2006 landes bioscience. do not distribute. - Google Sites

[Cell Cycle 5:14, 1477-1480, 15 July 2006]; ©2006 Landes Bioscience

New Directions for Neurodegenerative Disease Therapy Extra View

Using Chemical Compounds to Boost the Formation of Mutant Protein Inclusions ABSTRACT

Original manuscript submitted: 05/23/06 Manuscript accepted: 05/24/06

ND

ACKNOWLEDGEMENTS

ES

.D

BIO

SC

Huntingtin polyglutamine Huntington’s disease Parkinson’s disease 5-[4-(4-chlorobenzoyl)-1piperazinyl]-8-nitroquinoline

A common feature among neurodegenerative diseases such as Huntington’s, Parkinson’s and Alzheimer’s diseases is the excessive accumulation of “misfolded” proteins. Two examples are mutant Huntingtin (Htt) with expanded polyglutamine (polyQ) in Huntington’s disease (HD) and α-synuclein in Parkinson’s disease (PD). As new proteins are made, cells must make triage decisions. Mutant proteins that fail to fold properly are either refolded by chaperones or destroyed by the proteasome.1-3 In neurodegenerative diseases, these quality control mechanisms are overwhelmed, possibly partly due to natural declines in function as neurons age.4,5 Ultimately, misfolded protein accumulates and disrupts cellular function, resulting in neuronal degeneration.

IEN

Huntington’s disease, Parkinson’s disease, neurodegenerative diseases, aggregation, inclusion body, misfolded protein

NEURODEGENERATIVE DISEASES ARE MARKED BY ACCUMULATION OF MISFOLDED PROTEIN

CE

KEY WORDS

ABBREVIATIONS

UT E

ON

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2929

RIB

*Correspondence to: Ruth A. Bodner; Center for Cancer Research; Massachusetts Institute of Technology; E18-505; 77 Massachusetts Avenue; Cambridge, Massachusetts 02139 USA; Tel.: 617.253.0261; Fax: 617.253.5202; Email: [email protected]

IST

2MassGeneral Institute for Neurodegenerative Disease; Massachusetts General Hospital and Harvard Medical School; Charlestown, Massachusetts USA

OT D

1Center for Cancer Research; Massachusetts Institute of Technology; Cambridge, Massachusetts USA

Htt polyQ HD PD B2

Neurodegenerative diseases such as Huntington’s, Parkinson’s and Alzheimer’s diseases are marked by neuronal accumulation of toxic misfolded protein. Developing therapies for these misfolding diseases requires finding chemical compounds that can either clear toxic misfolded protein, or can protect neurons from their impact. Such compounds could not only provide the starting points for potential drugs, but could also provide valuable research tools for untangling the complexities of the disease process. Until now, chemical screens for these diseases have focused on finding compounds that prevent aggregation of mutant protein. We recently published a compound, B2, which promotes the formation of large inclusions by mutant Huntingtin and α-synuclein, while rescuing some of the toxic effects of these proteins. As inclusions were long believed to be toxic to cells, this contradicts previous therapeutic approaches. At the same time, the results support growing evidence for the protective effects of inclusions. In this review, we discuss these results, and place them in the context of ongoing therapeutic discovery efforts for Huntington’s disease and other neurodegenerative diseases.

.

Ruth A. Bodner1,* David E. Housman1 Aleksey G. Kazantsev2

©

20

06

LA

Ruth Bodner is supported by a postdoctoral fellowship from the Hereditary Disease Foundation. Aleksey Kazantsev receives gift support from Discovery of Novel Huntington's Disease Therapeutics, MIND-MGH

www.landesbioscience.com

MISFOLDED PROTEINS TAKE MANY CONFORMATIONS: WHICH IS THE MOST TOXIC?

While we know that misfolded protein accumulates, it is unclear which conformation of mutant protein is most toxic. Misfolded proteins can adopt many conformations and assemblies, including monomers, small aggregates, fibrils and large inclusion bodies that contain many aggregates.6 These large inclusions are a striking feature of multiple neurodegenerative diseases, and at first seemed toxic, as their presence appeared to correlate with pathology.7-10 However, further studies have dissociated inclusion formation and pathology, in cell culture models, animal models and even human brain samples.11-16 Studies in cultured cells have supported the idea that inclusions are not merely irrelevant for pathology, but actually cytoprotective.17,18 More recently, these cell culture studies were elegantly extended by the use of automated microscopy to follow inclusion formation and cell viability; inclusion formation appeared to extend survival for cells expressing a mutant Htt construct.19 Possible beneficial effects of large inclusion bodies include decreasing the amount of available toxic surface area or directing mutant protein for destruction, perhaps by macroautophagy.20,21 Thus inclusions are increasingly thought to be either irrelevant or even protective for cells. It is possible that monomers or oligomers are the toxic species, but this has not been definitively determined.22,23 Despite changes in our conception of the toxic species, drug discovery efforts have focused on breaking up large inclusions of aggregated protein.24-29 Cell Cycle

1477

Chemical Promotion of Inclusion Formation

APPROACHING DRUG DEVELOPMENT WITHOUT KNOWING THE TOXIC CONFORMATION Traditional drug discovery involves finding chemical compounds that bind to and alter the function of well-understood target proteins, such as receptors, channels or enzymes. Drug development for neurodegenerative diseases has been stymied by our lack of understanding of the drug targets involved. Despite much data on the identity, properties and toxic consequences of the mutant proteins involved in these diseases, we continue to struggle with functional understanding, structural conformation of the toxic species, and order of pathological events. Because large inclusions of aggregated Htt are easy to visualize by microscopy, and because they were considered the prime toxic suspects, numerous compounds have been isolated that act as aggregation inhibitors in models of HD.25,26,28,29 However, due to the complexities of the proposed aggregation pathway for mutant Htt, aggregation inhibitors could act at very different points in this pathway and with divergent consequences.30 For example, suppose that oligomers or other folding intermediates are indeed the most toxic species, and that their sequestration into inclusions is protective (Fig. 1). In that scenario, an aggregation inhibitor that prevented the creation of oligomers would be beneficial, yet one that prevented oligomers from ultimately forming inclusions could be quite harmful. Alternatively, if monomers are most toxic, an aggregation inhibitor might be beneficial if it promoted clearance of monomers, but harmful if it merely prevented monomers from aggregating. Without understanding the mutant Htt misfolding pathway or knowing the most toxic species of Htt, it is risky to base therapeutic efforts solely on aggregation inhibitors. Aggregation inhibitors still hold much promise, and may ultimately result in effective therapies. However, complementary approaches are needed: compounds that rescue particular pathological phenotypes, compounds that promote other conformations of mutant Htt and compounds that promote clearance of toxic species.

COMPOUND B2 PROMOTES INCLUSION FORMATION, BUT IS PROTECTIVE

The new data on inclusions calls into question whether disrupting inclusions is an appropriate therapeutic strategy for neurodegenerative diseases. We have focused on instead finding chemical compounds that block downstream pathological effects of misfolded mutant protein, regardless of effects on inclusions. In exploring this we came to a surprising finding: compounds that promote inclusion formation could attenuate pathological features in models of both Huntington’s and Parkinson’s diseases.31 Thus, creating larger clumps of aggregated protein could be therapeutic for affected neurons. Our research started with compounds that increased the size and number of inclusions of mutant Htt protein, which is the culprit in Huntington’s disease. These compounds were originally isolated in a screen of 37,000 compounds for their ability to increase the levels of a polyQ-GFP reporter. We considered that these compounds could either alter Htt conformation by acting directly on the mutant Htt fragments, or by interacting with an entirely distinct target. To answer this, we tested the effects of the most potent compound (5-[4-(4-chlorobenzoyl)-1-piperazinyl]-8-nitroquinoline, or “B2”) on a distinct neurodegenerative disease protein, α-synuclein. We reasoned that any effects on α–synuclein would support the target of B2 being a protein other than Htt. Cells were transfected with 1478

Figure 1. A model: different points for therapeutic intervention. Mutant proteins can adopt multiple folding conformations and assemblies. It is not known which is the most toxic species, but current evidence points towards monomers or oligomeric assemblies. Sequestration of these toxic species into inclusion bodies may be protective for cells. Thus, in this model, cytoprotection could arise from pharmacological interception at different points in the folding pathway. Aggregation inhibitors such as C2-8 that prevent the accumulation of toxic intermediates would be beneficial for cells.29 However, once toxic intermediates had formed, compounds like B2 that boost inclusion formation would be most advantageous.

α-synuclein and treated with B2 or DMSO, then examined by immunofluorescence for α-synuclein. In DMSO-treated cells, most cells had many smaller cytoplasmic aggregates, while a subset of cells had a large inclusion. When treated with B2, the large-inclusion phenotype doubled in frequency. Thus B2 appears to promote the formation of inclusions of multiple neurodegenerative disease proteins, and it is unlikely to act by binding Htt directly. The isolation of B2 provided the opportunity to test whether pharmacologically induced inclusions could protect cells from the harmful effects of mutant Htt or α–synuclein. We began by testing the effects of B2 on proteasome dysfunction, a pathological feature seen in many neurodegenerative diseases.32-38 The proteasome is a garbage disposal for the cell. When overwhelmed by an abundance of mutant protein, the proteasome loses its ability to destroy cellular garbage, with devastating effects for the cell. We tested the five inclusion-promoting compounds in a cell-based assay for Htt-mediated proteasome dysfunction. This cell line is stably transfected with both a reporter of proteasome function (GFP fused with the CL1 degron,39 which directs the GFP for proteasomal degradation) and an ecdysone-inducible fusion of the first exon of mutant Htt (with 97Q) and monomeric red fluorescent protein. Thus, when mutant Htt expression is induced, GFP accumulates in cells instead of being degraded. When treated with the inclusion-promoting compounds, fewer cells were GFP-positive, consistent with the relief of proteasome dysfunction by B2. As in the original screen, B2 was the most potent compound. Thus inclusion-promoting compounds relieve at least one pathological consequence of mutant Htt expression. Confirming the specificity of the effect, analogs of B2 behaved similarly in both the screen and the proteasome assay; structural modifications of B2 that lowered or eliminated activity in the screen also did so in the proteasome assay. Thus in a model of HD, driving more inclusion formation by Htt is protective for cells. Since B2 also promotes inclusion formation by α–synuclein, we decided to test if B2 altered toxicity of α–synuclein in a model of PD. Alpha–synuclein accumulates in Lewy bodies in PD, and one

Cell Cycle

2006; Vol. 5 Issue 14


hereditary form of PD is caused by a triplication in the α-synuclein locus.40 Alpha–synuclein overexpression is toxic to cells. B2 treatment partially rescued toxicity in cells that were transfected with SynT, a tagged version of α-synuclein.41,42 This is consistent with inclusionpromotion being protective in a second neurodegenerative disease model. Because B2 does not seem to act directly on Htt or α-synuclein, we speculated that it might alter the activity or levels of chaperones.43 Induction of a heat shock response (which results in the upregulation of several chaperones) has been shown to affect aggregation of multiple neurodegenerative disease proteins. However, B2 treatment did not cause an increase in the levels of Hsp70 or Hsp27, which would be elevated in the case of a heat shock response. B2 was also tested in an in vitro assay of chaperone activity, and failed to show any effects on chaperones. Thus, we do not believe that B2 directly acts on refolding of mutant proteins by chaperones.

WHAT CAN WE LEARN FROM B2?

This research on B2 has several important implications. First, the results bolster the idea that inclusions are protective for cells. Second, promoting inclusion formation is an entirely novel therapeutic approach, with implications for many neurodegenerative diseases. Finally, the discovery of compounds that promote inclusion formation or block aggregation provides tools for new insight into the Htt aggregation process. Each of these compounds produces its effects by interacting with a target protein, and affinity labeling of the compounds will allow us to discover the identity of these targets. The identification of the targets may provide surprises about the biology of the aggregation process. It may also provide new drug targets for future drug discovery efforts. However, finding the target of B2 will require discovering a more potent analog through structure activity relationship analysis. Once more potent analogs have been determined, we would like to test their effects in animal models of HD. The results of this study could have implications for preclinical studies of other potential HD drugs. If promoting inclusions is shown to be beneficial in animal models, then it may be more prudent to focus on other outcome measures, such as survival, motor function or brain weight, and not to give much weight to aggregate load.

DIFFERING THERAPEUTIC APPROACHES MAY PROVE EFFECTIVE

Ultimately, the therapeutic strategy for protein misfolding diseases must involve lowering the levels of toxic species, either by preventing them from forming in the first place, or by clearing them from the cell once formed. Aggregation inhibitors that act early on the aggregation pathway may prevent toxic species from forming, and allow cells to clear the mutant protein before it can do much damage. Alternatively, once toxic conformations have formed, chemicals that help cells sequester toxic folding intermediates into inclusions might be most helpful. Once the pathological species are trapped into inclusions and thus unable to inflict further damage on cells, cells may be able to recover and slowly degrade insoluble precipitates. We cannot predict which of these strategies will be more therapeutically effective. Complicating factors might include the nature of the disease proteins, the age of onset, and the duration of disease. Initial animal model studies suggest that aggregation inhibitors may not be therapeutic, even when these compounds reach high levels in the brain.27 To test if chemically induced sequestration of mutant proteins in inclusions is a viable therapeutic strategy, analogs of B2 www.landesbioscience.com

or similarly acting chemicals must be tested in animal models of misfolding diseases. References 1. Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: Destruction for the sake of construction. Physiol Rev 2002; 82:373-428. 2. McClellan AJ, Tam S, Kaganovich D, Frydman J. Protein quality control: Chaperones culling corrupt conformations. Nat Cell Biol 2005; 7:736-41. 3. Young JC, Agashe VR, Siegers K, Hartl FU. Pathways of chaperone-mediated protein folding in the cytosol. Nat Rev Mol Cell Biol 2004; 5:781-91. 4. Keller JN, Gee J, Ding Q. The proteasome in brain aging. Ageing Res Rev 2002; 1:279-93. 5. Soti C, Csermely P. Aging and molecular chaperones. Exp Gerontol 2003; 38:1037-40. 6. Ross CA, Poirier MA. Opinion: What is the role of protein aggregation in neurodegeneration? Nat Rev Mol Cell Biol 2005; 6:891-8. 7. Becher MW, Kotzuk JA, Sharp AH, Davies SW, Bates GP, Price DL, Ross CA. Intranuclear neuronal inclusions in Huntington’s disease and dentatorubral and pallidoluysian atrophy: Correlation between the density of inclusions and IT15 CAG triplet repeat length. Neurobiol Dis 1998; 4:387-97. 8. Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, Ross CA, Scherzinger E, Wanker EE, Mangiarini L, Bates GP. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 1997; 90:537-48. 9. DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin N. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 1997; 277:1990-3. 10. Ordway JM, Tallaksen-Greene S, Gutekunst CA, Bernstein EM, Cearley JA, Wiener HW, Dure LST, Lindsey R, Hersch SM, Jope RS, Albin RL, Detloff PJ. Ectopically expressed CAG repeats cause intranuclear inclusions and a progressive late onset neurological phenotype in the mouse. Cell 1997; 91:753-63. 11. Cummings CJ, Reinstein E, Sun Y, Antalffy B, Jiang Y, Ciechanover A, Orr HT, Beaudet AL, Zoghbi HY. Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice. Neuron 1999; 24:879-92. 12. Gutekunst CA, Li SH, Yi H, Mulroy JS, Kuemmerle S, Jones R, Rye D, Ferrante RJ, Hersch SM, Li XJ. Nuclear and neuropil aggregates in Huntington’s disease: Relationship to neuropathology. J Neurosci 1999; 19:2522-34. 13. Klement IA, Skinner PJ, Kaytor MD, Yi H, Hersch SM, Clark HB, Zoghbi HY, Orr HT. Ataxin-1 nuclear localization and aggregation: Role in polyglutamine-induced disease in SCA1 transgenic mice. Cell 1998; 95:41-53. 14. Kuemmerle S, Gutekunst CA, Klein AM, Li XJ, Li SH, Beal MF, Hersch SM, Ferrante RJ. Huntington aggregates may not predict neuronal death in Huntington’s disease. Ann Neurol 1999; 46:842-9. 15. Saudou F, Finkbeiner S, Devys D, Greenberg ME. Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell 1998; 95:55-66. 16. Slow EJ, Graham RK, Osmand AP, Devon RS, Lu G, Deng Y, Pearson J, Vaid K, Bissada N, Wetzel R, Leavitt BR, Hayden MR. Absence of behavioral abnormalities and neurodegeneration in vivo despite widespread neuronal huntingtin inclusions. Proc Natl Acad Sci USA 2005; 102:11402-7. 17. Tanaka M, Kim YM, Lee G, Junn E, Iwatsubo T, Mouradian MM. Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective. J Biol Chem 2004; 279:4625-31. 18. Taylor JP, Tanaka F, Robitschek J, Sandoval CM, Taye A, Markovic-Plese S, Fischbeck KH. Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein. Hum Mol Genet 2003; 12:749-57. 19. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 2004; 431:805-10. 20. Iwata A, Christianson JC, Bucci M, Ellerby LM, Nukina N, Forno LS, Kopito RR. Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation. Proc Natl Acad Sci USA 2005; 102:13135-40. 21. Rideout HJ, Lang-Rollin I, Stefanis L. Involvement of macroautophagy in the dissolution of neuronal inclusions. Int J Biochem Cell Biol 2004; 36:2551-62. 22. Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, Stefani M. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 2002; 416:507-11. 23. Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 2003; 300:486-9. 24. Apostol BL, Kazantsev A, Raffioni S, Illes K, Pallos J, Bodai L, Slepko N, Bear JE, Gertler FB, Hersch S, Housman DE, Marsh JL, Thompson LM. A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila. Proc Natl Acad Sci USA 2003; 100:5950-5. 25. Heiser V, Engemann S, Brocker W, Dunkel I, Boeddrich A, Waelter S, Nordhoff E, Lurz R, Schugardt N, Rautenberg S, Herhaus C, Barnickel G, Bottcher H, Lehrach H, Wanker EE. Identification of benzothiazoles as potential polyglutamine aggregation inhibitors of Huntington’s disease by using an automated filter retardation assay. Proc Natl Acad Sci USA 2002; 99:16400-6.

Cell Cycle

1479


26. Heiser V, Scherzinger E, Boeddrich A, Nordhoff E, Lurz R, Schugardt N, Lehrach H, Wanker EE. Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: Implications for Huntington’s disease therapy. Proc Natl Acad Sci USA 2000; 97:6739-44. 27. Hockly E, Tse J, Barker AL, Moolman DL, Beunard JL, Revington AP, Holt K, Sunshine S, Moffitt H, Sathasivam K, Woodman B, Wanker EE, Lowden PA, Bates GP. Evaluation of the benzothiazole aggregation inhibitors riluzole and PGL-135 as therapeutics for Huntington’s disease. Neurobiol Dis 2006; 21:228-36. 28. Wang J, Gines S, MacDonald ME, Gusella JF. Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation. BMC Neurosci 2005; 6:1. 29. Zhang X, Smith DL, Meriin AB, Engemann S, Russel DE, Roark M, Washington SL, Maxwell MM, Marsh JL, Thompson LM, Wanker EE, Young AB, Housman DE, Bates GP, Sherman MY, Kazantsev AG. A potent small molecule inhibits polyglutamine aggregation in Huntington’s disease neurons and suppresses neurodegeneration in vivo. Proc Natl Acad Sci USA 2005; 102:892-7. 30. Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med 2004; 10:S10-7. 31. Bodner RA, Outeiro TF, Altmann S, Maxwell MM, Cho SH, Hyman BT, McLean PJ, Young AB, Housman DE, Kazantsev AG. Pharmacological promotion of inclusion formation: A therapeutic approach for Huntington’s and Parkinson’s diseases. Proc Natl Acad Sci USA 2006; 103:4246-51. 32. Bence NF, Sampat RM, Kopito RR. Impairment of the ubiquitin-proteasome system by protein aggregation. Science 2001; 292:1552-5. 33. Bennett EJ, Bence NF, Jayakumar R, Kopito RR. Global impairment of the ubiquitinproteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol Cell 2005; 17:351-65. 34. Holmberg CI, Staniszewski KE, Mensah KN, Matouschek A, Morimoto RI. Inefficient degradation of truncated polyglutamine proteins by the proteasome. EMBO J 2004; 23:4307-18. 35. Jana NR, Zemskov EA, Wang G, Nukina N. Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release. Hum Mol Genet 2001; 10:1049-59. 36. Venkatraman P, Wetzel R, Tanaka M, Nukina N, Goldberg AL. Eukaryotic proteasomes cannot digest polyglutamine sequences and release them during degradation of polyglutaminecontaining proteins. Mol Cell 2004; 14:95-104. 37. Verhoef LG, Lindsten K, Masucci MG, Dantuma NP. Aggregate formation inhibits proteasomal degradation of polyglutamine proteins. Hum Mol Genet 2002; 11:2689-700. 38. Ross CA, Pickart CM. The ubiquitin-proteasome pathway in Parkinson’s disease and other neurodegenerative diseases. Trends Cell Biol 2004; 14:703-11. 39. Gilon T, Chomsky O, Kulka RG. Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae. EMBO J 1998; 17:2759-66. 40. Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Hulihan M, Peuralinna T, Dutra A, Nussbaum R, Lincoln S, Crawley A, Hanson M, Maraganore D, Adler C, Cookson MR, Muenter M, Baptista M, Miller D, Blancato J, Hardy J, Gwinn-Hardy K. α-Synuclein locus triplication causes Parkinson’s disease. Science 2003; 302:841. 41. McLean PJ, Kawamata H, Hyman BT. α-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons. Neuroscience 2001; 104:901-12. 42. Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin RC, Di Iorio G, Golbe LI, Nussbaum RL. Mutation in the α-synuclein gene identified in families with Parkinson’s disease. Science 1997; 276:2045-7. 43. Muchowski PJ, Wacker JL. Modulation of neurodegeneration by molecular chaperones. Nat Rev Neurosci 2005; 6:11-22.

1480

Cell Cycle

2006; Vol. 5 Issue 14