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Using Drosophila to identify therapeutic targets for Neurodegenerative Disease MCBU June 2012

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.

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Using Drosophila to identify therapeutic targets for Neurodegenerative Disease MCBU June 2012

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  1. Using Drosophila to identify therapeutic targets forNeurodegenerative 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

  2. 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

  3. Huntington’s disease is a dominant, late onset neurodegenerative disease Normal brain HD brain

  4. 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 Feany, Bonini Alzheimer’s/Tauopathies Jackson; Suzuki, Feany , Wittman

  5. Protein Misfolding diseases - a common theme Alpha synuclein ‘Lewy Bodies’ in Parkinson’s ß amyloid plaques in Alzheimer’s Neurofibrillary tangles of Tau protein inside nerve-cells of the Alzheimer’s brain Poly Q inclusion in SCA3, HD and other polyQ diseases

  6. 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 80 60 Age at Neurologic Onset 40 20 0 0 10 20 30 40 50 60 70 80 90 100 120 Number of CAG Repeat Units

  7. Can we‘humanize’ a fly to mimic the neurodegeneration seen in manWill this speed target identification for testing in mammals? Modeling HD in Drosophila

  8. Htt polyQ U A S U A S U A S Q22 Q22 UAS Q93 Q93 Htt exon 1 UAS Q108 Q108 UAS Gal4 Gal4 Elav Elav TATA TATA Drosophila can be engineered to express foreign genes anywhere, anytime X tissue specific promoters Htt polyQ U A S U A S U A S

  9. normal HD Normal eye structure Human brain Compound eye SEM section pseudopupil Huntington’s disease can be mimicked in flies Fly eye normal polyQ108 Httex1Q93 day 12 Photoreceptor neurons degenerate

  10. a KCB a’ a KCB g g b b’behind b OK107>GFP OK107>Httex1Q93;GFP Expression of human Htt in flies causes widespread degeneration Mushroom body of adult fly brain Renderings by L.Chang; A.Chaing, NTHU

  11. wt photoreceptor neurons 7 wt 1 5 7 12 6 5 4 Degeneration is progressive elav>Htt exon1Q93 Days post eclosion day 1 day 3 day 6 Q48

  12. The role of chromatin modifications on transcriptional dysregulation and disease pathogenesis in vivo

  13. Transcription is dysregulated in HD patients

  14. Ac Ac CHD1 SAGA HAT complex Transcription is regulated by modifying histones. e.g. H3K4;9 Me Me Hum H3 n-ARTKQTARKSTGGK… 14 9 4 Factor binding Activation Su(var)3-3? K9ac HATs,CBP K41,2me Sin3A,Rpd3, Sir2 K9 K4 Ash1, Rtf1,Trr, Trx? K43me Ash1,Egg,G9a,Su(var)3-9? Cg8165,15835,33182? K9me Lid/JARID1C HP1

  15. HATs e.g CBP Ac-CoA HDACs Pol II complex H H H on HATs Ac-CoA HDACs polyQ Can we target HDACs? Pol II complex H H off H Does acetylation homeostasis contribute to pathology in vivo? normal HD Steffan et al. Nature. 413:739 (2001).

  16. 7.0 6.6 6.2 5.8 5.4 5.0 Genetic reduction of HDAC activity slows degeneration Sin3A is a general cofactor for class I & II HDACs Normal HD & Sin3A+/- Photoreptor neurons HD CyO Sin3A+/-

  17. 1 2 3 4 5 6 7 Day 1 Day 6 Day 6 Q48 Q48 wt Q48 + butyrate 100 mM Pharmacologic inhibition of HDACs slows progressive degeneration 50 @ 6 days Q48+inhibitor 40 Q48 30 % ommatidia 20 10 0 Steffan et al. Nature. 413:739 (2001).

  18. Normal-treated-sick

  19. Does HDAC therapy translate to mammals?

  20. 300 200 100 0 HDAC inhibitors slow progressive degeneration in mice HELP! All in a day’s work, I suppose Time on rod (secs) +/+ R6/2 + SAHA P = 0.0001 P = 0.0006 R6/2 +/+ HD -R6/2 Normal butyrate in R6/2 R6/2[HD] 4 8 10 12 Age/weeks Ferrante,. et al. J Neurosci 23, 9418 (2003). Hockly, E. et al. PNAS. 100, 2014 (2003).

  21. But which HDACs are relevant?

  22. Rpd3 is most effective at relieving pathology among the class I, II, IV HDACs 6.4 6.4 Photoreceptor # 6 6 5.4 5.4 5.4 5.2 5.2 5.2 5.6 5.6 5 5 5 5.2 5.2 4.8 4.8 4.8 4.6 4.6 4.6 4.8 4.8 4.4 4.4 4.4 4.4 4.4 HDAC6- + Ctl HDAC3- + HDAC4- + HDAC11- + Ctl Ctl Rpd3- + Ctl Ctl elav>Httex193Q photoreceptor degeneration - day 7 Class II Class IV Class I

  23. 60 60 50 50 40 40 30 30 20 20 10 10 0 0 5.4 5.5 5.3 5.2 5.1 5 4.9 4.8 4.7 4.6 4.5 4.4 Genetic reduction of Sir2 improves pathology sir2 GOF sir2 LOF Photoreptors survival % of control Ctl Sir2 -/+ Ctl Sir2 EP(oe) Pallos et al HMG 2008

  24. Selisistat (SEL), an indole based inhibitor of Sir2 exhibits dose dependent rescue of retinal neurons 5.5 5 Photoreceptor neurons 4.5 4 100mM Butyrate 0 µM 0.1 µM 1.0 µM 10 µM SEL (uM)

  25. V e n t r i c u l a r e n l a r g e m e n t Striatal degeneration is suppressed by Selistat in R6/2 mice 6 0 0 0 0 0 R6/2 Veh R6/2 Selisistat 5 mg/kg 5 0 0 0 0 0 4 0 0 0 0 0 * arbitrary units 3 0 0 0 0 0 2 0 0 0 0 0 1 0 0 0 0 0 0 Veh 5mg/kg Siena Biotech

  26. Pathology is sensitive toRpd3 and Sir2 Pallos et al HMG 2008

  27. 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?

  28. 7.0 100 7.0 Ac Ac 80 6.6 6.6 60 6.2 6.2 Survival 40 5.8 5.8 Photoreptors Photoreptors 5.4 20 5.4 5.0 5.0 0 CHD1 SAGA HAT complex CyO Sin3A+/- CyO Sin3A+/- 80 TM3 SUV39+/- 60 40 % expected 20 0 CyO lid+/- Transcription is regulated by modifying histones. H3K4;9 – a control node for therapeutic intervention in HD? Me Me Hum H3 n-ARTKQTARKSTGGK… 14 9 4 Factor binding Activation Su(var)3-3? K9ac HATs,CBP K41,2me Sin3A,Rpd3, Sir2 K9 K4 Ash1, Rtf1,Trr, Trx? K43me Ash1,Egg,G9a,Su(var)3-9? Cg8165,15835,33182? K9me Lid/JARID1C HP1

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