Neurobiology of Disease 4, 387397 (1998)

Article No. NB980168

Intranuclear Neuronal Inclusions in Huntingtons

Disease and Dentatorubral and Pallidoluysian
Atrophy: Correlation between the Density of
Inclusions and IT15 CAG Triplet Repeat Length
Mark W. Becher,*,1 Joyce A. Kotzuk,* Alan H. Sharp,
Stephen W. Davies, Gillian P. Bates, Donald L. Price,*,,(
and Christopher A. Ross,(,**
*Department of Pathology, Division of Neuropathology, Department of Psychiatry,
Department of Neurology, and (Department of Neuroscience, and the **Program in Cellular
and Molecular Medicine, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205-2196,; Department of Anatomy and Developmental Biology,
University College London, London WC1E 6BT, United Kingdom; and the Division of Medical
and Molecular Genetics, UMDS, Guys Hospital, London SE1 9RT, United Kingdom
Received September 23, 1997; accepted for publication November 21, 1997

Huntingtons disease (HD) is caused by CAG triplet repeat expansion in IT15 which leads to polyglutamine
stretches in the HD protein product, huntingtin. The pathological hallmark of HD is the degeneration of
subsets of neurons, primarily those in the striatum and neocortex. Specific morphological markers of
affected cells have not been identified in patients with HD, although a unique intranuclear inclusion was
recently reported in neurons of transgenic animals expressing a construct encoding the N-terminal part
(including the glutamine repeat) of huntingtin (Davies et al., 1997). In order to understand the importance of
this finding, we sought for comparable nuclear abnormalities in autopsy material from patients with HD. In all
20 HD cases examined, anti-ubiquitin and N-terminal huntingtin antibodies identified intranuclear inclusions
in neurons and the frequency of these lesions correlated with the length of the CAG repeat in IT15. In addition,
examination of material from the related HD-like triplet repeat disorder, dentatorubral and pallidoluysian
atrophy, also revealed intranuclear neuronal inclusions. These findings suggest that intranuclear inclusions
containing protein aggregates may be a common feature of the pathogenesis of glutamine repeat
neurodegenerative disorders. r 1998 Academic Press

INTRODUCTION
Huntingtons disease (HD) is a fatal, progressive,
inherited neurodegenerative disease (Folstein, 1989;
Ross et al., 1997b). The IT15 gene on chromosome four
was first identified in 1993 by positional cloning
techniques and affected individuals were found to
have CAG triplet repeat expansions (Huntingtons
Disease Collaborative Research Group, 1993). This
discovery placed HD into the group of disorders
1To

whom correspondence and reprint requests should be addressed at Division of Neuropathology, Department of Pathology,
The Johns Hopkins University School of Medicine, 558 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196.
Fax: (410) 955-9777. E-mail: mbecher@welchlink.welch.jhu.edu.
0969-9961/98 $25.00
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associated with CAG triplet repeats which code for

polyglutamine stretches (La Spada et al., 1994; Ross,
1995; Warren, 1996). All of these disorders affect the
nervous system and are characterized by selective
vulnerability of neurons in distinct, but sometimes
overlapping regions of the central nervous system. In
HD, neuronal degeneration most severely affects the
medium spiny neurons of the caudate nucleus and
putamen and is accompanied by reactive astrocytosis
(Vonsattel et al., 1985; Robitaille et al., 1997; Ross et al.,
1997a). In addition, there is evidence of selective
neuronal loss in neocortical layers III, V, and VI
(Hedreen et al., 1991; Sotrel et al., 1991), and variable
degeneration in thalamic nuclei, the subthalamic
nucleus, locus ceruleus, and cerebellum (Rodda, 1981;
Robitaille et al., 1997; Ross et al., 1997a). Purkinje cell

387

388
loss is often a prominent feature in juvenile onset HD
(Jervis, 1963; Byers et al., 1967; Rodda, 1981). Except for
neuronal loss and astrocytosis, histopathological markers of disease in HD have not been recognized.
The HD gene transcript contains a long open reading frame (3145 codons) with the CAG repeat very
near to the predicted N-terminus of huntingtin (Huntingtons Disease Collaborative Research Group, 1993).
Huntingtin mRNA and protein are widely expressed
in the central nervous system and systemic organs (Li
et al., 1993; Sharp et al., 1995; DiFiglia et al., 1995;
Trottier et al., 1995a; Ferrante et al., 1997). Although
cellular localization and protein association studies
have suggested a possible role of huntingtin in cytoskeletal function and vesicle trafficking, the normal function of huntingtin is still uncertain (DiFiglia et al., 1995;
Gutekunst et al., 1995; Kalchman et al., 1996; Colomer et
al., 1997). Huntingtin with expanded repeats can be
separated on gels from the protein encoded by the
normal allele (Schilling et al., 1995; Aronin et al., 1995;
Trottier et al., 1995b). The mechanisms by which the
abnormal gene product damages specific subsets of
neurons and causes cell dysfunction and death are
unknown.
Dentatorubral and pallidoluysian atrophy (DRPLA)
is another neurodegenerative disorder which is caused
by an expanding CAG triplet repeat coding for polyglutamine (Smith et al., 1958; Li et al., 1993; Nagafuchi et
al., 1994; Koide et al., 1994). DRPLA is more frequent in
Japan than in European or North American countries,
but several cases have recently been described in
non-Japanese pedigrees (Pfeiffer et al., 1990; Burke et
al., 1994; Warner et al., 1994; Norremolle et al., 1995;
Potter et al., 1995; Becher et al., 1997). The clinical
features of DRPLA are often similar to those of HD,
and, like HD, adult onset cases differ from juvenile
onset cases (Ikeuchi et al., 1995; Nance, 1997). The
neuropathological changes of DRPLA vary considerably from case to case, but neuronal loss is most severe
in the dentate nucleus and dentatofugal and pallidofugal systems including the globus pallidus and superior
cerebellar peduncle (Smith et al., 1958; Takahashi et al.,
1988; Warner et al., 1994; Becher et al., 1997; Robitaille et
al., 1997). Other affected regions include the red nucleus,
subthalamic nucleus, and olivary nuclei in the medulla
(Becher et al., 1997; Robitaille et al., 1997). Unlike HD,
the neocortex and striatum are generally unaffected.
The most promising pathogenic hypothesis for glutamine repeat disorders involves the genetic gain-offunction mechanism (Huntingtons Disease Collaborative Research Group, 1993; Ambrose et al., 1994; Ross,
1995; MacDonald et al., 1996; Sharp et al., 1996).
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Becher et al.

Support for this hypothesis comes from a recent

animal model of HD in which exon one of the human
HD gene with an extremely long CAG repeat was
introduced as a transgene in mice (Mangiarini et al.,
1996). These animals display a striking behavioral
phenotype which has some similarities to patients
with HD, and transgenic animals have loss of both
brain and body weight (Mangiarini et al., 1996). Although there was no discernable loss of neurons in the
brains of these animals, a novel inclusion was described in the nuclei of neurons (Davies et al., 1997).
These neuronal nuclear inclusions, which are present
prior to the onset of the clinical phenotype, were found
by immunocytochemical studies with antibodies to the
N-terminus of huntingtin and with antibodies to ubiquitin (Davies et al., 1997). Prior to these studies,
analogous abnormalities had not been recognized in
human HD material, although early work on HD
biopsy material suggested the presence of increased
nuclear membrane folding and other nonspecific
nuclear abnormalities in neurons from HD patients
(Roizin et al., 1976).
In the present report, we demonstrate that intranuclear inclusions are present in neurons in the brains
of individuals with HD and one case of DRPLA. These
inclusions, readily identifiable in routinely processed,
formalin-fixed archival material, show ubiquitin immunoreactivity and, in HD cases, N-terminal huntingtin
immunoreactivity. In HD material, we studied the
distribution of lesions in HD patients with particular
attention to the involvement of the neocortical layers
and striatum and to the frequency of these inclusions
in HD subjects with different CAG repeat lengths in
IT15 and varying grades of severity.

MATERIALS AND METHODS

Subjects
This study takes advantage of archival brain tissue
from: 20 individuals with genetically confirmed HD;
one case of DRPLA (case 1, pedigree 922 II:2 (Becher et
al., 1997)); controls; and cases of other types of neurodegenerative diseases (i.e., Alzheimers disease and
Amyotrophic Lateral Sclerosis), which were accessioned over many years to the Johns Hopkins HD
Brain Bank and Neuropathology Brain Resource Center. All HD cases were followed in the Baltimore HD
Center clinic and longitudinal data were available for
most individuals. Following autopsy consent, brain
specimens were removed at our institution or at other
locations under our direction. All brains were immer-

389

Neuronal Inclusions in Huntingtons Disease and DRPLA

sion-fixed in 10% neutral-buffered formalin for 514

days, sliced in the coronal plane, and sampled for
routine paraffin processing. Genetic confirmation of
CAG expansion in IT15 (41-.100 for HD cases) or the
DRPLA gene (54 CAG repeats in CTG-B37 for DRPLA
case) was confirmed in life from a blood sample or
from frozen brain at autopsy (Huntingtons Disease
Collaborative Research Group, 1993; Stine et al., 1993;
Becher et al., 1997). All 20 HD cases (979 years of age;
mean 47 years) were previously examined neuropathologically by standard methods and the severity of HD
was graded [grade 0 (0); grade 1 (1); grade 2 (3); grade
3 (6); grade 4 (10)], according to the Vonsattel grading
system (Vonsattel et al., 1985). Samples from six Alzheimers disease cases (5487 years of age; mean 72
years), four Amyotrophic Lateral Sclerosis cases (2769
years of age; mean 50 years), and four control cases
without neurological disease (4066 years of age; mean
55) were obtained in a similar fashion. Neocortex and
striatum were available on all cases; additional areas
were examined in selected cases (Table 1).

Immunocytochemistry
Polyclonal antibodies to ubiquitin (DAKO, Carpinteria, CA), glial fibrillary acidic protein (GFAP; DAKO),
the N-terminal 117 amino acids of huntingtin (AP78
and AP194) (Sharp et al., 1995), and an internal epitope
of huntingtin (AP81; aa 650663) (Sharp et al., 1995)
were used in this study. Paraffin sections (8 m) were
stained with ubiquitin, GFAP, AP78, or AP194 antibodies by standard immunocytochemical techniques, with
minor modifications. Briefly, sections were deparaffinized, treated with hydrogen peroxide/methanol,
microwaved (650 Watt) in water for several minutes,
blocked with 3% normal goat serum, and then incubated with primary antibody at room temperature
overnight (1620 h). Following the primary incubation,
avidinbiotin complex reagents (Vector Laboratories,
Burlingame, CA) were used and antibody reactions
were visualized with 3,38-diaminobenzidine (DAB;
Fluka Chemicals, Ronkonkoma, NY) or 3-amino-9ethylcarbazole (AEC; Sigma, St. Louis, MO) chromagens with a brief hematoxylin counterstain. AP81stained material was available from previous studies
which used tissue which had been fixed in 4% paraformaldehyde, cryoprotected, cryostat sectioned, and
stained by free-floating immunocytochemistry. Technique control sections included omission of the primary antibodies or competition controls with preabsorbed antibodies treated with excess peptides or
antigen.

Two double label immunocytochemical techniques

were used. To demonstrate astrocytes and neuronal
inclusions, we performed sequential immunocytochemical staining of ubiquitin (DAB) followed by
GFAP (AEC) with standard methods. In order to
colocalize AP78 and ubiquitin, we first stained for
AP78 with AEC chromagen, photographed cells of
interest, removed the color precipitate with alcohol,
rephotographed the same cells to verify the loss of
reaction product, blocked possible crossreactivity of
the deactivated ABC complex with standard Avidin D
and biotin reagents (Vector), then stained for ubiquitin
with DAB chromagen. Control sections included omission of the second primary antibody or reapplication
of the first chromagen following AEC removal.

Results
Characterization of HD Neuronal Inclusions
All 20 cases of HD with confirmed triplet repeat
expansions (41.100 CAG repeats) in IT15 contained
ubiquitin and N-terminus huntingtin immunoreactive
inclusions in nuclei of neurons (Fig. 1). These inclusions are distinct intranuclear structures that are not
identifiable on routine hematoxylin and eosin staining,
although double labeling reactions in which the first
chromagen is removed show that some of the inclusions are foci of chromatin clearing (Fig. 1C, middle
panel). The majority of inclusions appeared as round
bodies approximately the size of the nucleolus; on
occasion, inclusions were elongated and rod-shaped
(Fig. 1B). Rarely, there was more than one inclusion
within the same nucleus. No cytoplasmic inclusions or
continuity between the nuclear inclusion and the
cytoplasmic domain were identified with the antibodies used in this study. The ubiquitin antibody identified more nuclear inclusions than did the N-terminal
huntingtin antibodies. Sequential double labeling with
first AP78 followed by removal of chromagen, then
staining with ubiquitin, showed that the N-terminal
huntingtin antigen colocalizes directly with ubiquitin
immunoreactivity (Fig. 1C). Light, punctate cytoplasmic huntingtin immunoreactivity was seen in neurons
with AP78, AP81, and AP194, but antibody dilutions
were established to preferentially label inclusions over
cytoplasmic huntingtin. No nuclear inclusions were
identified with AP81, the anti-huntingtin antibody
raised against an internal epitope, even when antibody
concentrations were increased to heavily label the
endogenous cytoplasmic huntingtin. In contrast to
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390

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Becher et al.

391

Neuronal Inclusions in Huntingtons Disease and DRPLA

TABLE 1
Clinical, Genetic, and Neuropathological Features of Huntingtons Disease Cases
Semiquantitative neuronal inclusion score*
HD
case

IT15 CAG
expanded
repeat

VS
grade

Age of
onset
(yrs)

Age of
death
(yrs)

A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T

41
42
42
43
44
44
49
50
50
50
53
53
55
59
59
60
63
69
74
.100

3
3
2
2
4
1
3
4
2
3
4
3
4
4
4
4
3
4
4
4

51
49
63
58
33
41
36
37
35
46
27
29
25
23
25
17
25
13
11
2

74
65
77
60
51
79
52
60
54
61
43
41
36
35
34
32
38
27
17
9

Neocortex
Frontal
2
2
2
0
1
2
3
1
3
4
2
4
2
2
0
4
4

Striatum

Other**

1
1
2
2
1

3
4
2
2
4
4

Caudate
1
1
1
3
0
2
3
0
0
1
2
2
1
1
2
2
1
0
2
4

Cerebellum

Putamen

1
3
0
2
2
0
1
1
2
1

2
1
2
4

Purkinje cells

Dentate

0
0
0

0
0
0

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
1
0

0
0
0
0

0
1
0
1

Note. VS, Vonsattel severity grade (04); *, semiquantitative assessment score of ubiquitin-positive intranuclear neuronal inclusions from none
(0) to numerous (4); **, Other neocortex sample was from Insula except cases G, J, and O, which were Temporal, and case E was Inferior
Parietal.

AP78 and AP194, AP81 was best visualized with

paraformaldehyde-fixed sections stained via freefloating immunocytochemistry, not formalin-fixed, paraffin-embedded tissue. Neurons from Alzheimers disease, Amyotrophic Lateral Sclerosis, and control
subjects lacked identifiable intranuclear inclusions with
any of the antibodies used in this study.

Distribution of HD Neuronal Inclusions

Inclusions appeared to be limited to neurons and
were widely distributed. In the majority of HD cases,

the most numerous inclusion-bearing neurons were

found in the neocortex (Table 1). No distinct lobar
distribution was discernable, with inclusions found in
frontal, temporal, parietal, occipital, primary motor,
and insular cortex neurons. The laminar neocortical
distribution of inclusions was studied by medium
power photomicroscopic montages (not shown) of
ubiquitin-stained sections of frontal cortex. In this area,
neocortical inclusions were most frequently found in
pyramidal neurons in layers III, V, and VI. Inclusions
were also identified in the striatum within nuclei of
medium-sized neurons in the caudate nucleus (Fig.

FIG. 1. Intranuclear inclusions in Huntingtons disease. Inclusions within nuclei of cortical neurons from an HD patient with 74 CAG repeats
in IT15 (AC) are strongly immunoreactive with antibodies to ubiquitin (A, B). These inclusions (A, arrow) are distinct from the nucleolus (A, B,
open arrow) and range from round to rod-shaped (B, arrowheads). Sequential double-antibody-labeling of the same neuron (see Materials and
Methods) shows colocalization of an N-terminal huntingtin epitope (AP78 with AEC chromagen; C, left panel) and ubiquitin (DAB chromagen;
C, right panel) with confirmation of loss of the first chromagen and chromatin clearing at inclusion site (C, middle panel). Inclusions were also
found in the striatum, amygdala, hippocampus, brainstem, and dentate nucleus of the cerebellum, including medium-sized caudate neurons (D,
ubiquitin), which appear similar to those in the caudate nucleus of N-terminus transgenic mice (inset D, AP78), and neurons in the red nucleus
(E, ubiquitin) from an HD patient with 63 CAG repeats (DF). Reactive astrocytosis includes highly atypical astrocytes (F, arrowhead),
occasionally with enlarged processes around an inclusion-bearing (F, arrow) cortical neuron (Ubiquitin-GFAP). Hematoxylin counterstain. Bars:
A, C, 5 m; B, inset D, DF, 10 m.

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392

Becher et al.

1D) and putamen. In addition, nuclear inclusions were

seen in lower frequencies in the amygdala, hippocampus, red nucleus (Fig. 1E), and dentate nucleus of the
cerebellum. Neuronal inclusions were not identified in
thalamus, substantia nigra, olivary complex in the
medulla, or cerebellar Purkinje cells. GFAP/ubiquitin
double labeling failed to identify nuclear inclusions in
astrocytes, although populations of reactive astrocytes
were readily demonstrated. Numerous astrocytes were
seen in the cortex, including many reactive astrocytes
adjacent to neurons with inclusions and an occasional
abnormal astrocyte whose cellular processes were
enlarged and wrapped around an inclusion-bearing
neuron (Fig. 1F).

IT15 CAG Repeat Length and Severity of HD

A semiquantitative assessment [none (score 0), rare
(score 1), inclusions present (score 2), many inclusions
(score 3), numerous inclusions (score 4)] was used to
determine the relative frequencies of ubiquitin immunoreactive inclusions in selected areas from HD subjects with different triplet repeat lengths (Table 1). All
ratings were performed by two investigators (M.W.B.,
J.A.K.) without knowledge of clinical or genetic data.
Numerous inclusions (score 4) in any area were identified only in cases with CAG repeats greater than 50
and were more likely in cases of high grade severity
(Vonsattel grade 4).
Neocortex. Based on the semiquantitative assessment of neocortical inclusion frequency, the mean
inclusion score increases with increasing length of the
CAG triplet repeat in IT15 (Fig. 2) and there was a
significant correlation of neocortical nuclear inclusion
rating with CAG repeat length (r 5 0.65; F 5 12.6; P ,
0.005). Furthermore, when the cases were divided into
two groups of equal numbers of cases (median split),
the mean rating of neocortical nuclear inclusions in
cases with 50 or less CAG repeats was significantly
different (P , 0.001) than that in cases with greater
than 50 repeats (Table 2). The frequency of cortical
inclusions varied greatly throughout the range of
Vonsattel severity grades, but severe cases (Vonsattel
grade 4) tended to have higher frequencies of inclusions in cortex than less severe (Vonsattel grade 2 and
3) cases (Table 1). The only case (Case D) in which
cortical inclusions were not identified was Vonsattel
grade 2.
Striatum. In the striatum, the frequencies of inclusions did not clearly vary with respect to CAG repeat
length (Table 1) or by Vonsattel grade. Although the
total number of cases in each severity grade is limited,
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FIG. 2. Frequency of intranuclear neuronal inclusions increases

with expansion of CAG triplet repeat length. Based on the semiquantitative assessment of inclusions in the neocortex by ubiquitin, the
mean inclusion score increases with increasing length of the CAG
triplet repeat in IT15.

there was no trend for cases with lower repeat lengths

(3950 CAG repeats) to have relatively more or less
inclusions in striatum than cases with greater than 50
CAG repeats. Only two cases (Cases D and G) were
found to have a higher frequency of intranuclear
inclusions in striatum than neocortex, but had unremarkable repeat lengths and severity ratings. The one
case with numerous widespread intranuclear inclusions in both caudate and putamen (Case T) was a
juvenile-onset case with onset at a very young age, a
very high repeat length, and widespread neuronal
degeneration including marked Purkinje cell loss in
the cerebellum. It is important to stress that the

TABLE 2
Correlation of IT15 CAG Repeat Length and Mean Cortical
Nuclear Inclusion
Number of IT15 CAG
Repeats

Mean Cortical
Inclusion Score

4150 (ie. #50)

5374 (ie. .50)

1.7 6 0.7*
3.3 6 0.9

Note. A semiquantitative score of intranuclear inclusion frequency

was obtained by the assessment of neurons with ubiquitin-positive
intranuclear inclusions on a 0 (none) to 4 (numerous) scale. Students
t test on the mean score for cases less than or equal to 50 CAG repeats
and greater then 50 CAG repeats showed significant correlation
(*P , 0.001).

Neuronal Inclusions in Huntingtons Disease and DRPLA

analysis of the frequencies of inclusions in striatal

neurons in different grades of severity is inherently
difficult due to the marked gradation of neuronal loss
with increasing Vonsattel grade.

Dentatorubral and Pallidoluysian

Atrophy Inclusions
In the DRPLA case, ubiquitin immunoreactive intranuclear inclusions were clearly identified in neurons in
the neocortex, caudate nucleus, and dentate nucleus of
the cerebellum (Fig. 3).

DISCUSSION
To date, the histopathological diagnosis of HD has
been entirely based on the presence of a typical pattern
of atrophy of the basal ganglia and, to a lesser extent,
neocortex with variable degrees of loss of medium
spiny neurons in the striatum, less extensive neuronal
loss in the globus pallidus and neocortex, and variable
local astrocytosis, which appears to be directly related
to the degree of neuronal degeneration (Vonsattel et al.,
1985; Robitaille et al., 1997). Taking advantage of recent
observations in vitro and in vivo (Scherzinger et al.,
1997; Davies et al., 1997), this study demonstrates the
presence of ubiquitin- and N-terminal huntingtinimmunoreactive intranuclear neuronal inclusions in
postmortem tissue from 20 HD cases and ubiquitinimmunoreactive nuclear inclusions in one case with
DRPLA. Similar appearing inclusions were present in
all HD brains studied, regardless of Vonsattel severity
grade or the length of the expanded CAG triplet
repeat. Intranuclear ubiquitin immunoreactive inclusions were not identified in cases of Alzheimers
disease, Amyotrophic Lateral Sclerosis, or neurologically normal control subjects.
In nearly all HD cases examined, intranuclear inclusions were present in the striatum and were only
identified within the nuclei of medium-sized neurons,
the cells primarily at risk in HD. A population of these
cells with inclusions was found in all grades of severity
as based on the Vonsattel grading scale (Vonsattel et al.,
1985). Nuclear inclusions were not found in the large
striatal interneurons, which are typically unaffected in
HD, nor in astrocytes, which are thought to respond to
neuronal degeneration. The majority of HD cases in
this study had a higher frequency of intranuclear
inclusions in neocortex than in striatum. This finding
may reflect, in part, the survival of affected neurons in
cortex in contrast to the severe neuronal loss in

393
striatum. However, the selective involvement of neocortex was also observed in HD cases with Vonsattel
grade 2 in which medium-sized striatal neurons were
relatively preserved compared to cases of higher grades.
In addition, the frequency of cortical neuronal inclusions strongly correlated with the length of the CAG
triplet repeat. Similar to the selective vulnerability of
neurons in the striatum, the distribution of inclusions
in neocortical neurons also showed selectivity. The
number of neurons with inclusions was greatest in the
pyramidal neuron populations of layers III, V, and VI,
the lamina most affected in HD (Hedreen et al., 1991;
Sotrel et al., 1991). Although individual cases varied,
we found neuronal inclusions in frontal, parietal,
temporal, and occipital regions as well as limbic
(insular) cortex and primary motor cortex.
Double label immunocytochemistry to demonstrate
nuclear inclusions and reactive astrocytes showed
highly atypical astrocytes directly associated with
inclusion-bearing neurons. This finding indicates that
the neurodegeneration in HD is more widespread than
the obvious loss of neurons and suggests that there
may be significant functional defects in some of the
affected, but histologically intact neurons. Furthermore, the astrocytic response to inclusion-bearing
neurons suggests that aggregates of abnormal huntingtin protein in the nucleus somehow produce a local
cellular response that is recognized by astrocytes.
While our DRPLA material was from only a single
case, ubiquitin immunoreactive intranuclear inclusions were clearly visible in a variety of populations of
neurons, including those selectively vulnerable in
DRPLA.
The inclusions found in our HD patients are very
similar to those first described in transgenic mice
which express a construct coding for the N-terminal
portion of huntingtin with a greatly expanded CAG
repeat (Mangiarini et al., 1996; Davies et al,.1997). In the
transgenic animals, N-terminal huntingtin immunoreactive nuclear inclusions were present before the onset
of the clinical phenotype (Davies et al., 1997), which
suggested a relationship between the presence of
inclusions, neuronal dysfunction, and behavioral manifestations. In the present postmortem study, it is not
possible to establish a causal relationship between the
presence of inclusions and the development of disease,
although the correlation between frequency of inclusions and CAG repeat length indicates a relationship
between inclusions and severity of disease. The study
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394

FIG. 3. Intranuclear inclusions in DRPLA. Ubiquitin-immunoreactive inclusions in neuronal nuclei from a case of genetically confirmed DRPLA are present in the dentate nucleus (A), which is
severely affected by neuronal loss, as well as neocortex (B) and
caudate nucleus (C), which are generally not vulnerable neuronal
populations in this disorder. Ubiquitin immunocytochemistry with
hematoxylin counterstain. Bar: AC, 10 m.

cally confirmed Vonsattel grade 0 or 1 cases will be

instrumental to determine the time course of these
lesions in the naturally occurring disease state.
The inclusions in the N-terminus transgenic mice
were not immunoreactive with antibodies to other
regions of huntingtin, suggesting that the huntingtin
protein present in these aggregates is derived only
from the transgene and that the endogenous huntingtin protein is not incorporated into these inclusions
(Davies et al., 1997). In addition, ubiquitin immunoreactivity developed later in the disease course, suggesting
that ubiquitinization takes place after the mutant
fragment enters the nucleus. Similarly, the nuclear
inclusions in our HD patients were reactive with
antibodies to the N-terminus of huntingtin (aa 117),
but not to an antibody raised against an internal
epitope (aa 650663). This suggests that the inclusions
in HD patients also contain an N-terminal truncation
of huntingtin, although we cannot rule out the possibility of masking of the internal epitope such as would
occur with a structural change in the protein. Double
label studies showed that the same inclusion is immunoreactive for both ubiquitin and N-terminal huntingtin with colocalization of the reaction products. In our
material, ubiquitin immunoreactive inclusions were
more numerous than those identified by N-terminal
huntingtin antibodies. This finding may indicate that
N-terminal huntingtin epitopes are only found in a
subset of ubiquitin-positive inclusions. More likely,
these differences in antigenicity most likely relate more
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Becher et al.

to the crosslinking modifications that result from

lengthy formalin fixation and processing of routine
human postmortem tissue and reflect differences in the
sensitivities of the two antibodies.
Following the initial submission of this manuscript,
a description of huntingtin immunoreactive intranuclear inclusions in HD patients was reported by
DiFiglia and colleagues (1997). They showed, in a
limited number of juvenile- and adult-onset HD cases,
that intranuclear neuronal inclusions can be identified
with antibodies to the N-terminus of huntingtin (antibody Ab1, aa 117; similar to AP78 and AP194) and
with antibodies to ubiquitin in 40-m frozen sections
from postmortem tissue fixed in 4% paraformaldehyde
(DiFiglia et al, 1997). The present study validates these
findings, correlates the presence of inclusions with
length of expanded CAG triplet repeats, and extends
these investigations to include DRPLA, another triplet
repeat disorder which we show to be associated with
intranuclear inclusions. In addition, DiFiglia et al.
(1997) demonstrated the presence of similarly immunoreactive extranuclear neurites. These structures are not
evident in our archival paraffin embedded material,
probably due to differences in fixation, tissue preparation, or staining parameters. Recently, intranuclear
inclusions immunoreactive with disease-specific antiprotein antibodies and ubiquitin have also been found
to be present in neurons from patients with spinocerebellar ataxia type 1 (SCA1) (Skinner et al., 1997) and
spinocerebellar ataxia type 3 (SCA3 or MachadoJoseph disease) (Paulson et al., 1997), suggesting that
these abnormal protein aggregates may be a unifying
feature of many triplet repeat disorders.
The exact sequence and structure of the N-terminal
truncated protein which aggregates in the nucleus in
HD has not yet been determined, but the recent
findings described above by several investigators have
focused attention upon investigations into the mechanisms of polyglutamine-containing protein aggregation and cleavage. In the present study, the intranuclear inclusions appear to contain a truncation
product, but its size cannot be determined by our
reagents. However, we suspect that it is shorter than
663 amino acids (e.g., N-terminal to the site of the AP81
peptide). The study of DiFiglia et al. (1997) suggested
that the nuclear and neuritic inclusions consist of a
40-kDa N-terminal fragment of huntingtin, based on
Western blot experiments. In vitro experiments, related
to the N-terminus transgenic mice of Bates and colleagues, have shown that exon 1 of huntingtin with an
expanded repeat can form insoluble aggregates with
some characteristics of beta-pleated sheets (Scherz-

395

Neuronal Inclusions in Huntingtons Disease and DRPLA

inger et al., 1997). Perutz and colleagues have hypothesized that glutamine repeats may form a stable
beta-pleated sheet via a so-called polar zipper held
together by hydrogen bonds (Perutz, 1994; Perutz et al.,
1994; Stott et al., 1995). The lesions described in the
current study and other recent publications could be
composed of aggregates of this sort, though why they
should form preferentially in the nucleus is not known.
Huntingtin can be cleaved by the apoptosis-related
proteolytic enzyme caspase 3, with an efficiency increasing with the expansion of the glutamine repeat (Goldberg et al., 1996). Several caspase 3-sensitive sites are
predicted to be present in the first 600 amino acids
(Goldberg et al., 1996). However, it is also possible that
other proteolytic enzymes may also be important.
Huntingtin also interacts with a ubiquitin ligase E2
enzyme (Kalchman et al., 1996), raising the possibility
of ubiquitin-triggered proteolytic cleavage. In the Nterminal transgenic animals, the ubiquitinization appears to follow the appearance of inclusion bodies
with cleaved huntingtin, suggesting that cleavage of
huntingtin, by an unknown mechanism, may precede
ubiquitin conjugation.
In summary, intranuclear neuronal inclusions are
not limited to the recently described N-terminal transgenic mouse model of HD, but are a previously
unrecognized component of several naturally occurring triplet repeat disorders including HD and DRPLA.
The identification of this unusual intranuclear inclusion in neurons from routinely fixed and processed
material from HD and DRPLA patients provides a new
marker for the study of the polyglutamine repeat
disorders which will be very useful for future studies
of both human tissue and transgenic animal models.
Recognition of these nuclear abnormalities in naturally
occurring triplet repeat diseases suggests that intranuclear inclusions may be a unifying feature of the
pathogenesis of polyglutamine triplet repeat disorders
and has provided us with numerous avenues of
investigation which should clarify the mechanisms
leading to degeneration of nerve cells in these disorders.

ACKNOWLEDGMENTS
We thank the patients and families of the Baltimore Huntingtons
Disease Center for their support and generosity. This research was
supported by NIH NS16375, NS 34172, the Hereditary Disease
Foundation; including the Lieberman Award to G.P.B., and the
Huntingtons Disease Society of America.

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