MINIREVIEW Huntington’s disease: revisiting the aggregation hypothesis in polyglutamine neurodegenerative diseases Ray Truant, Randy Singh Atwal, Carly Desmond, Lise Munsie and Thu Tran Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada Keywords huntingtin; Huntington’s disease; polyglutamine; protein aggregation; protein misfolding; Spinocerebellar ataxia Correspondence R Truant, Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, HSC 4H24A, Hamilton, Ontario L8N3Z5, Canada Fax: +1 905 522 9033 Tel: +1 905 525 9140 ext 22450 E-mail: truantr@mcmaster.ca Website: http://www.RayTruantLab.ca After the successful cloning of the first gene for a polyglutamine disease in 1991, the expanded polyglutamine tract in the nine polyglutamine disease proteins became an obvious therapeutic target Early hypotheses were that misfolded, precipitated protein could be a universal pathogenic mechanism However, new data are accumulating on Huntington’s disease and other polyglutamine diseases that appear to contradict the toxic aggregate hypothesis Recent data suggest that the toxic species of protein in these diseases may be soluble mutant conformers, and that the protein context of expanded polyglutamine is critical to understanding disease specificity Here we discuss recent publications that define other important therapeutic targets for polyglutamine-mediated neurodegeneration related to the context of the expanded polyglutamine tract in the disease protein (Received March 2008, revised 21 April 2008, accepted 12 May 2008) doi:10.1111/j.1742-4658.2008.06561.x The toxic aggregate hypothesis in polyglutamine diseases With the identification of expanded CAG repeats of the X-linked spinal and bulbar muscular atrophy (SBMA or Kennedy’s disease) gene at the androgen receptor in 1991 [1], followed by the Huntington’s disease (HD) gene in 1993 [2], and the cloning of the spinocerebellar ataxia type gene [3], the expanded polyglutamine tract as the result of a CAG DNA expansion became the focus of intense interest to investigators in these diseases Two seminal papers appeared near that time that presented hypotheses concerning the pathogenic mechanism of polyglutamine expansion One was from Nobel laureate Max Perutz, demonstrating the concept of polyglutamine ‘polar zipper’ interactions with the side groups of glutamine residues [4] Perutz focused on the fact that the genetics of some (but not all) polyglutamine diseases demonstrated that the minimal length of polyglutamine expansion required for disease was 37 repeats, and that a repeat length beyond 37 led to earlier disease onset That paper demonstrated that polyglutamine alone was toxic to Escherichia coli and Chinese hamster ovary cells, and concluded that polyglutamine had the ability to adopt a pleated b-sheet structure that could cause a displacement of water molecules and hence render the protein insoluble This theory was consistent with the genetic gain-of-function seen with mutant proteins in HD, in the ataxin-1 protein in spinocerebellar ataxia (SCA) type 1, and other polyglutamine diseases Polar zippers were predicted to form tighter interactions with increasing polyglutamine length, thus potentially affecting the severity of disease Abbreviations AR, androgen receptor; FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonant energy transfer; GFP, green fluorescent protein; HD, Huntington’s disease; NLS, nuclear localization signal; SCA, spinocerebellar ataxia; SMBA, spinal and bulbar muscular atrophy; YAC, yeast artificial chromosome 4252 FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS R Truant et al Consistent with this hypothesis, aggregates of protein are not seen in proteins expressing polyasparagine, an amino acid that differs from glutamine by only one methyl group [5] Although what exactly polyglutamine aggregates were doing to trigger toxicity was not hypothesized by the authors, they did conclude that this toxic property was universal to all cell types and species The second seminal paper concerning the prediction of aggregation of polyglutamine disease proteins was the report on the first HD model mouse using transgenic insertion technology [6] For this study, the authors expressed the first exon of mutant human huntingtin as a transgene in the mouse, thus expressing the expanded polyglutamine tract The resultant ‘R6 ⁄ 2’ mouse lines developed severe disease in as little as weeks, and obvious movement disorders that resembled the chorea seen in HD, as well as some brain mass loss and total body weight loss Brain slice imaging from these mice revealed the abundance of ubiquitinrich inclusions of huntingtin fragments in many areas of the brain, suggesting that these inclusions may be the toxic trigger of cell death and dysfunction leading to the HD-like phenotype in these mice As a result of these two papers, HD research was focused on what the gain-of-function was of the polyglutamine aggregates Published work on this small fragment of huntingtin has implicated its role in sequestering important proteins in aggregates [7,8], blocking cell vesicle trafficking [9], inhibiting proper proteasome function [10], and toxic titration of chaperones away from the rest of the cell [11] The important distinction of this work is that they define mutant huntingtin aggregates as static, misfolded, precipitated proteins that the cell clearance machinery has a problem in dealing with The central theme is that the toxic nature of huntingtin depends upon the formation of protein ‘aggregates’ Although these ubiquitin-rich inclusions are evident in the huntingtin exon mouse models and other small-fragment HD models [12,13], they can become cleared in conditional expression models correcting the disease phenotype to normal, for both huntingtin exon [13] and SCA1 [14] models The conditional expression models are the most promising for treatment of these diseases, implying that even at the point of severe phenotypic manifestation, the toxic effects can be reversed by stopping production of the mutant protein, either by the alleviation of dysfunction in neurons, or through the brain’s inherent plasticity Protein aggregation in neurodegenerative disease is not unique to polyglutamine diseases, and is a common theme with other amyloid diseases, including transmitted spongiform encephalopathies, Parkinson’s Revisiting the aggregation hypothesis disease, and Alzheimer’s disease [15] Polyglutamine diseases have often been historically considered as amyloid diseases New models, new insights One problem with huntingtin exon mouse models is that these models express only a fragment of mutant huntingtin protein that comprises roughly 3% of the total protein, and controls for observations in this mouse model are difficult, as a wild-type exon transgenic mouse is not typically used, and controls related to the positional effects of transgene insertion in genomic DNA are difficult to construct More genetically accurate huntingtin mouse models now exist that express the polyglutamine expansion in a full-length (3144 amino acid) context, with control wild-type length strains, using a wide variety of technologies, including: yeast artificial chromosomes (YACs) [16]; human CAG expansion knock-in to the mouse huntingtin allele [17]; conditional mutant huntingtin knock-outs [18,19]; and expanded polyglutamine knock-in to the mouse huntingtin allele [20] The phenotypes of these mice are generally much more attenuated, with little impact on animal longevity at years The incidence of visible aggregates is much lower, and aggregates cannot be detected in the early stages of disease in the mouse when there are measurable phenotypic changes as compared to wild-type mice In the absence of any early biomarkers for HD to date, the huntingtin exon model is still the mouse model in use for drug development, due to the relatively fast and severe phenotype In full-length huntingtin HD genetic mouse models, aspects of the disease phenotype seem more similar to the human disease, with the exception of specific striatal cell loss These models caused a rethinking of aggregates in polyglutamine disease, raising the possibility that whereas they can be seen in induced disease models and HD brains, they may not be the pathogenic trigger of disease One of the conceptual problems regarding the pathology of aggregates in exon models is that the pathogenic mechanisms implied not explain disease specificity in certain neuronal populations Many of the polyglutamine disease proteins are expressed in many cell types, even outside the brain, but pathology is typically restricted to specific cell loss in a few brain areas The most striking example of this is in SCA17, where the affected protein is the TATA box-binding protein, which is ubiquitously expressed and required for RNA polymerase II transcription initiation at most promoters, but only manifests as ataxia when expanded beyond 60 repeats FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS 4253 Revisiting the aggregation hypothesis R Truant et al [21] SCA17 challenges many aspects of the hypotheses concerning polyglutamine toxicity, as TATA box-binding protein has normal polymorphic polyglutamine tract lengths that can exceed 40 repeats with no disease, and is a normal nuclear protein The manifestation of the nine specific human diseases challenges the concept that expanded polyglutamine expression alone is toxic to all cells Unfortunately, to the nonexpert, understanding the field of polyglutamine diseases can be hampered by inconsistent and inaccurate terminology Huntingtin exon model system studies often conclude that effects are observed solely due to polyglutamine, and imply similar mechanisms in other polyglutamine diseases, but are rarely actually tested ‘Polyglutamine’ is often mislabeled mutant exon huntingtin, and the term ‘aggregates’ can actually refer to any puncta of inclusions of polyglutamine-containing protein, whether proven to be misfolded or not This is an important distinction, given the role of huntingtin in vesicular interactions [22,23] Even the term ‘huntingtin’ is often inaccurately used when only the exon fragment has been tested, leading to the assumption that all properties of exon huntingtin can be attributed to full-length huntingtin in HD One conceptual milestone that investigators will have to deal with is whether all the related pathology in HD can be recapitulated with only the first exon fragment of this protein, and that the remaining 97% of the protein may not be relevant to this disease Polyglutamine and protein context One of the first groups to design elegant, proofof-principle experiments in the mouse to test the universal toxicity of expanded polyglutamine was the long-term collaboration of the Orr and Zoghbi laboratories on SCA1 mouse models In both HD and SCA1, inclusions of polyglutamine-expanded protein can be seen within nuclei Orr’s group defined the nuclear localization signal (NLS) in ataxin-1 protein, inactivated it by point mutation, and expressed this NLS mutant (Q84) ataxin-1 in the mouse [24] The mice did not develop any disease, despite high expression of NLS mutant (Q84) ataxin-1 in the cerebellum Thus, two important conclusions could be drawn from this model: that expression of expanded polyglutamine in the mouse brain was in itself not sufficient for degeneration; and that the normal function of the polyglutamine disease protein probably contributed to the disease pathology This work was extended further by the definition of a phosphoserine near the NLS in ataxin-1 at position 776 that, when mutated to alanine, also did not lead to disease, but still allowed 4254 nuclear entry of polyglutamine-expanded ataxin-1 [25] Thus, nuclear localization of polyglutamine is not in itself sufficient to cause disease, and, perhaps of greatest interest to the polyglutamine diseases community, a serine kinase signaling pathway could modulate the toxicity of SCA1, defining another, potentially better drug target for a polyglutamine disease outside of the polyglutamine tract This single serine mutant also affected the ability of ataxin-1 to form nuclear inclusions, suggesting that functions in the host protein could affect the inclusion or aggregation ability of that protein The concept of targeting protein function for a polyglutamine disease is best illustrated with SBMA or Kennedy’s disease and the polyglutamine-expanded protein androgen receptor (AR) [26] Males with SBMA typically exhibit more severe disease than sibling females, owing to higher levels of circulating testosterone, leading to increased nuclear signaling of the AR Male mice treated with the gonadotropinreleasing hormone antagonist leuprorelin showed reduced levels of circulating testosterone and a dramatic decrease in the SBMA-like phenotype, a result that has now directly translated to the clinic with treatment of SBMA patients [27] Thus, SBMA represents a success story for the therapeutic development of treatment that does not target polyglutamine and aggregation, but targets the well-described known function of the AR SCA1 and SBMA are two striking examples of the importance of the protein context of polyglutamine mediating its toxic effects But what of universal polyglutamine toxicity? A major aspect of polyglutamine-mediated toxicity that was not considered in early biochemical work, and in typical longer-term cell overexpression models in HEK293, CHO, or Cos7 cell lines, is the level of huntingtin exon fragment required to see effects, typically in these cell lines orders of magnitude in molarity above the levels of endogenous huntingtin This is particularly evident in biochemical studies in vitro In tissue culture cell models with typical very strong cytomegalovirus-promoted expression vectors and relatively large amounts of protein expressed (relative to endogenous huntingtin), quantifiable in vivo by green fluorescent protein (GFP) fusions, the incidence of visible aggregates of mutant huntingtin fragments decreases dramatically with the increased length of huntingtin protein the expanded polyglutamine tract is expressed within Whereas visible aggregates are very frequent with huntingtin 1–81 or 1–171 fragment expression, they not appear in the context of larger huntingtin fragments, regardless of expression levels (J Xia, McMaster University, unpublished observations) FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS R Truant et al Revisiting the aggregation hypothesis What are the spots in polyglutamine diseases? If polyglutamine-expanded proteins form insoluble, static and precipitated protein, then quantitative biophysical methods such as fluorescence recovery after photobleaching (FRAP) in living cells could establish that once polyglutamine-expanded protein enters an inclusion, it does not exit, consistent with the original aggregate hypothesis Three groups, including ours, have independently used FRAP in the context of mutant huntingtin exon 1, ataxin-1 and ataxin-3 proteins Some polyglutamine-expanded proteins in puncta can exchange back to the soluble phase, others appear to be static and sequester soluble protein, and some can move from inclusion to inclusion [7,28,29] Thus, the effect of polyglutamine expansion on protein dynamics is not universal for all proteins This suggests that a third species of soluble, mutant protein can exist, and that this protein can exist in both the soluble and insoluble states and move between those two states (Fig 1) FRAP studies also confirm that fusions of GFP to polyglutamine disease proteins are not misfolded when in inclusions, as they continue to fluoresce quantitatively as protein is localized to the inclusions, even when in excess of lm in diameter In the case of ataxin-1, normal ataxin-1 function dictates the formation of nuclear ataxin bodies, which exist even in the complete absence of the polyglutamine tract [28] Ataxin-1 inclusion formation is dictated by signaling and post-translational phosphorylation of a single serine in ataxin-1 at position 776, regardless of polyglutamine tract length [25] These live cell dynamic observations and mouse model data obtained with ataxin-1 are inconsistent with the hypothesis that polyglutamine has a universal effect on protein misfolding and insolubility, rendering all proteins ‘amyloid’ Another inconsistency with the amyloid hypothesis for HD is in a YAC mouse model of HD that resulted from a cloning artefact that was carefully characterized The ‘shortstop’ mouse expressed only 120 amino acids of huntingtin on a YAC, or roughly 35 amino acids beyond exon in a polyglutamine-expanded context, and displayed large visible aggregates throughout the brain, but this mouse had no measurable disease [30] The corresponding full-length mutant huntingtin YAC construct does show a slow, progressive HD-like phenotype, but without large visible aggregates [16] These models demonstrate that with HD, as with SCA1, other sequences within the polyglutamine disease protein may be able to modulate toxicity, but that the formation of aggregates is not necessarily correlated with disease Correlation between aggregates and toxicity The connection between visible protein aggregates and polyglutamine diseases has been largely circumstantial In human brains, the incidence of aggregates is impos- Bleach area Ataxin-1 Q82-GFP A C 30 s D B 60 s E 120 s F 240 s 480 s G Loss Gain Fig Polyglutamine-expanded protein can exist in two reversible states FRAP experiment with overexpressed ataxin-1–GFP All of the protein is bleached except for one mutant ataxin-1 body in the nucleus Gain of fluorescence is first seen in the same inclusions bleached prior to recovery closest to the unbleached inclusion; the corresponding loss of fluorescence over time is seen in the unbleached inclusion Thus, polyglutamine-expanded mutant ataxin-1 can move from one inclusion of highly concentrated protein to another through a soluble phase FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS 4255 Revisiting the aggregation hypothesis R Truant et al sible to follow with disease in any one individual, although increased aggregates are noted in more severe stages or grades of HD [31,32] In order to directly follow the fate of individual neurons expressing a small fragment of polyglutamine-expanded huntingtin, Arrasate and colleagues transfected huntingtin exon 1– GFP expression plasmids in primary neuronal cultures, and used robotic 4D fluorescent microscopy to track the fate of single cultured neurons over time, imaging them repeatedly [33] From this work, they observed an inverse correlation between huntingtin exon fragment inclusion size and cell death; that is, the larger the aggregate, the more likely the neuron was to survive longer than a neuron expressing mutant huntingtin without any visible aggregates This work took advantage of recent technology and trends in cell biology towards quantitative measurement of effects This data thus indicated that large aggregates of huntingtin fragments may constitute a cellular protective mechanism to localize the toxic soluble mutant protein to insoluble and inactive protein reservoirs (Fig 2) This localization to large inclusions may also contribute to the loss-of-function seen in HD [34], whereas the soluble mutant protein can participate in normal protein functions with an additional gain-of-function We know that mutant huntingtin protein can assume the functions of wild-type protein, as it can lead to normal development in mutant homozygous mice and humans [17] The concept of neuroprotection of aggregates of polyglutamine disease proteins is not limited to HD In SCA7, two groups independently showed an inverse correlation of aggregate formation of ataxin-7 with toxicity, both in cultured neurons and in a mouse model [35,36] Ataxin-7 has a known role as a component of the transcription mediator complex known as STAGA, and when polyglutamine-expanded, can affect the proper recruitment and composition of this complex [37] Therefore, with ataxin-7, it is likely that the toxic version of the protein is not that found in 36 Repeats er In on t? ti er c In -fun f -o ss Lo Gain of structure Gain of toxicity Biological or chemical modulators Highly stable structure highly toxic protein Fig Polyglutamine expansion lengths may disrupt the equilibria between toxic and healthy protein and between toxic soluble species and inert insoluble species Polyglutamine lengths beyond 37 repeats in HD are predicted to form a structure leading to gain of toxic function Mutant protein can exist in three states: soluble and without structure (healthy); soluble with a structure leading to gain of toxic function; and insoluble with a structure leading to loss of normal function Longer expansion lengths can skew this equilibrium to essentially two conformers, either loss-of-function or gain-of-function, both contributing to the manifestation of disease Biological or chemical modulators are able to skew equilibria in vivo, suggesting that the optimal modulator may be a molecule that can push all mutant protein into the insoluble, unstructured and hence inert state This modulator may not necessarily need to interact with polyglutamine, and may be different for different protein contexts related to biological functions 4256 FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS R Truant et al aggregates, but rather the soluble mutant protein that is able to participate in complexes with STAGA to exert dominant effects over wild-type protein In SCA2, although aggregates can be seen in a small number of neurons, they are not seen within the nucleus, as they are in HD or SCA1 [38] In SCA3 ⁄ Machado–Joseph disease, as in HD and SCA7, an inverse correlation is seen between nuclear inclusions of ataxin-3 protein and cell death, both by examination of brain slices [39,40] and in tissue culture models [41] These newer data can therefore allow us to revisit the early brain pathology data obtained with HD patients from another perspective One of the hallmarks of HD in humans, but not as much in mouse models, is the striking loss of the striatum, and up to 30% of total brain mass, prior to death [31] In the neurons that remain to be seen post mortem, aggregates of huntingtin N-terminal fragments can be seen [32] One hypothesis was that these aggregates may be the cause of cell death, and when they were visualized, they were in neurons en route to death However, from a revisionist perspective, one can also hypothesize that these neurons may have survived longer than the missing striatal neurons, due to the presence of the aggregates The consideration of aggregates in HD follows many of the conundrums seen with polyglutamine diseases and the struggle to understand what is cause and what is effect in these diseases Hunting the elusive toxic polyglutamine conformer A thorough search of crystallographic databases reveals that polyglutamine tracts seen in a variety of normal cellular proteins are either annotated as ‘unstructured’ or have to be removed to facilitate crystallization Obtaining structural information on polyglutamine in proteins is technically difficult, as even wild-type polyglutamine lengths can tend to be insoluble at the high concentrations required for crystallographic or NMR studies Wetzel’s group has focused on the identification of the toxic structure of polyglutamine Led by the antiparallel b-sheet model originally proposed by Perutz [4], they inserted proline–glycine substitutions in pure polyglutamine tracts to induce a b-strand structure, and found that even short lengths of polyglutamine could form aggregates similar to pure Q45 lengths when b-strands and b-turns were induced [42] The group of Ross then showed that these structured constructs were similarly toxic in primary cultured neurons and tissue culture models [43] This work led to the concept that the Revisiting the aggregation hypothesis genetic gain-of-function of polyglutamine could be tied to a gain of structure [44], but that this structural gain did not necessarily have to exert toxicity by the formation of aggregates Recently, Onodera’s group confirmed the parallel b-sheet model or cylindrical b-sheet of polyglutamine in atrophin-1 by the use of fluorescence resonant energy transfer (FRET) studies in vivo This FRET-based ‘spectroscopic ruler’ tool allowed the investigators to distinguish between soluble expanded polyglutamine oligomers, soluble monomer and inclusion bodies in live cells In neuronal cell culture toxicity assays, they demonstrated that the toxic species appeared to be soluble oligomers, and not the protein in aggregates [45] The caveat of this work is that the authors assume that polyglutamine in the context of atrophin-1 fragments has the same structure in all polyglutamine disease proteins, but given the importance of flanking sequences to polyglutamine structure, this model needs to be tested in other polyglutamine disease contexts Biophotonic methods such as FRET and fluorescence correlation spectroscopy have led, and will probably continue to lead, to major biochemical insights into polyglutamine folding in vivo [46] With small huntingtin fragments, many groups, including ours, have independently reported the importance of flanking sequences next to the polyglutamine tract in huntingtin exon as modulators of toxicity In the yeast toxicity model, the positioning of flag-tags on the expression constructs modulated toxicity and the nature of aggregated protein, with tight, compact aggregates being benign, but amorphous aggregates being much more toxic [47] Another group observed modulation of polyglutamine aggregation by the use of structured chimeras with the cellular retinoic-acid binding protein in E coli [48] Again revisiting the seminal Perutz paper [4], investigators have shown that the glutathione S-transferase fusion to polyglutamine does affect the aggregation dynamics, and may not be an innocuous purification tag, as it was once considered to be Aggregation may occur through formation of a reservoir of soluble intermediates whose populations and stabilities increase with polyglutamine length [49] However, these sequences were exogenous to huntingtin exon 1, and toxicity was not assayed in mammalian cells Deletion of the proline-rich region in huntingtin exon greatly increases the toxicity of exon fragments in yeast, which are otherwise innocuous [50] Therefore, the proline-rich region appears to be protective against the effects of expanded polyglutamine The effects of polyproline in cis, in vitro can be seen to affect the structure of expanded polyglutamine [51] FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS 4257 Revisiting the aggregation hypothesis R Truant et al The first 17 amino acids of huntingtin, prior to the polyglutamine tract, are highly conserved (100% similarity) in all vertebrate species, and were originally annotated as unstructured [4] However, by exhaustive mutational analysis in vivo and CD spectroscopy in vitro with peptides, our group has determined the first 17 amino acids to be an amphipathic a-helix, with membrane-associating properties with regard to the endoplasmic reticulum [23] Like the proline-rich tract, this region of huntingtin, present in all mouse models of HD, was shown to modulate the toxicity of Q138 huntingtin 1–171 in a structure-dependent manner A single point mutant in the middle of the helix, shown to disrupt the a-helical structure, resulted in three surprising phenotypes: constitutive nuclear entry of full-length huntingtin, or any huntingtin small N-terminal fragments; the complete abrogation of any visible aggregates of polyglutamine-expanded huntingtin 1–171, even in the context of 250 repeats; and a corresponding increase of toxicity of this fragment of huntingtin in a polyglutamine-dependent manner of close to four-fold over Q138 huntingtin 1–171 Thus, loss of structure in regions adjoining the polyglutamine tract on either side of the tract can lead to increased huntingtin toxicity, with an inverse correlation with aggregation These results predict that regions on either side of the polyglutamine tract in huntingtin may interact with each other, with a critical component of normal interaction being the flexible region of at least four glutamine residues seen in all vertebrate huntingtin proteins Huntingtin 1–17 and the prolinerich region adjacent to the polyglutamine tracts are both involved in targeting vesicular populations [23,52] In HD, the gain-of-structure may perturb huntingtin functions in vesicular trafficking by a ‘rusty hinge’ model, where important on–off interactions may be stuck on or off by the structure gained as a result of polyglutamine expansion (Fig 3) Similar models may apply to other polyglutamine disease proteins, with different consequences Basic residues in the ataxin-3 protein form an interaction motif with VCP ⁄ p97 protein, and this interaction can modulate ataxin-3 aggregation and toxicity in Drosophila models [53] Serine mutations in the N-terminus of the AR can modulate polyglutamineexpanded AR’s ability to aggregate, with increased aggregation but less toxicity being seen in a Drosophila model [54] Thus, many different sequences flanking polyglutamine tracts can affect polyglutamine tractmediated toxicity and the potential to form aggregates The importance of the structure on either side of an expanded polyglutamine tract may be due to imprinting of structure on polyglutamine by adjoining 4258 Fig The ‘rusty hinge’ hypothesis of gain of structure leading to toxic function in HD We speculate that there is an overall superhelical structure of huntingtin, owing to the large number of HEAT repeats throughout the entire 3144 amino acid protein The normal polyglutamine tract, present in all vertebrates with least four glutamines, provides an important flexible region in the huntingtin scaffold for factors that can interact with the first 17 amino acids and downstream regions With increasing polyglutamine lengths, the pool of total mutant protein is skewed towards b-sheet structured polyglutamine, leading to a loss of flexibility and the ability of huntingtin 1–17 to interact with the rest of huntingtin via factors or complexes Normal interactions that should switch on or off will then be stuck in either the on or off position or pools of either position, both of which may be toxic Normal interaction between the proline-rich region and huntingtin 1–17 influences the structure of expanded polyglutamine in cis, leading to increased toxicity if the normal structures of these regions are disrupted sequences that interact with the flexible polyglutamine tract in cis This is consistent with peptides or small molecules in trans that are able to mediate the aggregation potential of polyglutamine tracts and skew the equilibrium distribution of polyglutamine-expanded protein towards soluble or insoluble Some of the factors that may be able to affect this equilibrium may include normal interacting proteins, such as chaperones, or the HYPK protein interaction with huntingtin’s N-terminus modulating its ability to form aggregates [55,56] Modifiers of polyglutamine structure and toxicity Even if large visible ‘aggregates’ are not the actual targets of therapeutic development in HD and other polyglutamine diseases, proteins, small molecules or other factors that affect polyglutamine-dependent aggregation may have important effects on the toxic soluble species of polyglutamine-expanded proteins Early high-throughput (biochemical) assays used filtertrapped aggregates as the readout for screening of small molecules Benzothiazole compounds were FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS R Truant et al identified as being able to prevent aggregation or solubilize aggregates [57] The small molecule C2–8 was identified from high-content screening (cell biological) as an inhibitor of polyglutamine aggregate growth [58], but its efficacy in mouse models was modest, despite it crossing the blood–brain barrier effectively [59] One surprising finding from a FRET-based high-content screen of a kinase inhibitor library is that the Rho kinase inhibitor, Y-27632, could prevent huntingtin exon fragment-mediated aggregation [60] What is not known is what the mechanism of this inhibition is, but Rho kinase inhibition suggests that other functions of huntingtin exon fragment, perhaps in actin association, may be necessary for the formation of aggregates These classes of small molecules that affect huntingtin aggregation now allow cell biologists to use these molecules as tools of ‘chemical biology’ in the study of huntingtin function and mutant huntingtin pathology One of the strongest lines of evidence for a soluble oligomeric or misfolded toxic species of polyglutamine, and effects of peptides in trans, comes from the studies of the polyglutamine-binding trytophan-rich peptide QBP1 (WKWWPGIF) Although it was originally described as a suppressor of polyglutamine-mediated toxicity through the suppression of aggregation [61,62], more detailed studies have shown that this peptide can inhibit the transition of polyglutamine from an unstructured state to the toxic soluble b-sheet monomer structure [63], consistent with independent work on the b-sheet structure of polyglutamine from many other groups Another look at the amyloid hypothesis In the past, it has been tempting to place polyglutamine diseases into the category of amyloid diseases, a family of neurodegenerative disorders caused by misfolded proteins leading to large protein ultrastructures within or outside affected neurons However, recent research evidence from Alzheimer’s and Parkinson’s diseases is starting to cast doubt on the universality of the amyloid hypothesis in those diseases as well In Alzheimer’s disease, genetic mutations in familial Alzheimer’s disease reveal that Alzheimer’s disease in those cases may be caused by a redox imbalance, leading to the effect of amyloid plaques [64] In Parkinson’s disease, a-synuclein accumulation, like mutant huntingtin aggregation, can be seen to be neuroprotective [65] Small molecules that encourage aggregation appear to be effective in toxicity assays for many amyloid diseases and HD [66,67] Although it now appears that understanding Revisiting the aggregation hypothesis polyglutamine disease probably cannot be achieved without the disease protein context, important lessons have been learned from huntingtin small-fragment models and studies focusing on the toxic species of polyglutamine in different disease contexts Proof-ofconcept successes with SCA1 pointing to serine kinase inhibition as a therapeutic strategy, and clinical success with the treatment of SBMA by leuprorelin, underscore the importance of analysis of huntingtin toxicity in the full protein context and the importance of elucidating the normal biological function of huntingtin From that milestone, HD researchers can then have a new vantage point from which to consider alternative or coincident therapeutic strategies related to huntingtin function along with antiaggregation compounds The hallmark of any good drug is selective toxicity for its target, and thus expanded polyglutamine remains a valid target in polyglutamine diseases, with the appeal that drug toxicity will be specific to the mutant, and not wildtype, protein Acknowledgements The Truant laboratory is supported by current and past grants from the Hereditary Disease Foundation, (HDF) USA, the Cure Huntington’s Disease Initiative (CHDI) USA, the Huntington’s disease Society of America (HDSA), the Huntington’s Society of Canada and the Canadian Institutes of Health Research (CIHR), Genetics and Mental Health and Addiction Institutes R Truant is Chair of the Huntington’s disease Society of Canada (HSC) scientific advisory board References La Spada AR, Wilson EM, Lubahn DB, Harding AE & Fischbeck KH (1991) Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy Nature 352, 77–79 The Huntington’s Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes Cell 72, 971–983 Orr HT, Chung MY, Banfi S, Kwiatkowski TJ Jr, Servadio A, Beaudet AL, McCall AE, Duvick LA, Ranum LP 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formation: a therapeutic approach for Huntington’s and Parkinson’s diseases Proc Natl Acad Sci USA 103, 4246–4251 FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS ... 2008 The Authors Journal compilation ª 2008 FEBS R Truant et al Revisiting the aggregation hypothesis What are the spots in polyglutamine diseases? If polyglutamine- expanded proteins form insoluble,... with the original aggregate hypothesis Three groups, including ours, have independently used FRAP in the context of mutant huntingtin exon 1, ataxin-1 and ataxin-3 proteins Some polyglutamine- expanded... polyglutamine from many other groups Another look at the amyloid hypothesis In the past, it has been tempting to place polyglutamine diseases into the category of amyloid diseases, a family of neurodegenerative