Using Drosophila to identify
therapeutic targets for
Neurodegenerative Disease
MCBU June 2012
Larry Marsh; Laszlo Bodai
Dept of Developmental and Cell Biology
German Enciso
Dept of Mathematics
Alex Ihler
ICS Information and Computer Science
Outline
Gene expression is regulated by
chromatin marks such as acHistone
Neurodegenerative diseases cause
abnormal gene expression patterns and
altered Histone acetylation patterns
Can we correlate particular chromatin
modifying proteins with particular gene
sets and can we identify the most
therapeutically attractive?
System: Human disease genes
expressed in Drosophila
Huntington’s disease is a dominant,
late onset neurodegenerative disease
Normal
brain
HD
brain
HD is one of several
Protein Misfolding diseases
5’
3’
CAG - polyQ (glutamine)
HD (Huntingtin -Htt) Marsh, Benzer, Jackson
DRPLA (Atrophin-1)
SCA-1 (Ataxin-1) Fernandez-Funez..Botas
SCA-2 (Ataxin-2)
SCA-3 (Ataxin-3) Warrick…Bonini
SBMA (Androgen Receptor) Takeyama..Kato
SCA-6 (Ca2+channel)*
SCA-7 (Ataxin-7)
Parkinson’s
Alzheimer’s/Tauopathies
Feany, Bonini
Jackson; Suzuki, Feany , Wittman
Protein Misfolding diseases - a common theme
Alpha synuclein ‘Lewy Bodies’
in Parkinson’s
Poly Q inclusion in SCA3, HD and
other polyQ diseases
ß amyloid plaques in Alzheimer’s
Neurofibrillary tangles of Tau protein inside
nerve-cells of the Alzheimer’s brain
Expansion of PolyQ above a
threshold causes disease
Normal Htt = 6-34 Qs
Adult onset = 37-40 Qs
≥41-121 always disease
≥ 70 Qs = juvenile onset
e.g. ≤ 21years
Age at Neurologic Onset
80
60
40
20
0
0
10 20 30 40 50 60 70 80 90 100
Number of CAG Repeat Units
120
Modeling HD in Drosophila
Can we‘humanize’ a fly
to mimic the neurodegeneration
seen in man
Will this speed target
identification for testing in
mammals?
Drosophila can be engineered to
express foreign genes
anywhere, anytime
tissue
specific
promoters
X
Elav
Elav
TATA
TATA
Gal4
Gal4
U U U
A A A
S S S
U U U
A A A
S S S
Htt polyQ
Htt polyQ
Q22 UAS Q22
Q93 Htt exon 1 UAS
Q93
Q108 UAS
Q108
Huntington’s disease can be mimicked
in flies
HD
Human brain
normal
Fly eye
Normal eye structure
Compound eye
SEM
section pseudopupil
normal
polyQ108
Httex1Q93
day 12
Photoreceptor neurons degenerate
Expression of human Htt in flies causes
widespread degeneration
Mushroom body of adult fly brain
a’
a
a
KCB
g
b
OK107>GFP
KCB
g
b’ behind
b
OK107>Httex1Q93;GFP
Renderings by L.Chang; A.Chaing, NTHU
Degeneration is progressive
photoreceptor neurons
wt
day 1
day 3
Q48
day 6
elav>Htt exon1Q93
7
6
5
4
wt 1
5
7
12
Days post eclosion
The role of chromatin modifications
on transcriptional dysregulation
and disease pathogenesis in vivo
Transcription is dysregulated in HD patients
Transcription is regulated by modifying histones.
e.g. H3K4;9
Me
Ac
Me
Ac
Hum H3 n-ARTKQTARKSTGGK…
4
9
Su(var)3-3?
K41,2me
Ash1, Rtf1,Trr, Trx?
K4
K43me
CHD1
SAGA
HAT complex
Lid/JARID1C
14
K9ac
Factor binding
Activation
K9
K9me
HP1
Does acetylation homeostasis contribute to
pathology in vivo?
normal
Ac-CoA
HATs
e.g CBP
HDACs
Pol II complex
H
H
H
on
HD
Can we target
HDACs?
HATs
Ac-CoA
polyQ
HDACs
H
H
H
Pol II complex
off
Steffan et al. Nature. 413:739 (2001).
Genetic reduction of HDAC activity
slows degeneration
Sin3A is a general cofactor for class I & II HDACs
Photoreptor neurons
7.0
Normal
6.6
6.2
HD
&
5.8
Sin3A+/-
5.4
5.0
HD
CyO
Sin3A+/-
Pharmacologic inhibition of HDACs slows
progressive degeneration
% ommatidia
50
40 Q48
30
wt
Q48
Q48+inhibitor
20
10
0
Day 1
@ 6 days
1
2
3
4
5
6
7
Day 6
Day 6
Q48
Q48 + butyrate 100 mM
Steffan et al. Nature. 413:739 (2001).
Normal-treated-sick
Does HDAC therapy
translate to mammals?
Time on rod (secs)
HDAC inhibitors slow progressive
degeneration in mice
HELP!
All in a
day’s work,
I suppose
300
+/+
200
P = 0.0001
R6/2 + SAHA
P = 0.0006
100
R6/2
+/+
HD -R6/2
Normal butyrate in R6/2
0
4
R6/2[HD]
8 10 12
Age/weeks
Hockly, E. et al. PNAS. 100, 2014 (2003).
Ferrante,. et al. J Neurosci 23, 9418 (2003).
But which HDACs are relevant?
Catalytic gene products
Type
Drosophila
human
Class I
Rpd3
HDAC1, HDAC2
Usually
ubiquitous &
nuclear
HDAC3
HDAC3, (8)
HDAC11
HDAC11
Class II
HDAC4
HDAC4, 5, 7, 9
Tissue specific &
shuttle
HDAC6
HDAC6, (10)
Class III
sirtuins
Sir2
sirt1
NAD dependent
Sirt2
sirt2
Co-repressors
Sin3A
Mi-2/Nurd
Bin1
class 4
a; b
Sin3A
NcoR
SMRT
Rpd3 is most effective at relieving pathology
among the class I, II, IV HDACs
Photoreceptor #
elav>Httex193Q photoreceptor degeneration - day 7
5.4
6.4
6.4
5.4
5.4
5.2
6
6
5.2
5.2
5
5.6
5.6
5
5
4.8
5.2
5.2
4.8
4.8
4.6
4.8
4.8
4.6
4.6
4.4
4.4
4.4
4.4
Ctl
Rpd3+
Ctl
HDAC3+
Class I
Ctl HDAC4+
Ctl
HDAC6-
Class II
+
4.4
Ctl HDAC11+
Class IV
Genetic reduction of Sir2 improves pathology
% of control
survival
Photoreptors
5.4
sir2 GOF
sir2 LOF
5.5
5.2
5.3
5
5.1
4.8
4.9
4.6
4.7
4.4
4.5
60
60
50
50
40
40
30
30
20
20
10
10
0
Ctl
Sir2 -/+
0
Ctl
Sir2 EP(oe)
Pallos et al HMG 2008
Photoreceptor neurons
Selisistat (SEL), an indole based inhibitor of Sir2
exhibits dose dependent rescue of retinal
neurons
5.5
5
4.5
4
0 µM
0.1 µM
1.0 µM
SEL (uM)
10 µM
100mM
Butyrate
Striatal degeneration is suppressed by
Selistat in R6/2 mice
R6/2 Veh
arbitrary units
600000
Ventricular enlargement
500000
400000
*
300000
R6/2 Selisistat 5 mg/kg
200000
100000
0
Veh
5mg/kg
Siena Biotech
Pathology is sensitive to
Rpd3 and Sir2
Type
Catalytic gene products
Rescue
Drosophila (5+) Human (11+)
Class I
Rpd3
HDAC1, HDAC2
Usually ubiquitous
& nuclear
HDAC3
HDAC3, (8)
HDAC11
HDAC11
Class II
HDAC4
HDAC4, 5, 7, 9
Tissue specific &
shuttle
HDAC6
HDAC6, (10)
Class III
Sir2
sirt1
sirt2
sirt2
NAD dependent
Y
Y
Y
Pallos et al HMG 2008
Mining the data
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 overexpression?
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)?
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
question is based on the observation that altered genes do not appear to be
randomly distributed on the chromosome .
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
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
domains based on chromatin modification marks. Can the dysregulated genes in
Htt challenged animals be found to correlate with a specific chromatin domain
identified by previous studies?
Transcription is regulated by modifying histones.
H3K4;9 – a control node for therapeutic intervention in HD?
Me
Ac
Me
Photoreptors
7.0
Ac
Hum H3 n-ARTKQTARKSTGGK…
4
9
6.2
5.8
5.4
5.0
CyO
14
Su(var)3-3?
K9ac
K9
Sin3A+/-
Factor binding
Activation
7.0
Photoreptors
K41,2me
Ash1, Rtf1,Trr, Trx?
K4
K43me
6.6
6.6
6.2
5.8
5.4
5.0
CHD1
SAGA
HAT complex
CyO
K9me
Lid/JARID1C
60
Survival
% expected
80
40
20
0
lid+/-
CyO
HP1
100
80
60
40
20
0
TM3 SUV39+/-
Sin3A+/-