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
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 1 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 polygluta-
mine 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 dis-
eases demonstrated that the minimal length of polyglu-
tamine expansion required for disease was 37 repeats,
and that a repeat length beyond 37 led to earlier dis-
ease onset. That paper demonstrated that polygluta-
mine 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

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 con-
text of the expanded polyglutamine tract in the disease protein.
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
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 trans-
genic insertion technology [6]. For this study, the
authors expressed the first exon of mutant human hun-
tingtin 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
3 weeks, and obvious movement disorders that resem-
bled 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 ubiquitin-

not unique to polyglutamine diseases, and is a com-
mon theme with other amyloid diseases, including
transmitted spongiform encephalopathies, Parkinson’s
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 1 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 1 trans-
genic mouse is not typically used, and controls related
to the positional effects of transgene insertion in geno-
mic 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 phe-
notypes of these mice are generally much more attenu-
ated, with little impact on animal longevity at 3 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

ease, and is a normal nuclear protein. The manifesta-
tion 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 1 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 1 huntingtin, and the term
‘aggregates’ can actually refer to any puncta of inclu-
sions 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 1 fragment has
been tested, leading to the assumption that all proper-
ties of exon 1 huntingtin can be attributed to full-length
huntingtin in HD. One conceptual milestone that inves-
tigators 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, proof-
of-principle experiments in the mouse to test the uni-
versal toxicity of expanded polyglutamine was the
long-term collaboration of the Orr and Zoghbi labora-

glutamine 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 gonadotropin-
releasing hormone antagonist leuprorelin showed
reduced levels of circulating testosterone and a dra-
matic decrease in the SBMA-like phenotype, a result
that has now directly translated to the clinic with treat-
ment 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 1 fragment required to see effects,
typically in these cell lines orders of magnitude in molar-
ity above the levels of endogenous huntingtin. This is
particularly evident in biochemical studies in vitro.In
tissue culture cell models with typical very strong cyto-
megalovirus-promoted expression vectors and relatively
large amounts of protein expressed (relative to endoge-

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 fluo-
resce quantitatively as protein is localized to the inclu-
sions, even when in excess of 5 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 poly-
glutamine tract length [25]. These live cell dynamic
observations and mouse model data obtained with
ataxin-1 are inconsistent with the hypothesis that poly-
glutamine 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 character-
ized. The ‘shortstop’ mouse expressed only 120 amino
acids of huntingtin on a YAC, or roughly 35 amino
acids beyond exon 1 in a polyglutamine-expanded con-
text, 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

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 1 frag-
ment 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 hun-
tingtin 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 contrib-
ute 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

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 con-
formers, 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.
Revisiting the aggregation hypothesis R. Truant et al.
4256 FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 inclu-
sions of ataxin-3 protein and cell death, both by exam-
ination 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 hall-
marks 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, aggre-
gates 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

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 solu-
ble expanded polyglutamine oligomers, soluble mono-
mer 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 polyglu-
tamine 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 impor-
tance of flanking sequences next to the polyglutamine
tract in huntingtin exon 1 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

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-ter-
minal fragments; the complete abrogation of any
visible aggregates of polyglutamine-expanded hun-
tingtin 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 compo-
nent of normal interaction being the flexible region of
at least four glutamine residues seen in all vertebrate
huntingtin proteins. Huntingtin 1–17 and the proline-
rich 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

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 filter-
trapped aggregates as the readout for screening
of small molecules. Benzothiazole compounds were
Fig. 3. The ‘rusty hinge’ hypothesis of gain of structure leading to
toxic function in HD. We speculate that there is an overall superhe-
lical 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 gluta-
mines, provides an important flexible region in the huntingtin scaf-
fold 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 hun-
tingtin 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 posi-
tion, 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.
Revisiting the aggregation hypothesis R. Truant et al.
4258 FEBS Journal 275 (2008) 4252–4262 ª 2008 The Authors Journal compilation ª 2008 FEBS
identified as being able to prevent aggregation or solu-
bilize aggregates [57]. The small molecule C2–8 was

Another look at the amyloid hypothesis
In the past, it has been tempting to place polygluta-
mine diseases into the category of amyloid diseases,
a family of neurodegenerative disorders caused by
misfolded proteins leading to large protein ultrastruc-
tures within or outside affected neurons. However,
recent research evidence from Alzheimer’s and Par-
kinson’s diseases is starting to cast doubt on the uni-
versality 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 accumula-
tion, like mutant huntingtin aggregation, can be seen
to be neuroprotective [65]. Small molecules that
encourage aggregation appear to be effective in toxic-
ity assays for many amyloid diseases and HD
[66,67]. Although it now appears that understanding
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-of-
concept successes with SCA1 pointing to serine
kinase inhibition as a therapeutic strategy, and clini-
cal success with the treatment of SBMA by leupro-
relin, underscore the importance of analysis of
huntingtin toxicity in the full protein context and the
importance of elucidating the normal biological func-

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