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The Journal of Neuroscience, November 1, 2001, 21(21):8473-8481
Huntingtin Aggregate-Associated Axonal Degeneration is an Early
Pathological Event in Huntington's Disease Mice
He
Li1, 3,
Shi-Hua
Li1,
Zhao-Xue
Yu1,
Peggy
Shelbourne2, and
Xiao-Jiang
Li1
1 Department of Genetics, Emory University School of
Medicine, Atlanta, Georgia 30322, 2 Division of Molecular
Genetics, Institute of Biomedical and Life Sciences, University of
Glasgow, Glasgow G11 6NU, United Kingdom, and 3 Department
of Histology and Embryology, Tongji Medical School, Huazhong University
of Science and Technology, Wuhan 430030, People's Republic of China
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ABSTRACT |
Huntington's disease (HD) is characterized by the selective loss
of striatal projection neurons. In early stages of HD,
neurodegeneration preferentially occurs in the lateral globus pallidus
(LGP) and substantia nigra (SN), two regions in which the axons of
striatal neurons terminate. Here we report that in mice expressing
full-length mutant huntingtin and modeling early stages of HD, neuropil
aggregates form preferentially in the LGP and SN. The progressive
formation of these neuropil aggregates follows intranuclear
accumulation of mutant huntingtin and becomes prominent from 11 to 27 months after birth. Neuropil aggregates, but no intranuclear
inclusions, were observed in the LGP and SN, suggesting that huntingtin
aggregates are formed in the axons of striatal projection neurons. In
the LGP and SN, we observed degenerated axons in which huntingtin aggregates were associated with dark, swollen organelles that resemble
degenerated mitochondria. Neuritic aggregates also form in cultured
striatal neurons expressing mutant huntingtin, block protein transport
in neurites, and cause neuritic degeneration before nuclear DNA
fragmentation occurs. These findings suggest that the early
neuropathology of HD originates from axonal dysfunction and
degeneration associated with huntingtin aggregates.
Key words:
Huntingtin; polyglutamine; axon; degeneration; transport; mitochondria
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INTRODUCTION |
Studies of postmortem brains from
patients with Huntington's disease (HD) have indicated that medium
spiny neurons are selectively degenerated (Ferrante et al., 1985 ;
Graveland et al., 1985; Vonsattel et al., 1985). The majority of these
neurons extend their axons to the lateral globus pallidus (LGP), the
medial globus pallidus (MGP) (also known as the internal globus
pallidus), and the substantia nigra (SN). Striatal neurons projecting
to the LGP are enriched in enkephalins, whereas striatal neurons
projecting to the MGP and SN are enriched in substance P (Graybiel,
1990). The decreased density of enkephalin-immunoreactive fibers in the
LGP and substance P-immunoreactive neuropils in the SN is found in
early HD patients. In addition, the LGP and SN are more significantly
degenerated than the MGP during early stages of HD (Reiner et al.,
1988; Richfield et al., 1995). More importantly, such early neuropil
degeneration has also been observed in presymptomatic HD patients
(Albin et al., 1990, 1992), suggesting that degeneration of striatal
projection neurons may be initiated from their axons.
The widespread expression of the HD protein, huntingtin, provides no
clues to the selective HD pathology. In HD, mutant huntingtin accumulates in the nucleus, in contrast to the predominantly
cytoplasmic distribution of normal huntingtin. Dystrophic neurites,
which are reported to be extracellular structures containing axonal processes, also contain mutant huntingtin in HD patient brains (DiFiglia et al., 1997). Although intranuclear accumulation of mutant
huntingtin affects gene expression in HD animal and cell models (Cha et
al., 1998; S. H. Li et al., 1999; Luthi-Carter et al., 2000;
Nucifora et al., 2001), nuclear polyglutamine inclusions (NIs) do not
correlate with neurodegeneration (Klement et al., 1998; Saudou et al.,
1998; Kuemmerle et al., 1999; Warrick et al., 1999; Kazemi-Esfarjani
and Benzer, 2000). Recently, we have identified small huntingtin
aggregates in dendrites and axons and named them neuropil aggregates
(Gutekunst et al., 1999; H. Li et al., 1999, 2000). Similarly, in the
brains of HD patients, N-terminal huntingtin accumulates and forms
puncta in corticostriatal fibers (Sapp et al., 1999). In the R6/2 HD
mouse model that expresses N-terminal mutant huntingtin, the formation
of neuropil aggregates is highly correlated with disease progression in
the absence of obvious neurodegeneration (H. Li et al., 1999).
Although our previous study showed that huntingtin aggregates are
enriched in striatal medium spiny neurons in HD mice (H. Li, et
al., 2000), it is unclear whether neuropil aggregates are selectively
localized in those neurons that are preferentially affected by HD and
whether these aggregates are associated with early neuropathological
changes. HD-repeat knock-in mice that express full-length mutant
huntingtin under the endogenous mouse Hdh
promoter do not show obvious neurological symptoms (Levine et al.,
1999; Shelbourne et al., 1999; Wheeler et al., 2000), providing a good
animal model to uncover neuropathological changes in presymptomatic or
early HD. Using these HD mice, we demonstrate that huntingtin
aggregates are preferentially formed in the striatal projection neurons
that degenerate during the early stages of HD. In cultured neurons,
huntingtin aggregates cause neuritic degeneration before nuclear DNA fragmentation.
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MATERIALS AND METHODS |
Light microscopic examination. R6/2 mice (B6CBA-TgN
(HDexon1)62) that express exon1 of the human mutant HD gene with
115-150 CAGs under the human HD gene promoter (Mangiarini et
al., 1996). These mice were obtained from The Jackson Laboratory (Bar
Harbor, ME) at 4-12 weeks of age. HD-repeat knock-in mice that express 72-80 CAGs in the endogenous mouse HD gene were generated as described previously (Shelbourne et al., 1999). Mice were anesthetized and then
perfused intracardially with PBS, pH 7.2, for 30 sec followed by
4% paraformaldehyde in 0.1 M phosphate buffer
(PB) at a pH of 7.2. Brains were removed, cryoprotected in 30% sucrose
at 4°C, and sectioned at 40 µm using a freezing microtome.
Free-floating sections were preblocked in 4% normal goat serum (NGS)
in PBS, 0.1% Triton X-100, and avidin (10 µg/ml) and then
incubated with EM48, a rabbit polyclonal antibody against the
first 256 amino acids of human huntingtin (Gutekunst et al., 1999; H. Li et al., 2000), at 4°C for 48 hr. The EM48 immunoreactive
product was visualized with an avidin-biotin complex kit (Vector
Laboratories, Burlingame, CA).
Electron microscopic immunocytochemistry. Immunogold
labeling was performed as described previously (H. Li et al., 1999,
2000). Briefly, mice were fixed by perfusion with PBS containing 4%
paraformaldehyde and 0.2% glutaraldehyde. After perfusion, the brain
was removed, post-fixed with 4% paraformaldehyde in PB for 6-8 hr,
and sectioned using a vibratome. Brain sections were incubated with
EM48 in PBS containing 4% NGS for 24-60 hr at 4°C and then with Fab
fragments of goat anti-rabbit secondary antibodies (1:50) conjugated to 1.4 nm gold particles (Nanoprobes Inc., Stony Brook, NY) in PBS with
4% NGS overnight at 4°C. After rinsing in PBS, sections were fixed
again in 2% glutaraldehyde in PB for 1 hr, silver intensified using
the IntenSEM kit (Amersham International, Buckinghamshire, UK),
osmicated in 1% OsO4 in PB, and stained
overnight in 2% aqueous uranyl acetate.
All sections used for electron microscopy (EM) were dehydrated
in ascending concentrations of ethanol and propylene oxide/Eponate 12 (1:1) and embedded in Eponate 12 (Ted Pella, Redding, CA). Ultrathin
sections (60 nm) were cut using a Leica Ultracut S ultramicrotome (Leica, Nussloch, Germany). Thin sections were counterstained with 5% aqueous uranyl acetate for 5 min followed by Reynolds lead
citrate for 5 min and examined using a Hitachi H-7500 electron microscope (Hitachi, Tokyo, Japan).
Quantification of aggregates in the brain. We quantified the
immunoreactive aggregates in sections using light microscopy at 63×
magnification with a Zeiss microscope (Axioskop 2; Zeiss, Thornwood, NY) and video system (Dage-MTI Inc. Michigan City, IN). All
huntingtin aggregates in the captured images (8455 µm2/frame) were counted. The aggregates
were categorized as intranuclear or neuropil aggregates. Neuropil
aggregates are localized outside the cell body and their size is
usually smaller, whereas nuclear aggregates or NIs appeared as single
inclusions within the nucleus. Data analysis was performed using
SigmaPlot 4.11E and Student's t tests.
Primary neuronal culture and transfection. Neurons were
isolated from the striatum of embryonic day 14-16 rats and
cultured in neurobasal/B27 medium according to the method used in our
previous study (S. H. Li et al., 2000). After culturing for 3-4
d, the striatal neurons were transfected with huntingtin cDNAs using CaPO4 transfection. We obtained a 2-5%
transfection rate. Huntingtin exon1 cDNA encoding 20 (GFP-20Q) or 120 (GFP-120Q) glutamines was fused in-frame to the C terminus of green
fluorescent protein (GFP) in the GFP expression vector
(Clontech, Palo Alto, CA). After 24-48 hr of transfection, cells were
used for immunofluorescence analysis. Cotransfection of striatal
neurons with GFP-120Q and red fluorescent protein (RFP) (Clontech) was
also performed. For immunofluorescent double labeling, we used mouse
antibodies to tubulin (Sigma, St. Louis, MO) and rabbit antibody EM48
to huntingtin. To examine fluorescence in living cells, cell-cultured
dishes (60 mm) were placed on a temperature-controlled stage (20/20
Technology, Mississauga, Canada) and cells were maintained at 37°C
with a constant CO2 flow that maintained the
medium pH at ~7.4. A Zeiss fluorescent microscope (Axiovert S100) and
video system (Dage-MTI Inc.) were used to capture images. The captured
images were stored and processed using Adobe Photoshop software (Adobe
Systems, San Jose, CA). Quantitative analysis of neurons containing
aggregates and neuritic degeneration was performed by counting 324-435
GFP-120Q-transfected neurons and 113-215 GFP-20Q- or GFP
vector-transfected neurons in two to three independent experiments.
Data were expressed as the mean ± SD and the statistical analysis
was performed with the Student's t test.
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RESULTS |
Selective accumulation of huntingtin aggregates in striatal neurons
in HD mice expressing full-length mutant huntingtin protein
Previous studies have shown that HD transgenic mice expressing
N-terminal fragments of mutant huntingtin have more severe neurological
symptoms than HD-repeat knock-in mice, which express full-length mutant
huntingtin (Davies et al., 1997; Levine et al., 1999; Schilling et al.,
1999; Shelbourne et al., 1999; Wheeler et al., 2000). Because
N-terminal mutant huntingtin is toxic and forms aggregates in the brain
(Davies et al., 1997; DiFiglia et al., 1997; Gutekunst et al., 1999;
Hodgson et al., 1999; H. Li et al., 2000), we compared aggregate
formation in various brain regions of these two types of HD mice. For
immunocytochemical studies, we used the EM48 antibody, which
specifically recognizes mutant huntingtin and its aggregates (Gutekunst
et al., 1999; H. Li et al., 1999, 2000). In R6/2 mice, which express
the HD exon1 mutant huntingtin with 115-150 glutamines (Davies et al., 1997), nuclear huntingtin aggregates are widely distributed in all of
the brain regions examined, including the hippocampus and cerebellum,
two regions that are relatively unaffected in HD. In contrast,
HD-repeat knock-in mice, which express 72-80 CAGs in the endogenous
mouse Hdh gene (Shelbourne et al., 1999), show the highest
density of nuclear huntingtin aggregates in the striatum. The cortex,
hippocampus, and cerebellum display weak or no nuclear huntingtin
staining (Fig. 1). Thus, the context of
full-length mutant huntingtin clearly influences the cell-specific
formation of aggregates.
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Figure 1.
Selective accumulation of nuclear huntingtin
aggregates in striatal neurons in HD-repeat knock-in mice.
Low-magnification micrographs of brain sections from an R6/2 mouse at
12 weeks of age and a HD-repeat knock-in mouse at 24 months of age are
shown. The sections were stained with EM48. Nuclear huntingtin
aggregates are widely distributed in R6/2 mice, which express
N-terminal mutant huntingtin. In the HD-repeat knock-in mouse brain,
which expresses full-length mutant huntingtin, only the striatal
neurons (Str) were immunoreactive with EM48.
Ctx, Cortex; Hipp, hippocampus;
Cereb, cerebellum. Scale bars, 25 µm.
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Neuropil aggregates are formed in projection neurons
Next, we examined the distribution of neuropil aggregates in
the LGP, MGP, and SN of HD-repeat knock-in mice. All three of these
regions receive axons from striatal projection neurons. The SN is
composed of the substantia nigra pars reticulate (SNr) and the
substantia nigra pars compacta. Because the SNr constitutes the
major part of the SN, we focused on the SNr and refer to it as the SN.
We also included an R6/2 mouse brain for comparison.
As expected, both neuropil and intranuclear aggregates were observed in
the striatum (Fig. 2). Because striatal
neuronal cell bodies also contain huntingtin aggregates, it is
difficult to determine whether neuropil aggregates in the striatum are
from cortical projection neurons or from the neurons within the
striatum. However, only neuropil aggregates, but not intranuclear
inclusions, were seen in the striatal projection regions (LGP, SN, and
MGP) in HD-repeat knock-in mice. This is consistent with observations that intranuclear accumulation of N-terminal huntingtin does not occur
in the globus pallidus region of the human HD brain (Sapp et al.,
1999). The lack of intranuclear staining in the projection regions
suggests that neurons within these regions do not accumulate mutant
huntingtin fragments. Thus, neuropil aggregates seen in the projection
regions are likely to be the aggregates that are formed in the axons
from striatal projection neurons. Interestingly, neuropil aggregates
are more abundant in the LGP and SN than in the MGP. The LGP and SN are
reported to show the earliest degenerative changes in HD patient brains
(Reiner et al., 1988; Richfield et al., 1995). The high density of
neuropil aggregates in the LGP and SN suggests that striatal neurons
projecting to these two regions contain more axonal aggregates, whereas
the lower frequency of neuropil aggregates in the MGP suggests that
striatal neurons projecting to the internal globus pallidus form fewer
aggregates in their axons.
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Figure 2.
Selective distribution of neuropil aggregates in
striatal projection regions in the HD-repeat knock-in mouse brain.
Micrographs of the striatum (Str), LGP, MGP, and SN from
an R6/2 mouse at 12 weeks of age and a HD-repeat knock-in mouse at 21 months of age are shown. In the R6/2 mouse, intranuclear and neuropil
aggregates are widely distributed in all brain regions. In the
HD-repeat knock-in mouse, the LGP and SN contain only neuropil
aggregates. Note that the neuropil aggregates are more abundant in the
LGP and SN than in the MGP. Scale bar, 20 µm.
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Progressive formation of neuropil aggregates in striatal
projection neurons
If neuropil aggregates are involved in the progressive
neuropathology of HD, their formation may also be progressive and
precede neurodegeneration. Therefore, we examined the temporal
relationship between the formation of neuropil aggregates and the age
of HD-repeat knock-in mice. At 4 months of age, we only saw very faint
and diffuse nuclear staining in the striatum. No obvious neuropil aggregates were seen at this age. At 7-8 months, a few neuropil aggregates appeared in both the striatum and the LGP. At 11-12 months,
significant neuropil aggregates were observed in the striatal projection regions, such as the LGP and SN. At 21-24 months, neuropil aggregates were even more abundant. The formation of neuropil aggregates in the sections of the LGP is illustrated in Figure 3A. Quantification of neuropil
aggregates (Fig. 3B) confirms that these aggregates are much
more abundant in the LGP and SN than in other regions at all ages
examined. As in previous studies (H. Li et al., 2000; Wheeler et
al., 2000), nuclear aggregates were always more frequent in the
striatum compared with other regions. They are also more abundant than
neuropil aggregates (data not shown). These findings suggest that
mutant huntingtin first accumulates in the nucleus of striatal neurons
to form intranuclear inclusions and then forms aggregates in their
processes.
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Figure 3.
Age dependence of neuropil aggregates in HD-repeat
knock-in mice. A, EM48 immunostaining of the LGP in HD
knock-in mice at 4, 8, 12, and 24 months of age. The density of
neuropil aggregates is greater in older mice. Scale bar, 10 µm.
B, Quantification of neuropil aggregate density in
various brain regions from HD-repeat knock-in mice at different ages
(7-24 months). Values are expressed as the mean ± SD and were
obtained by counting aggregates in three to five images (8455 µm2/per image) for each brain region.
Ctx, Cortex; STR, striatum.
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Mutant huntingtin aggregates are associated with
axonal degeneration
Because the formation of neuropil aggregates correlates with the
regions in which early neurodegeneration occurs in HD, we investigated
whether any axonal or dendritic degeneration is associated with
neuropil aggregates. To do so, we used EM48 immunogold labeling, because this method allowed us to better reveal huntingtin aggregates and the ultrastructure of neuronal processes.
EM examination relies primarily on the preservation of brain tissue and
does not allow for a large-scale screen, so we focused on those brain
tissues that had a well-preserved ultrastructure. EM examination of
younger HD mice (<11 months) did not reveal any obvious neuropil
degeneration (data not shown). However, two of three HD mice aged
between 17 and 27 months showed neuropil degeneration in the LGP (Fig.
4) and SN (data not shown) regions. Several pieces of evidence suggest that these neuropils are degenerated axons of striatal projection neurons (Fig. 4). First, the plasma membrane around the neuropils is clearly visible, making these neuronal
processes distinct from other cellular structures and organelles.
Second, because nuclear accumulation of mutant huntingtin precedes the
formation of neuropil aggregates in these HD mice and because there is
no nuclear staining of mutant huntingtin in the LGP, neuropil
aggregates seen in the LGP should be in axons of striatal projection
neurons. Moreover, no huntingtin aggregates were found in postsynaptic
terminals in the striatal projection regions, suggesting that only
axons of striatal neurons to the LGP contain huntingtin aggregates.
Third, although degenerating axons might lose their identifying
synapses and vesicles, some synaptic vesicles were still visible in the
degenerated processes (Fig. 4C, arrow). Fourth,
many dark, swollen organelles were seen within the degenerated
processes, providing evidence for axonal degeneration. The size of
these dark structures suggests that they may be mitochondria that have
degenerated and lost their double membrane. These structures also
resemble autolysosomes, which contain degenerated and dark cytoplasmic
organelles including lysosomes and mitochondria (Dunn, 1990; Mizushima
et al., 2001) and were also found in huntingtin-transfected cells
(Kegel et al., 2000). In an axonal terminal containing clusters of
immunogold particles or microaggregates, a degenerated mitochondrion is
more clearly demonstrated in Figure
5B. A number of immunogold
particles are also associated with the surface of synaptic vesicles. We noticed that some degenerated axons had fewer huntingtin immunogold particles (Fig. 4C). It is possible that ultrathin sections
of the brain used in electronic microscopy may not always be in the correct plane or of sufficient thickness to reveal aggregates in
degenerated axons.
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Figure 4.
Degeneration of LGP axons containing huntingtin
aggregates. EM48 immunogold labeling shows three degenerated axons in
the LGP of a HD-repeat knock-in mouse at 17 months of age. The axons
contain immunogold-labeled aggregates and many dark and swollen
organelles, suggesting an association of huntingtin aggregates with
degenerated axons. Some of the degenerated organelles have double
membranes and resemble mitochondria (arrowheads). In
C, synaptic vesicles (arrow) are evident
in the degenerated axon. Scale bars, 0.5 µm.
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Figure 5.
Ultrastructure of EM48 immunoreactive aggregates
in axon terminals. Micrographs indicate that immunogold particles are
clustered in axon terminals in the LGP. A, A HD mouse at
27 months of age. A large huntingtin aggregate is present in an axon in
which the postsynaptic density (double arrows) is
evident. The aggregate has a fibrous structure and has EM48 immunogold
labeling. It occupies most of the area of the axonal terminal, and no
synaptic vesicles can be identified. In normal axonal terminals without
huntingtin aggregates, however, many synaptic vesicles
(arrows) are observed. B, A HD mouse at
17 months of age. Huntingtin aggregates are associated with degenerated
mitochondria (arrowhead) in an axonal terminal. Synaptic
vesicles in this terminal are indicated by arrows. The
postsynaptic terminal (double arrows) contains normal
mitochondria and no huntingtin aggregates. Scale bars, 0.5 µm.
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We noticed that small huntingtin aggregates often accumulate in axons.
In some cases, EM examination revealed large huntingtin aggregates that
were present in axonal terminals. More convincing evidence to support
the above ideas is that axon terminals, which show clear synapses and
vesicles, contain huntingtin aggregates with a filament profile (Fig.
5). A striking finding is that the size of a huntingtin aggregate can
be so large that it almost occupies the entire presynaptic terminal
(Fig. 5A). Often, these synapses with aggregates contained
fewer vesicles compared with those synapses without huntingtin
aggregates (Fig. 5A). The plasma membrane of the synapses
near the aggregates is often not intact or appears to be ruffled (data
not shown), suggesting that a morphological change may result from the
early degeneration of a synapse. The observation that degenerated
mitochondria are also evident in axonal terminals (Fig. 5B)
is consistent with the idea that degenerated mitochondria are often
associated with huntingtin aggregates (Fig. 4).
Several additional lines of evidence support the specific association
of these huntingtin aggregates with degenerated axons in the striatal
projection regions. First, similar degenerated axons in association
with huntingtin aggregates are also found in the SN (data not shown).
Second, we did not observe degenerated axons in the cortex and striatum
of the same HD mouse brain using EM examination or terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end
labeling. Third, we never saw enlarged axons with dark and
degenerated organelles in the brains of age-matched wild-type mice.
More importantly, we identified these degenerated axons by using EM48
that labels mutant huntingtin and its aggregates.
We compared neuropil aggregate distribution in different brain regions
(Table 1). The density of neuropil
aggregates is well correlated with the brain regions that degenerate in
early HD patients or show degenerated axons in HD-repeat knock-in mice. However, in R6/2 mice that normally die at 12-14 weeks, we did not
observe degenerated axons associated with huntingtin aggregates. The
context of full-length huntingtin and age-dependent changes in cellular
function may contribute to the axonal degeneration seen in HD-repeat
knock-in mice.
Huntingtin aggregates block neuritic transport in cultured
striatal neurons
The large-sized aggregates may affect neuronal transport in axons
and neuronal processes. To provide evidence for this idea, we examined
cultured striatal neurons that were transfected with GFP fusion
proteins containing the HD exon1 protein with a 120 glutamine repeat
(GFP-120Q). We observed that neuritic aggregates could be formed within
2 hr in living cells (Fig.
6A). In contrast, neurons transfected with huntingtin containing a 20 glutamine repeat
(GFP-20Q) did not show such neuritic aggregates. The relatively rapid
formation of aggregates allowed us to examine the effect of huntingtin
aggregates on protein transport in neurites of cultured cells. Because
fluorescence proteins are transported from the cell body to the nerve
terminals of cultured neurons, we cotransfected GFP-huntingtin with
RFP to examine the influence of huntingtin aggregates on the
transport of RFP in living cells. When mutant huntingtin was diffuse
and did not form large aggregates in the neurites, RFP was also
diffusely distributed through all neurites (Fig. 6B).
Once neurite aggregates were formed 6 hr later, RFP existed as puncta
that were colocalized with huntingtin aggregates. More importantly, no
diffuse RFP localization within neurites was seen. Thus, mutant
huntingtin aggregates might retain RFP by blocking its transport in
neurites.
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Figure 6.
Blockage of RFP transport by huntingtin aggregates
in neurites of cultured striatal neurons. A, Time course
recording the formation of neuritic aggregates in living striatal
neurons transfected with GFP-120Q. Newly synthesized aggregates
after 2 hr of observation are indicated by arrows. A
control cell expressing GFP-20Q after 2 hr of observation did not form
such neuritic aggregates. B, Time-course examination of
RFP and GFP-120Q distribution in living striatal neurons. Note that at
the beginning (0 hr) of the examination, most of the RFP and GFP-120Q
was diffuse in the neurites, with some huntingtin aggregates formed
(arrow). After 6 hr, GFP-120Q formed a number of
neuritic aggregates. RFP was colocalized with these aggregates, and no
obvious diffuse distribution of RFP and GFP-120Q in neurites was seen.
C, D, RFP- and GFP-120Q-transfected cells were fixed and
then examined. Intranuclear huntingtin aggregates did not affect RFP
distribution in neurites (C), but a large
neuritic aggregate (arrow) prevented the distribution of
RFP in the distal region of the neurite
(D).
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To confirm the observation on living cells, we also fixed cells and
examined the distribution of RFP in those cells that contained huntingtin aggregates (Fig. 6C,D). Huntingtin aggregates in
the nucleus or the cell body of striatal neurons did not significantly affect RFP distribution in neurites (Fig. 6C). In the
neurites in which large huntingtin aggregates were present, however,
there was much less RFP present in the region distal to huntingtin
aggregates (Fig. 6D). Degeneration of neurons became
obvious after transfection for 2 d, and the number of degenerated
neurons in GFP-120Q transfection is significantly higher than that in
GFP vector or GFP-20Q transfection. These findings suggest that
huntingtin aggregates block the transport of RFP from the cell body to
the neurites.
Neuritic degeneration precedes nuclear fragmentation in huntingtin
transfected striatal neurons
Next, we wanted to know whether neuritic aggregates caused
neuritic degeneration that might occur earlier than degeneration of the
cell body (Fig. 7). We used tubulin
staining to examine neurite integrity (Fig. 7B) and Hoechst
nuclear staining (Fig. 7D) to examine nuclear DNA
fragmentation that represents cell-body degeneration. After 17-24 hr
of transfection, transfected neurons containing huntingtin aggregates
in their cell body (Fig. 7A,E) often had intact tubulin
staining in their neurites and intact nuclear DNA staining. However, in
neurons that contained large neuritic aggregates, tubulin staining of
neurites was often discontinuous, reflecting the degeneration of
neurites. Despite such neuritic degeneration, these neurons did not
show obvious nuclear DNA fragmentation (Fig. 7D,F).
Thus, neuritic degeneration could occur in the absence of cell-body
degeneration.
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Figure 7.
Neuritic degeneration of huntingtin-transfected
striatal neurons before nuclear fragmentation. A-D,
Cells were transfected with GFP-120Q for 24 hr and then stained with
antibodies to huntingtin (A) and tubulin
(B). In the merged image
(C), neuritic aggregates are associated with the
fragmentation of neurites that display discontinuous tubulin labeling.
D, Nuclear staining did not show any DNA fragmentation
in the neuron that has degenerated neurites. Small nuclei (no
arrows) of glial cells are also shown. E, F, A
merged image of striatal neurons transfected with
GFP-120Q for 48 hr. The cells were stained with antibodies against
huntingtin (green) and tubulin
(red). Note that a cell containing intranuclear
huntingtin has intact nuclear staining (arrow) and
neuritic tubulin labeling. A transfected cell showing DNA fragmentation
(arrowhead), however, displays neuritic aggregates and
has lost neuritic tubulin staining. G, Quantitative
measurement of GFP-120Q-, GFP-20Q-, or GFP vector-transfected cells
that displayed neuritic degeneration and nuclear degeneration. The data
were obtained by counting transfected cells in two to three independent
transfections.
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To quantitatively examine the effect of huntingtin aggregates on
neurites, we counted huntingtin-transfected neurons that showed
neuritic degeneration or nuclear DNA fragmentation. The result revealed
that during early transfection time (days 1-2), more neuritic
degeneration was observed than nuclear degeneration (Fig.
7G). As transfection time was prolonged (day 4), the number of cells with nuclear DNA fragmentation was significantly increased. These findings suggest that N-terminal fragments of mutant huntingtin induce neuritic degeneration that occurs earlier than nuclear degeneration.
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DISCUSSION |
Selective neurodegeneration has been a puzzling issue in all
polyglutamine disorders. The present study demonstrates that the
context of full-length huntingtin protein confers a selective accumulation of mutant huntingtin in striatal projection neurons, precisely the neurons that are preferentially affected in HD. More
importantly, the distribution of huntingtin aggregates in the axons of
these striatal projection neurons is specifically associated with
axonal degeneration. In vitro experiments also show that
neuritic aggregates affect protein transport in neurites and cause
neuritic degeneration before neurons die. Thus, the present study
demonstrates for the first time the association between neuropil
aggregate formation and the selective neuropathology in early HD and
suggests that HD pathology originates in axons.
The above conclusion is supported by the comparison of huntingtin
aggregates in R6/2 mice that express N-terminal mutant huntingtin and
HD-repeat knock-in mice that express full-length mutant huntingtin. R6/2 mice show a widespread distribution of intranuclear and neuropil aggregates, whereas HD-repeat knock-in mice display a selective distribution of huntingtin aggregates in striatal neurons. It was shown
recently that the length of the huntingtin CAG repeat tends to expand
in striatal tissue in the HD-repeat knock-in mice that we examined in
this study, suggesting that the instability of this very large CAG
repeat may contribute to cell-specific neuropathology (Kennedy and
Shelbourne, 2000). Another possibility is that the selective processing
of full-length mutant huntingtin in striatal neurons may lead to the
accumulation and aggregation of N-terminal mutant huntingtin in these
neurons. Consistent with the latter idea, our previous study has shown
that N-terminal huntingtin fragments form huntingtin aggregates in
HD-repeat knock-in mice (H. Li et al., 2000).
Because the presence of nuclear inclusions does not correlate with
patterns of HD pathology (Saudou et al., 1998; Kuemmerle et al., 1999),
the present study focused on the role of mutant huntingtin in neuronal
processes. Neuropil aggregates appear to be different from nuclear
inclusions, because most of them are not labeled by anti-ubiquitin
antibodies (Gutekunst et al., 1999; H. Li et al., 1999). In addition,
we were unable to label these neuropil aggregates with antibodies
against heat shock protein 70 (Hsp70), Hsp40, or the
transcription factor cAMP response element binding protein binding
protein (data not shown). If neuropil aggregates are involved in
the selective neuropathology of HD, they should, at least, have the
following two features. First, the development and distribution of
neuropil aggregates should correlate with the early neurodegeneration
pattern. Second, aggregated huntingtin should associate with early
pathological changes. The first feature is supported in this study by
the temporal and regional studies of neuropil aggregate formation in
the regions in which striatal neurons extend their axons. We have
confirmed that the LGP and SN, which show degeneration earlier than
other regions, contain the highest density of neuropil aggregates.
Conversely, the MGP, which degenerates later, contains fewer neuropil
aggregates. It is possible that the MGP contains fewer axons from the
striatum so it has fewer neuropil aggregates. Nevertheless, the
preferential localization of huntingtin aggregates in axons of striatal
projection neurons agrees well with the degeneration of the striatal
projection neurons in HD patient brains, which was identified based on
the decreased enkephalin and substance P immunostaining of terminals within the LGP and SN (Reiner et al., 1988; Albin et al., 1990, 1992;
Richfield et al., 1995).
The second and most significant finding is that aggregates are
associated with the degeneration of axons. Electron microscopic examination revealed that many huntingtin aggregates were in axonal terminals but not in dendrites or postsynaptic terminals. Degenerated organelles, including mitochondria, are found to be present in the
axons containing huntingtin aggregates. Mitochondrial degeneration fits
well with the notion that mitochondrial dysfunction and oxidative stress are involved in HD pathogenesis (Browne et al., 1997; Sawa et
al., 1999; Schapira, 1999; Beal, 2000). Although it remains to be
investigated whether mutant huntingtin or its aggregates directly
affect the function of mitochondria and other organelles or whether
some of these degenerated organelles are autolysosomes, as reported by
others (Dunn, 1990; Kegel et al., 2000; Mizushima et al., 2001), our
finding provides the morphological evidence for mitochondrial
degeneration in early HD.
The presence of large-sized aggregates in axons and their terminals
suggests that huntingtin aggregates may block the transport of
organelles or vesicles in axons. This blockage could impair axonal
function, either by creating a physical barrier or through biochemical
interactions of mutant huntingtin with other proteins. Consistent with
this idea, huntingtin aggregates block RFP transport in neurites of
transfected striatal neurons. Blocking axonal transport could also
contribute to the degeneration of mitochondria and other organelles and
ultimately lead to cell-body degeneration. It is also possible that
mutant huntingtin may directly bind to synaptic vesicles and affect
synaptic transmission before it forms large aggregates. This may
explain why in the hippocampal region in HD-repeat-knock-in mice, where
no obvious huntingtin aggregates were observed, an impairment of
synaptic transmission was also observed (Usdin et al., 1999). Whether
aggregated huntingtin produces more adverse effects on synaptic
transmission in striatal neurons remains to be investigated. If these
pathological changes occur in a subset of neurons, they may not be
sufficient to induce obvious degeneration of cell bodies but may be
able to cause the behavioral phenotype observed in these HD mice
(Shelbourne et al., 1999) or the motor dysfunction observed in
presymptomatic HD patients (Smith et al., 2000). In HD exon1 transgenic
mice, however, the widely distributed and abundant neuropil aggregates
may contribute to their more obvious synaptic dysfunction (Murphy et
al., 2000) and mitochondrial abnormalities (Tabrizi et al., 2000).
Previous studies using HD transgenic mice expressing mutant huntingtin
under exogenous promoters showed that cell bodies of striatal neurons
were degenerating in symptomatic mice (Reddy et al., 1998; Hodgson et
al., 1999). Because these mouse models used different promoters and
constructs, the expression levels and localization of mutant huntingtin
in transgenic mice are not the same as that in HD knock-in mice. Mutant
huntingtin, when expressed at a higher level, could also affect
neuronal function or induce neuronal degeneration before forming
obvious aggregates (Reddy et al., 1998; Hodgson et al., 1999). The
present study demonstrates that when expression of full-length mutant
huntingtin is driven by endogenous mouse Hdh regulatory
elements, axonal degeneration selectively occurs in HD-affected neurons
in mice modeling early stages of the disease. Despite the abundance of nuclear aggregates in the striatum, striatal neurons do not show obvious cell-body degeneration. It is worthy pointing out that axonal
degeneration was found in corticostriatal pathways in the brains of HD
patients (Sapp et al., 1999). We did not observe such
degeneration in HD-knock-in mice, suggesting that axonal degeneration
of striatal neurons may occur earlier than neuronal degeneration in
other brain regions. All of these findings also point out the
possibility that HD pathology originates in axons of these striatal
neurons. This possibility is also supported by in vitro
experiments showing that neuritic degeneration precedes cell-body
degeneration in cultured striatal neurons. Moreover, many other studies
have shown that polyglutamine aggregates in the nucleus are not
associated with neurodegeneration (Klement et al., 1998; Saudou et al.,
1998; Kuemmerle et al., 1999; Warrick et al., 1999; Kazemi-Esfarjani
and Benzer, 2000). However, cytoplasmic polyglutamine aggregates can
induce apoptosis (Sanchez et al., 1999). The association of neuropil
aggregates with degenerated axons implies an important role of axonal
aggregates in the selective neurodegeneration of HD. The pathological
role of axonal aggregates is also supported by the fact that axonal
damage is the cause of a variety of nervous system diseases, such as
stroke (Sawlani et al., 1997), spongiform encephalopathies (Liberski
and Gajdusek, 1997), Guillain-Barré syndrome (Trojaborg, 1998),
insulin-dependent diabetic neuropathy (Said et al., 1992), and multiple
sclerosis (Arnold, 1999; Trapp et al., 1999). Because axonal injury
usually results in delayed cell death because of the relatively long
distance between the site of injury and the cell body, our findings
also suggest that prevention of this early neuropathologic change may provide an effective therapeutic approach to the treatment of HD.
| |
FOOTNOTES |
Received April 17, 2001; revised Aug. 17, 2001; accepted Aug. 21, 2001.
This work was supported by grants from the National Institutes of
Health (AG19206) and the Hereditary Disease Foundation. H.L. is a
recipient of the postdoctoral fellowship of the Hereditary Disease
Foundation and is partly supported by the National Natural Science
Foundation of China.
Correspondence should be addressed to Dr. Xiao-Jiang Li, Department of
Genetics, Emory University School of Medicine, 1462 Clifton Road
Northeast, Atlanta, GA 30322. E-mail: xiaoli{at}genetics.emory.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
