC

meme

Page 4: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Changing DNA methylation:demethylation and de novo methylation

• Pre‐existing methylation can be lost when DNA replicates in the absence of DNMT1p

• de novo gain of methylation at previously unmethylated sequences is catalyzed by theunmethylated sequences is catalyzed by the other methyltransferase enzymes – DNMT3A and DNMT3B (DNMT3A is most important inand DNMT3B (DNMT3A is most important in the brain)

Page 5: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Could DNA methylation be y“involved in AD”?

By Braak stage 2, tau tangles have accumulated further and have caused some neurons to burst apart and die. However, mental testing still shows minimal impairment. Tangles at this level or worse are found in the brains of about 60% of over‐65s. At this stage the tau tangles are much more extensive in the transitional entorhinal region (highlighted in yellow) and have begun to kill neurons here. This region is a "relay station" where sensory input is y ) g g y y pfiltered before being stored in the memory. Meanwhile, in the brain's hippocampus (pink) and neo‐cortex, tau protein is also beginning to aggregate but has not yet formed tangles.  

W k b t t iWe know about toxic Abeta aggregates and tau tangles – why do 

   

At Braak stage 2, tau tangles are causing cell death (yellow) in the transitional entorhinal region, a part of the brain that filters sensory 

input before passing it on to the memory. Tau protein is aggregating in th hi ( i k) hi h i i l f d i th t

we need another hypothesis?

the hippocampus (pink) which is crucial for memory, and in the cortex(blue) which is associated with conscious thought.  

Page 6: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Methods for profiling CpGp g pmethylation

• MSNP and related methods (methyl‐sensitive restriction enzymes; microarrays)y ; y )

• MeDIP/MIRA (affinity capture of methylated DNA; microarrays or Nextgen sequencing)DNA; microarrays or Nextgen sequencing)

• Illumina Infinium (bisulfite conversion; primer extension on Beadarrays)extension on Beadarrays)

• Nextgen bisulfite sequencing of targeted h l isequences or whole epigenomes

Page 7: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Promoter meth: major differences among tissues

Y‐axis: 2,300 genes with methylation fraction >.5 in at least 20% of the samples

Page 8: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Promoter meth: few differences in AD vs. control

Page 9: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Promoter meth: BA37 (temporal cortex)AD vs controlAD vs. control

Need to use lenient criteria to find anything:criteria to find anything:>10% absolute diff in methylation at a given gene;p<.05 by simple ANOVA (not Bonferoni (corrected)

Page 10: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

AD vs. control whole temporal cortex:h h l d i i dhypermethylated promoter regions associated with decreased mRNA expression in published 

d (AZ S H l h )data (AZ Sun Health group)

mRNA foldMethyl CpGfractional

gene symbolBrainregions 

mRNA P‐value

mRNA fold  change AD vs. control

fractional change AD vs. 

control

TERF2IP HC 2 1E‐06 ‐2 44 0 15TERF2IP HC 2.1E‐06 ‐2.44 0.15

KCNC3 EC 7.9E‐03 ‐3.34 0.12

SUMO3 HC 6.1E‐03 ‐1.63 0.10

MGMT MTC 1.5E‐04 ‐2.24 0.10

C16orf24 EC 4.4E‐04 ‐2.66 0.10

NPTX2 MTC 1.1E‐05 ‐4.68 0.10

Page 11: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Separating neuronal vs. glial cellsSeparating neuronal vs. glial cells

Dounce frozen (autopsy) brain tissue

Isolate nuclei using density digradient 

Label neuronal nuclei with NeuNtib dantibody

Separate neuronal and glial nuclei using FACS 

DNA extraction Determine differential methylation patterns using  microarrays, y p g y ,

Nextgen sequencing, etc.

Page 12: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Sorted neurons from autopsy brainsSorted neurons from autopsy brains

NeuN+

NeuNNeuN-

Page 13: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Sorted neurons:  very few differentially methylated genes in AD vs. controls

• Infinium 27K “first generation” Beadarrays; mostly analyzing promoter regions; not other genomicanalyzing promoter regions; not other genomic regions.

• AD and control neurons from BA9 (frontal) and BA37AD and control neurons from BA9 (frontal) and BA37 (temporal) cortex.

• Zero differentially methylated CpG’s in AD vs. control y y psorted neurons, even with lenient criteria.

Page 14: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

…but there are clear differences in neuronal DNA methylation between 

different neocortical regionsdifferent neocortical regions

ANOVA p 05;ANOVA p.05; require >15 % difference in methylation.methylation.BA9 control vs. BA37 control neurons.Compare samples on controls only; add AD and DLBD to the clustering.

Page 15: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Results from Infinium 27KResults from Infinium 27K• In whole grey matter a few interesting genes do have mildly 

l d h l laltered promoter CpG methylation in AD vs. control.

• In purified neurons we find almost no examples of genes with altered promoter methylation in AD vs. control.altered promoter methylation in AD vs. control.

• However, in purified neurons, promoter methylation varies substantially across different neocortical regions.  Some of the differentially methylated genes are interesting.

Conclusion/hypothesis: patterns of promoter region DNAConclusion/hypothesis:  patterns of promoter region DNA  methylation likely help to specify neuronal identities in the human cerebral cortex but, except for a few interesting genes, these methylation patterns are not strongly altered in the earlythese methylation patterns are not strongly altered in the early to middle stages of AD.

Page 16: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Assessing global methylation: anti‐me‐C

T111-5 control D t t

T111-5 control CA1

T111-5 control LGN

Assessing global methylation: anti me C

DentateCA1 LGN

10x

40x

Page 17: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Globally reduced me‐C in CA1 in AD?

T247 -5 ADT247-5 AD T247-5 AD

Globally reduced me C in CA1 in AD?

DentateCA1 LGN

10x

40x

Page 18: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

IHC using anti‐OHmeCT99 Control

IHC using anti OHmeC

DentateCA1 LGN

10x

40x

Page 19: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Globally reduced OH‐meC in CA1 in AD?

T247 AD

Globally reduced OH meC in CA1 in AD?

DentateCA1 LGN

10x

40x

Page 20: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Global meC and OH‐meC: work is still in progress

• Careful checking of technical issues (antigen accessibility) using other nuclear antigensy) g g

• Examining other brain regions in AD and control brainscontrol brains

• Careful correlations with neuropathology including plaques and tanglesincluding plaques and tangles

Page 21: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

DNA methylation and neuronal specification:  what happens in Down 

syndrome?syndrome?

• Methylation profiling and detailed studiesMethylation profiling and detailed studies comparing normal vs. Down syndrome cells– Blood cells (PBL T‐cells)Blood cells (PBL, T cells)

– Cortical grey matter and purified neurons

Page 22: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Down Syndrome (trisomy 21)

Cognitive disability

Down Syndrome (trisomy 21)

Cognitive disabilityCongenital heart defectsEarly‐onset Alzheimer’s disease (1.5X APP)Childhood leukemiasAutoimmune diseases (50‐fold increased)Recurrent infectionsRecurrent infectionsAltered lymphocyte and NK cell function

Page 23: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Altered patterns of CpG methylation in PBL from adults with DS

AML1

AML2

AML3

DS PBL Control PBL DS PBL Control PBL

Illumina Infinium 27KAffymetrix 250K MSNP

Page 24: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Validations: the TMEM131 locus

BstUI

U

M

DS PBL Normal PBL

HpyCH4IV 

M

U

M

DS PBL (TMEM131 short ) Normal PBL (TMEM131 short)

240 KbTMEM131 long

TMEM131 short

0.5 Kbrs6760008

CGI

Page 25: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Validations: the PLD6 and CD3Z genes

DS PBL Normal PBL DS PBL Normal PBL

g

S o a S o a

PLD6 (LOC201164)

Infinium probe CD247 (CD3Z)

Infinium probe

200bp 200bpCGI 

Page 26: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

MS-Pyroseq

DS PBLNormal PBLNormal cord bloodNormal PBMCN l PMNNormal PMN

Page 27: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Altered mRNA expression of the differentially th l t d i DS PBL

essi

on .

10 DS av=5.40Control av=1.68T t t <0 0001

40 DS av=2.92

Control av=16.5T t t 0 00012

methylated genes in DS PBL

M131-

shor

t exp

re

3

5

8

T-test p<0.0001

10

20

30

CF7

expr

essi

on

T-test p= 0.00012

TMEM

DSN=30

ControlN=20

0

3

DSN=24

ControlN=23

0

10TC

250 Kb (additional exons)TMEM131 long

CGI TMEM131 short

rs67600081 kb

TCF7

rs756699

5 kb 23 kb

CGI 1 kb

Page 28: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

The altered methylation patterns are functionally relevant: gene activation

JurkatPrimary PBMC,

0 40.60.81.01.21.4

shor

t mR

NA

-sho

rt m

RN

A

15202530

cells expanded with IL-150

0.20.4

TMEM131-

s

0 0.25 0.55aza-dC (M) TM

EM131-

0510

0 0.25 0.55aza-dC (M)

56

1012

012345

TCF7

mR

NA Primary

PBMC, expanded with PHA

02468

TCF7

mR

NA

Jurkatcells

00 0.25 0.5

5aza-dC (mM)

00 0.25 0.5

5aza-dC (mM)

Page 29: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

A programmed epigenetic response to a 

Altered patterns of 

simple chromosomal aneuploidy in humans

Trisomy 21DNA methylation

Altered 

Self‐renewing tissue:

Cellular selection

regulatory networks

ghematopoietic system

Outgrowth of clones of cells with stereotypical epigenetic abnormalities. Genes are involved in lymphocyte function – candidates for producing immune deficits and  increased autoimmunity in Down syndrome and in the general population, i.e. Down syndrome as an “experiment of nature”.

Page 30: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Recent publications from our groupgroup

Page 31: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Infinium 27K Methylation DS vsnormal cerebellum; ANOVA p.05; 1.2fold diff; 0 1 absolute diffdiff; 0.1 absolute diff

Page 32: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Infinium 27K Methylation; DS vscontrol cerebral cortex; 1.2fold; 0 05diff; ANOVA p 050.05diff; ANOVA p.05

Page 33: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

ESR1: preliminary validations of differential p ymethylation in DS  vs. normal brains

ESR140 kb 

ESR1

cg20627916

5965DS BRAIN

5983CONTROL BRAIN

5980DS BRAIN

ESR1 Methyl: .31 .26 .09

Page 34: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

ESR1 is a risk locus for AD in DS:potential convergence of genetic and epigenetic data

Page 35: DNA Methylation in Alzheimer’s and Down syndrome · 2011-04-25 · Down Syndrome (trisomy 21) Cognitive disability Congenital heart defects Early‐onset Alzheimer’s disease (1.5X

Current Research GroupCurrent Research Group

Ri h d M• Huferesh Darbary

• Martha Salas

• Richard Mayeux

• Nicole Schupf

• Sandra Barral

• Maite Mendioroz

• John Crary

• J‐P Vonsattel

• Alexis Temkin

• Kristi Kerkel

• Jerzy Wegiel

• Wayne SilvermanKristi Kerkel• Warren Zigman

• Ed JenkinsGrant Support from the NIA d NICHDNIA and NICHD