Using Drosophila to identify Using Drosophila to identify therapeutic targets for therapeutic targets for

Neurodegenerative Disease Neurodegenerative Disease MCBU June 2012 MCBU June 2012

Larry Marsh; Laszlo BodaiLarry Marsh; Laszlo BodaiDept of Developmental and Cell BiologyDept of Developmental and Cell Biology

German EncisoGerman EncisoDept of MathematicsDept of Mathematics

Alex IhlerAlex IhlerICS Information and Computer ScienceICS Information and Computer Science

OutlineOutline Gene expression is regulated by Gene expression is regulated by

chromatin marks such as acHistonechromatin marks such as acHistone Neurodegenerative diseases cause Neurodegenerative diseases cause

abnormal gene expression patterns and abnormal gene expression patterns and altered Histone acetylation patternsaltered Histone acetylation patterns

Can we correlate particular chromatin Can we correlate particular chromatin modifying proteins with particular gene modifying proteins with particular gene sets and can we identify the most sets and can we identify the most therapeutically attractive?therapeutically attractive?

System: Human disease genes System: Human disease genes expressed in Drosophilaexpressed in Drosophila

HuntingtonHuntington’’s disease is a s disease is a dominantdominant, , late onset neurodegenerative diseaselate onset neurodegenerative disease

NormalNormalbrainbrain

HDHDbrainbrain

HD is one of several HD is one of several Protein MisfoldingProtein Misfolding diseases diseases

CAG - polyQ (glutamine)CAG - polyQ (glutamine)HD (Huntingtin -Htt) HD (Huntingtin -Htt) Marsh, Benzer, JacksonMarsh, Benzer, Jackson

DRPLA (Atrophin-1) DRPLA (Atrophin-1) SCA-1 (Ataxin-1) SCA-1 (Ataxin-1) Fernandez-Funez..Botas Fernandez-Funez..Botas

SCA-2 (Ataxin-2) SCA-2 (Ataxin-2) SCA-3 (Ataxin-3) SCA-3 (Ataxin-3) Warrick…BoniniWarrick…Bonini

SBMA (Androgen Receptor) SBMA (Androgen Receptor) Takeyama..KatoTakeyama..Kato

SCA-6 (CaSCA-6 (Ca2+2+channel)*channel)*SCA-7 (Ataxin-7)SCA-7 (Ataxin-7)

5’ 3’

ParkinsonParkinson’’ss Feany, BoniniFeany, Bonini

AlzheimerAlzheimer’’s/Tauopathiess/Tauopathies Jackson; Suzuki, Feany , WittmanJackson; Suzuki, Feany , Wittman

Protein MisfoldingProtein Misfolding diseases - a common theme diseases - a common theme

Alpha synuclein Alpha synuclein ‘‘Lewy BodiesLewy Bodies’’ in in ParkinsonParkinson’’ss

ß amyloid plaques in Alzheimerß amyloid plaques in Alzheimer’’ss

Neurofibrillary tangles of Tau protein inside Neurofibrillary tangles of Tau protein inside nerve-cells of the Alzheimernerve-cells of the Alzheimer’’s brains brainPoly Q inclusion in SCA3, HD and Poly Q inclusion in SCA3, HD and

other polyQ diseasesother polyQ diseases

Expansion of PolyQ above a Expansion of PolyQ above a threshold causes diseasethreshold causes disease

Normal Htt = 6-34 QsNormal Htt = 6-34 Qs Adult onset = 37-40 QsAdult onset = 37-40 Qs

≥ ≥41-121 always disease41-121 always disease

≥ ≥ 70 Qs = juvenile onset 70 Qs = juvenile onset e.g. ≤ 21yearse.g. ≤ 21years

Number of CAG Repeat UnitsNumber of CAG Repeat Units

00 1010 2020 3030 4040 5050 6060 7070 8080 9090 100100 12012000

2020

4040

6060

8080

Ag

e at

N

euro

log

ic O

nse

tA

ge

at

Neu

rolo

gic

On

set

Can weCan we‘‘humanizehumanize’’ a fly a fly to mimic the neurodegeneration to mimic the neurodegeneration

seen in manseen in manWill this speed target Will this speed target

identification for testing in identification for testing in mammals?mammals?

Modeling HD in DrosophilaModeling HD in Drosophila

Drosophila can be engineered to Drosophila can be engineered to express foreign genes express foreign genes

anywhere, anytimeanywhere, anytime

tissue tissue specific specific

promoterspromoters

Q22Q22 UAS Q22Q22

Q108Q108 UAS

Q93 Htt exon 1Q93 Htt exon 1 UAS Q93Q93

Q108Q108

Gal4 TATAElavUAS

UAS

X

Htt polyQUAS

Gal4 TATAElavUAS

UAS

Htt polyQUAS

normal HD

Hu

ma

n b

rain

Httex1Q93Httex1Q93day 12day 12

polyQ108polyQ108normalnormal

Fly

eye

Fly

eye

HuntingtonHuntington’’s disease can be mimicked s disease can be mimicked in fliesin flies

Compound eye SEM section pseudopupilCompound eye SEM section pseudopupil

Normal eye structureNormal eye structure

Photoreceptor neurons degeneratePhotoreceptor neurons degenerate

Expression of human Htt in flies causes Expression of human Htt in flies causes widespread degenerationwidespread degeneration

Renderings by L.Chang; A.Chaing, NTHURenderings by L.Chang; A.Chaing, NTHU

Mushroom body of adult fly brainMushroom body of adult fly brain

KCBKCB

OK107>Httex1Q93;GFPOK107>Httex1Q93;GFP

’’behindbehind

’’

KCBKCB

OK107>GFPOK107>GFP

elav>Htt exon1Q93elav>Htt exon1Q93

Days post eclosionDays post eclosion

44

55

66

77

11 55 77 1212wtwtph

oto

rece

pto

r n

euro

ns

ph

oto

rece

pto

r n

euro

ns

wtwt

day 1 day 3 day 6Q48Q48

Degeneration is progressiveDegeneration is progressive

The role of chromatin modifications The role of chromatin modifications on transcriptional dysregulation on transcriptional dysregulation

and disease pathogenesis and disease pathogenesis in vivoin vivo

Transcription is dysregulated in HD patientsTranscription is dysregulated in HD patients

CHD1CHD1SAGASAGA

HAT complexHAT complex

Hum H3 n-ARTHum H3 n-ARTKKQTARQTARKKSTGGSTGGKK……44 99

K4K4 K9K9K4K41,2me1,2me

K4K43me3me

Ash1, Rtf1,Trr, Trx?Ash1, Rtf1,Trr, Trx?

Su(var)3-3?Su(var)3-3?

Lid/JARID1CLid/JARID1C

K9K9acac

K9K9meme

HATs,CBPHATs,CBP

Ash1,Egg,G9a,

Ash1,Egg,G9a,Su(var)3-9

Su(var)3-9??

Cg8165,15835,33182?

Cg8165,15835,33182?

Sin3A,Rpd3, Sir2

Sin3A,Rpd3, Sir2

MeMeMeMe

HP1HP1

Factor bindingFactor bindingActivationActivation

Transcription is regulated by modifying histones.Transcription is regulated by modifying histones.e.g. H3K4;9e.g. H3K4;9

AcAcAcAc

1414

Does acetylation homeostasis contribute to Does acetylation homeostasis contribute to pathology pathology in vivoin vivo??

HHH

Ac-CoAHDACs

onon

HATse.g CBP

Pol II complex

Ac-CoA

HDACs

HH

offoffH

Pol II complex

HATs

polyQCan we target Can we target

HDACs?HDACs?

normalnormal

HDHD

Steffan et al. Steffan et al. NatureNature. 413:739 (2001).. 413:739 (2001).

Genetic reduction of HDAC activity Genetic reduction of HDAC activity slows degenerationslows degeneration

Sin3A is a general cofactor for class I & II HDACsSin3A is a general cofactor for class I & II HDACs

Phot

orep

tor n

euro

nsPh

otor

epto

r neu

rons

HDHD

HD HD &&

Sin3A+/-Sin3A+/-

5.05.0

5.45.4

5.85.8

6.26.2

6.66.6

7.07.0

CyOCyO Sin3A+/-Sin3A+/-

NormalNormal

Pharmacologic inhibition of HDACs slows Pharmacologic inhibition of HDACs slows progressiveprogressive degeneration degeneration

% o

mm

atid

ia%

om

mat

idia

0

10

20

30

40

50

1 2 3 4 5 6 7

Q48 Q48+inhibitorQ48+inhibitor@ 6 days

Q48 + butyrate 100 mMQ48 Q48

Day 1 Day 6 Day 6

wt

Steffan et al. Steffan et al. NatureNature. 413:739 (2001).. 413:739 (2001).

Normal-treated-sickNormal-treated-sick

Does HDAC therapy Does HDAC therapy translate to mammals?translate to mammals?

All in a day’s work, I suppose

HELP!

R6/2 + SAHAR6/2 + SAHAP = 0.0001

00

200200

300300

100100

44 88 1010 1212

Tim

e o

n r

od

(s

ec

s)T

ime

on

ro

d (

se

cs)

Age/weeksAge/weeks

P = 0.0006

R6/2R6/2

+/++/+

+/++/+ HD -R6/2HD -R6/2

HDAC inhibitors slow progressive HDAC inhibitors slow progressive degeneration in micedegeneration in mice

Hockly, E. et al. Hockly, E. et al. PNASPNAS. 100, 2014 (2003).. 100, 2014 (2003).

Normal butyrate in R6/2 R6/2[HD]Normal butyrate in R6/2 R6/2[HD]

Ferrante,. et al. Ferrante,. et al. J NeurosciJ Neurosci 23, 9418 (2003). 23, 9418 (2003).

But which HDACs are relevant?But which HDACs are relevant?

TypeType

Catalytic gene products Catalytic gene products

Co-repressorsCo-repressorsDrosophilaDrosophila humanhuman

Class IClass I

Usually Usually ubiquitous & ubiquitous & nuclearnuclear

Rpd3Rpd3 HDAC1, HDAC2HDAC1, HDAC2 Sin3ASin3A

Mi-2/NurdMi-2/Nurd

Bin1Bin1HDAC3HDAC3 HDAC3, (8)HDAC3, (8)

HDAC11HDAC11 HDAC11 HDAC11 class 4class 4

Class IIClass IIa; ba; b

Tissue specific & Tissue specific & shuttleshuttle

HDAC4HDAC4 HDAC4, 5, 7, 9HDAC4, 5, 7, 9 Sin3A Sin3A

NcoRNcoR

SMRTSMRTHDAC6HDAC6 HDAC6, (10)HDAC6, (10)

Class IIIClass III

sirtuinssirtuinsNAD dependentNAD dependent

Sir2Sir2 sirt1sirt1

Sirt2Sirt2 sirt2sirt2

Rpd3 is most effective at relieving pathology Rpd3 is most effective at relieving pathology among the class I, II, IV HDACsamong the class I, II, IV HDACs

elav>Httex193Q photoreceptor degeneration - day 7elav>Httex193Q photoreceptor degeneration - day 7

Class IClass I Class IVClass IVClass IIClass II

Ph

oto

rec

ep

tor

#P

ho

tore

ce

pto

r #

4.44.4

4.64.6

4.84.8

55

5.25.2

5.45.4

CtlCtl Rpd3Rpd3--

++HDAC11HDAC11--

++CtlCtl

4.44.4

4.64.6

4.84.8

55

5.25.2

5.45.4

4.44.4

4.64.6

4.84.8

55

5.25.2

5.45.4

HDAC6HDAC6--

++CtlCtlHDAC3HDAC3--

++CtlCtl

4.4

4.8

5.2

5.6

6

6.4

4.4

4.8

5.2

5.6

6

6.4

CtlCtl HDAC4HDAC4--

++

Genetic reduction of Sir2 improves pathologyGenetic reduction of Sir2 improves pathology

sir2 LOFsir2 LOF

Sir2 -/+Sir2 -/+

sir2 GOFsir2 GOF

Sir2 EP(oe)Sir2 EP(oe)

surv

ival

surv

ival

% o

f c

on

tro

l

0

10

20

30

40

50

60

0

10

20

30

40

50

60

Phot

orep

tors

Phot

orep

tors

4.4

4.6

4.8

5

5.2

5.4

4.5

4.7

4.9

5.1

5.3

5.5

CtlCtlCtlCtlPallos et al HMG 2008Pallos et al HMG 2008

44

4.54.5

55

5.55.5

0 µM0 µM 0.1 µM0.1 µM 1.0 µM1.0 µM 10 µM10 µM 100mM100mMButyrateButyrateSEL (uM)SEL (uM)

Phot

orec

epto

r neu

rons

Phot

orec

epto

r neu

rons

Selisistat (SEL), an indole based inhibitor of Sir2 Selisistat (SEL), an indole based inhibitor of Sir2 exhibits dose dependent rescue of retinal exhibits dose dependent rescue of retinal

neuronsneurons

R6/2 Veh

R6/2 Selisistat 5 mg/kg

Ventricular enlargement

0

100000

200000

300000

400000

500000

600000

*

arb

itra

ry u

nit

sStriatal degeneration is suppressed by Striatal degeneration is suppressed by

Selistat in R6/2 miceSelistat in R6/2 mice

Siena BiotechSiena Biotech

Veh 5mg/kgVeh 5mg/kg

Pathology is sensitive toPathology is sensitive toRpd3 and Sir2Rpd3 and Sir2

TypeType Catalytic gene products Catalytic gene products RescueRescue

Drosophila (5+)Drosophila (5+) Human (11+)Human (11+)

Class IClass I

Usually ubiquitous Usually ubiquitous & nuclear& nuclear

Rpd3Rpd3 HDAC1, HDAC2HDAC1, HDAC2 YYHDAC3HDAC3 HDAC3, (8)HDAC3, (8) --HDAC11HDAC11 HDAC11HDAC11 --

Class IIClass IITissue specific & Tissue specific & shuttleshuttle

HDAC4HDAC4 HDAC4, 5, 7, 9HDAC4, 5, 7, 9 --HDAC6HDAC6 HDAC6, (10)HDAC6, (10) --

Class IIIClass IIINAD dependentNAD dependent

Sir2Sir2 sirt1sirt1 YYsirt2sirt2 sirt2sirt2 Y Y

Pallos et al HMG 2008Pallos et al HMG 2008

Mining the dataMining the data

1- Can one identify disease relevant HDAC’s by finding those that influence the 1- Can one identify disease relevant HDAC’s by finding those that influence the expression of a set of genes that are also altered by polyglutamine expression of a set of genes that are also altered by polyglutamine overexpression? overexpression?

2- Are there common sets of dysregulated genes seen in the different polyQ 2- Are there common sets of dysregulated genes seen in the different polyQ disease models (and possibly in other disease models like ALZ and PD)? disease models (and possibly in other disease models like ALZ and PD)?

3- Are there HDAC’s that exhibit a chromatin binding pattern that overlaps with the 3- Are there HDAC’s that exhibit a chromatin binding pattern that overlaps with the genomic location of the genes dysregulated upon polyglutamine expression? This genomic location of the genes dysregulated upon polyglutamine expression? This question is based on the observation that altered genes do not appear to be question is based on the observation that altered genes do not appear to be randomly distributed on the chromosome .randomly distributed on the chromosome .

4- Finally, some studies suggest that the genome can be described in terms of 5-9 4- Finally, some studies suggest that the genome can be described in terms of 5-9 different chromatin domain types (e.g. housekeeping genes vs developmentally different chromatin domain types (e.g. housekeeping genes vs developmentally regulated vs heterochromatin etc). For example, Filion et al describe 5 domains regulated vs heterochromatin etc). For example, Filion et al describe 5 domains based on binding data of transcription factors while Kharchenko et al , describe 9 based on binding data of transcription factors while Kharchenko et al , describe 9 domains based on chromatin modification marks. Can the dysregulated genes in domains based on chromatin modification marks. Can the dysregulated genes in Htt challenged animals be found to correlate with a specific chromatin domain Htt challenged animals be found to correlate with a specific chromatin domain identified by previous studies? identified by previous studies?

CHD1CHD1SAGASAGA

HAT complexHAT complex

Hum H3 n-ARTHum H3 n-ARTKKQTARQTARKKSTGGSTGGKK……44 99

K4K4 K9K9K4K41,2me1,2me

K4K43me3me

Ash1, Rtf1,Trr, Trx?Ash1, Rtf1,Trr, Trx?

Su(var)3-3?Su(var)3-3?

Lid/JARID1CLid/JARID1C

K9K9acac

K9K9meme

HATs,CBPHATs,CBP

Ash1,Egg,G9a,

Ash1,Egg,G9a,Su(var)3-9

Su(var)3-9??

Cg8165,15835,33182?

Cg8165,15835,33182?

Sin3A,Rpd3, Sir2

Sin3A,Rpd3, Sir2

MeMeMeMe

HP1HP1

Factor bindingFactor bindingActivationActivation

Transcription is regulated by modifying histones.Transcription is regulated by modifying histones.H3K4;9 – a control node for therapeutic intervention in HD?H3K4;9 – a control node for therapeutic intervention in HD?

Phot

orep

tors

Phot

orep

tors

5.05.0

5.45.4

5.85.8

6.26.2

6.66.6

7.07.0

CyOCyO Sin3A+/-Sin3A+/-

Surv

ival

Surv

ival

002020404060608080100100

TM3TM3 SUV39+/-SUV39+/-

AcAcAcAc

00

2020

4040

6060

8080

lid+/-lid+/- CyOCyO

% e

xpec

ted

% e

xpec

ted

Phot

orep

tors

Phot

orep

tors

5.05.0

5.45.4

5.85.8

6.26.2

6.66.6

7.07.0

CyOCyO Sin3A+/-Sin3A+/-

1414