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Regular Articles |
From the Departments of Pathology*
and
Neurology,
Brain Research Institute, Niigata
University, Niigata, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Another concern about the pathogenesis of CAG repeat diseases is the influence of NII formation on intranuclear structures. A recent in vitro study has indicated that large intranuclear aggregates, induced by mutant ataxin-1, sequester promyelocytic leukemia protein (PML) nuclear bodies and alter their normal nuclear distribution.8 The expression of mutant ataxin-3 in cultured cells also demonstrates the co-localization of intranuclear aggregates and PML nuclear bodies.23 PML is a nuclear-matrix-associated protein and a component of PML nuclear bodies.24,25 A typical mammalian nucleus has 10 to 20 PML nuclear bodies, which vary in size from 0.3 to 1 µm and are thought to be involved in growth regulation, transcriptional regulation, and apoptosis.24-26 Thus, the culture-based experiments suggest that in CAG repeat diseases, the alteration of intranuclear organizations may be induced by inclusion formation, which leads to nuclear dysfunction. To elucidate the effect of NII formation on intranuclear structures in human brains, in the present study we investigated the distribution of the PML nuclear body and the coiled body, the two most prominent subtypes of nuclear bodies.27 We show that in both DRPLA and MJD brains, PML reorganizes a specific structure around NII with a unique distribution pattern that has not been observed in previous in vitro studies. In addition, this study is the first to report that NIIs may be found in contact with coiled bodies. The interaction between coiled bodies and intranuclear aggregates is also confirmed in the brains of DRPLA transgenic mice and an in vitro study. The present study clarifies the significant nuclear events involved in the formation of NIIs, which may play a pivotal role in the pathological mechanisms of CAG repeat diseases in the human brain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Brains obtained at autopsy from a patient with MJD (female, Q83, age 32 years), a patient with DRPLA (female, Q59, age 79 years), and seven controls (ages 65 to 83 years; mean, 73.4 years) served as the materials for the present study. We also examined the brains of transgenic mice harboring a single copy of a full-length human mutant DRPLA gene with 129 CAG repeats.28 Because the occurrence of ubiquitinated NIIs has been detected in mice after 9 weeks of age, for the present study we examined the cerebral cortex of mice at 14 weeks of age.
Immunohistochemistry
Tissue fragments of the pontine nuclei from each human brain were obtained at autopsy, quick-frozen in cold isopentane, and kept in a deep freezer until use. Cryostat sections (8-µm thick) were made from the frozen materials, fixed with cold acetone (-20°C) for 7 minutes, and immunostained by the avidin-biotin-peroxidase complex (ABC) method with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA), using either a rabbit antibody against ubiquitin (1:800), c-Jun (1:2000), c-Fos (1:2000), Egr-1 (1:2000), Egr-2 (1:2000), or Egr-3 (1:2000) as a primary antibody. Sections were incubated with the primary antibody for 18 hours at 4°C. Cryostat sections were also immunostained with either a mouse monoclonal antibody against PML (1:200) or SUMO-1 (1:200). With the exception of the anti-ubiquitin antiserum, which was purchased from Dakopatts (Glostrup, Denmark), the primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Diaminobenzidine was used as the chromogen. After immunostaining, the sections were counterstained with hematoxylin. To investigate the interaction of NIIs with intranuclear structures, the cryostat sections were subjected to double-immunofluorescence staining. After incubation with 10% normal goat serum, sections were incubated with a mixture of a rabbit antibody against PML (1:50; Medical & Biological Laboratories, Nagoya, Japan) and a mouse monoclonal antibody against ubiquitin (1:50; Chemicon, Temecula, CA) for 18 hours at 4°C. For double-immunofluorescence staining of NIIs and coiled bodies, a rabbit antibody against PML was replaced with a rabbit antibody against p80-coilin29 (1:200; a gift from Dr. EK Chan, the Scripps Research Institute, La Jolla, CA). After washing with phosphate-buffered saline (PBS), the sections were incubated with a mixture of fluorescein-conjugated goat anti-mouse IgG (1:40; Cappel, Durham, NC) and rhodamine-conjugated goat anti-rabbit IgG (1:40; Cappel) for 1 hour at room temperature, and observed with the aid of a fluorescence microscope. For negative controls, the primary antibody was replaced with normal rabbit or mouse serum. A brain was obtained from a DRPLA transgenic mouse (14 weeks of age), which had been sacrificed by overdose inhalation of ether. The cerebrum was cut coronally, and quick-frozen in cold isopentane. Cryostat sections were made from the frozen tissues and immunostained in the same manner as the human material.
COS-7 Cell Transfection and Immunocytochemistry
As described previously,5 COS-7 cells were transfected with a full-length or truncated DRPLA cDNA encoding 19 or 82 glutamine residues. At 48 hours after transfection, cells were fixed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4), permeabilized with 0.1% Triton X-100 in PBS, and then treated with 10% normal goat serum to quench nonspecific staining. Double-immunofluorescence staining was performed using a mixture of mouse anti-FLAG M5 monoclonal antibody (1:400; Eastman Kodak, Rochester, NY) and rabbit anti-PML antibody (1:200), or a mixture of mouse anti-FLAG M5 antibody (1:400) and rabbit anti-p80-coilin antibody (1:200) for 18 hours at 4°C, followed by incubation with secondary antibodies in the same manner as the double-immunofluorescence staining of the human brain sections described earlier.
Electron Microscopic Observation
For conventional electron microscopic examination, tissue fragments of the pontine nuclei were obtained at autopsy from a brain of a MJD patient, fixed in 3% glutaraldehyde-1% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4, postfixed in 1% osmium tetroxide, dehydrated through a graded ethanol series, and embedded in Epon 812 resin (Polysciences, Warrington, PA). A DRPLA transgenic mouse was deeply anesthetized by inhalation of ether, and then perfused transcardially with PBS followed by 3% glutaraldehyde-1% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. After perfusion, the brain was removed from the cranium and immersed in the same fixative for a further 16 hours at 4°C. An age-matched nontransgenic mouse was prepared in the same way. The cerebral cortices of the mice were embedded in Epon 812 resin by the same method as the human brain tissue. Ultrathin sections were made from the tissue fragments, stained with uranyl acetate and lead citrate, and examined with a Hitachi-7100 electron microscope (Hitachi, Hitachinaka-city, Japan).
For immunoelectron microscopy, tissue fragments of the MJD pontine nuclei obtained at autopsy were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. A transgenic mouse was deeply anesthetized by inhalation of ether, and then perfused transcardially with the same fixative as was used to fix the human brain. The cerebral cortex was dissected out and prepared for analysis. The tissue fragments were dehydrated in a graded dimethylformamide series and embedded in LR White resin (London Resin Company, Berkshire, UK). Ultrathin sections were cut and mounted on nickel grids. After incubation with 10% normal goat serum for 10 minutes, the sections were incubated overnight at 4°C with a mixture of a rabbit antibody against PML (1:400) and a mouse monoclonal antibody against ubiquitin (1:50; Chemicon), or a mixture of a rabbit antibody against p80-coilin (1:200) and a mouse monoclonal antibody against ubiquitin (1:50; Chemicon). After washing with PBS, the sections were incubated with a mixture of a goat anti-rabbit IgG conjugated to 15-nm gold particles and a goat anti-mouse IgG conjugated to 10-nm gold particles (1:30; British BioCell International, Cardiff, UK) for 30 minutes at room temperature. The sections were then washed with PBS and incubated with 2% glutaraldehyde in 0.1 mol/L of sodium cacodylate buffer, pH 7.4. After washing with distilled water, the sections were stained with uranyl acetate and lead citrate, and examined with a Hitachi H-7100 electron microscope. For negative controls, the primary antibody was replaced with normal rabbit and mouse serum.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain the morphological details of NIIs, we investigated the
pontine nuclei of patients with MJD and DRPLA. In MJD, ubiquitinated
NIIs were observed in 54.4% of neurons. The number of NIIs per nucleus
varied, and the incidences of nuclei with a single, two (Figure 1a)
, three (Figure 1b)
, or more than
three NIIs was 25.3%, 73.4%, 0.6%, and 0.7%, respectively. In
DRPLA, ubiquitinated NIIs were observed in 8.5% of neurons, of which
the number of nuclei with one, two, or three NIIs was 67.6%, 30.9%,
and 1.5%, respectively. The NIIs varied in size from 0.8 to 3.7 µm
in MJD, and from 0.7 to 2.2 µm in DRPLA. NIIs were occasionally
present in the vicinity of the nucleoli. In the case of neurons with
two NIIs per nucleus, the inclusions frequently appeared as pairs or
doublets (70.3% in MJD and 71.5% in DRPLA, Figure 1a
).
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. SUMO-1-immunoreactive NIIs
were observed in 56.3% and 2.8% of neurons in MJD and DRPLA,
respectively. We examined further the interaction between transcription
factors and NIIs, and found that in both MJD and DRPLA, NIIs were
intensely immunopositive for c-Jun (Figure 1d)
and weakly
immunopositive for c-Fos (data not shown), but were immunonegative for
Egr-1, Egr-2, and Egr-3. In MJD and DRPLA, c-Jun-positive NIIs
were observed in 20.4% and 1.5% of neurons, respectively, and
c-Fos-positive NIIs were observed in 13.8% and 0.8% of neurons,
respectively. PML Nuclear Bodies in CAG Repeat Diseases, DRPLA Transgenic Mice, and Cultured Cells Transfected with DRPLA Protein with Expanded PolyQ Stretches
PML immunoreactivity appeared in normal neurons as 8 to 18 nuclear
dots, which varied in size from 0.2 to 0.8 µm (Figure 1e)
. In MJD and
DRPLA brains, PML labeling was rearranged in 47.1% and 11.0% of
neurons, respectively, and formed a single large body (Figure 1f)
,
which varied in size from 1.7 to 3.0 µm and from 1.5 to 2.3 µm,
respectively. Most of these large PML structures were present solely in
the neuronal nuclei; however, some bodies coexisted with a few PML dots
of normal size. In neuronal nuclei that lacked the formation of large
PML structures, there were no remarkable changes in their appearance
and distribution. Double-immunofluorescence staining of MJD and DRPLA
brains revealed that the large PML structures were co-localized with
ubiquitinated NIIs (Figure 1
; g, h, and i). In the case of the
single-type NIIs, the incidence of the co-localization was as high as
94.4% in MJD and 83.9% in DRPLA. Interestingly, in the case of
neurons harboring more than one NII per nucleus, the large PML
structures were co-localized with only one of the NIIs (Figure 1
; i to
l). In general, the PML-immunolabeling was larger than the
ubiquitin-labeling, and was accentuated in the peripheral region of the
NIIs, exhibiting a donut-like appearance.
We examined the PML nuclear bodies of neurons in DRPLA transgenic mice
carrying a single copy of a full-length human mutant DRPLA gene with
129 CAG repeats.28
At 3 weeks of age these mice exhibited
myoclonic movement. This was followed at up to 12 weeks of age by rapid
progression of ataxia, myoclonic movement, and epilepsy; these animals
all died by the age of 14 to 16 weeks. Neuropathologically, the brains
showed progressive atrophy beginning as early as 4 weeks, but disclosed
no apparent loss of neurons in any central nervous system (CNS) region
until death. The occurrence of ubiquitinated NIIs was first detectable
at 
9 weeks of age in restricted regions of the CNS such as the
subthalamic nucleus and the cerebellar nuclei. In the 14-week-old mouse
brain, ubiquitinated NIIs were present in a single form, and appeared
in more extensive regions of the CNS including the cerebral cortex
(layers II, III, IV, and VI), globus pallidus, thalamus, pontine
nuclei, and the brainstem tegmentum, showing similarities with the
pathology seen in human brains.3
In normal cerebral
cortical neurons in 14-week-old mice, PML labeling appeared as multiple
small dots in each nucleus, measuring from 0.3 to 0.6 µm in size, and
scattered throughout the nucleoplasm. In the DRPLA transgenic mouse
brain, cerebral cortical neurons lacking NIIs showed no change in the
PML-labeling pattern. Most of the cortical neurons harboring NIIs also
retained the scattered pattern of PML labeling (Figure 1, m and n)
, but
the association of a few PML dots was occasionally encountered around
some NIIs (Figure 1m)
. The large donut-like labeling of PML seen in
human brains was not observed in these young mouse brains.
We examined the distribution of PML nuclear bodies in COS-7 cells
expressing mutant DRPLA protein. As observed in our previous
study,5
the formation of aggregate bodies was restricted
to cells expressing truncated DRPLA protein with 82 glutamines, and
observed mostly in the perinuclear regions of the cytoplasm, and less
frequently in the nuclei. In cells lacking aggregate formation, PML
nuclear bodies appeared as multiple intranuclear dots 0.3 to 1.1 µm
in size, and were scattered throughout the nucleoplasm. In cells
harboring intranuclear aggregates, PML nuclear bodies retained their
multiple dot-like appearance, but some were recruited around the
aggregates (Figure 1o)
. Cells with only cytoplasmic aggregates showed
no apparent changes in the distribution of PML nuclear bodies. The
immunohistochemical studies using human and mouse brains, as well as
cultured cells, revealed no positive staining in the control tissue.
Localization of PML to the Ring-Like Structure Surrounding NIIs
To elucidate the precise localization of PML, we conducted an
ultrastructural analysis of the NIIs of a MJD patient. Conventional
electron microscopic examination revealed that NIIs lacked a limiting
membrane, were heterogeneous in composition, and contained a mixture of
granular and filamentous structures (Figure 2a)
. The filamentous composition was
straight or slightly curved, 12 to 15 nm in diameter, and usually
organized in random, but sometimes parallel arrays. Although we did not
observe neuronal nuclei with more than two inclusions per nucleus,
nuclei with two NIIs were occasionally encountered when viewed with the
aid of an electron microscope. These twin-type NIIs were also composed
of granular and filamentous structures similar to those of the
single-type NIIs. Occasionally, NIIs were surrounded by ring-like
structures, which were basically composed of granular materials and
exhibited various appearances from broad circles with an irregular and
vague contour, to a clear-ring architecture (Figure 2, b and c)
.
Interestingly, in the case of the nuclei harboring two inclusions per
nucleus, the formation of ring-like structures was restricted to only
one of the NIIs (Figure 2, b and c)
. Electron microscopic
immunohistochemistry using immunogold labeling revealed that PML
immunoreactivity was present in both types of NIIs, those lacking or
harboring the ring-like structures. In the case of the former type,
labeling was scattered rather broadly at the marginal region of the
NIIs (Figure 2d)
. In the case of the latter, however, PML labeling was
more concentrated on the ring architecture (Figure 2e)
. Ubiquitin
immunoreactivity was essentially present on the core structure of the
NIIs, and was rarely observed intermingled with PML labeling, even in
NIIs lacking a ring-like structure (Figure 2d)
.
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9
to 12 nm in diameter, and organized in random arrays. The ring-like
structures seen around the NIIs of the MJD brain were not observed in
DRPLA transgenic mice. No labeling for PML protein was detected on or
around NIIs (Figure 2f)
, as revealed using immunoelectron microscopy. Close Association of NIIs with p80-Coilin-Immunopositive Structures
To clarify the interaction of NIIs with intranuclear structures,
we investigated further, immunohistochemically, the localization of
p80-coilin, a constituent protein of coiled bodies,30
in
MJD and DRPLA brains. In the neuronal nuclei of normal human brains,
p80-coilin immunoreactivity appeared as dots measuring up to 1.7 µm.
Most neurons showed the presence of a single coiled body in a nucleus
(Figure 3a)
, but some had a few or more
smaller coiled bodies per nucleus (Figure 3b)
. In the MJD brain, no
apparent changes were observed in the distribution and sizes of
p80-coilin labeling; however, some neuronal nuclei showed paired
immunolabeling (Figure 3c)
similar to that observed with ubiquitin
immunohistochemistry. Double-immunofluorescence studies revealed that
most of the ubiquitinated NIIs were distinct from structures positive
for p80-coilin, but both were present in close contact with each other
(Figure 3
; d to g). This relationship was also confirmed in the DRPLA
human brain (Figure 3, h and i)
. In the case of the single-type NIIs,
the incidence of the adjoining of NIIs and coiled bodies was 76.7% in
MJD and 83.3% in DRPLA. The rest of the single-type NIIs mostly lacked
p80-coilin labeling in any nuclear regions of the examined section.
When observed with the aid of fluorescence microscopy, most of the
paired NIIs were associated with one or more coiled bodies (Figure 3, f and g)
. In some nuclei, p80-coilin lost its normal dot-like
distribution, and was co-localized with ubiquitinated NIIs (Figure 3
;
j, k, and l). Contacts or partial co-localization of NIIs with
p80-coilin labeling were also observed in the cerebral cortical neurons
of DRPLA transgenic mice (Figure 3, m and n)
, with the incidence
reaching as high as 93.1% of NIIs. Labeling for p80-coilin appeared as
a few dots (1.1 µm) in most of neuronal nuclei, and there were no
apparent changes between normal and transgenic mice in the size and
distribution of the labeled structures. In COS-7 cells,
p80-coilin-labeling appeared as a few discrete round dots measuring
from 0.4 to 1.0 µm. Although there were no apparent differences in
the size and distribution of coiled bodies between cells harboring or
lacking the aggregates of mutant protein, intranuclear aggregates were
frequently associated with p80-coilin-positive structures (Figure 3o)
.
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To elucidate the morphology of p80-coilin-immunopositive dots in
neuronal nuclei, we subjected NIIs in the MJD brain to immunoelectron
microscopic observation. Immunogold labeling for p80-coilin was
localized on granulofibrillar structures (Figure 4
; a, b, and c) that were round to oval
in shape, measured from 0.5 to 1.0 µm in diameter, and corresponded
morphologically to the coiled body.31,32
These labeled
structures were present in contact with NIIs (Figure 4a)
, and were
occasionally positioned between two NIIs (Figure 4, b and c)
. The
association of NIIs with coiled bodies was observed frequently, even
when viewed using conventional electron microscopy (Figure 2
; a, b, and
c, arrowheads).
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, and the incidence reached as high as 77.8% of NII sections
(n = 126). The morphology of the
granulofibrillar bodies was consistent with that of coiled bodies, and
the size varied, probably because of the level at which the bodies were
sectioned, reaching up to 0.9 µm in diameter. The bodies possessed
radially oriented filaments around the granulofibrillar core, and NIIs
were connected with these filamentous structures (Figure 4e)
. Electron
microscopic immunohistochemistry confirmed the granulofibrillar cores
to be positive for p80-coilin (Figure 4f)
. In the neuronal nuclei of
nontransgenic mouse brains, the granulofibrillar bodies were
encountered in 11.1% of nucleus sections. No apparent differences were
observed in the size and appearance of granulofibrillar bodies,
including the surrounding filaments, between the transgenic and
nontransgenic mice. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is interesting that in the present study PML was concentrated on one
NII per nucleus, even when more than one ubiquitinated NII was present
in a nucleus. This predilection was not observed in previous culture
experiments forming multiple intranuclear aggregates of mutant
proteins.8,23
As disclosed by the ultrastructural study,
NIIs were heterogeneous in composition. Therefore, a certain specific
type of NII may be present in each nucleus, which in turn may be
related to the predilection of PML for a particular NII, although we
were unable to describe the morphological features common to the NIIs
that were associated with PML. Alternatively, the concentration of PML
may depend on the order of NII formation. From our results, we
speculate that most of the PMLs are concentrated in the first NII that
is formed in a nucleus. In other words, the formation of the first NII
within a nucleus might exert a certain influence on the subsequent
distribution of PML and bring about the accumulation of the protein to
itself, even when another NII has been formed. At present, it remains
to be demonstrated whether in human brain pathology more than one NII
is formed simultaneously in a nucleus from the beginning. However, we
have shown that in DRPLA transgenic mice single NII formation is an
essential event in the brain during at least the period from 9 weeks
(an initial stage of NII formation) up to 16 weeks (maximal survival
time of the mice) of age.
In contrast to PML, SUMO-1 was distributed prevalently among NIIs. SUMO-1 is a protein with a ubiquitin-homology domain, and it covalently modifies specific nuclear proteins such as PML and Sp100, another PML nuclear body-associated protein.38 This modification by SUMO-1 (sumoylation) has been proposed to play a role in directing the proteins to specific subnuclear compartments such as PML nuclear bodies, rather than targeting them to proteasomal degradation.39 This implicates a role for SUMO-1 in the compartmentalization of certain proteins into NIIs. Alternatively, SUMO-1 may function in protecting specific proteins from degradation at NIIs. As indicated in recent studies,21,22,40,41 several transcription factors such as CBP and p53 are recruited into intranuclear inclusions. The present study also demonstrated the co-localization of c-Jun and c-Fos in NIIs. The fact that p53 and c-Jun are targets of ubiquitin and are also substrates for SUMO-1 that enable it to avoid ubiquitination,42 may suggest that SUMO-1 plays a role in protein stability in NIIs. It is likely, however, that the recruitment of many kinds of nuclear proteins into NIIs leads to gradual nuclear dysfunction, eventually resulting in neuronal atrophy or degeneration.
Another striking feature revealed by the present study is the close
association between NIIs with coiled bodies. This association was
observed in DRPLA and MJD brains, DRPLA transgenic mouse brains, and
cultured cells, forming intranuclear aggregates with mutant DRPLA
protein. The common occurrence of this phenomenon among different
diseases suggests that the contact is dependent on the expanded polyQ
stretches within the causative DRPLA protein or ataxin-3.
Ultrastructurally, NIIs and coiled bodies were observed either in
direct contact with each other or connected to each other by
filamentous structures (Figure 4e)
. The molecular basis of this
interaction is a matter of interest; however, it is unlikely that
p80-coilin mediates this connection, because the molecule does not
contain any clearly recognizable peptide motifs43-45
or
polyQ domains, the latter of which have the potential to interact with
mutant extended polyQ.20
Electron microscopic
immunohistochemistry for p80-coilin also failed to label either the
connecting filaments or the NIIs. Between proteins contained in coiled
bodies,27,46
TATA-binding protein may be one of the
candidates involved in the connection, because TATA-binding protein
contains a particularly long polyQ stretch and is recruited in the NIIs
of DRPLA and MJD brains.21
The function of coiled bodies is not fully understood. They contain small nuclear ribonucleoprotein particles, which are involved in splicing, and recent studies have suggested that these structures play a role in small nuclear ribonucleoprotein particle biogenesis.25,27 The coiled body is a highly dynamic structure that undergoes assembly and disassembly during the cell cycle, the number of changes being related to cell growth and gene expression level.25,47 Although in the present study the coiled bodies showed no apparent changes in their morphology and distribution, it is possible that their close interaction with NIIs may result indirectly in alterations in the function of coiled bodies. It is feasible that because of their relatively large size, NIIs interfere with the motility of coiled bodies.48 This interference may extend to the intimate relationship between the coiled body and the nucleoli.25,27 To elucidate the mechanisms that underlie neuronal cell death in CAG repeat diseases, it is now necessary to explore how the association between NIIs and coiled bodies affects the function of the latter. Aside from the influence of NIIs, the preferential association between NIIs and coiled bodies may explain the reason why NIIs are frequently present in paired or doublet form when more than one NII is present in a nucleus. As revealed by electron microscopy, coiled bodies were frequently observed sandwiched between two NIIs.
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Acknowledgements |
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Footnotes |
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Supported by a grant from the Research Committee for Ataxic Diseases, the Ministry of Health, Labor, and Welfare, Japan; and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Accepted for publication July 27, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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