CAG Expansion and Huntington’s Disease -

1. CAG Expansion and Huntington’s Disease - Could you repeat that, please? A research review on Huntington’s disease Image source: http://www.vh.org/Providers/Textbooks/BrainAnatomy/Ch5Text/Section01.html

2. Here’s the Plan • Introduction • Clinical symptoms • Pathology • Molecular basis • Current research • Treatment ideas • Future plans • Conclusions

3. Introduction Huntington’s Disease • Dr. George Sumner Huntington • Published paper in 1872 • Patients of East Hampton, Long Island http://www.uic.edu/depts/mcne/founders/page0048.html

4. Introduction Check Out My Figures… • Affects 30,000 people in the U.S. • Frequency of about 1 in 10,000 • 150,000 may develop the disorder • Strikes both men and women • 10% of the cases are children

5. Symptoms Symptoms: A Threefold Attack • Three stages of progression • Three areas affected • Mental: Dementia • Emotional: Mood swings • Physical: Chorea, unsteady gait, weight loss http://www.nlm.nih.gov/medlineplus/ency/article/003199.htm

6. Pathology Cerebral cortex Caudate Putamen Pathology: The Wreckage of HD • General loss of brain mass • Specific neuronal loss in the striatum (caudate nucleus and the putamen) and the cerebral cortex DebBurman, S.K.

7. Pathology Damage Report • Inhibitory and excitatory neurons • Loss of inhibitory control • Manifested in chorea

8. Molecular Basis CULPRIT: Huntingtin Gene & its Mutation • Normal huntingtin protein - vital, but function unknown • Gene identified as IT15 • Chromosome location 4p16.3 • Dynamic mutation • Expanded CAG repeat

9. Molecular Basis Nascent Strand 5’ …CAG... Template Strand Nascent Strand 5’ Template Strand DNA Polymerase Slippage How mutation happens: Expanded CAG Repeat Sequence

10. Molecular Basis DNA Polymerase Slippage Expanded CAG Repeat Translation Mutant Huntingtin Protein Manifestation Neuronal Cell Death Biological ModelMutant Huntingtin causes disease Huntingtin Gene IT15

11. Molecular Basis Longer the repeat, earlier the onset Persichetti et al., 1994

12. Molecular Basis CAG CAG CAG CAG CAG CAG Increasing Generations CAG CAG CAG CAG Paternal Anticipation • Longer CAG repeats are more unstable • Expansions occur more when passed through the male germ line • Meiotic transmission may cause the CAG expansion • Age of onset decreases with the increasing number of generations • Paternal anticipation is not fully understood Decreasing Age of Onset

13. Molecular Basis Conclusions • A dynamic mutation results in the CAG repeat • The mutation may be caused by DNA polymerase slippage • Inverse relation between length of CAG repeat and age at death • Paternal Anticipation results in longer CAG repeat and earlier age of onset

14. Demonstration Let Us Demonstrate An example of a sequence specific amplification

15. Gaps in Knowledge in 1997 • How does mutant huntingtin protein misfold? • What kind of cell death takes place? How does misfolded huntingtin cause cell death? • Who talks with huntingtin in health? How does that change in disease? • How can we prevent HD?

16. Current Research 1. How does Huntingtin misfold?Discovery of Neuronal Intranuclear Inclusions (NIIs) • N-terminus of htt accumulates in neuronal nuclei • PolyQ repeat leads to the formation of insoluble -sheets or barrels • Role of NIIs in HD pathology is presently unclear DiFiglia, 1997

17. Current Research Causal Role Of NIIs? • NIIs are hallmarks of polyQ diseases • Observed in the striatum and cortex • Appear before onset of HD • Ubiquitinated • Reduction NIIs decreases in vitro cell death Saudou, 1998

18. Current Research The Plot Thickens: Are NIIs Helpful or Deleterious? • htt induces cell death • Reduction in NII formation shows increased death from HD • NIIs: a possible protection mechanism? Saudou, 1998

19. Current Research Presently the role of NIIs in HD is unclear

20. Current Research 2. How Do Neurons Die? • Apoptosis- programed cell death • Non-apoptotic model - role for NIIs • Excitotoxicity - death by over-excitement

21. Current Research Caspase 1 Caspase 3 Apoptosis A. Role of Caspases in apoptosis • Degrade apoptotic cells • Triggers activation of other caspases • Overactivation of caspases in HD

22. Current Research B. A Non-Apoptotic Model

23. Current Research C. Excitotoxicity • High glutamate levels excite some neurons to death • Expected increased cell death in HD mice • Surprise! HD mice are resistant to the addition of quinolinic acid Hansson et al, 1999

24. Current Research So Which One is It? • No one knows! • Contradictory evidence from different laboratories and many unanswered questions • Still investigating each mechanism

25. Current Research Who does huntingtin talk with?Healthy & pathogenic Protein Interactions Molecular Chaperones & Transcription Factors

26. Current Research Chaperones: Fold With Me • Yeast model • Different levels of polyQ • Ubiquitination? • Still aggregation http://www.letterset.com/ufo.htm

27. Current Research Keep Foldin’, Foldin’, Foldin’ • Overexpression or deletion of HSPs • Increased HSPs, increased aggregation • Deletion of HSP104 - NO AGGREGATION http://www.ccc.nottingham.ac.uk/~mbzspd/tutorials/CSC.htm

28. Current Research Additional Folding Models • Mammalian model • HSPs increase aggregation • Worm model _ NIIs move to cytoplasm with more Hsps • Fly model • Hsp70 and Hsp40 together Inhibit fibril (aggregate) formation

29. Current Research Transcribe this…connecting with p53 • htt and p53 • p53 aggregation • Repressed transcription • Leads to Apoptosis? Perutz, 1994

30. Aggregation leads to Interfered transcription Essential protein synthesis disruption Cell death CA150 Transcriptional co-activator Rich in Glu-Ala Found with ubiquitinated htt in inclusions binds preferentially with htt Current Research What in the CA150??

31. Current Research So…… • HSPs are essential in htt misfolding • Possibility that the pathology involved htt aggregrating with transcription factors, which leads to cell death

32. Treatment Ideas 4. Therapeutic Possibilities • Addition of Minocycline • Turn off promoter to prevent htt translation • Fetal striatal tissue transplant

33. Treatment Ideas A. Minocycline Treatment • An antibiotic that can cross BBB • Will inhibit caspases • Delays disease progression • No inhibitory effect on the nuclear inclusions

34. Treatment Ideas B. Inhibiting transcription halts HD in mice • Transgenic mice developed with the ability to inhibit htt expression with addition of doxycycline (DOX)

35. Treatment Ideas C 18 On Off Lose Your Inhibitions • Inhibition of htt expression allows for neuronal recovery • Gene off mice show caudate regeneration Yamamoto, 2000

36. Treatment Ideas C. Transplanting the Seeds of Hope... with fetal transplants Freeman, et al., 2000 • Fetal striatal transplants by Freeman et al. • Used human subject • Only 18 months of data collected • Transplanted neurons unaffected

37. Conclusion The Underlying Theme • The field of HD has become increasingly controversial at the molecular level • Mechanisms are unknown even though progress has been substantial • HD does not follow suit with the rest of the trinucleotide repeat diseases

38. Future Plans What Next? • Combine treatments to target the different proposed mechanisms • Continued research to determine a definitive mechanism • Re-evaluate other trinucleotide repeat diseases

39. Future Plans We Wish We Knew... • How are the symptoms and pathology interrelated? • What is the exact mechanism of HD? • The role of NIIs: is it cause or effect? • What causes paternal genetic anticipation?

40. Acknowledgments We Would Like to Thank • Dr D • Aash Bhatt • Becky Bielang • the TA staff • Our Moms http://www.on-line-commerce.com/cgi-bin/nph-tame.cgi/posters/category.tam?rlk=569031