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The Journal of Neuroscience, October 1, 1998, 18(19):7674-7686
The Cellular and Subcellular Localization of
Huntingtin-Associated Protein 1 (HAP1): Comparison with Huntingtin in
Rat and Human
Claire-Anne
Gutekunst1,
Shi-Hua
Li2,
Hong
Yi1,
Robert J.
Ferrante3,
Xiao-Jiang
Li2, and
Steven M.
Hersch1
Departments of 1 Neurology and 2 Genetics,
Emory University School of Medicine, Atlanta, Georgia 30329, and
3 Geriatric Research Education Clinical Center, Bedford
Veterans Administration Medical Center, Bedford, Massachusetts 01730, and Department of Neurology, Boston University School of Medicine,
Boston, Massachusetts 02118
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ABSTRACT |
The cellular and subcellular distribution of HAP1 was examined in
rat brain by light and electron microscopic immunocytochemistry and
subcellular fractionation. HAP1 localization was also determined in
human postmortem tissue from control and Huntington's disease (HD)
cases by light microscopic immunocytochemistry. At the cellular level,
the heterogeneity of HAP1 expression was similar to that of huntingtin;
however, HAP1 immunoreactivity was more widespread. The subcellular
distribution of HAP1 was examined using immunogold electron microscopy.
Like huntingtin, HAP1 is a cytoplasmic protein that associates with
microtubules and many types of membranous organelles, including
mitochondria, endoplasmic reticulum, tubulovesicles, endosomal and
lysosomal organelles, and synaptic vesicles. A quantitative comparison
of the organelle associations of HAP1 and huntingtin showed them to be
almost identical. Within HAP1-immunoreactive neurons in rat and human
brain, populations of large and small immunoreactive puncta were
visible by light microscopy. The large puncta, which were especially
evident in the ventral forebrain, were intensely HAP1 immunoreactive.
Electron microscopic analysis revealed them to be a type of
nucleolus-like body, which has been named a stigmoid body, that may
play a role in protein synthesis. The small puncta, less intensely
labeled, were primarily mitochondria. These results indicate that the
localization of HAP1 and huntingtin is more similar than previously
appreciated and provide further evidence that HAP1 and huntingtin have
localizations consistent with roles in intracellular transport. Our
data also suggest, however, that HAP1 is not present in the abnormal
intranuclear and neuritic aggregates containing the N-terminal fragment
of mutant huntingtin that are found in HD brains.
Key words:
Huntington's disease; electron microscopy; immunogold; nucleolus-like bodies; cytoplasmic inclusions; stigmoid bodies
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INTRODUCTION |
The neuropathology of Huntington's
disease (HD) includes the selective loss of neurons that is most severe
in the neostriatum but also affects other brain regions. Excitotoxic,
oxidative, and apoptotic mechanisms of neuronal death have all been
implicated in HD (Hersch and Ferrante, 1997 ; Wellington et al., 1997);
however, none have yet been directly connected to the polyglutamine
expansion occurring in the expressed protein huntingtin. In human
neostriatum, neurons most vulnerable to neurodegeneration express the
highest levels of huntingtin, whereas the resistant ones express little or no huntingtin (Gutekunst et al., 1995; Ferrante et al., 1997). Although the normal function of huntingtin is unknown, a role in
intracellular transport has been hypothesized based on its subcellular
association with microtubules and membrane-bound organelles (DiFiglia
et al., 1995; Gutekunst et al., 1995). Recent studies have shown that
N-terminal fragments of mutant huntingtin can form aggregates (Davies
et al., 1997; DiFiglia et al., 1997; Scherzinger et al., 1997) that
might play a role in cell injury.
Other protein-protein interactions may also be important in the
pathogenesis of HD. Huntingtin interacts with several proteins including huntingtin-associated protein 1 (HAP1) (Li et al., 1995, 1996), glyceraldehyde phosphate dehydrogenase (Burke et al., 1996), an
unidentified calmodulin-associated protein (Bao et al., 1996), a
ubiquitin-conjugating protein (HIP2) (Kalchman et al., 1996), and a
protein homologous to the yeast cytoskeleton-associated protein Sla2p
(huntingtin-interacting protein 1 or HIP1) (Kalchman et al., 1997;
Wanker et al., 1997). Binding of both HAP1 and HIP1 varies with the
length of the polyglutamine tract; thus their normal function may be
altered by the HD mutation.
HAP1 was first identified by yeast two-hybrid screening (Li et al.,
1995). The highest levels of HAP1 are in the striatum, olfactory bulbs,
and brainstem, whereas lower levels were identified in the cerebral
cortex, hippocampus, and cerebellum (Li et al., 1995, 1996), regions
with the highest levels of huntingtin expression (Li et al., 1993;
Strong et al., 1993; Landwehrmeyer et al., 1995). Despite regional
differences in HAP1 and huntingtin expression, their distribution
within neurons may be quite similar. Subcellular fractionation studies
have shown that both HAP1 and huntingtin are present in soluble and
membrane-containing fractions (S.-H. Li et al., 1993; Sharp et al.,
1995; X.-J. Li et al., 1996). Two recent studies have shown that HAP-1
interacts with dynactin P150Glued (Engelender et
al., 1997; Li et al., 1998a), suggesting its possible role in
intracellular transport. HAP1 may be involved in trafficking of
organelles or proteins such as huntingtin. The HD mutation may alter
the normal transport of huntingtin via HAP1 and lead to abnormal
accumulation and aggregation in neuronal processes.
Using pre-embedding immunogold electron microscopy, we show that HAP1
and huntingtin have very similar cellular and subcellular localizations
and provide further evidence that HAP1 and huntingtin may have roles in
intracellular trafficking. We also show that huntingtin-containing
intranuclear and neuritic aggregates found in HD do not contain HAP1.
Finally we found HAP1 to be a marker of the neuronal structures
identified as stigmoid bodies.
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MATERIALS AND METHODS |
Antibodies. Rabbit polyclonal antibodies specific for
HAP1 were raised against a recombinant protein derived from a segment of the rat HAP1 (amino acids 287-452) fused to
glutathione-S-transferase (GST) and affinity purified, as
described previously (Li et al., 1995). Rat monoclonal antibodies
specific for huntingtin (mHD549) were raised against a segment of the
human huntingtin (amino acids 549-659) fused to GST, as described
previously (Gutekunst et al., 1995). To detect N-terminal huntingtin
aggregates, we also used a polyconal antibody (EM48) developed against
the first 253 amino acids of huntingtin (Li and Li, 1998). Polyclonal
antibodies were used at 1:1000, and mHD549 was used at 1:100 in the
following immunocytochemistry experiments. Mouse anti-human
mitochondrial monoclonal antibodies MAB1273 (Chemicon, Temecula, CA)
and MCA151A (Serotec, Oxford, UK) were obtained commercially and used
in human brain tissue at 1:500-750.
Immunocytochemistry. Adult male Sprague Dawley rats
(n = 6) were used for light microscopic
immunocytochemistry. Each rat was deeply anesthetized with an overdose
of 4% chloral hydrate, injected intraperitoneally with 300 IU of
heparin, and then perfused intracardially with 0.9% saline containing
0.005% sodium nitroprusside for 1 min, followed by 3%
paraformaldehyde in 0.1 M phosphate buffer at pH 7.2 (PB)
for 10 min at a rate of 20 ml per min, followed by 10% sucrose in 0.1 M PB (200 ml for 10 min). Brains were removed and
cryoprotected in 30% sucrose at 4°C. Brains were sectioned at 40 µm using a freezing microtome, collected in PB, and rinsed in 0.05 M Tris-buffered saline (TBS), pH 7.2. Free-floating
sections were preblocked in 4% normal goat serum (NGS) in TBS, 0.1%
Triton X-100, and avidin (10 µg/ml) for 1 hr at 4°C. Sections were
incubated in primary antibodies in TBS containing 2% NGS, 0.1% Triton
X-100, and biotin (50 µg/ml) at 4°C for 48 hr. Sections were then
rinsed in TBS for a total of 1 hr and incubated overnight in
biotinylated goat anti-rabbit antibody for HAP1 and goat anti-rat
antibody (ABC Elite; Vector Laboratories, Burlingame, CA) for HD549 in TBS containing 2% NGS. After several rinses in TBS, the sections were
incubated in avidin-biotin complex (ABC Elite; Vector) for 1 hr and
rinsed several times in TBS. Immunoreactivity was visualized by
incubation in 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB;
Sigma, St. Louis, MO) and 0.01% hydrogen peroxide in 0.05 M Tris buffer, pH 7, until a dark brown reaction product
was evident (5-10 min). Controls included the omission of primary
antisera and preabsorption of antibody with excess respective GST
fusion proteins (HAP1 or huntingtin) for 1 hr at room temperature.
Five additional rats were used for both immunoperoxidase and
pre-embedding immunogold electron microscopy experiments. These rats
were anesthetized as described above and perfused with 200 ml of 3%
paraformaldehyde and 0.2% glutaraldehyde in 0.1 M PB at pH
7.2. Brains were sectioned at 50 µm using a vibratome (Pelco, Redding, CA). Immunoperoxidase staining was performed as described above except that Triton X-100 was omitted from the blocking steps and
from antibody solutions. After DAB visualization, sections were
osmicated (1% OsO4 in 0.1 M cacodylate
buffer), rinsed, and stained overnight in 2% aqueous uranyl
acetate.
For more precise spatial resolution, we used pre-embedding immunogold
labeling of HAP1 and huntingtin. Vibratome sections were preblocked in
5% NGS and 0.05% Triton X-100 in TBS for 30 min and incubated at
4°C on a shaker platform in primary antibodies in 2% NGS in TBS for
60 hr. Sections were then rinsed in six changes of TBS for a total of
60 min and were incubated overnight in Fab fragments of goat anti-rat
secondary antibodies (1:50) conjugated to 1.4 nm gold particles
(Nanoprobes, Stony Brook, NY) in TBS with 2% NGS. After rinsing in TBS
and then PB, sections were fixed with 2% glutaraldehyde in 0.1 M PB. After several washes in PB, sections were silver
intensified following a modification (Yi and Hersch, 1997) of the Burry
et al. (1992) method, post-fixed for 10 min in 0.5% OsO4
in PB, and processed for electron microscopy as described below.
Two additional animals were perfused with 3% paraformaldehyde and
2.0% glutaraldehyde to provide optimally fixed tissue for examining
unlabeled cytoplasmic inclusions. All the sections used for electron
microscopy were dehydrated in ascending concentrations of ethanol and
propylene oxide (1:1) and embedded in Eponate 12 (Ted Pella, Redding,
CA). Ultrathin sections (90 nm) were cut with a Leica Ultracut S
ultramicrotome and examined with a JEOL 100C electron microscope. Thin
sections were counterstained with 5% aqueous uranyl acetate for 5 min,
followed by Reynolds lead citrate (Reynolds, 1963) for 5 min.
To examine the normal distribution of HAP1 in the human, we performed
immunocytochemistry on postmortem striatal and cortical tissue from 23 male and female humans with no evidence of neurological disease. Ages
of the controls ranged from 54 to 79 years (mean age, 64.3 years). We
also performed immunocytochemistry on four adult-onset grade 3 and
grade 4 HD cases to determine whether HAP1 was contained within the
recently described protein aggregates containing N-terminal fragments
of huntingtin. Ages of the HD cases ranged from 42 to 78 years (mean
age, 68.6 years). Intact hemispheres were placed in cold (4°C) 2%
paraformaldehyde-lysine-periodate solution overnight. Hemispheres
were then sliced coronally (1 cm thick) and fixed another day in fresh
fixative solution. Tissue slices were rinsed in 0.1 M
sodium phosphate buffer and placed in cold cryoprotectant in increasing
concentrations of 10 and 20% glycerol and 2% DMSO solution. Blocks
including the regions of interest were dissected, and frozen serial
sections were cut at 50 µm intervals. The cut sections were stored in
0.1 M sodium phosphate buffer and 0.08% sodium azide at
4°C for subsequent immunocytochemistry. Immunocytochemistry was
performed as recently described (Ferrante et al., 1997).
Immunogold quantification. Quantification of the immunogold
labeling within dendrites was performed in the frontal cortex so that
longitudinally sectioned apical dendrites of pyramidal cells could be
used to maximize the visualization of microtubules. A similar
comparison was not performed for axon terminals because too few
well-labeled huntingtin-immunoreactive terminals were identified in any
brain region. For each antigen, one block each from the frontal cortex
from two different brains was selected, thin sectioned, mounted on mesh
grids, and stained as described previously. Three to four grid squares
containing the tissue/plastic interface were scanned, and every
dendritic profile containing more than five immunogold particles was
photographed at 13,000×. The electron micrographs were examined, and
the localization and associations of each individual immunogold
particle were assessed. Potential associations were with the plasma
membrane, microtubules, mitochondria, and tubulovesicular elements
(smooth endoplasmic reticulum and vesicles). Immunogold particles not
associated with any structure were designated cytoplasmic, even though
they could be in contact with structures above or below the plane of
the section. When clusters of particles (usually two to four) were encountered, an effort was made to assess each particle individually. Particles were considered to be associated with an organelle if they
were in direct contact. If there was any space at all between an
immunogold particle and an organelle, they were not considered to be
associated. Because dendritic profiles varied greatly in size and
labeling density, statistical treatments in which individual profiles
are treated as comparable counting objects could not be used. The
association data for each antibody were summed, and the proportions of
immunogold particles associated with a given organelle or compartment
were calculated.
Subcellular fractionation and microtubule preparation.
Subcellular fractions of rat brain were prepared essentially as
described previously (Huttner et al., 1983). Tissue homogenate was
centrifuged for 10 min at 1000 × g to produce a crude
nuclear fraction (P1). The supernatant (S1) was then centrifuged at
10,000 × g for 15 min to produce a pellet (P2) and a
supernatant (S2). S2 was centrifuged at 100,000 × g
for 2 hr to give pellet (P3) and soluble (S3) fractions. The P2
fraction was resuspended in a small volume of buffered sucrose and
hypotonically lysed by addition of 10 vol of ice-cold water on ice for
30 min. The P2 lysate was centrifuged for 20 min at 25,000 × g to yield the lysate pellet (LP1) and lysate supernatant
(LS1). The LS1 fraction was then centrifuged at 165,000 × g for 2 hr to give a crude synaptic vesicle pellet (LP2) and supernatant (LS2). Equal amounts of protein from each fraction (65 µg/lane) were resolved in 4-12% SDS gels, and Western blot analysis
was conducted with antibodies to HAP1 (Li et al., 1995), huntingtin
(Gutekunst et al., 1995), and synaptophysin (Boehringer Mannheim,
Indianapolis, IN).
Rat brain microtubule pellets were prepared as described previously
(Vallee, 1982). Microtubules in the rat brain cytosolic extract in
PIPES buffer (50 mM PIPES, 1 mM EGTA, and 1 mM MgSO4, pH 6.9) were polymerized in
the presence of 1 mM GTP and 20 mM taxol
(Sigma) for 20 min at room temperature. Polymerized microtubles were
pelleted by centrifugation at 100,000 × g for 30 min
at room temperature. The microtubules were resuspended in the same
PIPES buffer and precipitated again by centrifugation through a 20% sucrose cushion. The cytosolic fraction and microtubule pellets (80 µg of protein per lane) were subjected to Western blot analysis with
antibodies to HAP1 and huntingtin.
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RESULTS |
Cellular distribution of HAP1
Although HAP1 immunoreactivity was widespread in the rat brain,
there were regional differences (Fig. 1)
with the most intense labeling being evident in the striatum, globus
pallidus, amygdala, hypothalamus, and much of the ventral forebrain.
Individual laminae were intensely stained in other areas, such as layer
V of the cerebral cortex, the hippocampal pyramidal cell layer, and the Purkinje cell layer of the cerebellum. There were HAP1-immunoreactive neurons in every region of the brain examined, with minimal labeling of
glia and only modest labeling of white matter tracts (Figs. 1,
2). In all cases, immunoreactivity was
abolished when primary antibodies were either omitted or preabsorbed
against GST-HAP1 fusion protein (Fig. 1F).
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Figure 1.
Regional distribution of HAP1 in the rat brain.
A-E, Representative coronal sections from
HAP1-immunostained tissue are shown from rostral to caudal.
F, The immunolabeling is abolished after preabsorption
over excess GST-HAP1 fusion protein. bs, Brainstem;
cb, cerebellum; cx, cortex;
hip: hippocampus; poa, preoptic area;
st, striatum thal, thalamus. Scale bar, 1 mm.
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Figure 2.
Similarities in HAP1 and huntingtin distribution
in the rat brain. Light micrographs showing HAP1
(A-D) and huntingtin Hp549
(E-H) immunoreactivity are shown. Both HAP1 and
huntingtin immunolabeling filled neuronal perikarya and dendrites.
Throughout the brain, HAP1 cellular distribution was very similar to
that of huntingtin; however HAP1 labeled more neurons than did
huntingtin. A, E, In somatosensory
cortex, HAP1 (A) and huntingtin
(E) are found in pyramidal neurons.
B, F, In striatum, both medium-sized
neurons and larger neurons (arrows) are labeled with
HAP1 (B) and huntingtin
(F). C, G, In
globus pallidus, neurons and dendrites are well stained by both HAP1
(C) and huntingtin (G).
D, H, In the cerebellum, Purkinje cells
(arrows) are more intensely stained by both HAP1
(D) and huntingtin
(H) than are neurons in the granule cell
layer (g). Scale bars: A,
E, 200 µm; B, D,
F, H, 50 µm; C,
G, 25 µm.
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The cellular pattern of immunostaining for HAP1 was
heterogeneous in most rat brain regions with both intensely labeled and lightly labeled neurons evident (Fig. 2A-D). This
pattern of heterogeneity was very similar to that of huntingtin
immunolabeling from contiguous sections (Fig.
2E-H). HAP1 immunocytochemistry, however,
often revealed more neurons than did huntingtin immunocytochemistry. In
the cerebral cortex (Fig. 2A,E),
layer V pyramidal cells and scattered layer III and VI neurons were
most intensely stained by both antibodies; however, more lightly
stained neurons in all layers were evident with HAP1
immunocytochemistry. Few nonpyramidal cells were evident with either
antibody. In the striatum (Fig. 2B,F), populations of light-
and dark-stained neurons were evident with both antibodies, but more
neurons were evident with HAP1 immunocytochemistry. More medium-sized
neurons and more neurons with the appearance of striatal interneurons
appeared to be HAP1 immunoreactive, in comparison with huntingtin. In
the globus pallidus (Fig. 2C,G), neurons and
dendrites were similarly well stained by HAP1 and huntingtin
immunocytochemistry. In both the striatum and pallidum, the neuropil
was more intensely stained by HAP1 than by huntingtin
immunocytochemistry. Regional heterogeneity consistent with striosome
or patch and matrix organization was not evident with HAP1
immunocytochemistry. In the hippocampus, both pyramidal and granule
cells stained for both HAP1 and huntingtin. In the cerebellum (Fig.
2D,H), Purkinje cells were
densely stained by both HAP1 and huntingtin antibodies, whereas the
granule cell layer was relatively lightly stained. Individual
HAP1-immunoreactive neurons contained reaction product primarily in
their perikarya and proximal dendrites (Fig. 2A-D).
In the most intensely labeled neurons, however, extensive labeling of
dendritic trees and proximal axon segments was evident.
HAP1 labeling of cytoplasmic puncta
By light microscopy, the HAP1 immunoreaction product was not only
diffusely present in neuronal perikarya and dendrites but was also
evident as intensely labeled cytoplasmic puncta (Fig. 3). These puncta were observed in neurons
throughout the brain and could be divided into two types based on their
size, labeling intensity, and number per neuron. The first type of
puncta (Fig. 3A-D) was intensely labeled and ranged from
0.5 to 4 µm in diameter. These puncta were evident in pyramidal
and nonpyramidal neurons of the cerebral cortex (Fig. 3A),
in medium-sized striatal neurons (Fig. 3B), and in
scattered neurons in most brain regions. Neurons containing the largest
and greatest numbers of these puncta were in the medial preoptic area
(Fig. 3C), the lateral septum (Fig. 3D),
many hypothalamic nuclei, the bed nucleus of the stria terminalis, and
the medial amygdala. Less than 12 of these puncta were observed in the
cytoplasm of the neurons containing them. The second type of puncta was
less intensely labeled, much more frequent, and ranged from 0.5 to 1 µm in diameter. They were very numerous with >50 countable per
neuron where they were observed in perikarya and proximal dendrites.
These smaller puncta were widely distributed but were especially
discernible in larger, well-stained cells such as globus pallidus
neurons (Fig. 3E), pontine motor neurons (Fig.
3F), and Purkinje cells (Fig. 3G).
The large and small puncta often coexisted in individual neurons (Fig.
3F). The nature of these puncta was subsequently
determined using immunogold electron microscopy.
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Figure 3.
HAP1 labeling of large and small inclusions in the
rat brain. Light micrographs showing HAP1 immunolabeling of inclusions
in neurons from the cortex (A), striatum
(B), preoptic area (C), and
the septal region (D) are shown. In globus
pallidus (E), brainstem neurons
(F), and Purkinje cells
(G), HAP1 immunoperoxidase labels organelles that
are smaller and more numerous than stigmoid bodies and may primarily be
mitochondria. Scale bar, 10 µm.
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HAP1 labeling in human brain
To determine whether the punctate pattern of labeling was also
present in the human, we performed HAP1 and huntingtin
immunocytochemistry in sections from the putamen (Fig.
4A,C,
respectively) and frontal cortex (Fig.
4B,D, respectively) of adult human
postmortem brains. The visualization of inclusions in the human was
aided by Nomarski optics. Both sizes of puncta could be distinguished
with both HAP1 and huntingtin immunocytochemistry. The first type was
large and thick-walled by Nomarski optics. Most neurons contained zero to five of these, and they ranged from ~1 to 4 µm in diameter. These were considerably darker and easier to visualize in HAP1 material. The second type was smaller (<1 µm in diameter) and very
numerous and was visible as hollow ovoids by Nomarski optics. This
latter type of inclusion was readily observed with both HAP1 and
huntingtin antibodies and was similar in size and shape to mitochondria
that were labeled for comparison by the use of specific antibodies
(Fig. 4E).
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Figure 4.
HAP1 immunostaining in the human and comparison
with huntingtin and mitochondrial membrane markers. Nomarski imaging of
HAP1 (A, B), huntingtin
(C, D), and mitochondria
(E) in human brain is shown. Large and small
inclusions (thick and thin arrows,
respectively), similar to those visualized in the rat, are visible in
these neurons from the putamen (A, C) and
cerebral cortex (B, D). The human
mitochondrial marker MAB1273 is shown (E)
labeling inclusions (arrows) similar in size to the
small HAP1- and huntingtin-immunoreactive inclusions.
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Recent studies have demonstrated the presence of intranuclear and
neuritic protein aggregates containing the N-terminal portion of
huntingtin in HD postmortem brain tissue (Becher et al., 1997; DiFiglia
et al., 1997). To determine whether HAP1 was present in these
aggregates, we performed HAP1 immunocytochemistry on sections from
several advanced HD patients including the cortex, striatum,
hippocampus, and cerebellum. We first confirmed the presence of
huntingtin aggregates in these cases by performing immunocytochemistry
using a fusion protein antibody specific for the N-terminal portion of
huntingtin (Li and Li, 1998). Staining with this antibody labeled many
aggregates in a variety of regions in all HD cases (Fig.
5A). Staining of adjacent
sections with the HAP1 antibody did not label aggregates, suggesting
that huntingtin aggregates do not contain a significant level of HAP1
(Fig. 5B). In HD cases, HAP1 immunoreaction product filled
perikarya and proximal dendrites, with a staining pattern very similar
to that seen in control cases.
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Figure 5.
HAP1 is not present in N-terminal huntingtin
aggregates. A, Light micrograph showing EM48 labeling in
cortex from an HD case. Em48 immunostained many aggregates located
mostly in the neuropil but also found in neuronal nuclei.
B, An adjacent section immunolabeled with HAP1.
Immunostaining is localized to neuronal perikarya and proximal
processes and is diffuse in the neuropil. There is no labeling of
aggregates. Scale bar, 20 µm.
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Subcellular localization of HAP1
The subcellular localization of HAP1 was examined in the rat by
immunoperoxidase and pre-embedding immunogold electron microscopy. The
regions examined included the neostriatum, frontal cortex, lateral
septum, and cerebellum. There were no major differences in HAP1
localization between each of these areas. Immunolabeling was almost
exclusively found in neurons and neuronal processes. The majority of
identifiable astrocyte and oligodendrocyte somata were devoid of
labeling. In each region examined, DAB reaction product or immunogold
particles were observed labeling neuronal somata, dendrites of all
calibers, and axon terminals. Although more processes were labeled with
immunoperoxidase, immunogold provided superior spatial resolution, and
thus the descriptions and illustrations that follow will be from this
material.
Subcellular associations of HAP1 within perikarya (Fig.
6A) were very similar
to those reported previously for huntingtin. Immunogold particles were
primarily cytoplasmic, often found in regions rich in polysomes.
Because the immunogold particles were much larger than were individual
ribosomes, it was impossible to determine whether they were directly
labeled. HAP1 labeling was also present in neuronal nuclei where it
associated with nuclear rods (Fig.
7A). Nuclear rod labeling was
also evident in human tissue (Fig. 7B). Many immunogold
particles were in contact with the outer surface of membrane-bound
organelles, including rough and smooth endoplasmic reticulum,
tubulovesicular profiles, and mitochondria. A type of large
nonmembrane-bound cytoplasmic body, when observed, was especially well
labeled, as described more fully below. Labeling associated with the
Golgi apparatus was rare, as were immunogold particles in contact with
the nuclear envelope or plasma membrane. Immunogold labeling of
perikaryal microtubules was also evident.
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Figure 6.
Subcellular localization of HAP1.
A, Electron micrograph showing the cell body of a
HAP1-immunolabeled neuron in the septal region. Most immunogold
particles were cytoplasmic, whereas a few were also found in the
nucleus (n). Most particles were either free in
the cytoplasm in regions rich in polysomes or in contact with the outer
surface of rough endoplasmic reticulum (thick arrows),
mitochondria (asterisks), and tubulovesicular elements
(thin arrows). A few rare particles were found in
contact with the plasma membrane or within regions rich in Golgi
apparatus (g). B, Longitudinal
section through a dendrite (d) showing immunogold
particles in contact with microtubules (arrowheads) or a
mitochondrion (asterisk). C,
Cross-section through a dendrite (d) in which
immunogold particles can be seen in contact with microtubule
cross-sections (arrowheads). Scale bars, 250 nm.
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Figure 7.
Association of HAP1 to nuclear rods.
A, Electron micrograph showing HAP1 immunogold in the
nucleus (n) of a preoptic area neuron.
Gold particles are located along an intranuclear rod
(arrow). The cytoplasm (C) is separated
from the nucleus by the nuclear envelope. B, Light
micrograph of a coronal section through normal human cortex showing
HAP1 immunostaining. In pyramidal neurons, the DAB reaction product is
present in perikarya and proximal dendrites but is also found along
intranuclear rods (arrows). Scale bars:
A, 300 nm; B, 10 µm.
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Within dendrites (Fig. 6B,C),
immunogold particles were rarely out of contact with organelles. Most
were in contact with microtubules, mitochondria, and tubulovesicles.
Rare immunogold particles were in contact with the plasma membrane, and
almost none were associated with dendritic synaptic specializations.
Immunogold particles observed in dendritic spines (Fig.
8A) were cytoplasmic,
although sometimes associated with the spine apparatus. Axon terminals (Fig. 8A,B) were frequently labeled
with immunogold particles, most of which were observed on the outer
surface of synaptic vesicles. The external surface of mitochondria were
also labeled in axon terminals. The plasma membranes and presynaptic
densities of axon terminals were not labeled. Some myelinated axons
(Fig. 8C) in the examined neuropils also contained
immunogold particles that were usually associated with the axonal
cytoskeleton or with mitochondria.
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Figure 8.
Subcellular localization of HAP1.
A, B, Electron micrographs showing HAP1
immunogold particles in four axon terminals (a)
and in three of their postsynaptic dendritic spines
(s). In the axon terminals, immunogold particles
are located amid the synaptic vesicles, frequently contacting their
cytoplasmic surfaces (arrowheads) but also contacting
the outer membrane of mitochondria (asterisk).
Immunogold particles in dendritic spines were free in the cytoplasm. In
both axon terminals and dendritic spines, immunogold particles were not
found in association with synaptic junctional complexes.
C, Electron micrograph of a myelinated axon
(a) showing a few immunogold particles either
amid cytoskeletal elements or in contact with a mitochondrion
(asterisk). Scale bars, 250 nm.
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Sections from control experiments in which the primary antibody was
omitted were examined to assess the extent of background immunogold
labeling. In these sections, immunogold particles were often present,
scattered throughout the tissue. When present, these occurred in any
ultrastructural compartment. These were at a very low density, however,
with most cells and processes being completely unlabeled. When labeled,
axon terminal and dendrite profiles contained no more than two
particles. These immunogold particles did not occur in clusters and did
not show the selectivity described above for the surfaces of
microtubules or organelles.
Quantitative comparison of HAP1 and huntingtin
immunogold labeling
The subcellular associations of HAP1 with membranous organelles
and microtubules are very similar to what we have reported for
huntingtin (Gutekunst et al., 1995). The major qualitative differences
were that HAP1 immunogold particles were more readily found in axon
terminals and much more densely labeled a certain type of cytoplasmic
inclusion. To provide a more quantitative comparison of these
associations, we made blinded counts of all the immunogold particles
visible in electron micrographs of dendrites from the cerebral cortex
labeled for either HAP1 (n = 22 dendrites containing
276 immunogold particles) or huntingtin (n = 20 dendrites containing 197 immunogold particles). Because the labeling of one organelle is not really comparable with the labeling of another because of differences in surface area and frequency, this analysis does not enable the determination of the relative labeling of different
organelle populations. However, it does permit fairly sensitive
comparisons of the extent to which the two markers label a given
organelle. The results are remarkably similar for the two markers (Fig.
9), indicating their subcellular
associations to be similarly distributed within dendrites.
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Figure 9.
Comparison of the proportional labeling of
organelles and membranes by HAP1 and huntingtin immunogold particles.
Histograms showing the proportions of immunogold particles observed to
be associated with particular subcellular compartments in frontal
cortex dendrites are shown. These data indicate that the proportional
distribution of HAP1 and huntingtin among the compartments is very
similar. Because the frequency and surface areas of each compartment
are not comparable, however, these data should not be interpreted as
giving any indication of the relative abundance of HAP1 or huntingtin
in each.
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Electron microscopic analysis of
HAP1-immunoreactive puncta
The large intracellular puncta observed by light microscopy to be
densely HAP1 immunoreactive were examined using electron microscopy
(Fig. 10). Because they were often so
densely labeled that their substructure was obscured by immunogold
particles, we also examined comparable structures in material not
labeled by immunocytochemistry (Fig.
10D,E). These puncta appear
identical to a group of nonmembrane-bound cytoplasmic organelles that
have been identified in brain and peripheral tissues and variously termed "nucleolus-like body" (Navarro et al., 1995),
"nematosomes" (Hindelang-Gertner et al., 1974), "basophilic
intracellular granules" (Hindelang-Gertner et al., 1974; Holmgren et
al., 1987), "stigmoid bodies" (Shinoda et al., 1993), and
"botrysomes" (Kind et al., 1997). Based on the classification
proposed by Shinoda et al. (1993), we will use the term stigmoid
bodies. HAP1-immunoreactive stigmoid bodies were found to be
heterogeneous electron-dense organelles that were ovoid to circular in
shape, 0.5 to 4 µm in diameter, and not enclosed by a limiting
membrane. The largest ones were most easily found in perikarya (Fig.
10A). Rough endoplasmic reticulum and polyribosomes
were often observed in the immediate vicinity of HAP1-immunoreactive
stigmoid bodies. Smaller stigmoid bodies were also observed in large to
medium caliber dendrites (Fig. 10E) and, in rare
instances, in axon terminals (Fig. 10F). The
ultrastructure of stigmoid bodies consists of closely packed collections of electron-dense particles and fibrils frequently surrounding a central or eccentric electron-lucent core (Fig. 10B-D). Many of the individual elements
making up the electron-dense shell were circular, semicircular, or
tubular elements ~20-50 nm in diameter surrounding their own
electron-lucent core. Because these elements were only rarely
elongated, we interpret them to represent spherical
collections of protein or other molecules. These elements did not
appear to be vesicular because we could not discern them to have
trilaminar membrane structure. Other smaller particles were similar in
size and density to ribosomes. HAP1 immunogold particles most densely
labeled the periphery of the stigmoid bodies (Fig.
10A-C). Immunogold particles were also found throughout the electron-dense shell but only rarely in the electron-lucent centers. The content of the electron-lucent centers was
similar to that of the cytoplasm (Fig.
10B,C) and contained occasional
tubulovesicles, fibrils, larger dense particles, and small particles
that could be ribosomes. Unlabeled stigmoid bodies were not observed in
the immunolabeled tissue.
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Figure 10.
Labeled and unlabeled stigmoid bodies in neurons.
A-F, Electron micrographs of stigmoid bodies
demonstrating their structure, location, and HAP1 immunoreactivity.
Stigmoid bodies do not have a limiting membrane and are made up of
heterogeneous electron-dense material, often surrounding a large
electron-lucent core (c). Much of the dense
material appears to be made up of individual rounded electron-dense
elements with electron-lucent centers. A, Electron
micrograph showing two immunolabeled stigmoid bodies
(arrows) in the perikaryon of a neuron in the lateral
septum. This perinuclear positioning is very frequent. Immunogold
particles are visible both around and within the stigmoid bodies.
B, C, Immunogold-labeled stigmoid bodies
in neurons from the preoptic area and the lateral septum seen at higher
magnification. Label is concentrated on the surface of the stigmoid
bodies but also is found deep within the electron-dense material.
D, Electron micrograph of unlabeled stigmoid bodies in
the perikaryon of a preoptic neuron showing stigmoid ultrastructure
unobscured by immunogold particles. E, F,
Unlabeled stigmoid (arrow) in a medium caliber dendrite
(d) and a labeled stigmoid in an axon terminal
(a) demonstrating that they can be found in more
distal locations. Scale bars, 500 nm.
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The population of smaller HAP1 puncta seen by light microscopy appears
to be made up of several different types of organelles, all of which
are membrane bound. Most of these were mitochondria (see Fig. 5)
that were frequently labeled with immunogold particles, although not
nearly as densely as were stigmoid bodies. Although a single section
through a mitochondrion may only show labeling by a few immunogold
particles, the dozens of such particles that would cover an entire
mitochondrion make them more visible by light microscopy. Other types
of labeled organelles with the size and shape of the small inclusions
were less frequent and included multivesicular bodies and
lysosomes.
Subcellular fractionation
To investigate further the apparent associations of HAP1 with
microtubules and organelles, as well as that of huntingtin, we examined
their subcellular fractionation in rat brain and examined whether HAP1
and huntingtin are enriched in purified microtubule fractions.
Fractionation results for HAP1 and huntingtin were the same (Fig.
11A). HAP1 cloning
has revealed two isoforms (HAP1A and HAP1B) resulting from alternative
splicing at the C-terminal region (Li et al., 1995). The two HAP1
isoforms and huntingtin were present in membranous (P2 and P3) and
cytosolic fractions (S3). Subfractionation of P2 using a hypotonic
lysis method (Huttner et al., 1983) resulted in an LP2 fraction
enriched in synaptic vesicles. HAP1 and huntingtin were present in the
LP2 fraction along with the synaptic vesicle protein synaptophysin.
After purification of microtubules (Fig. 11B) from
rat brain cytosolic fractions (S3), the HAP1 isoforms remained highly
enriched. In contrast, huntingtin was not as well retained in the
purified microtubule fraction.
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Figure 11.
Subcellular distribution of HAP1 and huntingtin.
A, Subcellular fractions from rat whole brain were
prepared according to the method of Huttner et al. (1983). Western blot
analysis was conducted with antibodies for HAP1, huntingtin, and
synaptophysin. B, Coomassie blue staining and Western
blot analysis of the rat brain microtubule (MT)
fraction that was obtained by polymerization of tubulin in the
cytosolic fraction (S3) are shown. Western blot analysis
was conducted with antibodies to HAP1 and huntingtin.
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DISCUSSION |
Our results include several major findings concerning the cellular
and subcellular localization of HAP1. First, in the rat brain HAP1 is
heterogeneously expressed in neurons in a pattern similar to but more
widespread than that of huntingtin. Second, light and electron
microscopy analyses show that HAP1 is mostly localized in the cytoplasm
but is also found in nuclei where it specifically associates with
nuclear rods. In the cytoplasm it strongly associates with distinct
populations of large and small puncta. Although these two populations
of puncta are found in human brains, they are distinct from the
intranuclear and cytoplasmic protein aggregates containing the
N-terminal portion of huntingtin that have been described in HD brain
tissue. In fact HAP1 was not found to stain huntingtin-containing
aggregates in HD tissue. At the subcellular level, HAP1 associates with
microtubules and most types of membrane-bound organelles including
mitochondria, endoplasmic reticulum, tubulovesicles, lysosomes, and
synaptic vesicles. We also found HAP1 to be a specific marker for large nonmembrane-bound cytoplasmic bodies, recently named stigmoid bodies.
The subcellular localization was confirmed by fractionation studies and
is consistent with a role for HAP1 in intracellular transport.
Furthermore, quantification in cortical dendrites revealed a
distribution of organelle associations remarkably similar to that of
huntingtin. These results show that there is a great correspondence between the subcellular distribution of HAP1 and huntingtin in rat and
normal human brains, further confirming a potential functional association between these two proteins.
The cellular localization of HAP1
Our immunocytochemistry confirms earlier findings that HAP1 is
selectively expressed in neurons with little or no labeling of glia.
HAP1 staining was also more intense in striatum, brainstem, and ventral
forebrain than in cerebral cortex and cerebellum. This is dissimilar to
huntingtin immunoreactivity in which the cerebral cortex and cerebellum
are the most intensely stained regions and the striatum is only
moderately stained (DiFiglia et al., 1995; Gutekunst et al., 1995;
Sharp et al., 1995; Trottier et al., 1995). Within a given brain
region, however, the cellular patterns of HAP1 and huntingtin
immunostaining were the same or very similar. With both markers, the
most intensely stained neurons in the cerebral cortex were layer V and
scattered layer VI pyramidal cells. Furthermore, both markers
heterogeneously label populations of medium-sized striatal neurons. The
differences in both these regions are that more neurons appear to be
HAP1 immunoreactive than are huntingtin immunoreactive. Thus, it seems
likely that huntingtin-immunoreactive neurons are a subset of
HAP1-immunoreactive neurons. For example, nitric oxide
synthase-containing neurons are HAP1 immunoreactive (Li et al., 1996)
but appear to be devoid of huntingtin (Ferrante et al., 1997; Kosinski
et al., 1997). Such differences suggest that HAP1 may have a function
that does not always require the presence of huntingtin. In other
regions, such as the cerebellum and globus pallidus, no differences in the cellular pattern of HAP1 and huntingtin immunoreactivity were discernible.
The subcellular localization of HAP1
The main focus of this study has been the subcellular localization
of HAP1 within individual neurons as revealed by pre-embedding immunogold and subcellular fractionation. The most prominent
associations found for HAP1 were with microtubules, a variety of
membrane-bound organelles, and nonmembrane-bound organelles recently
named stigmoid bodies. Associations with membrane-bound organelles were
supported by subcellular fractionation findings in which HAP1 was found to be enriched in membrane- and synaptic vesicle-containing fractions. Because immunogold electron microscopy did not show HAP1 to be associated with nuclear and plasma membranes, the fractionated membranes must originate from organelles. Similarly, copurification with microtubules indicates that the HAP1/microtubule association observed by EM is specific. Immunogold localization of the
microtubule-based transport protein kinesin (Hirokawa et al., 1991) and
of MAP1 and MAP2 (Leterrier et al., 1994) has shown similar labeling of microtubules and membrane-bound organelles. By the use of the same
pre-embedding immunogold technique to study the subcellular localization of cytoplasmic proteins and mRNA, neither microtubules nor
membrane-bound organelles were labeled (Kachidian and Pickel, 1993;
Trembleau et al., 1994; Feng et al., 1997). The observed associations
with particular membrane-bound organelles also seem to be specific
because not all cellular membranes are labeled (the nuclear membrane,
Golgi apparatus, and plasmalemma were labeled at background levels). We
thus believe that these localizations for HAP1 are specific and suggest
a role for HAP1 in the microtubule-based localization or transport of
organelles. We recently reported that HAP1 specifically interacts with
the accessory motor protein dynactin P150Glued (Li
et al., 1998a). These interactions are further evidence that HAP1 may
play a role in intracellular transport.
HAP1 is a marker for stigmoid bodies
The ultrastructural features of the large cytoplasmic puncta
labeled with the HAP1 antibodies were identical to those of a subtype
of nucleolus-like bodies that have been named stigmoid bodies (Shinoda
et al., 1993). Further evidence that the large HAP1-immunoreactive
puncta are identical to stigmoid bodies is that the brain regions in
which they are most abundant are the same regions in which stigmoid
bodies have also been reported to be concentrated (Shinoda et al.,
1992). Although their function is not yet well established, the work of
several groups suggested a potential role of stigmoid bodies in protein
synthesis (Weakley, 1969; Kishi, 1972; Takeuchi and Takeuchi, 1982).
Given the remarkable density of HAP1 labeling observed in stigmoid
bodies, HAP1 may serve an important role in transporting the molecular
constituents or products of these bodies. This possibility is supported
by the finding that transfection of HAP1 cDNA into HEK293 cells results in the de novo formation of HAP1-immunoreactive inclusions
with a very similar ultrastructure to that of stigmoid bodies (Li et al., 1998a,b). Alternatively, HAP1 may be synthesized or stored in
stigmoid bodies. Because high levels of HAP1 are found in stigmoid bodies and the binding of huntingtin to HAP1 is enhanced by the polyglutamine repeat expansion, stigmoid bodies might have a role in HD
pathology. This seems unlikely, however, because stigmoid bodies are
not huntingtin immunoreactive in control and HD brain and because they
are most prominent in brain regions with limited HD neuropathology.
HAP1 and huntingtin
The subcellular associations of HAP1 were very similar to those we
and others have reported for huntingtin and were consistent with the
identified associations of these two proteins (Li et al., 1995, 1996).
We have further substantiated their similarities by quantifying the
relative proportions of HAP1 and huntingtin immunogold particles found
to associate with particular organelles. The relative proportions of
immunogold labeling of different organelles with each antibody were
extremely similar, strongly supporting their association. Because the
numbers and surface areas of each type of organelle are not comparable,
this analysis does not address the relative density of immunogold
labeling of the different organelles. For example, although
microtubules were the most labeled organelle in dendrites, they also
are the most numerous and have the greatest surface area. Several of
the associations are quite interesting, however. Both HAP1 and
huntingtin significantly labeled mitochondria. Quantification of
immunogold labeling brought out the huntingtin labeling of mitochondria
that had not been reported previously (DiFiglia et al., 1995; Gutekunst
et al., 1995). Presumably, this association was overlooked because the
nonquantitative methods used allowed the 10% association we report
here to be missed. Given that the total mitochondrial surface area is
relatively small, this 10% likely reflects a significant association.
The potential importance of this finding relates to the relevance of
mitochondria to oxidative injury, excitotoxicity, and apoptosis as
mechanisms of neuronal death in HD. Perhaps HAP1 and/or huntingtin play
roles in the transport, anchoring, or even turnover of mitochondria or
mitochondrial constituents. Similarly, the association of HAP1 and
huntingtin with synaptic vesicles suggests possible roles in vesicular
trafficking. Although little is known about the transport of proteins
and protein complexes, an additional possibility is that HAP1 serves as
a carrier for a heteromultimer containing huntingtin and the other
proteins with which it associates. Thus, HAP1 could also be involved in
the assembling or transporting of macromolecular complexes.
We have not detected HAP1 within the abnormal nuclear and cytoplasmic
protein aggregates formed by the N-terminal fragments of huntingtin in
HD cases. Lack of HAP1 within intranuclear protein aggregates could
reflect its limited nuclear localization. Lack of HAP1 within
cytoplasmic protein aggregates was more surprising because HAP1 binds
more tightly to huntingtin with an expanded polyglutamine repeat than
to normal huntingtin (Li et al., 1995) and the cytoplasmic subcellular
localizations of both proteins are otherwise so similar in normal
tissue. Although it is possible that our antibody does not penetrate
the aggregate, this seems unlikely because other antibodies are able to
penetrate them and because HAP1 immunoreactivity was not even present
at the periphery of the aggregates. Another possibility is that HAP1
antigenicity may be altered by its binding to huntingtin fragments
within the aggregates. At least in transfected cells, however, HAP1 and
an N-terminal huntingtin fragment can be colocalized using the same HAP1 antibody, suggesting that binding does not interfere with antibody
recognition. Finally, and most likely, there is no HAP1 protein in the
protein aggregates. HAP1 may be unable to bind to aggregated
huntingtin; perhaps aggregation blocks the HAP1 binding site that is
close to the polyglutamine tract (Li et al., 1995). It remains
possible, however, that HAP1 interacts with and transports N-terminal
fragments of Huntingtin that have not yet aggregated and may even
deliver them to forming aggregates.
| |
FOOTNOTES |
Received April 20, 1998; revised July 15, 1998; accepted July 17, 1998.
This work was supported by a Markey Center for Neurological Sciences
grant (C.-A.G.), by the Huntington's Disease Society of America and
the Veterans Administration (R.J.F.), by the Hereditary Disease
Foundation and the Wills Foundation and Whitehall Foundation (X.-J.L.),
and by National Institutes of Health Grants NS01624 (S.M.H.), NS35255
(S.M.H., C.-A.G., and R.J.F.), and NS36232 (X.-J.L.). We thank Ryan
Berglund and Shannon Mulroy for their assistance with
illustrations.
Correspondence should be addressed to Dr. Steven M. Hersch, Department
of Neurology, WMRB Suite 6000, Emory University School of Medicine,
Atlanta, GA 30322.
| |
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Top
Abstract
Introduction
