Oligomeric and polymeric aggregates formed by
proteins containing expanded polyglutamine
S. Iuchi*, G. Hoffner†‡, P. Verbeke†‡, P. Djian†, and H. Green*§
*Department of Cell Biology, Harvard Medical School, Boston, MA 02115; and†Unite ´ Propre de Recherche 2228, Centre National de la Recherche
Scientifique, Unite ´ de Formation et de Recherche Biome ´dicale, Universite ´ Rene ´-Descartes, 75006 Paris, France
Contributed by H. Green, December 16, 2002
polyglutamine (polyQ) are characteristically associated with insol-
uble neuronal inclusions, usually intranuclear, and neuronal death.
We describe here oligomeric and polymeric aggregates formed in
cells by expanded polyQ. These aggregates are not dissociated by
concentrated formic acid, an extremely effective solvent for oth-
cells by expanded polyQ can be completely dissolved in concen-
trated formic acid, but a soluble protein oligomer containing the
expanded polyQ and released by the formic acid is not dissociated
to monomer. In Huntington’s disease, a formic acid-resistant oli-
gomer is present in cerebral cortex, but not in cerebellum. Cortical
nuclei contain a polymeric aggregate of expanded polyQ that is
insoluble in formic acid, does not enter polyacrylamide gels, but is
retained on filters. This finding shows that the process of poly-
merization is more advanced in the cerebral cortex than in cultured
cells. The resistance of oligomer and polymer to formic acid
suggests the participation of covalent bonds in their stabilization.
taining an expanded series of glutamine repeats. Such a protein,
different in each disease, confers a dominant gain of function
whose most striking feature is the formation of insoluble aggre-
gates, largely confined to inclusions in neuronal nuclei. These
inclusions contain a variety of proteins, in addition to the mutant
protein bearing the expanded polyglutamine (polyQ).
Two general mechanisms of stabilization of such protein
aggregates have been proposed.
(i) Stabilization by noncovalent bonds: It was proposed by
Perutz and coworkers (4) that aggregates are stabilized by
hydrogen bonds between main-chain and side-chain amides of
expanded polyQ aligned in the form of a ?-sheet (polar zipper).
Experiments performed in numerous laboratories have given
some support to this interpretation, although it does not easily
explain the association of expanded polyQ with proteins not
(ii) Stabilization by covalent bonds: Studies of involucrin, an
epidermal protein that becomes incorporated into a polymeric
structure by the action of transglutaminase, revealed that the
protein is rich in glutamine (5) and frequently contains glu-
tamine repeats (6, 7). This finding led to the proposal that any
protein containing glutamine repeats could be a very good
glutamine donor in a transglutaminase-catalyzed reaction with
any protein whose lysine residues can participate as amine
solid-phase synthesis showed that increasing the number of
consecutive glutamine residues led to increased reactivity of
as many as 80% of the total glutamine residues participated in
the reaction (9). Experiments by others showed that the addition
of polyQ to the protein GST greatly increased its reactivity as a
transglutaminase substrate (10). Increasing the length of syn-
thetic polyQ increased the reactivity per Q residue in transglu-
taminase-catalyzed coupling to spermine (11). Our experiments
ennedy disease (1), Huntington’s disease (2), and a number
of other neuronal diseases (3) result from a protein con-
on full-length huntingtin containing glutamine repeats of dif-
ferent length showed that, as in the peptides, the activity of
huntingtin as a substrate for transglutaminase increased sharply
with length of the polyQ (12).
Numerous other reports have provided supporting evidence
linking diseases of polyQ to the action of transglutaminase (13–24).
The role of transglutaminase has also been challenged (25). What
is lacking to prove the role of transglutaminase in these diseases is
a direct demonstration of covalently bonded polymers in the
inclusions and ultimately the identification of the cross-linking
isodipeptide in the polymers. We describe here the presence, in the
inclusions of cultured cells making expanded polyQ and in the
cortical neurons in Huntington’s disease, of oligomers and polymer
resistant to dissociation by concentrated formic acid.
The use of concentrated formic acid as a solvent for proteins
insoluble in solutions of SDS appears to date from the work of
Mokrasch (26). The ability of concentrated formic acid to
dissolve aggregates containing polyQ was first demonstrated by
Hazeki and coworkers (27). They showed that Cos cells tran-
siently transfected with a construct expressing huntingtin exon 1,
encoding expanded polyglutamine, accumulated aggregates of
large molecular weight, which could be dissolved by a brief
treatment with 100% formic acid. After removal of the formic
acid by drying in vacuo, there remained, in addition to the
monomeric huntingtin fragment, an expanded polyQ-containing
protein of size thought to correspond to a dimer (27).
Construction of Cell Line PC12-Q205, Which Conditionally Produces
Expanded PolyQ. The plasmid pSG5hAR-M4 (28) contains a
synthetically expanded polyQ flanked by the residues adjacent to
the polyQ-containing region of the androgen receptor. This
construct was kindly provided by H. Nakajima (Osaka Medical
College, Osaka). The plasmid was altered to produce Met-(c-
Myc)-56AA-205gln-17AA-(6xHis), and then ligated to an up-
stream ecdysone-inducible promoter derived from pIND (In-
vitrogen), whose neomycin-resistance gene had been substituted
by a blastocidin-resistance gene. This final plasmid was desig-
nated as pIBQ205G.
PC12 (ATCC CRL-1721), derived from a pheochromocytoma
of the rat adrenal gland, was transfected with plasmid pVgRXR
(Invitrogen) and selected for growth in medium containing
Zeocin. From the culture, a single colony was isolated twice and
regrown. This line expressed the mammalian retinoid x-receptor
mRNA as highly as the EcR 293 cells of Invitrogen. The
zeocin-resistant PC12 clone was then transfected with
pIBQ205G and selected for growth in the medium with blasti-
cidin and Zeocin. After two consecutive colony isolations, a
single cell was isolated under direct vision and expanded to
obtain the line PC12-Q205, whose expression of Q205was induced
with ponasterone A.
Abbreviations: polyQ, polyglutamine; 2-ME, 2-mercaptoethanol.
‡G.H. and P.V. contributed equally to this work.
§To whom correspondence should be addressed. E-mail: email@example.com.
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Purification of Inclusions from Cultured Cells. PC12-Q205cells were
cultivated for 6 days in the presence of 10 ?M ponasterone A.
The cells were then collected, washed, and suspended in isotonic
Hepes buffer. The suspension was centrifuged and the pellet was
suspended with the aid of sonication in Tris?HCl buffer. DNase
I, RNase (Roche Applied Science), and 0.2% Triton X-100 were
added and incubated for 1 h at 37°C. A solution of 6.6 M
guanidine hydrochloride was added to the suspension with
stirring. The suspension was cleared by centrifugation at 800 ?
g, and the supernatant was then centrifuged at 72,000 ? g. The
pellet was washed once in 5 M guanidine and once in PBS,
suspended in a solution of 1.4 M NaCl containing 2% SDS and
5% 2-mercaptoethanol (2-ME), and boiled for 5 min. The
inclusions, which were resistant to all of these treatments, were
collected by centrifugation for 30 min at 100,000 ? g, washed,
and resuspended in water.
Analysis of Expanded PolyQ in the Brain of Patients with Huntington’s
Disease. Frozen brain samples from patients with juvenile Hun-
tington’s disease were obtained from the Harvard Brain Tissue
Resource Center, which is supported in part by Public Health
Service Grant MN?NS 31862. Patients were a 16-year-old, grade
3 female, 3123; a 14-year-old, grade 3 female, 3815; an 18-year-
old, grade 4 female, 3177; a 22-year-old, grade 4 female, 3482;
and a 28-year-old, grade 3 male 3535. The human lymphoblas-
huntingtin (Q17?Q79), was provided by the Banque d’ADN et
de Cellules de l’Unite ´ 289 de Institut National de la Sante ´ et de
la Recherche Me ´dicale (Ho ˆpital de la Pitie ´-Salpe ˆtrie `re, Paris).
Western blots. For analysis of huntingtin, extracts of cortex (50 ?g
of protein) and cerebellum (80 ?g of protein) were analyzed by
unless specified otherwise. Huntingtin was stained by an anti-
N-terminal antibody (30), diluted 1:1,500, and by the 1C2
antibody specific for expanded polyQ (31), diluted 1:2,500.
Isolation of nuclei. Briefly, the gray matter of cortex or cerebellum
was homogenized in isotonic sucrose buffer containing Brij. The
homogenate was filtered through a 70-?m nylon mesh. The nuclei
the supernatant was collected as a source of cytoplasmic proteins.
The crude nuclear pellet was resuspended and sedimented again
under the same conditions. The nuclei in the resulting pellet were
further purified by centrifugation through discontinuous gradients
of either iodixanol (32) or sucrose (33), using a modification of a
procedure described in ref. 34.
For scoring of inclusions, the crude homogenate was stained
with the anti-N-terminal antibody and Hoechst 33258. The
proportion of nuclei containing inclusions was determined by
Detection of polymer in nuclei isolated from cerebral cortex of Hunting-
ton’s disease by filter retention assay. After formic acid treatment of
isolated nuclei, undissolved polymer was deposited on cellulose
acetate, by using the well-known filter retention assay (35).
Quantitation of Oligomer in Cultured Cells and Oligomer and Polymer
in Cerebral Cortex of Huntington’s Disease by IR Fluorescence. Ni-
trocellulose membranes for Western blots and cellulose acetate
filters with retained polymer were incubated with 1C2 antibody,
then with Alexa Fluor 680-labeled goat anti-mouse IgG and
finally washed in Tris-buffered saline containing 0.05% Tween
20. This near IR fluorophore was excited at 680 nm and the
emission at 702 nm was quantitated in channel 700 of the
LI-COR Infrared Imaging System (Odyssey, Lincoln, NE).
Signal intensity was proportional to the amount of expanded
A complete description of the methods is available in Sup-
porting Methods, which is published as supporting information on
the PNAS web site, www.pnas.org.
Solubility in Concentrated Formic Acid of Peptides Containing PolyQ.
Peptides containing more than six consecutive Q residues made
by solid-phase synthesis become increasingly insoluble in water.
This is demonstrated for Q20and p(pyro)EA Q20IV (36) (Fig.
1). Each peptide was added in amounts sufficient to make a
2-mM solution in 100 ?l of water or of a solution containing SDS
and incubated for 5 min at 100°C. In both cases, most of the
peptide remained insoluble. The addition of formic acid to 90%,
instead of SDS, dissolved the peptides completely. The solvent
effect of formic acid was appreciable at concentrations of ?45%
and complete at 90% (Fig. 1).
It may be concluded that if the insolubility of peptides
containing polyQ is caused by the formation of hydrogen-
bonded ?-sheet, as proposed by Perutz et al. (4), or of secondary
bonds of some other kind (37), these bonds are dissociated in
90% formic acid. The resistance of any polyQ-containing ag-
gregate to 90% formic acid must be ascribed to stronger bonds.
For example, epidermal corneocyte envelopes are stabilized by
isopeptide cross-links of N?(?-glutamyl) lysine (38) introduced
by transglutaminase (39). We prepared cornified envelopes from
the tail and the ear of an adult mouse, by boiling the tissues in
a solution containing 2% SDS and 2% 2-ME (40). The envelopes
were then sedimented and incubated in 94% formic acid for 2 h
at 37°C (106envelopes per 200 ?l). No appreciable difference
either in the number or the morphology of the envelopes was
detected after incubation in the formic acid. In what follows, we
will designate as oligomer an aggregate of polyQ that is soluble
but not converted to monomer by concentrated formic acid and
resolved as a band of higher molecular weight in gel electro-
phoresis. We designate as polymer a larger aggregate that is
insoluble in formic acid and unable to pass through a cellulose
acetate filter of pore size 0.2 ?m.
Synthesis of expanded polyQ by PC12-Q205cells was initiated
with ponasterone A. At different times, samples of the cells were
stained with antibody to tetra-His, present in the C-terminal tag,
and with antibody 1C2 (Fig. 2).
added to water and a solution of 2% SDS in an amount sufficient to make a
2-mM solution. The suspensions were centrifuged at 14,000 rpm for 5 min in
an Eppendorf centrifuge. Any sediment was resuspended in water, and an
aliquot was allowed to evaporate on a glass plate. The residue on the plate
was photographed against a black background with overhead illumination.
(A) Q20and pEA Q20IV were largely insoluble in water or in a solution of
SDS. When 90% formic acid (FA) was substituted as solvent, both peptides
of formic acid (FA).
Solubility of Q20peptides in formic acid. Q20and pEA Q20IV were
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Within 1 day, 1C2 revealed abundant cytoplasmic staining of
staining was reduced, but few stained inclusions were revealed.
Intact inclusions are known to be poorly stained by 1C2 (41).
Staining by anti-tetra-His showed a different pattern, in which
staining of inclusions predominated. These could be seen as
early as day 1, but the inclusions steadily increased in size and
number until day 3. The number decreased by day 6, when the
inclusions were considerably larger and generally reduced to one
per cell. The inclusions were associated mainly with nuclei, but
were likely perinuclear rather than intranuclear.
Purification of Inclusions. Inclusions were purified on the basis of
their insolubility and identified by staining with antibody to
c-Myc. We found that a commercial anti-rabbit IgG-FITC
(Roche Applied Science) can stain cellular proteins nonspecifi-
cally, and we used it to monitor the purity of the inclusions. The
sonicated cell suspension contained inclusions associated with
abundant contaminating protein (Fig. 3A). Extraction with
guanidinium chloride removed most contaminating protein (Fig.
3B) and subsequent extraction with SDS?2-ME removed nearly
all of it (Fig. 3C).
Presence in the Inclusions of Insoluble Monomer and Oligomer Con-
taining Expanded PolyQ. Purified inclusions were submitted to
electrophoresis. There was no detectable signal after staining
with 1C2 antibody (Fig. 4). This finding is not surprising because
the inclusions had been insoluble in SDS?2-ME during their
purification and could therefore not be expected to enter a
polyacrylamide gel. When the inclusions were first treated with
90% formic acid for 1 h at 37°C, the formic acid removed under
vacuum, and the residue neutralized, electrophoresis of the
proteins showed an intense band of monomer and a smaller
discrete band with mobility equaling 21% that of the monomer.
Longer incubations in the presence of formic acid resulted in no
further change. The effect of formic acid pretreatment may be
explained by its ability to dissociate noncovalently stabilized
aggregates otherwise unable to enter the gel, as well as by its
ability to enhance immunostaining (42). After filtration of
formic acid-treated extract through cellulose acetate filters, no
retained aggregate could be detected.
In contrast to the insolubility of the oligomer and monomer of
the inclusions in the absence of formic acid pretreatment, both
monomer and oligomer could readily be detected in the cyto-
plasm without formic acid pretreatment.
To see whether these results depended on the cell line used,
we carried out transient transfections of 293 cells (human
embryonic kidney) and examined the distribution of expanded
polyQ by electrophoresis with and without formic acid pretreat-
ment. Only after formic acid treatment were monomer and
oligomer bands strongly stained. The ratio of the mobility of the
oligomer to that of the monomer was 15%, a value similar to that
obtained from PC12 cells. We concluded that formation of
oligomer did not depend on properties exclusive to PC12 cells.
The oligomer did not result from polyubiquitination of the
monomer, because an antiubiquitin antibody (Dako), at a
1:1,000 dilution, failed to stain any proteins in the region of the
oligomer, whereas it easily detected free ubiquitin and ubiqui-
tinated proteins of red blood cells and lymphoblasts.
As in the case of PC12-Q205cells, water-soluble monomer and
oligomer could be detected in the cytosol.
Absence of Reassociation of Monomer After Removal of Formic Acid.
To be certain that oligomer dissociated by formic acid does not
reform after removal of the formic acid, the dried residue after
formic acid treatment of inclusions was redissolved in buffer
containing SDS with or without neutralization and allowed to
stand for various periods. The sample that still retained some
acidity was then neutralized and both samples were subjected to
cells. Expression of the construct encoding Q205, flanked by 67 N-terminal and
17 C-terminal residues, followed by 6xHis, was induced from an ecdysone-
inducible promotor, using ponasterone A. The 1C2 antibody, which is known
to stain inclusions poorly, showed mainly diffuse cytoplasmic staining, declin-
ing with time, whereas staining by anti-tetra-His showed mainly inclusions,
beginning as early as day 1, first increasing in number with time but later only
in size, the number then being reduced.
Appearance and number of inclusions after induction of PC12-Q205
of PC12-Q205cells were purified. The inclusions were stained with anti-c-Myc
coupled indirectly to Cy3 (red) and nonspecific proteins were stained with
anti-rabbit IgG-FITC (green). (A) No purification. (B) After extraction with
guanidinium chloride. (C) After extraction with SDS?2-ME. (Magnifications:
Purification of inclusions. Inclusions harvested 6 days after induction
polyQ. Purified inclusions, with or without formic acid (FA) treatment, were
amide. After transfer to nitrocellulose, the proteins were stained with the
antibody 1C2. In the absence of formic acid pretreatment, neither monomer
nor oligomer was detected. After pretreatment with 90% formic acid for 1 h
an apparent molecular mass of ?100 kDa; the true molecular mass should be
36,340, but expanded polyQ reduces mobility in gel electrophoresis. In addi-
tion to the monomer, an oligomer with a considerably higher molecular mass
the oligomer, but estimation of staining intensity by IR fluorescence showed
that the oligomer was present in a range from 0.5% to 3% of monomer. The
ratio of the mobility of the oligomer to that of the monomer was 0.21.
Synthesis by PC12-Q205cells of an oligomer containing expanded
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electrophoresis. During storage of the formic acid-treated ma-
terial at 4°C for up to 72 h under either condition, there was no
detectable increase in the amount of oligomer. It was also found
that when the migration was slowed during electrophoresis by
lowering the applied voltage, there was similarly no tendency to
reaggregation. These results showed that the dissociation of
oligomer in formic acid is indeed stable (27). It is not clear why
this should be. In addition to disaggregation of proteins, treat-
ment by concentrated formic acid is known to result in formy-
lation of serine residues (43, 44). There are five serine residues
and three threonine residues in the flanking regions of the Q205.
We have found that increasing the incubation time in formic acid
resulted in more acidity after drying and dissolving in SDS
solution. Perhaps this finding suggests that some kind of formy-
lation had occurred during incubation.
The Partition of Expanded PolyQ Between the Cytosolic and Particu-
late Cell Compartments. There have been numerous previous
studies of the inclusions resulting from proteins with expanded
polyQ (37, 45–47). None of these studies dealt with the role
of oligomers containing expanded polyQ. We have studied
the kinetics of the transfer of monomer and oligomer from the
cytosol into the particulate compartment, which contains the
inclusions induced in the cell line PC12-Q205. At intervals,
duplicate cultures were trypsinized. The cells were washed in
PBS containing a mixture of protease inhibitors (Complete,
Roche Applied Science) and 100 mM EDTA and were sonicated
for 2–3 s twice in 10 mM Tris?HCl buffer, pH 7.5, containing 1
mM EDTA, the same protease inhibitors and 1% Triton X-100.
A cytosolic fraction and a particulate fraction containing the
inclusions were then separated by centrifugation at 100,000 ? g.
Each fraction was dissolved in 90% formic acid and taken to
dryness in vacuo. The proteins were then incubated in buffered
SDS, neutralized, heated to 100°C for 5 min, and subjected to
electrophoresis in SDS. After transfer to nitrocellulose and
staining with the 1C2 antibody, the amounts of expanded polyQ
in the monomer and oligomer bands were quantitated by near IR
At 4 h after induction, monomer and oligomer both could be
detected in the cytosol and the particulates (Fig. 5). In the
cytosol, the amount of monomer reached its maximum at 12 h
and then declined sharply (and continued to do so in longer term
experiments), whereas monomer continued to increase in the
particulates. The amount of oligomer in the youngest inclusions
(4–8 h) equaled 30% of the oligomer in the entire cytosol. This
pattern shows that oligomer participates in inclusion formation
from the very beginning of the process; with time, both oligomer
and monomer were removed from the cytoplasm into the
inclusions, where the amount of monomer ultimately became 50
times larger than the amount of oligomer.
Soluble Oligomer Containing Expanded Huntingtin in the Cerebral
Cortex of Patients with Juvenile Huntington’s Disease. It has been
reported (12, 31, 48) that in the cortex of patients with Hun-
tington’s disease, expanded huntingtin is replaced by a broad
band or smear containing huntingtin and extending upward from
the expected position of expanded huntingtin. We prepared
extracts of cortex and cerebellum from four patients with
juvenile Huntington’s disease, obtained from the Harvard Brain
particulates of induced PC12-Q205cells. One day after inoculation, PC12-Q205
cells were induced by 10 ?M ponasterone A and harvested by trypsinization
after 0, 4, 8, 12, and 24 h of induction. Sonicated extracts were fractionated
into soluble and particulate fractions (containing the inclusions) and both
fractions were analyzed for Q205by Western blotting. The amount of Q205
of induction. The amount of monomer always exceeded the amount of
oligomer, but the particulates account for an appreciable part of the total
oligomer at the earliest time points (see text).
Kinetics of appearance of monomer and oligomer in cytosol and
Huntington’s disease. (A) Crude extracts of cortex and cerebellum were
treated with 96% formic acid. Western blots prepared after electrophoresis
were stained consecutively with anti-N-terminal antibody (30) and 1C2. All
none of the cortical specimens, produced a clear band of monomeric ex-
panded huntingtin. (B) Blots prepared after electrophoresis of formic acid-
treated crude brain extracts were photographed after staining with the
N-terminal antibody and again after staining with the 1C2 antibody. The
antibody. The broad band of oligomer in the merged figure clearly extends
above the corresponding monomer.
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Tissue Resource Center. We then dried them and incubated the
residue in 96% formic acid for 1 h at 37°C. The free formic acid
was evaporated, and the samples were dissolved in gel loading
by PAGE and immunoblotting, using sequential staining by the
anti-N-terminal antibody and the 1C2 antibody (Fig. 6A).
huntingtins were detected as expected. But the cortical extract
gave a broad band of formic acid-resistant oligomeric protein
extending over a molecular mass range of ?100 kDa and with
mean mobility of 0.90 ? SD 0.03 that of the expanded huntingtin
of the cerebellum (Fig. 6A). In the absence of formic acid
treatment, the appearance of this band was unchanged. The
mean mobility of the oligomeric form was not reduced as much
with respect to monomeric huntingtin as was the mobility of the
oligomer of cultured cells with respect to its monomer.
Sequential staining with the anti-N-terminal and 1C2 anti-
bodies of blots prepared after electrophoresis of crude cortical
extracts of cases 3482 and 3535 showed that a small amount of
expanded monomer could be detected below the broad band of
oligomer (Fig. 6B). We had previously shown by Southern
analysis that the expanded cortical allele of case 3482 contained
63 CAGs, or only one more CAG than the corresponding allele
of the cerebellum (49). This finding establishes that the broad
band of oligomer cannot be explained as the result of cortical
mosaicism. The oligomer was not appreciably stained by anti-
body to the N-terminal sequence (Fig. 6B) or by mAb 2166,
directed against sequence C-terminal of the polyQ, or by an
Quantitative analysis of the oligomer was carried out by IR
fluorescence. The amounts of expanded polyQ in the form of
oligomer in cortices 3482 and 3177 were 9.8- and 4.5-fold,
respectively, the amount of expanded monomeric huntingtin of
the cerebellum. After normalization for unequal content of
normal huntingtin monomer in the lanes of cortex and cerebel-
lum (a factor of 1.74 for 3482 and 1.4 for 3177), the values for
the ratio of oligomer to monomer were 5.6 and 3.2. (see Table
2, which is published as supporting information on the PNAS
To determine the subcellular location of the polymer, we
isolated cortical nuclei by centrifugation through density gradi-
ents of sucrose or iodixanol. In the absence of formic acid
pretreatment, the oligomer containing expanded polyQ could be
detected only in the cortical cytoplasm. But if the isolated nuclei
were first treated with formic acid, subsequent electrophoresis
revealed a band corresponding to oligomer, another broad band
of lower molecular weight, also stained by 1C2 antibody and
presumably consisting of fragmented huntingtin, and a promi-
nent band located at the top of the gel. These components are
most likely located in the nuclear inclusions (see Fig. 8, which is
published as supporting information on the PNAS web site).
Polymer Insoluble in Formic Acid Recovered from the Nuclei of
Cerebral Cortex. To examine and quantitate the component
located at the top of the gel, we isolated nuclei from the cortex
of patients 3815 and 3123 by density gradient centrifugation,
incubated the nuclei in 96% formic acid, and submitted the
samples to the filter retention assay (35, 50). In cortex of patient
3815, which contained inclusions in 20% of the nuclei, the
presence of nuclear polymer insoluble in 96% formic acid and of
size permitting its retention on the filter was revealed by a dark
spot stained with the 1C2 antibody (Fig. 7). In contrast, nuclei
prepared from the cerebellum of the same patient contained no
inclusions and virtually no stainable polymer. Quantitative assay
by IR fluorescence gave a value for cortex ?100 times than that
of cerebellum (Table 1). The lymphoblastoid cell line SALDAV
080010 and rat cortex gave low values.
Cortex 3123 was found to contain many fewer inclusions than
cortex 3815. Counts of inclusions in 1,068 nuclei of cortex 3123
revealed their presence in only 1.96% of nuclei. Chemilumines-
cent staining was consistent with this difference (Fig. 7). Quan-
titation by IR fluorescence showed that the value for polymer of
cortex 3123 was about one-tenth that of cortex 3815. Accord-
ingly, 120,000 nuclei were used for its accurate measurement,
and the sensitivity of the instrument had to be increased. In two
independent experiments under these conditions, cortex 3123
gave highly significant values for retained polymer but the
control values were appreciable for SALDAV 080010 and rat
brain, whose huntingtin contains only Q8(51) (Table 1). Because
the value for IR fluorescence of rat brain did not increase with
the number of nuclei applied, this value could be subtracted as
background correction. When this was done, values for cortex
were further elevated with respect to those of cerebellum. The
cerebellar values, although quite low, may be real, whereas those
for SALDAV 080010 were reduced to background. We may
conclude that the nuclear inclusions in the cortex of juvenile
cases of Huntington’s disease contain a formic acid-insoluble
Huntington’s disease. Purified nuclei of cortex and cerebellum of brain
3815 and of controls were extracted with 96% formic acid. The suspension
was diluted with a 10-fold excess of Tris?SDS?2-ME, boiled for 5 min, and
was visualized by chemiluminescence by using antibody 1C2 and the ECL
plus kit. Control SALDAV 080010 was a lymphoblast line possessing a
huntingtin with 79 glutamine residues. Rat cortex, whose huntingtin pos-
sesses Q8(51), should not be stainable by 1C2. Cortex 3815: 20,000 nuclei
gave a strong signal. The corresponding cerebellum, SALDAV 080010, and
rat cortex gave weak signals. Cortex 3123: 120,000 nuclei produced a
weaker signal than 20,000 nuclei of cortex 3815. Arrow indicates control
without nuclei. Because chemiluminescence does not provide a quantita-
tive measure of the amount of expanded polyQ, quantitation was per-
formed by using IR fluorescence (see Table 1).
Polymeric state of expanded huntingtin in cerebral cortex in
Table 1. Formic acid-insoluble polymer of expanded polyQ in
cortical nuclei of Huntington’s disease: Quantitation of the
polymer retained on cellulose acetate filters
Source of polyQ
IR fluorescence, units
Brain 3123, 120,000 nuclei
For brain 3123, two independent determinations were done.
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Concluding Remarks Download full-text
Using 90–96% formic acid to dissociate aggregates stabilized by
noncovalent bonds, we have demonstrated the existence of three
types of aggregate-containing expanded polyQ: (i) an oligomer
released by formic acid from inclusions produced by cultured
cells synthesizing Q205; (ii) a water-soluble oligomer present in
the cytoplasm of cerebral cortex affected by juvenile Hunting-
ton’s disease but not in the cytoplasm of the corresponding
cerebellum; this oligomer is also present in an insoluble form in
nuclei (presumably their inclusions), from which it is released by
formic acid; and (iii) a formic acid-insoluble polymer of too large
a size to pass through a 0.2-?m filter, present in the nuclei of the
same cortices, but not in the corresponding cerebellum or in
lymphoblasts or in cultured cells synthesizing Q205. The amount
of this polymer correlates with the number of nuclear inclusions.
The oligomer of the nuclei is likely to be a precursor of this
This work was aided by a consortial grant from the National Institute
of Neurological Disorders and Stroke (5 RO1 NS38566-03) (to H.G.
1. La Spada, A. R., Wilson, E. M., Lubahn, D. B., Harding, A. E. & Fischbeck,
K. H. (1991) Nature 352, 77–79.
2. The Huntington’s Disease Collaborative Research Group (1993) Cell 72,
3. Gusella, J. F. & MacDonald, M. E. (2000) Nat. Rev. Neurosci. 1, 109–115.
4. Perutz, M. F., Johnson, T., Suzuki, M. & Finch, J. T. (1994) Proc. Natl. Acad.
Sci. USA 91, 5355–5358.
5. Eckert, R. L. & Green, H. (1986) Cell 46, 583–589.
6. Delhomme, B. & Djian, P. (2000) Gene 252, 195–207.
7. Djian, P., Phillips, M., Easley, K., Huang, E., Simon, M., Rice, R. H. & Green,
H. (1993) Mol. Biol. Evol. 10, 1136–1149.
8. Green, H. (1993) Cell 74, 955–956.
9. Kahlem, P., Terre ´, C., Green, H. & Djian, P. (1996) Proc. Natl. Acad. Sci. USA
10. Cooper, A. J., Sheu, K. R., Burke, J. R., Onodera, O., Strittmatter, W. J., Roses,
A. D. & Blass, J. P. (1997) J. Neurochem. 69, 431–434.
11. Gentile, V., Sepe, C., Calvani, M., Melone, M. A. B., Cotrufo, R., Cooper, A. J.,
Blass, J. P. & Peluso, G. (1998) Arch. Biochem. Biophys. 352, 314–321.
12. Kahlem, P., Green, H. & Djian, P. (1998) Mol. Cell 1, 595–601.
13. Chun, W., Lesort, M., Tucholski, J., Faber, P. W., MacDonald, M. E., Ross,
C. A. & Johnson, G. V. (2001) Neurobiol. Dis. 8, 391–404.
14. Igarashi, S., Koide, R., Shimohata, T., Yamada, M., Hayashi, Y., Takano, H.,
Date, H., Oyake, M., Sato, T., Sato, A., et al. (1998) Nat. Genet. 18, 111–117.
15. Lesort, M., Attanavanich, K., Zhang, J. & Johnson, G. V. (1998) J. Biol. Chem.
16. de Cristofaro, T., Affaitati, A., Cariello, L., Avvedimento, E. V. & Varrone, S.
(1999) Biochem. Biophys. Res. Commun. 260, 150–158.
17. Karpuj, M. V., Garren, H., Slunt, H., Price, D. L., Gusella, J., Becher, M. W.
& Steinman, L. (1999) Proc. Natl. Acad. Sci. USA 96, 7388–7393.
18. Jeitner, T. M., Bogdanov, M. B., Matson, W. R., Daikhin, Y., Yudkoff, M.,
Folk, J. E., Steinman, L., Browne, S. E., Beal, M. F., Blass, J. P. & Cooper, A. J.
(2001) J. Neurochem. 79, 1109–1112.
19. Cariello, L., de Cristofaro, T., Zanetti, L., Cuomo, T., Di Maio, L., Campanella,
G., Rinaldi, S., Zanetti, P., Di Lauro, R. & Varrone, S. (1996) Hum. Genet. 98,
20. Cooper, A. J., Wang, J., Pasternack, R., Fuchsbauer, H. L., Sheu, R. K. & Blass,
J. P. (2000) Dev. Neurosci. 22, 404–417.
21. Mastroberardino, P. G., Iannicola, C., Nardacci, R., Bernassola, F., de Lau-
renzi, V., Melino, G., Moreno, S., Pavone, F., Oliverio, S., Fesus, L. &
Piacentini, M. (2002) Cell Death Differ. 9, 873–880.
23. Karpuj, M. V., Becher, M. W., Springer, J. E., Chabas, D., Youssef, S., Pedotti,
R., Mitchell, D. & Steinman, L. (2002) Nat. Med. 8, 143–149.
24. Dedeoglu, A., Kubilus, J. K., Jeitner, T. M., Matson, S. A., Bogdanov, M.,
Kowall, N. W., Matson, W. R., Cooper, A. J., Ratan, R. R., Beal, M. F., et al.
(2002) J. Neurosci. 22, 8942–8950.
25. Chun, W., Lesort, M., Tucholski, J., Ross, C. A. & Johnson, G. V. (2001) J. Cell
Biol. 153, 25–34.
26. Mokrasch, L. C. (1965) Biochim. Biophys. Acta 107, 608–610.
27. Hazeki, N., Tukamoto, T., Goto, J. & Kanazawa, I. (2000) Biochem. Biophys.
Res. Commun. 277, 386–393.
28. Nakajima, H., Kimura, F., Nakagawa, T., Furutama, D., Shinoda, K., Shimizu,
A. & Ohsawa, N. (1996) J. Neurol. Sci. 142, 12–16.
29. Laemmli, U. K. (1970) Nature 227, 680–685.
30. Hoffner, G., Kahlem, P. & Djian, P. (2002) J. Cell Sci. 115, 941–948.
31. Trottier, Y., Lutz, Y., Stevanin, G., Imbert, G., Devys, D., Cancel, G., Saudou,
F., Weber, C., David, G. & Tora, L. (1995) Nature 378, 403–406.
32. Kegel, K. B., Meloni, A. R., Yi, Y., Kim, Y. J., Doyle, E., Cuiffo, B. G., Sapp,
33. Valenzuela, S. M., Martin, D. K., Por, S. B., Robbins, J. M., Warton, K.,
Bootcov, M. R., Schofield, P. R., Campbell, T. J. & Breit, S. N. (1997) J. Biol.
Chem. 272, 12575–12582.
34. Vakakis, N., Hearn, M. T., Veitch, B. & Austin, L. (1991) J. Neurochem. 57,
35. Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenbach, B.,
Hasenbank, R., Bates, G. P., Davies, S. W., Lehrach, H. & Wanker, E. E. (1997)
Cell 90, 549–558.
36. Parameswaran, K. N., Velasco, P. T., Wilson, J. & Lorand, L. (1990) Proc. Natl.
Acad. Sci. USA 87, 8472–8475.
37. Chen, S., Berthelier, V., Yang, W. & Wetzel, R. (2001) J. Mol. Biol. 311,
38. Rice, R. H. & Green, H. (1977) Cell 11, 417–422.
39. Rice, R. H. & Green, H. (1979) Cell 18, 681–694.
40. Sun, T. T. & Green, H. (1976) Cell 9, 511–521.
41. Sieradzan, K. A., Mechan, A. O., Jones, L., Wanker, E. E., Nukina, N. & Mann,
D. M. (1999) Exp. Neurol. 156, 92–99.
42. Kitamoto, T., Ogomori, K., Tateishi, J. & Prusiner, S. B. (1987) Lab. Invest. 57,
43. Orlando, R., Kenny, P. T. & Zagorski, M. G. (1992) Biochem. Biophys. Res.
Commun. 184, 686–691.
44. Klunk, W. E. & Pettegrew, J. W. (1990) J. Neurochem. 54, 2050–2056.
45. Hazeki, N., Nakamura, K., Goto, J. & Kanazawa, I. (1999) Biochem. Biophys.
Res. Commun. 256, 361–366.
46. Kazantsev, A., Walker, H. A., Slepko, N., Bear, J. E., Preisinger, E., Steffan,
J. S., Zhu, Y.-Z., Gertler, F. B., Housman, D. E., Marsh, J. L. & Thompson,
L. M. (2002) Nat. Genet. 30, 367–376.
47. Scherzinger, E., Sittler, A., Schweiger, K., Heiser, V., Lurz, R., Hasenbank, R.,
Bates, G. P., Lehrach, H. & Wanker, E. E. (1999) Proc. Natl. Acad. Sci. USA
48. Schilling, G., Sharp, A. H., Loev, S. J., Wagster, M. V., Li, S. H., Stine, O. C.
& Ross, C. A. (1995) Hum. Mol. Genet. 4, 1365–1371.
49. Kahlem, P. & Djian, P. (2000) Neurosci. Lett. 286, 203–207.
50. Wanker, E. E., Scherzinger, E., Heiser, V., Sittler, A., Eickhoff, H. & Lehrach,
H. (1999) Methods Enzymol. 309, 375–386.
51. Holzmann, C., Maueler, W., Petersohn, D., Schmidt, T., Thiel, G., Epplen, J. T.
& Riess, O. (1998) Biochem. J. 336, 227–234.
www.pnas.org?cgi?doi?10.1073?pnas.0437660100Iuchi et al.