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The Journal of Neuroscience, October 1, 2001, 21(19):7608-7619
Neurons Lacking Huntingtin Differentially Colonize Brain and
Survive in Chimeric Mice
Anton
Reiner1,
Nobel
Del Mar1,
Christopher A.
Meade1,
Huaitao
Yang1,
Ioannis
Dragatsis2,
Scott
Zeitlin3, and
Daniel
Goldowitz1
1 Department of Anatomy and Neurobiology, College of
Medicine, The University of Tennessee, The Health Science Center,
Memphis, Tennessee 38163, and Departments of 2 Genetics and
Development and 3 Pathology, Columbia University, New York,
New York 10032
 |
ABSTRACT |
To determine whether neurons lacking huntingtin can participate in
development and survive in postnatal brain, we used two approaches in
an effort to create mice consisting of wild-type cells and cells
without huntingtin. In one approach, chimeras were created by
aggregating the 4-8 cell embryos from matings of Hdh
+/
mice with wild-type 4-8 cell embryos.
No chimeric offspring that possessed homozygous Hdh
/ cells were obtained thereby, although
statistical considerations suggest that such chimeras should have been
created. By contrast, Hdh / ES
cells injected into blastocysts yielded offspring that were born and in
adulthood were found to have Hdh
/ neurons throughout brain. The
Hdh / cells were, however, 5-10
times more common in hypothalamus, midbrain, and hindbrain than in
telencephalon and thalamus. Chimeric animals tended to be smaller than
wild-type littermates, and chimeric mice rich in Hdh
/ cells tended to show motor
abnormalities. Nonetheless, no brain malformations or pathologies were evident.
The apparent failure of aggregation chimeras possessing Hdh
/ cells to survive to birth is likely
attributable to the previously demonstrated critical role of huntingtin
in extraembryonic membranes. That Hdh
/ cells in chimeric mice created by
blastocyst injection are under-represented in adult telencephalon and
thalamus implies a role for huntingtin in the development of these
regions, whereas the neurological dysfunction in brains enriched in
Hdh / cells suggests a role for
huntingtin in adult brain. Nonetheless, the lengthy survival of
Hdh / cells in adult chimeric
mice indicates that individual neurons in many brain regions do not
require huntingtin to participate in normal brain development and to survive.
Key words:
basal ganglia; cortex; development; Huntington's
disease; HD gene; colonization
| |
INTRODUCTION |
Huntington's disease (HD) is a
dominant hereditary neurodegenerative disorder characterized by
progressive cognitive decline and motor dysfunction (Bruyn and Went,
1986
; Wilson et al., 1987; Albin and Tagle, 1995). The major site of
neuron loss in HD is the striatal part of the basal ganglia, and it is
this loss that accounts for the progressive movement disorder
(Vonsattel et al., 1985; De La Monte et al., 1988; Hedreen et al.,
1991; Storey et al., 1992). The gene and the specific mutation
responsible for HD have been known for several years (Huntington's
Disease Collaborative Research Group, 1993). The gene product,
huntingtin, is of unknown function, although several lines of evidence
suggest that it is involved in vesicular trafficking (DiFiglia et al.,
1995; Sharp et al., 1995; Wood et al., 1996). The mutation in the HD
gene involves an expansion of a CAG repeat at the 5' end of the gene beyond its normal 10-35 repeat range (Albin and Tagle, 1995). The
means by which this mutation causes preferential destruction of the
striatum is uncertain. Several lines of evidence suggest that, whatever
its precise mechanism of action, the HD mutation acts in a
"gain-of-function" manner. First, HD shows autosomal dominant
inheritance. Second, in a single fortuitously discovered case, one copy
of the gene coding for huntingtin was found to be inactivated by a
translocation, yet this individual did not have HD symptoms despite
having only 50% of normal HD gene expression (Ambrose et al., 1994;
Persichetti et al., 1996). Third, homozygous knock-out of the gene
coding for huntingtin in mice (Hdh) is embryonically lethal
(Duyao et al., 1995; Nasir et al., 1995; Zeitlin et al., 1995), whereas
homozygous mutation of the HD gene in humans is not (Wexler et al.,
1987; Myers et al., 1989; Gusella and MacDonald, 1996). Finally, humans
homozygous for the HD mutation show no clear differences from
heterozygotes in disease onset or progression (Wexler et al., 1987;
Myers et al., 1989; Gusella and MacDonald, 1996).
The possibility remains, however, that the HD mutation does, at least
in part, act as a loss-of-function mutation, and this effect for some
unknown reason is not manifest until well after birth. For example, it
could be the case that the mutated version of huntingtin in HD
heterozygotes comes to neutralize the function of the normal protein
because of unknown age-related changes in the behavior of either the
mutant or normal protein (Cattaneo et al., 2001). If in fact this type
of event occurs and contributes to HD pathogenesis, one would expect
that neurons lacking huntingtin should survive poorly in mice
engineered to possess an Hdh deletion. To examine this
possibility, we overcame the fact that homozygous deletion of
Hdh is embryonically lethal by a chimeric strategy (Goldowitz et al., 1992; Duyao et al., 1995; Nasir et al., 1995; Zeitlin et al., 1995; Dragatsis et al., 1998). Our results show that
neurons devoid of huntingtin can participate in normal brain formation
and survive in adult mouse brain. Nonetheless, huntingtin does appear
to play some role in neural functioning, because motor impairment was
evident in many chimeric mice for which the brains were enriched in
Hdh / neurons. Additionally,
huntingtin appears to be required for proliferation or survival of
neurons in some brain regions during early development, because we
observed that Hdh / cells
preferentially colonize hypothalamus, midbrain, and hindbrain and are
scarce in telencephalon and thalamus.
| |
MATERIALS AND METHODS |
Subjects. Hdh
+/ mice (Duyao et al., 1995) and
ROSA26 mice were obtained from the Jackson Laboratories (Bar
Harbor, ME) and bred in our colony at the University of Tennessee. The
Hdh disruption in the Duyao et al. (1995) mice involves a
deletion of exons 4 and 5, resulting in the abolition of expression for
Hdh. These mice were used together with the
ROSA26 mice to create chimeras by the method of early embryo
aggregation (with the embryos from both lines being at the 4-8 cell
stage at the time of aggregation), as described below. Hdh
/ 
Hdh
+/+ chimeric mice also were created
in the laboratory of Dr. Scott Zeitlin by injecting embryonic stem (ES)
cells nullizygous for Hdh into wild-type blastocysts
(Dragatsis et al., 1998). The Hdh nullizygous ES cells
possessed a homozygous-targeted disruption of both Hdh
alleles (Dragatsis et al., 1998). The Hdh disruption in this
case involved deletion of the promoter, exon 1, and flanking intronic
sequences (Hdhtm1Szi), again resulting in
the loss of Hdh expression (Zeitlin et al., 1995). So that
their detection could be facilitated in chimeric tissues, the Hdh
/ ES cells also bore a lacZ
transgene that had been introduced into their genome by the mating
strategy described below. All studies were conducted in accordance with
National Institutes of Health and Society for Neuroscience policies on
the ethical use of animals in research.
Production of aggregation chimeras. Aggregation chimeras
were created by standard methods (Goldowitz et al., 1992), with the goal of creating mice that consisted of a mixture of Hdh
/ cells and wild-type cells. Briefly,
Hdh +/ females were superovulated
and mated with Hdh +/ males.
Plug-positive females were taken 2 d later, and their oviducts
were flushed with medium to harvest the 4-8 cell stage embryos. The
wild-type component of the chimera was derived from the 4-8 cell stage
embryos of the ROSA26 strain of mice, which allows for
identification of cells from the wild-type contribution to the chimeras
by an X-gal histochemical procedure for the lacZ transgene
product (Friedrich and Soriano, 1991). Note that, in chimeras created
by the method of aggregating two 4-8 cell stage embryos, the Hdh
/ cells would be able equally, in
principle, to colonize all parts of the embryo, including the
extraembryonic membranes (Le Dourain and McLaren, 1984).
Production of chimeras by blastocyst injection. Hdh
//ROSA26 ES cells were
obtained by dissociating and culturing the inner cell mass from
embryonic day 3.5 (E3.5) embryos that were the products of a mating
strategy designed to produce embryos that possessed a homozygous
Hdh deletion and also possessed the ROSA26 transgene. In the first step of this mating strategy Hdh
+/ female mice were mated with males
homozygous for the ROSA26 lacZ transgene. In the second step
of the mating strategy the Hdh
+//ROSA26 offspring were
intercrossed, and E3.5 blastocysts were obtained from this mating, 75%
of which bore the ROSA26 transgene. ES cell lines were
derived from these blastocysts, and Southern and Western analyses were
performed to identify those ES cell lines that were Hdh
/. Hdh
//ROSA26 ES cells were
injected into wild-type C57BL/6 (B6) host E3.5 blastocysts via standard
procedures, and the injected blastocysts were transferred into the
uterine horns of pseudopregnant females. Any of three different lines
of Hdh / ES cells described
previously (A, B, or I) were used for these injections (Dragatsis et
al., 1998). These procedures have been described in detail previously
(Dragatsis et al., 1998). After birth, the chimeric mice were monitored
and killed at various time points up to ~1 year of age. Note
that, because ES cells preferentially colonize the embryonic ectoderm
and poorly colonize extraembryonic tissues derived from the inner cell
mass and completely fail to colonize extraembryonic tissues derived
from trophectoderm, in the chimeras made by blastocyst injection the
Hdh / cells are completely
absent from trophectoderm derivatives and are mainly absent from the
extraembryonic membrane derived from inner cell mass tissue (Beddington
and Robertson, 1989).
PCR genotyping of Hdh +/ mice used to
generate embryos for aggregation chimeras. PCR genotyping was
performed as described previously to identify Hdh
+/ mice for breeding purposes (Duyao et
al., 1995; Chen et al., 1997; Fusco et al., 1999). Genomic DNA
extracted from tail biopsies was used to detect mice bearing an
Hdh knock-out allele in their genome by PCR genotyping mice
for Hdh and for the neomycin resistance selection cassette
(neo) introduced during the creation of the Hdh
deletion. The primers for the detection of huntingtin DNA in these PCR
assays were 5'-CAAATGTTGCTTGTCTGGTG-3' and
5'-GTCAGTC-GAGTGCACAGTTT-3'. The amplified Hdh DNA
fragment has a size of 150 bp. The primers for the detection of
neo DNA were 5'-CTTGGGTGGAGAGGCTATTC-3' and 5'-AGGTGAGATGACAGGAGATC-3'. The amplified neo PCR
product has a size of 280 bp. One-half of a microliter of DNA template
(250 ng/µl genomic DNA) was used, and the PCR for Hdh and
neo for each animal was run simultaneously in the same
thin-walled PCR tube. The PCR reaction solution contained the
following: 2.0 µl of 10× PCR buffer (10 mM
Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton
X-100), 1.6 µl of 25 mM
MgCl2, 0.4 µl of 10 mM
dNTPs, 2.0 µl of 10 µM amounts of the four
primers, 2.76 µl of DNA tracking dye, 4.66 µl of
dH2O, and 0.08 µl of Taq polymerase.
Contamination from extraneous DNA was checked by replacing the cellular
template with water. Amplification was performed on a thermal cycler
(MJ Research, Watertown, MA), typically under the following cycle conditions: denaturation at 94°C for 30 sec, annealing at 55°C for
45 sec, and extension at 72°C for 1 min for a total 30 cycles. After
PCR amplification, aliquots of reaction product were analyzed by
electrophoresis on ethidium bromide-impregnated 3% agarose gels.
PCR genotyping of aggregation chimeras to detect Hdh
/ cells. So that cells that are homozygous
for the Hdh knock-out could be detected, it is necessary to
detect the presence of neo and find no evidence for the
presence of Hdh. For distinguishing aggregation chimeras
bearing homozygous Hdh knock-out cells from those bearing a
hemizygous Hdh deletion, PCR genotyping by tail biopsy is
inadequate, because both Hdh
+/ and Hdh
/ cells possess neo, and
the possible presence of Hdh in wild-type cells in the tail
biopsy would hide evidence of any Hdh
/ cells potentially contributing to
the formation of the chimera. Thus to detect the presence of Hdh
/ cells in the aggregation chimeras,
we excised pure populations of ROSA26-negative cells (which
come from the Hdh knock-out line) from the fixed livers of
aggregation chimeras postmortem. Liver slices (50 µm thick) from the
aggregation chimeras that had shown a tail-snip PCR signal for
neo were prepared and reacted with X-gal as described below.
The liver slices were mounted on slides and examined with a dissecting
microscope. Patches of cells that were devoid of X-gal labeling and
were therefore from the Hdh knock-out strain were removed
from the tissue with a glass micropipette. These cells were lysed with
Proteinase K, and 1-2 µl of the digested material was assayed by PCR
to determine whether it was negative for Hdh. Three primers
were used that allowed us to detect differentially the normal
Hdh gene and the truncated form of the allele created by the
targeted disruption of Hdh: (1)
5'-ACGTGAGCTGTCCAGGTGAGCC-3'; (2) 5'-TATAGAGTTCTAACTGTAGCCTTG-3';
and (3) 5'-TCGCCGCTCCCGATTCGCA-GCATCG-3'. The first and second of
these primers amplify a 350 bp fragment of wild-type Hdh
containing intron 3 and exon 4, whereas the first and third primers
amplify a 650 bp fragment of disrupted Hdh containing intron
3 and the neo insert. The PCR reaction solution contained the following: 2 µl of 10× PCR buffer, 1.2 µl of 25 mM MgCl2, 0.2 µl of 20 mM dNTPs, 1 µl of 20 µM
amounts of the three primers, 2.76 µl of DNA tracking dye, 10.7 µl
of dH2O, and 0.1 µl of 7.5 U Taq
polymerase. Contamination from extraneous DNA was checked by replacing
the cellular template with water. Amplification was performed on a
thermal cycler (MJ Research), as described above, and aliquots of
reaction product were analyzed by electrophoresis on ethidium
bromide-impregnated 3% agarose gels. To ensure that chimeric mice
genotyped as Hdh +/+ by tail biopsy
were in fact Hdh +/+, we
performed genotyping of non-ROSA26 liver cells from
these animals as well.
Behavioral assessment of chimeras produced by blastocyst
injection. Several simple motor/behavioral tests were performed on the chimeras produced by blastocyst injection of Hdh
//ROSA26 ES cells
(Dragatsis et al., 1998). These included a limb-clasping assessment, a
wire rod hanging test, an evaluation of the ability to cling to a wire
cage being rotated 180 degrees, and a gait analysis. These tests have
been shown to be sensitive to the neurological/motor abnormalities seen
in mutant mouse models of neurological disease (Mangiarini et al.,
1996; Dragatsis et al., 2000). Mice typically were tested once a
month. For limb clasping, chimeric mice and nonchimeric littermates
were suspended 30 cm above an open cage and over a 1 min period were
lowered toward the bottom of the cage. Mice that clasped their limbs
within 5 sec of suspension and maintained the clasping during the
entire descent were scored as showing a positive limb-clasping
response. For the wire rod hanging test, mice were allowed to grasp a
narrow wire rod (diameter, <0.25 cm) suspended 30 cm above a padded
work surface and were observed for 1 min. Normal mice can maintain a
grip with their forelimbs for the full test duration, so mice that fell
within 30 sec of the onset of the test thus were scored as positive for neurological impairment. For the wire cage rotation test, mice were
placed on top of a wire cage held ~30 cm above a padded work surface.
Then the cage was rotated 180° over a period of 1 min and held for an
additional 30 sec in the inverted position. Mice scoring positive for
neurological impairment in this test were unable to climb to a position
at the top of the rotating frame as it was turned and fell to the work
surface (Crawley, 1999). For gait analysis, the hindpaws of mice were
dipped in nontoxic fingerpaint and placed on a strip of paper between
two guide walls. In the comparisons of chimeras and controls, the
distance between successive paw prints and the width between each pair
of prints were measured. Body weight was measured in the chimeric mice
and a set of 10 age-matched C57BL/6 males and 10 age-matched C57BL/6 females at 1, 3, 6, and 9 months.
Tissue fixation. Under deep Avertin anesthesia, mice used
for histological analysis were perfused transcardially with PBS (0.1 M sodium phosphate buffer, pH 7.4, with 0.9% NaCl),
followed by 4% paraformaldehyde in 0.1 M PB. These mice
included all chimeric mice created by the aggregation method, six ES
blastocyst injection chimeras and their two B6 controls, and five
nonchimeric ROSA26 mice used as controls to demonstrate the
ability of the lacZ gene to be expressed ubiquitously
throughout the nervous system in mice (Friedrich and Soriano, 1991).
The brain and liver were removed and stored in a 20% sucrose/10%
glycerol solution at 4°C. Livers from aggregation-chimera mice
created by using ROSA26 as the wild-type strain were
sectioned with a vibratome at 50 µm and used in genotyping, as noted
above. The brains of the six ES blastocyst injection chimeras, their
two B6 controls, and the control ROSA26 mice were sectioned
frozen on a sliding microtome in the transverse plane at 35 µm. Each
of these brains was collected as 6-12 separate series in 0.1 M PB and 0.02% sodium azide and stored until
processed for histochemistry, histology, or immunohistochemistry. For
four additional ES blastocyst injection chimeras the brains were
removed and immersion fixed overnight in 0.2% paraformaldehyde in 0.1 M PIPES, pH 6.9, plus 2 mM
MgCl2 and 5 mM EGTA, washed
in PBS and MgCl2, cryo-preserved in 30% sucrose
plus MgCl2, embedded in Tissue-Tek OCT compound
(Fisher Scientific, Pittsburgh, PA), and frozen. These brains were
sectioned in the sagittal plane with a cryostat and mounted on
Superfrost Plus glass slides. A series of brain sections for each
chimeric mouse created by the blastocyst injection method (and the B6
controls) was stained with cresyl violet or neutral red to study normal
brain histology. Additional series were processed by using fluoro-jade
labeling to detect any degenerating cells (Schmued et al., 1997). As
positive controls in the fluoro-jade studies, C57BL/6 mice were
injected intraperitoneally with 1 mg of kainic acid per 30 gm of body
weight. This dose was chosen because it gives stage 3 seizures and
consistent hippocampal cell death (Schauwecker and Steward, 1997).
These animals were perfusion fixed by using the same fixative as for the chimeric mice. Brains were cryosectioned, mounted on glass slides,
and processed in parallel with the chimeric tissue for the fluoro-jade
demonstration of degenerating neurons.
Demonstration of cell genotype via 
-galactosidase
histochemistry. One series of sections through the brain and/or
liver of chimeric mice, the B6 control mice, and the nonchimeric
ROSA26 control mice was processed for the -galactosidase
marker by using the procedure of Oberdick et al. (1994), in which
sections are incubated at 30-35°C overnight in buffer (containing 5 mM of potassium ferricyanide and ferrocyanide, 2 mM magnesium chloride, 0.02% Nonidet P-40, and
0.01% sodium deoxycholate) with 0.1% X-gal substrate in dimethyl
sulfoxide (Boehringer Mannheim, Indianapolis, IN). Slides were rinsed
and counterstained with neutral red to identify and quantify labeled
and unlabeled cells. Tissue was dehydrated and cleared in xylenes;
coverslips were applied with Permount. This approach identified the
cells from the wild-type strain in aggregation chimeras in which the
ROSA26 line was used as the wild-type strain and knock-out
cells themselves in the blastocyst injection chimeras (because
Hdh / cells bore a
lacZ transgene expressing -galactosidase attributable to
the mating strategy described above).
Immunohistochemistry. Immunohistochemical labeling was
performed to confirm the absence of huntingtin in X-gal-labeled
perikarya in the chimeras created by blastocyst injection and to
characterize the effects of the homozygous Hdh deletion on
these cells by assaying for perturbation in neuropeptide and
neurotransmitter expression as well as for the expression of markers of
regional or cellular stress. Conventional immunofluorescence or the
peroxidase-anti-peroxidase (PAP) procedures were used (Anderson and
Reiner, 1990, 1991; Figueredo-Cardenas et al., 1994). Tyramide signal
amplification was used to enhance labeling for huntingtin (Fusco et
al., 1999). In some cases the immunolabeling also was enhanced by
pretreating the free-floating sections with a 30 min immersion in 10 mM sodium citrate buffer, pH 9.0, at 85°C (Jiao
et al., 1999). To detect huntingtin, we used a mouse monoclonal
antibody (Mab2170, Chemicon, Temecula, CA). The antibody was generated
against amino acids1247-1646 of human huntingtin; its specificity for
huntingtin in rodents and humans has been demonstrated previously
(Bessert et al., 1995; Trottier et al., 1995; Fusco et al., 1999).
Antigens that were screened to assess the normalcy of the blastocyst
injection chimera brains possessing Hdh
/ cells included vasoactive intestinal
polypeptide (VIP), tyrosine hydroxylase (TH), calbindin (CALB),
parvalbumin (PARV), glial fibrillary acidic protein (GFAP), substance P
(SP), methionine-enkephalin (ENK), neuropeptide Y (NPY), choline
acetyltransferase (ChAT), and vasopressin (VP) (Loren et al., 1979;
Kiyama et al., 1990; Reiner and Anderson, 1990; Armstrong et al., 1994;
Figueredo-Cardenas et al., 1994, 1998; Kawaguchi et al., 1995; Karle et
al., 1996; Elmquist et al., 1999). The specificity and sources of the
antisera that were used have been described in previous studies
(Reiner, 1991; Armstrong et al., 1994; Figueredo-Cardenas et al., 1994, 1998; Karle et al., 1996).
| |
RESULTS |
Aggregation chimeras
From our efforts to create aggregation chimeras containing
cells with a homozygous deletion of the huntingtin gene, 21 chimeric animals were born. Note that we would expect only 25% of our
aggregation chimeras to possess Hdh
/ cells, because only 25% of the
embryos from our mating of Hdh +/
females with Hdh +/ males would be
expected to be Hdh /. To
distinguish chimeras bearing Hdh
/ cells from those bearing Hdh
+/ cells, we believed that PCR
genotyping of DNA isolated from a tail biopsy would be inadequate,
because wild-type cells in the tail biopsy would hide evidence of
homozygous Hdh knock-out cells. Thus we used the strategy
described in Materials and Methods whereby we excised pure populations
of cells from the knock-out line from the fixed liver of postmortem
chimeric mice. Of our 21 chimeric animals created by aggregation
methods, 12 showed a positive signal for the neo cassette by
PCR. The remaining mice (9) were chimeric assemblages of Hdh
+/+ cells from the knock-out strain and
the wild-type strain. Of the 12 showing a positive signal for the
neo cassette (indicating at least one Hdh null
allele), none contained cells that were nullizygous for Hdh,
and all neo-positive cells thus appeared to be Hdh
+/. The failure of aggregation chimeras
with homozygous Hdh knock-out cells to be born is
extremely unlikely to have occurred by chance, because of the 21 chimeric mice that were phenotyped, approximately five would have been
expected to possess Hdh / cells
(p < 0.05, by 
2
analysis). Based on these considerations, it seems that the chimeric mice possessing Hdh / cells
presumably created by the aggregation method must not have come to
term. It is of note that, of the 21 chimeric mice created by
aggregation methods that were born, the percentage of contribution from
the embryos derived from the Hdh
+/ cross ranged from 5 to 95%. If the
same percentage of chimerism can be assumed for the homozygous
Hdh knock-out chimeras, who are presumed to have died
in utero, it would suggest that even a very
small percentage of Hdh /
cells is incompatible with development to term.
Blastocyst injection chimeras: Characteristics of Hdh
/ chimeras
By contrast, blastocyst injection of Hdh
/ ES cells yielded viable chimeric
mice (10 males, 13 females) that were able to live well into adulthood
(Table 1). Chimeras were generated with
any of three Hdh nullizygous ES cell lines (two female
lines, clones I and B, and one male line, clone A). Southern blot data
from organs of some killed chimeras suggested that the coat color
chimerism tended to reflect overall bodily chimerism at the DNA level
(the ES cell lines were derived from the 129 Sv/Ev
background and gave rise to agouti animals, whereas the host
blastocysts were obtained from C57BL/6 females with black fur). The
chimeras were estimated to range from 20 to 75% in their contribution
from the Hdh / ES-derived
mouse strain by their coat color. The body weight of these chimeras
ranged from 60 to 100% of wild-type mice, and body weight was
significantly inversely correlated (r = 0.81), with the
percentage of ES cell-derived contribution indicated by coat color
(i.e., the more Hdh /cells,
the lower the body weight of the chimera). The male chimeras never
mated successfully; there were no pregnancies, and no vaginal plugs
were detected in wild-type females housed with male chimeras. By
contrast, female chimeras generated by blastocyst injection of
Hdh / ES cells were able to mate
successfully with wild-type males, because two transmitted the
Hdh mutation to their progeny. Five male and two female
chimeras showed a characteristic set of abnormal motor traits, notably
clasping of the forelimbs and hindlimbs and curling of the body during
the clasping, beginning at weaning (4 weeks of age). In addition, four
of these chimeras, beginning at 4 weeks, could not grasp or support
themselves well on an elevated wire rod, and, when placed on a wire
cage that then was rotated slowly, the chimeras invariably fell off the
cage (whereas control animals did not). By 20 weeks of age all seven of
these chimeras failed the rod grasping and rotating cage grasping
tests. A simple gait analysis that used inked hindpaws did not,
however, reveal differences from controls. The abnormal animals also
tended to be attacked and wounded by their littermates when housed with them, implying the symptomatic chimeric mice were less able to defend
themselves.
Of the seven animals that exhibited the suite of severe behavioral
abnormalities noted above, six had a chimeric contribution 
45%
(based on coat color). By contrast, of the 16 chimeras that did not
exhibit the severe behavioral abnormalities, 13 had a chimeric
contribution <45% (based on coat color; Table 1). Among the three
mice with a chimeric contribution 45% that did not show the suite of
severe behavioral/motor abnormalities, two in fact showed mild clasping
(one mouse with 60% coat color chimerism and one with 65%) but were
unaffected in the other tests (Table 1). One further chimeric mouse
(with 40% coat color chimerism) also had a mild clasping tendency but
was unaffected in the other tests as well (Table 1). The data therefore
suggest that the magnitude of the overall Hdh
/ contribution, as inferred from coat
color, was associated with an increased likelihood of motor
abnormalities, either severe or slight. To assess this apparent
association, we assigned a score of 0 for no motor deficit; a 1 for a
slight clasping defect; a 2 for showing clasping, poor rod grasping,
and poor rotating cage grasping by 20 weeks; and a 3 for showing
clasping, poor rod grasping, and poor rotating cage grasping by 4 weeks. With the use of this motor scoring scale, the percentage of ES
cell contribution indicated by coat color is correlated significantly (r = 0.73) with the motor deficit. Nonetheless, it is
evident that the severity of the behavioral abnormalities was not
predicted inexorably by the percentage of coat color chimerism. For
example, severe motor abnormalities were observed in one animal with
only 30% coat color chimerism but were absent in three with 45%
coat color chimerism. Such deviations from a strictly linear
relationship between coat color chimerism and the magnitude of the
motor abnormalities may have occurred because the extent of
colonization by Hdh / cells of
the key neural and/or the key extraneural regions that were needed to
produce the observed suite of behavioral abnormalities was not
reflected accurately in all cases by the abundance of Hdh
/ cells in the coat. Such an
explanation is consistent with the evidence that the relative
colonization of bodily tissues by the genotypically different lineages
comprising a chimeric animal can differ from tissue to tissue and
animal to animal (Le Dourain and McLaren, 1984; Goldowitz et al., 1992;
Kuan et al., 1997).
The seven mice showing the suite of behavioral abnormalities also
exhibited severe weight loss and severe hypoactivity by 6-9 months of
age and were killed at that time. The brains of four were immersion
fixed overnight, as described in Materials and Methods, and stored
frozen until processed as slide-mounted cryostat sections. Six of the
chimeric mice that survived slightly beyond 1 year without showing the
suite of severe behavioral abnormalities were perfused transcardially
with fixative, as described in Materials and Methods, and the brains
were processed histologically as free-floating slide-mounted sections.
Blastocyst injection chimeras: Histological analysis of Hdh
/ chimeras
The 10 chimeras and two B6 control mice were sectioned and
analyzed histologically and histochemically. The brains of the chimeras
appeared normal in gross morphology, cytoarchitecture, regional
neuronal abundance, and ventricular outline (Fig.
1). The brains of the chimeras, however,
typically were slightly smaller than those of the controls. In the 10 chimeras, numerous X-gal-labeled cells derived from the Hdh
nullizygous ES cells injected at the blastocyst stage were found
throughout the brain (Table 2; Fig. 1).
No systematic differences were observed in the distribution of these
Hdh / cells for the three
injected lines of Hdh /
embryonic stem cells (A, B, and I). Side-by-side comparisons of
X-gal-labeled tissue and huntingtin-labeled tissue showed that regions
rich in X-gal-labeled cells were poor in huntingtin-labeled cells,
thereby confirming that X-gal-labeled cells were indeed Hdh
/. This was particularly evident in
the cornu ammonis of the hippocampus, in which alternating bands of
X-gal-labeled and huntingtin-labeled neurons could be observed (Fig.
2). No X-gal-labeled cells were observed
in the B6 control mice. Although X-gal-labeled Hdh
/ cells were found throughout the
brain of all 10 chimeric mice, the Hdh
/ cells tended to be relatively scarce
in the telencephalon (except for the hippocampus, in which they were
consistently present), mainly absent from the thalamus and Purkinje
cell layer of the cerebellum, and abundant in the epithalamus, preoptic
region, hypothalamus, midbrain, granule cell layer of the cerebellum, and hindbrain (Table 2; Fig. 3). The
abundance of these cells in these regions, however, varied from case to
case. For example, in some cases Hdh
/ cells in hypothalamus and brainstem
made up at least 50% of the cells, which was especially evident for
the motoneuron pools in the case of the brainstem, whereas in others
Hdh / cells made up no more than
~25% of the cells in hypothalamus or brainstem (Table 2). In the
latter cases Hdh / cells were
nearly absent in the cerebral cortex, basal ganglia, and thalamus,
whereas even in the former they typically did not include >10% of
telencephalic and thalamic cells. By contrast, in the nonchimeric
ROSA26 control mice, X-gal labeling was observed in all
cells throughout the brain, consistent with the previous observations
of others (Friedrich and Soriano, 1991; Goldowitz et al., 2000).
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Figure 1.
Image of a sagittal section from one of the
chimeric mice created by blastocyst injection of Hdh
/ ES cells, stained to reveal the
location of the X-gal-positive Hdh
/ ES cell progeny that have colonized
the brain. The tissue was counterstained with neutral red. This animal
(ES13) was among those chimeric mice possessing Hdh
/ cells that were killed before 1 year
of age because of signs of morbidity. The green-blue X-gal
labeling shows that Hdh / cells
are found throughout brain but are most abundant in hippocampus,
preoptic area, hypothalamus, midbrain, and hindbrain.
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Table 2.
Blastocyst injection chimeras: Distribution of
Hdh/ cells in brain and traits of chimeric
mice examined histologically
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Figure 2.
Image of CA2 in a transverse section from one of
the chimeric mice created by blastocyst injection of Hdh
/ ES cells, stained to reveal the
localization of huntingtin-containing wild-type cells (visualized by
DAB immunolabeling; A) and X-gal-positive ES cell
progeny that have colonized the hippocampus (B).
The X-gal-labeled tissue also was counterstained with neutral red. This
animal was among those that lived up to 1 year of age with no signs of
morbidity. The green-blue X-gal labeling shows that
Hdh / CA2 pyramidal cells in
regions lacking in X-gal labeling are immunostained for huntingtin
(arrow indicates a large cluster of huntingtin-labeled
neurons). By contrast, regions of CA2 richly labeled for X-gal
(arrowhead) are poor in or devoid of huntingtin
immunolabeling.
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Figure 3.
High-magnification images showing the distribution
and abundance of Hdh / cells in
several brain regions in the same chimeric mouse created by blastocyst
injection of Hdh / ES cells, as
shown in Figure 1. The green-blue X-gal labeling shows that
Hdh / cells are scarce in
striatum (A) and highly abundant in the preoptic
region (B) and dorsal pons
(C). The image presented in D
shows that the vast majority of the neurons in the facial nucleus in
this case was Hdh /. The section
from which these images were taken had been counterstained lightly with
neutral red.
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Because the X-gal reaction product in the chimeric mice in many cases
clearly could be localized to neurons (particularly in the case of
large neurons for which the shape was unmistakable because the X-gal
reaction product followed the cellular outline; Fig. 3), our results
show that Hdh / neurons can
survive in the chimeric brain for >1 year and that, up to an abundance
of 50% Hdh / cells in midbrain
and hindbrain, brain development seems normal. Three of the four
chimeras showing the behavioral abnormalities and morbidity that were
examined histologically, however, were enriched in hypothalamic and
brainstem Hdh / cells (estimated
to be 50-75% for the brainstem motoneuron pools, for example, in
these mice). By contrast, none of the chimeric mice who showed no
morbidity up to 1 year after birth and who were examined histologically
possessed >50% Hdh knock-out cells in both hypothalamus
and motoneuron pools (Table 2). This suggests that >50% colonization
of hypothalamus and brainstem by Hdh
/ cells may have played a role in the
behavioral abnormalities and morbidity observed in at least some
chimeric animals.
We used various immunomarkers to assess the normalcy of brain
functional architecture. In light of the prominent involvement of the
basal ganglia in HD, the possible effect of Hdh
/ cells on the striatum and its
projection systems was of particular interest. Although the chimeric
animals varied in their abundance of Hdh
/ cells in the striatum (from
negligible to ~15%), we observed no evident differences between
chimeric mice and wild-type mice in the distribution or abundance of
calbindin-containing projection neurons or parvalbumin-containing
interneurons in the striatum (Figs. 4,
5), both of which are affected in HD
(Kiyama et al., 1990; Harrington and Kowall, 1991). Additionally, the
abundance of enkephalinergic striatal fibers in the globus pallidus and of substance P-containing fibers in the entopeduncular nucleus and
substantia nigra appeared no different from those in the B6 control
mice (see Fig. 4), suggesting further that striatal projection neurons,
which are affected prominently in HD (Reiner et al., 1988; Albin et
al., 1990a,b, 1992; Richfield et al., 1995; Sapp et al., 1995), were
unaffected in the chimeric mice. Similarly, a high abundance of
Hdh / cells in the hypothalamus
was not associated with any evident abnormality in the hypothalamic
distributions of vasopressin, VIP, tyrosine hydroxylase, or NPY, and a
prevalence of Hdh / neurons in
brainstem motoneurons was not associated with any evident loss of
choline acetyltransferase labeling of these neurons or any alteration
in the abundance of these neurons (Figs.
6, 7).
Consistent with the absence of any brain abnormalities, as assayed by
neuropeptide or neurotransmitter-related enzyme content, neither
immunolabeling for GFAP (see Figs. 5, 7) nor staining with fluoro-jade,
a marker of neurodegenerative changes, revealed any signs of pathology
in the chimeric brains. It is noteworthy that this was true for both
the chimeras showing morbidity by 6-9 months as well as those
surviving with no signs of ill health until the time of death at ~1
year. By contrast, the hippocampus of the kainate-injected C57BL/6 mice
showed large numbers of degenerating fluoro-jade-positive pyramidal
neurons.
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Figure 4.
High-magnification images of transverse sections
through dorsomedial striatum showing the distribution and abundance of
Hdh / cells (as visualized by
X-gal labeling, followed by a light neutral red counterstain) in the
striatum of a chimeric mouse created by blastocyst injection of
Hdh / ES cells. Shown is the
striatum of a chimeric mouse, who displayed no ill health up to 1 year
of age (A), compared with the striatum of a
wild-type mouse in which no Hdh /
cells are present (B). C,
D, The presence of Hdh /
cells in the striatum of the chimeric mouse shown in A
has not produced any evident abnormality in the enkephalinergic
striatal output fibers (ENK; visualized by DAB
immunolabeling) within the ipsilateral globus pallidus
(GP) of the chimeric mouse (C).
Medial is to the left and dorsal to the
top in all images.
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Figure 5.
High-magnification images of transverse sections
through dorsomedial striatum of the same chimeric and wild-type animals
as shown in Figure 4. The presence of Hdh
/ cells in the striatum of the chimeric
mouse has not produced any evident abnormality in the labeling of
calbindergic striatal perikarya (A, B) or
any evidence of neuropathology in the striatum, as shown by the absence
of any upregulation of glial fibrillary acid protein
(GFAP) in the striatum (C,
D). Medial is to the left and dorsal to
the top in all images.
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Figure 6.
High-magnification images of transverse sections
through the paraventricular nucleus of the hypothalamus showing the
distribution and abundance of Hdh
/ cells in striatum of a chimeric mouse
created by blastocyst injection of Hdh
/ ES cells (ES8). Shown is the striatum
of a chimeric mouse, who displayed no ill health up to 1 year of age
(A), compared with the paraventricular nucleus of
a wild-type mouse in which no Hdh
/ cells are present
(B). The Hdh
/ cells in A are
visualized by X-gal labeling, and neuronal cytoarchitecture in both
A and B is visualized by neutral red
counterstaining. C, D, The presence of
Hdh / cells in the
paraventricular nucleus of the chimeric mouse shown in A
has not produced any evident abnormality in the vasopressinergic
(VP) neurons (visualized by DAB immunolabeling) of the
ipsilateral paraventricular nucleus of the chimeric mouse. Medial is to
the left and dorsal to the top in all
images.
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Figure 7.
High-magnification images of transverse sections
through pons showing the distribution and abundance of Hdh
/ cells in the facial nucleus in a
chimeric mouse created by blastocyst injection of Hdh
/ ES cells. Shown is the facial nucleus
in a chimeric mouse, who displayed no ill health up to 1 year of age
(A), compared with facial nucleus of a wild-type
mouse in which no Hdh / cells are
present (B). The Hdh
/ cells in A are
visualized by X-gal labeling, and neuronal cytoarchitecture in both
A and B is visualized by neutral red
counterstaining. C, D, The presence of
Hdh / cells in the facial nucleus
of the chimeric mouse shown in A has not produced any
evident abnormality in the facial motoneurons (visualized by DAB
immunolabeling for choline acetyltransferase) within the ipsilateral
facial nucleus of the chimeric mouse. High-magnification images of
transverse sections through facial nucleus of the same chimeric and
wild-type animals as shown in A and B
reveal that the presence of Hdh /
cells in the facial nucleus of the chimeric mouse has not produced any
upregulation of GFAP in the facial nucleus (E,
F). Medial is to the left and
dorsal to the top in all images.
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DISCUSSION |
To understand better the significance of huntingtin in neuronal
function, we overcame the embryonic lethality of homozygous knock-out
of the HD gene homolog in mice by a chimeric strategy. Our findings
reveal several important points regarding the role of huntingtin in
neural development and neuronal survival, as discussed below.
Comparison to previous findings by others
Our failure to produce viable aggregation chimeras possessing
cells with homozygous Hdh deletion may stem from the
previously demonstrated role of huntingtin in the extraembryonic
membranes, particularly those derived from trophectoderm, during early
development (Dragatsis et al., 1998). The apparent participation of
huntingtin in vesicular trafficking (DiFiglia et al., 1995; Sharp et
al., 1995; Wood et al., 1996) may be important in the transport of nutrients across the extraembryonic membranes, and disruption of this
function may be the basis of the embryonic lethality of homozygous
huntingtin deletion (Dragatsis et al., 1998). Huntingtin with a
polyglutamine expansion in the range causing HD does not disrupt this
function critically, because humans with a homozygous HD mutation do
not exhibit embryonic lethality (Wexler et al., 1987; Myers et al.,
1989; Gusella and MacDonald, 1996) and because a transgene expressing
huntingtin with such a polyglutamine expansion rescues Hdh
null mice from embryonic lethality (Hodgson et al., 1999; Leavitt et
al., 2000). Additionally, one normal huntingtin allele is sufficient in
humans or mice for normal development and postpartum life (Ambrose et
al., 1994; Duyao et al., 1995; Nasir et al., 1995; Zeitlin et al.,
1995; Persichetti et al., 1996). By contrast, our inability to produce
viable chimeras containing Hdh /
cells by using the method of early embryo aggregation suggests that
even limited colonization of the extraembryonic membranes by Hdh
/ cells may impair their function so
as to be lethal to the developing embryo. Note, however, that we did
not investigate the stage beyond which our aggregation chimeras with
Hdh / cells failed to develop.
Deficiencies in huntingtin also are known to promote defects in
proliferation (White et al., 1997), and it may be that such a defect
placed the 4-8 Hdh / cells from
the Hdh knock-out strain at a disadvantage relative to the
4-8 Hdh +/+ cells from the
wild-type strain in their ability to proliferate and populate chimeras
after aggregation.
The results of our studies on chimeras created by the blastocyst
injection method, in which the lethal effects of Hdh
/ cells in the extraembryonic
membranes are avoided (Dragatsis et al., 1998), demonstrate that
Hdh / cells can participate in
the normal formation of the brain and can develop into neurons with
normal morphology and transmitter content that survive for >1 year
with no evident signs of brain pathology. These results extend on
recent data showing that Hdh /
ES cells transformed into a neuronal phenotype in vitro can
survive for several weeks and show typical neuronal
electrophysiological traits (Metzler et al., 1999). We did, however,
find evidence that Hdh / cells
do not colonize equally and/or thrive in all brain areas. In
particular, we observed that neurons and/or glia derived from the
Hdh / ES cells tended to
colonize preferentially the hypothalamus, midbrain, granule cell layer
of cerebellum, and hindbrain, with only a sparse occupancy of cerebral
cortex, basal ganglia, thalamus, and Purkinje cell layer of cerebellum.
Our studies of lacZ expression in control ROSA26
mice indicate that this paucity of X-gal-labeled cells in some brain
regions in the blastocyst injection chimeras is not a false negative
stemming from poor lacZ expression by ES cells that had
colonized these regions. Thus our findings raise either of two
possibilities. First, it may be that some peculiarity of our ES cells
independent of their Hdh genotype resulted in this
differential colonization. Although there is evidence that ES cells can
show a tendency to colonize CNS differentially in chimeric animals
(Kuan et al., 1997), the pattern has been found to vary randomly from
one chimeric animal to the next even for the same ES cell line. By
contrast, we observed the same consistent pattern of preferential
hypothalamic, midbrain, and hindbrain colonization by ES cells for each
of our three independently derived lines of Hdh
/ ES cells, and the consistency of
preferential colonization was greater than reported to occur for a
single ES cell type (Kuan et al., 1997). Thus the second, and more
likely, interpretation of the consistent paucity of Hdh
/ ES cell progeny in cerebral cortex,
basal ganglia, thalamus, and the Purkinje cell layer of cerebellum is
that huntingtin may be needed for cells to migrate to, proliferate in,
and/or survive extensively within these regions.
Thus individual cells in many brain regions may not need huntingtin to
differentiate into neurons that survive and function normally, whereas
in other brain regions many cells may need to express normal levels of
huntingtin if development is to proceed normally for that region. For
example, brain development may have been seemingly normal even in the
hypothalamus and brainstem of chimeras in which 50% of the resident
neurons were Hdh /, because
huntingtin plays no major role in the development and/or functioning of
these regions. By contrast, huntingtin clearly seems to be critical for
cortical and striatal development, because mouse mutants in which
huntingtin is expressed at one-third of wild-type levels exhibit
defective neurogenesis, profound malformations of cortex and striatum,
ventricular enlargement, and agenesis of fiber tracts (White et al.,
1997). Consistent with a regionally differential role of huntingtin in
neural development, cortex and striatum express higher levels of
huntingtin than do hypothalamus and brainstem (Li et al., 1993;
Landwehrmeyer et al., 1995; Sharp et al., 1995; Bhide et al., 1996;
Sapp et al., 1997; Fusco et al., 1999). The absence of forebrain
developmental abnormalities in our chimeras with Hdh
/ cells may be a consequence of the
relatively low colonization of cortex and striatum by Hdh
/ cells. This low colonization could
stem from impaired proliferation of Hdh
/ cells during development or
from the death of these cells. Note, however, that we did not see
signs that cell loss had occurred recently in cortex, basal ganglia,
thalamus, or Purkinje cell layer in the chimeras. Thus if the
paucity of Hdh / neurons in
telencephalon, thalamus, and Purkinje cell layer reflects their
failure to survive in these regions, this loss had to occur at an early
enough point in development for the Hdh
/ neuroblasts to have been replaced by
wild-type neuroblasts. It is also possible that the reduced brain size
seemingly typical of the chimeras masked evidence of early neuron loss.
The chimeric mice created by the blastocyst injection method showed a
reduced body size proportional to the bodily abundance of Hdh
/ cells implied by coat color, and the
chimeras most enriched in Hdh /
cells additionally tended to show motor abnormalities and morbidity before 1 year of age. The basis of these abnormalities is uncertain, but the findings do suggest that an excess of Hdh
/ in some key neural (or extraneural)
regions compromises their function in some currently unknown way. For
example, the reduced body size characteristic of the blastocyst
injection chimeras could stem from abnormalities in feeding and growth
regulation because of colonization by Hdh
/ cells of neural and extraneural
regions critical to these processes. The mice with the behavioral
abnormalities and premature morbidity tended to be those in which
Hdh / cells in general, as
inferred from coat color, made up 45% of the cells in the body.
Given, however, that colonization of any individual tissue by Hdh
/ cells may have deviated from the
degree of colonization suggested by coat color (Le Dourain and McLaren,
1984; Goldowitz et al., 1992), the bodily tissues critical to the
occurrence of the abnormalities may not have been colonized highly in
all chimeras that were 45% Hdh
/ by coat color, or they may have, in
fact, been colonized highly in chimeras with <45% Hdh
/ by coat color. This may explain the
absence of abnormalities in some chimeras 45% Hdh
/ by coat color and their presence in
one with <45% Hdh / by coat
color. The identity of the neural or extraneural regions for which the
high colonization by Hdh / cells
is critical to the occurrence of the behavioral/motor abnormalities in
the blastocyst injection chimeras is uncertain. In three of the four
mice with the behavioral abnormalities that were examined histologically, >50% of the neurons in hypothalamus and brainstem motoneuron pools were found to be Hdh
/, suggesting their involvement in the
abnormalities in at least some chimeric mice. The precise basis of the
behavioral/motor abnormalities, however, will require focused study of
the relationship between the percentage of colonization of various
bodily tissues and the abnormalities in a larger number of animals than
was possible in the present study.
Finally, the infertility of the male blastocyst injection chimeras may
be behavioral, or it also could be attributable to problems in the
testes. In this light, it is interesting that mice with a conditional
deletion of Hdh under the control of a calmodulin-dependent
kinase II (CaMKII) promoter (CaMKII is expressed late in development
and thereby allows these mice to evade the embryonic lethality of
homozygous Hdh knock-out) are infertile because of a very
low sperm count (Dragatsis et al., 2000). Similarly, mice possessing
transgenic insertions of CAG-expanded forms of the human HD gene
against a nullizygous Hdh background have been shown to
undergo spermatocyte degeneration (Leavitt et al., 2000). By contrast,
Hdh +/ males mated with wild-type
females can pass on the Hdh mutation to their offspring,
implying that sperm (which are haploid) lacking huntingtin can survive
and fertilize eggs (Duyao et al., 1995; Nasir et al., 1995; Zeitlin et
al., 1995). Thus huntingtin may not be needed for the viability of
sperm, but it may play some critical role in the viability of
spermatogonia or primary spermatocytes or in spermatogenesis itself.
Implications for the pathogenesis of HD
A gain of function associated with the HD mutation is the
aggregation of the N-terminal fragment of mutated huntingtin within neuronal nuclei and cytoplasm (DiFiglia et al., 1997; Li and Li, 1998;
Martindale et al., 1998; Gutekunst et al., 1999; Maat-Schieman et al.,
1999). Although considerable attention has focused on the possibility
that these aggregates are a key pathogenic event in HD (Davies et al.,
1997; DiFiglia et al., 1997; Kim and Tanzi, 1998; Saudou et al., 1998;
Sisodia, 1998), the means by which they might lead to neuronal death
remains uncertain (Cha et al., 1998; Hackham et al., 1998; Sisodia,
1998). The possibility that the aggregates may act, at least in part,
by inactivating both mutant and normal huntingtin has been raised by
recent evidence that the aggregates that form in HD and in transgenic
animal models of HD can sequester normal-length
polyglutamine-containing proteins, including huntingtin (Cha et al.,
1999; Narain et al., 1999; Preisinger et al., 1999; Wheeler et al.,
1999; Cattaneo et al., 2001). This has led to the recent suggestion
that the HD mechanism of action might receive a contribution from a
late-onset inactivation of normal huntingtin (Cattaneo et al., 2001).
Such a possibility is consistent with the finding that huntingtin
appears to exert an anti-apoptotic effect in cultured striatal neurons
subjected to serum deprivation or metabolic stress (Rigamonti et al.,
2000
TOP
ABSTRACT
INTRODUCTION
