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Laboratory of Molecular Neurobiology, Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7
1Correspondence: Department of Pharmacology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. E-mail: har1{at}is.dal.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: glia • lactation • Sprague-Dawley • epidermal growth factor receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The mutation that
causes HD is an expansion of a trinucleotide (CAG) repeat in exon 1 of
the IT15 gene encoding the protein, huntingtin (2,
3)
. The function of
neither the normal or mutated form of huntingtin is known. Although
alterations in the expression of this gene have been reported to be
induced by excitotoxins (4,
5)
, its regulation during naturally
occurring physiological changes has not been demonstrated.
The arcuate nucleus of the hypothalamus, in response to gonadal
hormones, undergoes marked physiological alterations during the late
stages of pregnancy, parturition, and postparturition (6
7
8
9)
. For
example, the activity of the tuberoinfundibular dopamine neurons of the
arcuate nucleus is suppressed in lactating animals, an effect that is
enhanced and maintained by the suckling stimulus (10,
11)
. The
mechanisms by which these changes occur remain unknown. In this report,
we demonstrate that huntingtin protein and mRNA are increased in the
arcuate nucleus of the hypothalamus in postpartum, lactating animals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemistry
Brains were blocked and cut into 50 µM thick coronal sections
on a vibratome. Sections were collected through the rostrocaudal axis
of the hypothalamus and processed for huntingtin immunohistochemistry
and in situ hybridization analysis. For
immunohistochemistry, free-floating sections were washed for 10 min in
0.1 M phosphate-buffered saline containing 0.1% Triton-X (PBS-TX).
This was followed by a 15 min incubation in 1% hydrogen peroxide (to
inactivate endogenous peroxidase activity) and subsequent 3 x 10
min washes in PBS-TX at room temperature. The sections were then
incubated in a 1:500 dilution of a monoclonal antibody to the
huntingtin protein (Chemicon, El Segundo, Calif.; MAB2166) for 16–24 h
at 4°C. After incubation with the primary antibody, the sections were
washed 3 x 10 min in PBS-TX and incubated in a 1:500 dilution of
horse anti-mouse secondary antibody (Vector Laboratories, Burlingame,
Calif.) for 1–2 h at room temperature. Excess antibody was removed by
washing 3 x 10 min in PBS-TX and the bound secondary was
visualized using the avidin-biotin technique (ABC Elite; Vector
Laboratories) by using diaminobenzidine (DAB; Sigma, St. Louis, Mo.) as
the chromogen. The sections were mounted on gelatin-coated slides,
air-dried, dehydrated in a graded alcohol series, delipidated in
xylene, and coverslipped using Entellan adhesive (Merck, Rahway, N.J.).
Sections were also studied by using dual label immunohistochemistry for huntingtin and glial fibrillary acidic protein (GFAP). The primary incubation media contained both the monoclonal antibody to huntingtin (1:500) and a rabbit polyclonal antibody to GFAP (1:2000; DAKO, Carpenteria, Calif.) in PBS-TX. After incubation (16–24 h) with the primary antibodies, the sections were washed 3 x 10 min in PBS-TX and subsequently incubated in CY2-conjugated donkey anti-mouse and CY3-conjugated donkey anti-rabbit (1:400; BioCan) for 16–24 h at 4°C. The sections were then washed 3 x 10 min in PBS-TX, mounted on gelatin-coated slides, and coverslipped by using Citifluor (Marivac). Fluorochromes were visualized using filter sets to detect CY2 (Zeiss, catalog no.487710) and CY3 (Zeiss, catalog no.487715) immunofluorescence.
Analysis of huntingtin mRNA
Northern blot analysis
The hypothalamus was removed from adult rats (postpartum,
n=3; naive female, n=3; male, n=3) and
stored in liquid nitrogen until further processing. Total cellular RNA
was isolated from individual samples using TRIzol reagent (Gibco-BRL,
Grand Island, N.Y.) according to the manufacturer's protocol. A
Northern blot was prepared by fractionating 10 µg aliquots of RNA on
a 1% denaturing agarose gel, followed by transfer of the RNA to
Zetaprobe (Bio-Rad, Hercules, Calif.) membrane using standard
methodology (12)
.
Northern hybridization probe
A 45-base oligonucleotide probe
(5'-CTT-GTT-CTA-CAA-TCC-CTC-TGA-TCA-TGC-TCA-ACT-TTC-TTC-CAA-ATC-3') was
designed that was complementary to bases 7570–7614 of the huntingtin
mRNA (GenBank accession no. U18650). The oligonucleotide (5 pmol) was
radiolabeled with [32P]
-dATP using terminal
transferase (3'-end labeling kit; Amersham). Unincorporated
radionucleotides were removed from the labeled probe by centrifugation
through a Microspin-G25 column (Pharmacia Biotech, Piscataway, N.J.).
Northern hybridization conditions
The Northern blot was prehybridized in buffer containing 5X
standard saline citrate (SSC), 5X Denhardt's solution, 50 mM sodium
phosphate, 1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 10 µg/ml
tRNA, 50 µg/ml denatured salmon sperm DNA, 50 µg/ml denatured yeast
RNA, and 50% formamide at 42°C for 6 h. After prehybridization,
the blot was transferred to a hybridization buffer [5X SSC, 1X
Denhardt's solution, 20 mM sodium phosphate, 2% sodium dodecyl
sulfate (SDS), 5 mM EDTA, 10 µg/ml tRNA, 100 µg/ml denatured salmon
sperm DNA, 100 µg/ml denatured yeast RNA, 50% formamide and 100
mg/ml dextran sulfate] containing 2 x 106
counts/ml of radiolabeled oligonucleotide. The blot was hybridized for
a minimum of 18 h at 42°C, after which it was washed for 15 min
in 2X SSC/0.1% SDS (42°C), 2X SSC/0.1% SDS (50°C), then twice in
1X SSC/0.1% SDS (50°C). The blot was subsequently washed in 0.5X
SSC/0.1% SDS at 50°C and exposed to Biomax MR autoradiographic film
(Kodak, Rochester, N.Y.) for 4 days at -70°C.
In situ hybridization and emulsion autoradiography
In situ hybridization was performed on fixed and
fresh-frozen brain tissue. Fixed tissue was sectioned coronally (50
µM), mounted on Superfrost Plus slides and air-dried overnight. The
fixed sections were dehydrated and cleared in a graded alcohol series,
followed by delipidation in xylene. After rehydration, sections were
washed 3 x 5 min in 1X PBS, followed by 3 x 5 min in 2X SSC
and air-dried. Frozen tissue was sectioned coronally (14 µM) on a
cryostat, mounted on Superfrost Plus slides and stored at -70°C. The
frozen sections were allowed to reach room temperature, fixed for 5 min
in 4% paraformaldehyde and rinsed 2 x 3 min in 1X PBS, followed
by 1 x 20 min in 2X SSC and air dried.
In situ hybridization probe
The 45-base huntingtin oligonucleotide probe used in the
Northern blot analysis was also used for in situ
hybridization. The oligonucleotide (5 pmol) was radiolabeled at the
3'-end with [33P]-dATP, as described above.
In situ hybridization conditions
The sections were covered with hybridization buffer [5X SSC, 1X
Denhardt's solution, 20 mM sodium phosphate, 2% sodium dodecyl
sulfate (SDS), 5 mM EDTA, 10 µg/ml tRNA, 100 µg/ml denatured salmon
sperm DNA, 100 µg/ml denatured yeast RNA, 50% formamide, and 100
mg/ml dextran sulfate] containing 5 x 106
counts/ml of radiolabeled huntingtin probe and incubated at 38°C in a
humidified chamber for a minimum of 18 h. After hybridization, the
slides were washed for 4 x 15 min each in 1X, 0.5X, 0.25X SSC at
50°C, rinsed in dd H2O, and air-dried
overnight. The slides were exposed to Biomax MR (Kodak)
autoradiographic film for 7 days. The slides were subsequently immersed
in autoradiographic emulsion (Kodak NTB2), allowed to dry, and exposed
for 4 wk at 4°C prior to developing with Kodak D19 developer and
fixer.
After the processing of slides for autoradiographic emulsion, the sections were counterstained for Nissl substance. Briefly, the sections were dehydrated and rehydrated in a graded alcohol series, then washed in cold water. Slides were immersed in 1% (w/v in H2O) Cresyl Violet (ICN Biochemicals, Irvine, Calif.) for ~1 min. Excess stain was removed by washing in dilute acetic acid and the slides were dehydrated, delipidated in xylene, and coverslipped.
Statistical analysis
Densitometric analysis of in situ autoradiographs was
performed by using Molecular Analyst software (Bio-Rad) to determine
the optical density (OD) of the radiolabel in hypothalamic regions.
Autoradiographic films were scanned into the computer using a Bio-Rad
GS-690 Imaging Densitometer. The sections were numbered in a random
fashion, with the observer blind to the animal group from which they
were obtained. There were four animals per group included in this
analysis. Optical density units were calculated by blocking individual
arcuate regions for each animal and calculating these values for three
to four sections of the hypothalamus per animal to obtain a total of
6–8 OD values per animal. To account for nonspecific radioactivity in
the sections, the OD value for the ventromedial nucleus of the thalamus
was taken as the background signal and subtracted from all values
obtained from the arcuate nucleus in each section. The mean value of
this difference was calculated for each animal and used as a
representation of the intensity of hybridization signal in the arcuate
nucleus.
Once all densitometric values had been calculated, the number code for the animals was revealed and the mean OD values were grouped as postpartum female, naive female, or male. These values were then subject to a one-way analysis of variance, followed by a post hoc Newman-Keuls test. Significance was assumed at P<0.05. Data was converted to relative OD values by expressing the raw data as a percentage of the highest OD value obtained in the postpartum group.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Previously, it has been shown, by using
immunohistochemistry and Western blot analysis that MAB 2166
immunolabels neuronal perikarya, neuropiles, varicosities, and nerve
endings in human brain tissue. Western blot analysis demonstrated that
this antibody also recognizes rat huntingtin in neural and nonneural
tissue (3,
13)
. As expected, huntingtin-immunoreactivity
(huntingtin-IR) was observed in neurons of several rat brain regions,
including cortex, thalamus, hippocampus, and hypothalamus. In
postpartum animals, the arcuate nucleus exhibited typical levels of
huntingtin-IR in neurons, but also had robust expression in a
population of cells that did not appear to be neurons (Fig. 1
A, B). Naive female (Fig. 1C, D
) and male (Fig. 1E, F
) animals had similar low levels of neuronal expression
of huntingtin-IR in the arcuate nucleus compared with postpartum
females, but markedly less immunostaining of the nonneuronal cells.
Omission of the primary antibody eliminated immunostaining in all
animal groups (Fig. 1G, H
), excluding the possibility that
the DAB reaction product observed in the arcuate nucleus, was a result
of endogenous peroxidase activity. Peroxidase activity is affected by
changes in estradiol concentrations in various estrogen target tissues
(14
15
16)
.
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Specificity of the huntingtin oligonucleotide
To confirm that the 45-base oligonucleotide that was designed
against the huntingtin mRNA would hybridize exclusively to huntingtin
transcripts, we examined its specificity in Northern blot analysis
against electrophoretically fractionated total RNA isolated from rat
hypothalamus. In each of three individual RNA samples per group that
were subjected to Northern blot analysis, the radiolabeled
oligonucleotide annealed exclusively with discrete bands of ~11 kb
and 13 kb (Fig. 2
A and data not shown). The size of these hybridizing bands
correspond to those previously reported for the huntingtin transcripts
(17
18
19)
. These results confirmed that the oligonucleotide probe used
for subsequent in situ hybridization analyses was specific
for transcripts whose sizes correspond to those of the huntingtin
mRNA.
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Localization of huntingtin mRNA in the hypothalamus
Consistent with the protein expression, examination of
autoradiographs from all animal groups revealed pronounced
huntingtin-radiolabeling in the piriform cortex, hippocampus and the
hypothalamus (Fig. 2B
). Postpartum, lactating animals also
showed intense radiolabeling in the arcuate region of the hypothalamus
while expression in naive female and male animals was low. In
postpartum animals, in agreement with the observed huntingtin-IR,
hybridization signal was present throughout the rostrocaudal axis of
the arcuate nucleus (data not shown). Densitometric analysis of the
huntingtin hybridization signal in the arcuate region revealed an
increase of approximately sevenfold (P<0.05) in the
postpartum animals compared with naive female and male animals. No
significant difference (P=0.902) in the huntingtin-specific
hybridization signal was observed between the arcuate nucleus of naive
female and male rats (Fig. 2C
).
Examination of the arcuate nucleus after emulsion autoradiography with
the huntingtin probe revealed a clustered distribution of silver
grains. This pattern was observed in postpartum (Fig. 3
) and, to a lesser extent, in naive female and male animals (data not
shown). Nissl staining revealed that silver grains associated with the
huntingtin-specific hybridization signal were consistently found in
close apposition to neuronal soma. However, the silver grain clusters
were not directly overlying, but appeared juxtaposed to, these neuronal
soma (Fig. 4
A). Comparison of mRNA and protein distributions in the
arcuate nucleus revealed similar patterns of localization, relative to
neuronal perikarya of this region (Fig. 4A, B
).
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Colocalization of huntingtin and GFAP
Because of the stellate appearance of the cells expressing
huntingtin-IR and the huntingtin mRNA in postpartum animals, we
performed immunofluorescent dual label immunohistochemistry for
huntingtin and the astrocytic protein, GFAP. The expression of GFAP was
ubiquitous and immunofluorescence was intense in several regions of the
brain. To ensure that the immunofluorescence of the huntingtin signal
(CY2) was only observed under the appropriate filter and not with the
filter used to view the GFAP signal (CY3), we examined various regions
of the brain. For example, when the dentate gyrus of the hippocampus
was examined for huntingtin (Fig. 5
A) and GFAP (Fig. 5B
) expression, a composite
image of the two signals revealed that the individual fluorochromes did
not fluoresce when the tissue was viewed with a filter that was
specific for the emission wavelength of the other fluorochrome (Fig. 5C
).
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Huntingtin immunofluorescence was observed in the arcuate nucleus of
postpartum animals (Fig. 5D
). These cells were also
immunopositive for GFAP expression (Fig. 5E, F
). It appeared
that cells in the periphery of the arcuate nucleus exhibited robust
huntingtin expression, but had less GFAP immunoreactivity than cells in
the center of this nucleus (Fig. 5H, I
). Moreover,
examination of the pattern of silver grains after in situ
hybridization using the huntingtin mRNA-specific oligonucleotide and
emulsion autoradiography revealed that the cells of the arcuate nucleus
of postpartum females expressed huntingtin mRNA in the cell soma (Fig. 4A
). Overall, the cells appeared to have a stellate
morphology. Immunocytochemical analysis using fluorescent labeled
secondary antibodies showed a similar pattern of huntingtin protein
(Fig. 5D, F, G, I
). The stellate morphology of the cells,
however, was obscured using the avidin-biotin-DAB visualization method
(Fig. 4B
), most likely due to the multiplicity of binding of
avidin and biotin over the bound primary antibody. The emulsion
autoradiography together with dual label immunohistochemistry
demonstrated that cells positive for huntingtin had the morphology of
astrocytes and that many of these stellate cells also expressed the
astrocyte-specific marker, GFAP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In summary, astrocytes in the arcuate nucleus of postpartum lactating female rats had increased levels of the protein huntingtin and approximately sevenfold higher levels of the huntingtin mRNA than naive female or male rats. This regulation of huntingtin expression in the arcuate nucleus represents the only known example of regulation of huntingtin levels, an effect that was apparently produced at the level of transcription.
The arcuate nucleus is involved in the regulation of the release of
numerous hormones, including some that are involved in reproductive
processes (gonatotrophin-releasing hormones), parturition (oxytocin),
and care of the young (prolactin and oxytocin). There are also hormones
that could be involved in the changes in metabolism involved in
pregnancy. Much recent evidence indicates that glia may play a
significant role in the link between the endocrine and nervous systems.
Gonadal steroids appear to regulate astroglial morphology,
differentiation, and gene expression in different brain areas (20)
. The
actions of hormones on glia may have important consequences for
neuronal development, metabolism, and activity, for the formation of
synaptic connections, and for regulation of the release of hypothalamic
hormone releasing factors. For example, in mixed neuronal-glial
cultures from hypothalamus, both astrocytic shape and the distribution
of GFAP are modified by estradiol treatment (21
22
23
24
25
26
27)
. Estradiol
treatment produces a progressive differentiation of GFAP-immunoreactive
cells from a flattened epithelioid shape to bipolar, radial, and
stellate shapes. Although we cannot say that the cells expressing
huntingtin are the same cells that undergo differentiation in response
to estradiol, clearly they are similar in morphology and coexpress
GFAP. A similar situation exists in transforming growth factor
(TGF-) -induced secretion of luteinizing hormone-releasing hormone
(LHRH) from hypothalamus. This involves stimulation of local astrocytes
that secrete soluble factor(s), including prostaglandin E2, which
induces neuronal LHRH release (24,
27)
. Likewise, the
testosterone-dependent expression of growth hormone-releasing hormone
(GHRH) and the number of GHRH neurons in the arcuate nucleus appear to
involve effects on glia (20)
. In general, there is solid evidence that
glial cells play an essential role in the regulatory functions of the
arcuate nucleus.
In light of our observation of the proximity of the
huntingtin-expressing cells to neurons, it is interesting note that the
effects of estradiol on shape and GFAP expression observed in
vitro were not observed in pure hypothalamic glial cultures, but
required the presence of neurons (25)
. In fact, only in the presence of
hypothalamic neurons, but not cerebellar neurons or fibroblasts, would
hypothalamic glial cultures respond to estradiol with changes in shape
and GFAP expression. These results indicate that estradiol induction of
shape changes in hypothalamic astrocytes is dependent on the presence
of hypothalamic neurons and suggests that physical contact between
astrocytes and neurons is necessary for the manifestation of the effect
of this hormone. Others have reported that glia-to-neuron apposition in
the hypothalamus is influenced by gonadal steroids, suggesting that the
astrocytic regulation of neuronal function may require close physical
proximity (28,
29,
30)
. Thus, it is clear that changes in circulating
hormones such as estrogen and estradiol can have profound effects on
expression of trophic factors in the arcuate nucleus and that these
effects are mediated by astrocytes. Astrocytes in the hypothalamus
express TGF- and the epidermal growth factor receptor (EGFR) (31)
.
Estradiol has been shown to increase the expression of TGF- in
hypothalamic astrocytes, but not in astrocytes of the cerebellum,
indicating that hormonal responsiveness in these cells is greater in
endocrine regions of the brain (26)
. Estradiol-induced TGF- is
blocked by inhibition of EGFR. As estrogen receptors are expressed in
as few as 10% of hypothalamic glial cells, it is thought that the
stimulation of TGF- expression and release produces a
paracrine/autocrine enhancement of this factor in adjacent cells that
do not express estrogen response sequence (26,
32)
. Recently, Liu et
al. (33)
demonstrated that huntingtin may be an adaptor protein for EGF
receptor-mediated signaling and may be involved in the regulation of
Ras-dependent signaling pathways. Thus, although the transduction
mechanism in arcuate nucleus remains unclear as does the relationship
of hormonal activation to huntingtin expression, possible pathways do
exist for regulation of huntingtin expression in arcuate nucleus.
The present results suggest that metabolic alterations that occur in
the arcuate nucleus after parturition involve changes in astrocyte
function and that these changes in turn involve the increased
expression of huntingtin. The present study implicates the
Huntington's disease gene and its product huntingtin in the changes
that occur during a time of major restructuring in the hypothalamus.
Huntingtin is expressed in cells we know are undergoing major
hormone-induced alterations in the cellular mechanisms that mediate
transcriptional and, perhaps most significant, morphological change.
These results appear to be the only instance of regulation of the
transcription of this gene by physiological mechanisms. Understanding
the normal function of this essential gene (34,
35)
is importanct and
will help in determining the function associated with the lengthened
polyglutamine segment in the huntingtin produced from the Huntington's
disease allele.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication September 18, 1998.
Revision received January 25, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus. Proc. Natl. Acad. Sci. U. S. A. 87,9698-9702 (TGF) gene expression in astrocytes of the neuroendocrine brain. J. Neurosci. 14,5644-5651[Abstract]
with the secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone. Endocrinology 138,19-25 (TGF-) and epidermal growth factor-receptor (EGF-R) immunoreactivity in normal and pathologic brain. Prog. Neurobiol. 49,99-123[Medline]
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