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Huntington's disease gene Regional and cellular expression in brain of normal and affected individuals.

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Huntington's Disease Gene: Regonal and
Cellular Expression in Brain of Normal
and Affected Individuals
G. Bernhard Landwehrmeyer, MD," Sandra M. McNeil, P h D , t Leon S. Dure IV, MD," Pei G e , EdMJ
Hitoshi Aizawa, MD,S Qin Huang, MD, PhD," Christine M. Ambrosr:, P h D , i Mabel P. Duyao, PhD,?
Edward D. Bird, MD,"B Ernesto Bonilla, M D , PhD,I/? Margot de Young, MD,"
Alejandro J. Avila-Gonzales, MD,/I Nancy S. Wexler, PhD,"' Marian DiFiglia, PhD,*
James F. Gusella, PhD,,'"' Marcy E. MacDonald, PhD,f John B. Penney, MD,"
Anne B. Young, MD, PhD," and Jean-Paul Vonsattel, MDS
Huntington's disease (HD) is an autosomal dominant disorder characterized by involuntary movements, dementia, and
progressive, global, but regionally accentuated, brain atrophy. The disease affects the striatum most severely. An
expansion of a trinucleotide repeat on chromosome 4p16.3 within the coding region of a gene termed IT15 has been
identified as the mutation causing HD. The normal function of IT15 and the mechanisms by which the presence of
the mutation causes H D are unknown. Although IT15 expression has been detected in the brain, as well as in other
organ tissues, by Northern blot and in situ hybridization, it is not known whether a preferential regional or cellular
expression of IT1 5 exists within the central nervous system of normal, affected, and presymptomatic individuals.
Using quantitative in situ hybridization methods, we examined extensively the regional and cellular expression of
1T15. In controls, IT15 expression was observed in all brain regions examined with the highest levels seen in cerebellum, hippocampus, cerebral cortex, substantia nigra pars compacta, and pontine nuclei. Expression in the striatum
was intermediate and expression in the globus pallidus was low. IT15 was expressed predominantly in neurons; a low
but significant level of expression was seen in glial cells. Analysis of grain counts per square micrometer in neurons
showed that the regional differences in the level of mRNA expression were related to density and size of neurons in
a given region and not primarily to differences in levels of mRNA expression in individual cells after correction for
cell size. Neurons susceptible to degeneration in H D did not selectively express high levels of IT15 niRNA. In H D
brains (grades 2-4), the distribution and levels of IT15 mRNA were comparable with controls in all areas except in
neostriatum where the intensity of labeling was significantly reduced. Presymptomatic H D brains had a striatal
expression similar to controls and surviving striatal neurons in more advanced HD had an expression of IT1 iwithin
normal limits. It is apparent from these results that the presence of expanded trinucleotide repeats in H D does not
result in the absence of IT15 mRNA expression or in altered patterns or levels of expression. T h e lack of correlation
between the levels of 1T15 mRNA expression and susceptibility to degeneration in H D strongly suggests that the
mutant gene acts in concert with other factors to cause the distinctive pattern of neurodegeneration in HD.
Landwehrmeyer GB, McNeil SM, Dure LS IV, Ge P, Aizawa ti, Huang Q, Ambrose CM, Duyao MP.
Bird ED, Bonilla E, de Young M, Avila-Gonzales AJ, Wexler NS, DiFiglia M, Gusella JF,
MacDonald ME, Penney JB, Young AB, Vonsattel J-P. Huntington's disease gene: regional and cellular
expression in brain of normal and affected individuals. Ann Neurol 1095;_17:218-230
Huntington's disease (HD) is an autosomal dominant
neurodegenerative disorder characterized by the onset in midlife of personality changes and involuntary
movements 111. T h e gene mutant in HD has been
localized to chromosome 4 by genetic linkage 121 and
has been isolated by a positional cloning approach [3].
An expansion of an unstable, polymorphic (CAG), repeat in the 5' end of the coding region appears to cause
the disease; the number of repeats correlate inversely
with age of onset (3-61. T h e mechanisms by which
From the Departments o f 'Neurology and $Pathology, Massachusects General Hospital, anll '*Dcplutinenr of Genetics, Harvard
Received May 6, 1994. and i n revised form Aug 9. Accepted for
publicatioii Aug 18. 1091.
varil Medical School: Belmont, MA; IIUniversidad de Zulia, Maracaiho, a d PVenezuela Cooperative Research Project. Venezuela;
and #Departments ot Neurology and Psychiatry, College of Physicians and Surgeons. Columbia IJniversiry, New York. and Heredit,xy Disease Foundation. Santa Monica. CA.
218 Copyright 0 I005 hy the American Neurological Association
the expansion of CAG repeats in the HD gene cause
the HD phenotype are unknown. The expression
of the HD gene c ~ ~ 1 A~ 1, 5(for "interesting transcript 15"), in brain and other organs has been
ined with northern blot and in situ hybridization techniques 17- 101. Although relatively few brain regions
have been
thus far' these studies have sugSince the
gested that IT15 is
localization Of IT15 in
and HD brain has not
been examined, it is unclear to what extent the transcript is selectively expressed in neuronal and nonneuronal cell populations in different brain regions, particularly the striatum, where neuronal loss is most severe.
Moreover, there is no information on IT15 expression
in presymptomatic HD cases, which is required for a
better understanding of the pathogenesis of H D , since
selective neuronal vulnerability could be due to aberrant gene expression or overexpression early in the
illness. Therefore, we undertook a detailed and quantitative analysis of the regional and cellular expression
of IT15 in brains of normal subjects and HD patients
at various stages of the disease process [ll].
Materials and Methods
Postmortem Tissue
The brains used in this study were selected based on the
results of an initial screening for adequate preservation of
mRNA by in situ hybridization using probes for p-actin. Two
sets of brains were examined (Table 1). In the first set, which
included 4 control and 4 HD cases, 10 regions, including t h e
striatum, were examined. Control and HD brains did not
differ significantly in age ( p = 0.19 by analysis of variance
[ANOVA} and Scheffe test) or postmortem interval (PMI)
( p = 0.69; by ANOVA and Scheffe test). In the second set
of brains, the caudate/putamen in 14 control and 23 HD
subjects was studied. All HD brains were graded as previously described [ 111. Clinical details about the two presymptomatic cases studied have been published elsewhere [ 12,
HD. 0
HD, 1
HD. 2
HD. 3
HD. 4
Set A
Age ( y r )
PMI (hr)
Set B
PMI thr)
41 -c 5
18 ? 2
Frontal cortex (Brodmdnn areas 10. 4 ) , hippocampal formation, amygdala, clausrrum, caudare nucleus, nucleus accumb a s , putamen, lateral and medial globus pallidus, nucleus
basalis of Meynert. thalamus, mesencephalon, metencephalon. and cerehellum
49 i 9 5 1 c 1 49 2 5
Caudate nucleus, nucleus accumbens. putamen, lateral and medial globus pallidus
I n Situ Hybridization
Twenty-micrometer-thick frozen sections were cut on a cryostat (Lipshaw) and mounted on poly-L-lysine-coated slides,
fixed in 45X paraformaldehydei0.1 M phosphate-buffered saline (PBS), rinsed in PBS (3 x ), acetylated (0.25% acetic
anhydride in triethanolamine buffer, p H KO), dehydrated in
a graded series of ethanol, delipidated in chloroform, partially
rehydrated in 100% and 95%' ethanol, and air dried. Sections
were hybridized in a humid chamber for 5 hours at 50°C in
a buffer containing 50% formamide, 0.3 M NaCI, 20 mM
Tris-HCI, 5 mM EDTA, 0.5 mg/ml yeast tRNA, 1 x Denhardt's solution, 107; dextran and 100 mM dithiothreitol,
and 150,000 cpni of ?S-labeled probe per microliter of hybridization buffer. After hybridization, sections were rinsed
in four changes of 2 x standard saline citrate (SSC), treated
with RNase A (100 pgiml) at 37°C. rinsed, washed in three
changes of 0.1 x SSC for 3 hours at 70"C, and partially dehydrated in 7 0 g , and 957; ethanol. Slides were apposed to
p-max film (Amersham) for 6 to 10 days or dipped in nuclear
emulsion (NTB2, Kodak, 1:1, with distilled water), exposed
for I to 2 months, developed with D19 (Kodak), fixed, and
stained with hematoxyhdeosin (HE).
Data Atzalysis
Tubkr I . Characteristics of SubjrctslBrains Studied
Construction of KNA Probes
The RNA probes used for in situ hybridization histochemistry ( I S H H ) were constructed from cDNA subclones making
up the composite sequences of IT15 [ 3 ] . Probe 1 was an fragment from the ITl5B cDNA clone subcloned
into pBluescript SK I1 (Stratagene) spanning bases 3,019 to
4,159 of the published sequence [ 3 ] . Antisense and sense
probes were derived from opposite strands of this plasmid
C-lnStruCt by runoff transcription using RNA polymerase T 3
or T7. resDectivelv. after EroRI or BnmHI digestions. Probe
2 was a HindIlI-EmRI fragment spanning bases 7,571 to
8,913 from the ITl5A 2.8-kb EcoRI cDNA clone. The antisense and sense probes were transcribed using T3 and T7
RNA polymerase, following HirrdIII or EcuRI digestions.
Ii5S]CTP was incorporated using a Promega RNA labeling
kit. The size of the transcripts was reduced to an average
length of 300 bases by limited alkaline hydrolysis 141.
',Mean ? sem.
"PMl only known for 1 case.
PMI = postmortem interval; HD, 0; HD, 1 etc = Huntington's disease,
grade of neuropathological severity 0. 1, etc.
Film autoradiograms of the labeled sections were analyzed
using an image-processing system (MCID, Imaging Research,
St Catharine's, Ontario, Canada). Optical densities were assessed in each section and readings from the identical region
of RNase-pretreated sections were subtracted from the density of the antisense section. On emulsion-dipped slides, silver grains were visualized under bright-field illumination
using a 100 x water immersion lens (Leitz). Quantitative
assessment of IT1 5 expression was accomplished by rneasurement of grain densities over HE-stained cell bodies. A
computer-assisted quantitative image analysis system (Imaging Research) was used to define the borders of each neuron, measure the cross-sectional area of the neuronal profile
in square micrometers, and count the number of silver grains
overlying the cell. The total number of grains and the area
of profile of each neuron were used to calculate the number
o f grains per 1,000 p n 2 of neuronal area. Grains over a
total of 4,573 cellular profiles in cerebral cortex, hippocampal
Landwehrmeyer et al: Huntington's Disease Gene
formation, claustrum, striatum, globus pallidus, ventrolateral
thalamus, subthalamic nucleus, substantia nigra, and basal nucleus of Meynert were sampled. Neurons were sampled by
a continuous drive through the respective structures and
identified by morphological criteria (nucleolus, cell size).
Statistical comparisons using a two-way ANOVA combined with tests for multiple post hoc comparisons (Scheffe
test; BonferronilDunnett) were performed using StatView I1
software (Abacus Concepts, Inc, Berkeley, CA).
Specificity of Hybridization Signal
For all regions examined, a significant difference in hybridization signals was observed between the signal obtained with antisense probes and that obtained with
sense probes and following pretreatment with RNase
A (ANOVA: p < 0.0001 for all regions; Scheffk and
Fig 1 . IT15 mRNA expression in frontal cortex of normal hunun bruin visualized by in situ hybridization histochemistry using probe 1 (At and probe 2 (C). B and D show hybridization
signals obtuined by hybridizution with the sense styand of probe
1 a n d 2 , respectively. Bar = 5 mm.
220 Annals of Neurology
Vol 37
No 2
February 1995
BonferroniiDunnett). Hybridization signal under control conditions (sense probes and RNase A pretreatment) was close to background, that is, similar to the
signal obtained on film or slide around the tissue section (Fig 1). Hybridization with two distinct sets of
riboprobes gave identical results (see Fig 1A and C).
Expression of IT15 mRNA in N o m a f Brains
REGIONAL DISTRIBUTION. Hybridization signals were
detected throughout the brain and appeared highest in
the cerebellum and the dentate gyrus (Fig 2). In the
cerebral cortex, hybridization signal ranged from intermediate to high and was in register with the laminar
organization of the cortical areas studied. In visual cortex (Brodmann area 17; Fig 3A), for example, hybridization signal was intense in layers I1 to 111, IVc, and
VI, and lower in layers 1, IVb, and V. In temporal
(Brodmann area 36; see Fig 3A) and prefrontal cortices
(Brodmann area lo), hybridization signal was prominent in granular and multiform cell layers (layers 11,
IV, and VI) (see Figs 1 and 3A). Hybridization signals
0 25
0 05
Fig 2. Histogram of IT15 expression iti control ( n = 4, and
Huntington? disease ( H D i ( n = 41 brains deriwdfrom film
autoradiograms of in situ hybridization histochemistty. Absolute
optics( densitie.s minus nonspecifc signdl in the zmarious regions
were plotted for control (filled bani and H D (open baui. The
densities of expre.uion are tiot signifcantb different between
H D and controls except in the putamen umhrre IT1 5 expression
i.c decreased in H D cases and in the lateral globus pallidus
i d w e it is increased f*p < 0.05 by Mann-Whitneji U test).
ACC = niideuJ accumbens: A M G = amnygdafa:CA3 = stvatun1 pyramidale of CA3 region of hippocampus; C A 2 = Jtratum pyramidale of CA2; CAI = stratum pyramidale of C A l :
C A U = caudate nuileus; CBL = granule 1-ell laser of cerebellum: C L = claustvum; D G = dentate gyrus hippoiampi;
FCX = front01 cortex (Brodniann averl 10); LGP = lateral
globus pallidus: MGP = medial globus pallidids: NBM = nucleus basalis vf Meynert; PONS = pontine nuclei: PUT = putamen; SNC = substantia nigra pars iomparta: T H = thalamus.
in the hippocampal formation (Fig 3C) were very high
in the granular cell layer of the dentate gyrus and high
to intermediate in the pyramidal cell layer of the cornu
ammonis. In amygdala, hybridization signal was intermediate in the granular portion of the basal nucleus
and in the lateral nucleus and low in the central and
accessory basal nucleus. In striatum (caudate nucleus
and putamen; Fig 3E and F), the hybridization signal
was of intermediate density and lower than in cortex,
claustrum, hippocampal formation, subtantia nigra pars
compacta, pontine nuclei, and cerebellum. There was
no obvious gradient between posterior and anterior or
dorsomedial and ventrolateral striatum (see Fig 3E and
F; also see Fig 6). The hybridization signal was inhomogeneous, in particular in rostral areas of the striatum,
giving a mottled appearance. Striosomes as defined by
acetylcholinesterase histochemistry in serial sections
exhibited similar or slightly lower hybridization signals
than the matrix compartment (data not shown). Hybridization signal in globus pallidus and substantia nigra
pars reticulata was low, although small dots of high
signal intensities were visible within globus pallidus
(see Fig 3F; also see Fig 6C). In thalamus (Fig 3G),
punctuate signals were apparent in all subnuclei and
especially prominent in the lateral nuclei. A similar
pattern was observed in the subthalamic nucleus (see
Fig 3G). Intense signals were observed in substantia
nigra pars compacta (Fig 3H). Very intense signals
were present in the granular cell layer of the cerebellum (see Fig 3D) and moderate to low signals in the
cerebellar nuclei (see Fig 3D), red nucleus, and inferior
olive; the pontine nuclei were labeled with high intensity (see Fig 2). In spinal cord gray matter, structures
in anterior, intermediate, and posterior horn were labeled at low-intensity levels. Labeling in white matter
tracts (see Fig 3) was very low but significantly different
from signals following hybridization with sense probes
or RNase A pretreatment (ANOVA: p < 0.0001;
Scheffk and BonferrodDunnett).
CELLULAR EXPRESSION. Microscopic examination of
emulsion-dipped slides demonstrated high numbers of
silver grains associated with neuronal profiles (Fig 4).
Neuronal populations appeared labeled to varying degrees; large neurons in all brain areas tended to be
more densely labeled than medium sized and small
neurons. In all regions examined we also encountered
a small number of lightly labeled neurons. The mean
values and SEM based on counts of more than 2,500
neurons in control brain are summarized in Table 2.
There was a moderate but statistically significant correlation between the cross-sectional area and the number of silver grains overlying neurons (counts/cell: correlation coefficient, 0.58; 95% confidence interval,
0.56-0.60). Striatal neurons did not stand out as a
particularly intensely labeled population of neurons.
When corrected for cell size (dimension: grains per
1,000 pm') the hybridization signals across different neuronal populations were remarkably similar;
only pigmented neurons in substantia nigra pars compacts and macroneurons in the lateral globus pallidus
were labeled significantly more intensely than other
neurons (ANOVA; Scheffe test, p < 0.001).
Essentially all neurons in the striatum were labeled
Landwehrmeyer et al: Huntington's Disease G e n e
based on an analysis of 574 neurons sampled in two
continuous transits through the head of the caudate
nucleus (at the level of the nucleus accumbens) of a
control brain (Fig 5A). The labeling intensities were
normally distributed (see Fig 5A). Large striatal neurons (97th percentile: mean cross-sectional area, 199
pm2; range, 174-259 pm2) were labeled as intensely
(mean count per cell, 32 2 4; 159 2 19 grains/1,000
pm2) as smaller striatd neurons.
Ependymal cells appeared labeled. Grain counting
disclosed that grain numbers over oligodendrocytes
and astroglial cells, although low, were significantly
higher than over the same cell populations in control
sections hybridized with sense probes (astroglia antisense probe: 2.6
0.2 grains/cell [or 69
7 grains/
1,000 pm2} vs. sense probe: 0.5 2 0.1 grainsicell [or
14 2 4 grains/1,000 prn’]; ANOVA: p < 0.0001,
Scheffk test: oligodendroglia antisense probe: 2.0
0.1 grains/cell [68 -+ 5 grains/1,000 pm2] vs. sense
probe: 0.2 i 0.1 grains/cell[7 -+ 4 grains/1,000 pm’l;
ANOVA: p < 0.0001, Scheffk test). N o significant
Annals of Neurology Vol 37
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February 1995
Fig 3. Regional dijtribution of IT15 hybridization signal in
normal human brain. (A)occipital cortex. Cortical layers are indicated by roman numerals. Bar = 1 mm. (B) Temporal cortex.
Cortical layers are indicated by roman numerals. Bar = 1 mm.
( C ) Hippocampalformation. C A 1-4 = subregions of the
cornu ammonis: D G = dentate Ryrus: Sub = subiculum; a =
alveus: o = stratum oriens; p = stratum pyramidale: Y = stratum radiatum; I-m = stratum lacunosum moleculare; ml = molecular layer of the dentate gyrus: g l = granular cell layer of the
dentate gyrus; pl = polymorph cell layer of the dentate gyrus.
Bar = 2 mm. (0)Cerebellum. D N = dentate nucleus; g =
granular cell layer; m = molecular cell layer. Bar = 4 mm. (Ei
Caudate head. CAU = caudate nucleus. Bar = 5 mm. (Fi
Lentifom nucleus. GP = globus pallzdus. NBM = nucleus
basa1i.c of Meynert; PUT = putamen. Bar = 5 m m . (G)Thalamus. R N = red nucleus; S T N = subthalamic nucleus: V L =
ventrolateral nucleus. *Area of infarction. Bar = 5 mni. (HI
Rostra1 midbrain. I I I = nucleus of the third cranial nerve; C G
= central gray: R N = red nucleus; SC = superior rolliculus;
SN = substantia nigra. Bar = 5 mm.
labeling of endothelial or smooth muscle cells could
be detected.
Expression of IT1 5 mRNA in Huntington's
Disease Brains
REGIONAL DISTRIBUTION. With the exception of the
striatum and lateral globus pallidus, the distribution
and density of hybridization signals in grades 2 to 4
HD brains was similar to those observed in control
brains (see Fig 2). Hybridization signal in striatum was
significantly lower than in control brains, whereas signal in the lateral globus pallidus at the level of film
autoradiography was significantly higher ( + 37%) than
in controls (see Fig 2; Fig 6D). In striatum of an addi-
tional 21 HD brains and 14 control brains, we confirmed a significantly decreased hybridization signal in
caudate nucleus, putamen, and nucleus accumbens (Fig
7). Signal reduction was most marked in caudate nucleus ( - 50%), and similar in putamen ( - 4 5 % ) , but
less marked in nucleus accumbens (-36%). The decrease in signal intensity was related to the grade of
striatal atrophy; signal in striatum of 2 presymptomatic
HD patients was indistinguishable from control striarum (see Fig 7).
As in control brains, inspection
of emulsion-dipped slides of HD brain sections disclosed hybridization signals concentrated over neu-
Landwehrmeyer et al: Huntington's Disease Gene 223
F i g 4. Cellular distribution of 12’15 mRNA in normal bruin.
(A)Occipital cortex, layer I I . (Bi Occipital cortex, pyramidal
neurons layer VI. (Ci Striatum. medium sized neurons. (Di
Striaturn. large neuron. (El Basal nucleus of Meynert. ( F ) Substantia nigra pars sornpacta. Bar in F = 20 Fm and upplies
also to A to E.
Annals of Neurology
Vol 37 No 2
February 1995
Tuble 2. kbeling of Neuronal and Nonneuronal Cells
in Control Brains
Counts per
Criiuiar Prohie
(mean i SEM)
Cell type
2.6 t 0.jd
2.0 t O . l h
5 9 ? 0.6
17.4 t 0.6
3.6 t O.jh
12.2 i 0.4
26.0 2 3'
12.6 ? 0.5
25.3 2 1 . 7 ~
13.0 t 0.6
36.9 f 0.6'
I4 4 f 0.7
48.5 t 2.4'
Cerrbral cortex (average)
~, layer 1
~,layer 11
layer 111
~, layer IV
~, layer V
~, layer V1
Motor cortex, layer V
Hippocmpus, dentate gyrus
Hippocmpus. CA pyramidal cell
Glohus pallidus, lateral
Glohus pallidus, medial
Thalamus, ventrolateral, macroneurons
Thalamus. ventrolateral. microneurons
Suhthalamic nucleus
Basal nucleus of Meynert
Substantia nigra. pars compacta
15.8 t 0.6
17.1 t 0.4
76 5 i 3.2'
70.6 2 6.7'
60.9 ? 2.9'
Counts/ 1,000
(mean i- SEMI
69 t ?b
68 2 Sb
125 t 13
172 t 1
68 i- bb
179 t 5
I92 i 8
165 2 5
169.9 % 6.3
149 i 5.8
220 t 8
189 2 7
I48 5 7
Silver grains11 000 prn2
196 t 7
"'Significantly (ANOVA: ' p < 0.01, ' p < 0.05; Scheffk test) lower hybridization signal than in striatal neurons.
"Significantly (ANOVA: ' p < 0.01, ' p < 0.05; Scheffe test) higher hybridization signal than in striatal neurons.
125 ? 6
185 t 4
251 t 8'
205 i- 9
Silver grainsllOO0 prn2
analysis of variance
ronal profiles. The intensity of neuronal labeling in HD
brains was similar to that observed in control brains
(Fig 8). In striatum, grain counting disclosed that the
mean density of silver grains over the remaining striatal
neurons in HD did not differ significantly from controls (153 ? 4 grains/1,000 pm' in HD vs. 155
grains/1,000 km2 in control; ANOVA, NS). Labeling
intensity was similar for all grades of HD examined.
The frequency distribution of labeling densities disclosed a normal distribution indistinguishable from
control striatum (see Fig 5B). Reactive fibrillary
astrocytes in HD striatum did not differ significantly
in their labeling intensity from normal, nonreactive
astrocytes (fibrillary astrocytes 2.9 ? 0.3 counts/cell
or 51 ? 6 counts/1,000 pm'; ANOVA, N S compared
with nonreactive astrocytes of control brain).
As noted above, hybridization signal in the lateral
globus pallidus was increased at the film level. Due to
shrinkage, the density of neurons in the lateral pallidum was significantly higher in the HD brains examined than in controls (20
1 cells/mm' in HD vs. 9
k 0.5/mm2 in controls; ANOVA, p < 0.0001; Scheffk
test). The labeling of individual pallidal macroneurons
in HD, however, was slightly but not significantly
lower than in controls (59 ? 8 grains/cell in HD vs.
67 2 5 grains/cell in controls; see Fig 8).
Fig 5 , Freyrrrn~ydistribution of hybridization signal intensity
(dimensioiz = cuuiitsI1,OOO pm') i t 1 indiiidual neurons in caudate nucleus in (A) normal striatun1 and IBi Huntington? diseuse striaturn (see text).
The most important finding in the present study is the
demonstration that there is no correlation between the
regional and cellular expression of IT15 mRNA and
the relative selective susceptibility to degeneration in
HD. This study confirms in addition that IT15 is
widely expressed in brain and that it is enriched in
neurons but also detectable in nonneuronal cells 13, 8,
91, suggesting that IT1 5 serves a general or constitutive
function common to many cell types. Finally, this study
helps to narrow down the mechanisms by which the
mutant HD gene may cause the HD phenotype.
Neuropathologically, HD is characterized by atrophy, cellular loss, and gliosis involving especially the
striatum. Our study confirms previous reports 17-10]
that in normal brain IT15 expression is not more abundant in the striatum than in other cerebral regions. In
addition, this study demonstrates that the pattern of
striatal IT1 5 expression does not match the pathognomonic distribution pattern of neuronal loss in HD; striatal regions that are involved early and consistently
[ 111 in HD (tail of the caudate, mediodorsal caudate,
Landwehrmeyer et al: Huntingron's Disease G e n e
Fig 6. Comparison of IT15 mRNA espres.rioti in strintum ofa
coiitrol (A. C ) , grade I Hutitingtotij dij-ease ( H D ) ( B ) ,and a
grade 4 H D rtriatum 1D).CAU = caudate tiudeus: ACC =
nucleus acc-uttzlens;PUT = pzrtamen; C L = thustrurn; GP
= globus pallidus. Bars = 5 vwi.
and putamen) do not differ in hybridization signal intensity from regions that are typically relatively preserved in HD (nucleus accumbens). Furthermore, our
quantitative cellular analysis suggests that IT15 mFWA
is expressed by essentially all neurons in striatum. O n
microscopic examination, large putatively cholinergic
striatal interneurons (thought to be relatively spared in
HD [15, 161) were labeled at an intensity similar to
medium sized putative projection neurons (known to
be particularly vulnerable to the effects of the HD
226 Annals of Neurology
Vol 37
No 2 February 1995
mutation C12, 13, 15, 17-20)). Similarly in cerebral
cortex, neurons in all cortical layers express IT15
mRNA at comparable levels; i.e., large cortical neurons
contained higher absolute amounts of IT15 mRNA
than small neurons; correction for cell size, however,
disclosed that there was no significant difference
between cortical neurons of different cortical layers
(ANOVA, NS: see Table 2). Neuropathological
studies have demonstrated, however, significant volumetric reductions in HD cortices [21, 22) and a preferential degeneration of large (>20 p m in apical-basal
diameter) neurons in layers Ill, V, and VI in frontal
cortex, suggesting a relatively selective loss of cortical
projection neurons [23-25}. In addition, expression
levels of cortical pyramidal neurons were similar to
F i g 7.Comparison of in situ hybridization histochemistry signals (measured as optical densities) of the caudate nucleus, putamen, and nucleus accumbens b o r n 14 control brains (filled bars),
two grade 0 to 1 brains (hatched bars) of individuals with the
Huntington’s disease (HD! genotype but no symptoms at the
time of death and 21 grade 2 to 4 H D brains (open bars).
Data are reported as absolute optical density for each region minus nonspecific signal after 7 to 10-day exposures. Optical densities for the groups were compared using the Kruskal-Wallis test
f H = 14.821, p < 0.002 for caudate; H = 16.508. p <
0.001 for putamen; H = 8.323, p < 0.02 for accumbens).
**p < 0.001: “p < 0.02.
those of pyramidal neurons in all CA subregions of
hippocampus, a neuronal population thought to be relatively resistant to neuronal degeneration in HD, at
least in subregions CA2 and CA3 1261. A similar lack
of correlation between abundance of IT15 mFWA and
susceptibility to HD is demonstrated by the high levels
of expression of IT15 mRNA in subcortical projection
neurons (basal nucleus of Meynert [ N B M ] , substantia
nigra compacta, locus ceruleus, dorsal raphe), which
are thought to be preserved in HD 127-291. Thus it
can be concluded that regions or cells primarily affected in HD d o not express IT15 mRNA at higher
levels than relatively resistant cells. On the other hand,
resistance to degeneration in HD cannot be simply
attributed to the absence of IT15 mRNA expression;
in this context the presence of IT15 mRNA in oligodendrocytes is of interest, since oligodenrocytes appear
to be relatively resistant to the disease process [ l l , 24,
The presence of IT15 mRNA in essentially all cerebral regions and all neurons studied in both control
and HD brain puts the observation that HD is associated with a diffuse atrophy of the brain into a new
perspective. Although the brunt of neuropathological
changes are found in the striatum, pathological changes
are not restricted to the basal ganglia and pathological
alterations can be demonstrated outside the striatum
C22, 3 1-33]. Regions with well-documented extrastriatal pathological changes include cortical and hypothalamic areas with no known striatal connections 123,
341; pathological changes have also been described in
cerebellum 135, 361, particularly in infantile HD patients 137, 381. The ubiquitous expression of the mutant IT15 could account for the widespread neuronal
loss in multiple unconnected areas, particularly prominent in patients with very long repeat expansions.
However, since the cellular level of IT15 mRNA does
not predict whether a cell is vulnerable to degeneration
in H D , the prominent involvement of the striatum and
the characteristically selective pathology of HD cannot
be explained by the expression pattern of the HD-gene
The association of a disorder characterized by a relatively selective pathology with a mutation in an apparently ubiquitously expressed gene is not without precedent. Mutations of the Cu/Zn superoxide dismutase
gene that give rise to familial amyotrophic lateral sclerosis [391 show that a widely expressed gene may have
a regionally specific pattern of pathology. The selective
pattern of pathology may be due to an interaction of
the mutant gene with tissue components that are expressed exclusively in some tissues or may be due to
different compensatory capacities in various cell populations. The prominence of neuronal loss and gliosis in
striatum would indicate that striatal projection neurons
have some yet unknown unique properties that render
them particularly vulnerable to the effects of the mutant IT1 5 allele. Excitotoxic mechanisms or mitochondrial dysfunction may act in concert with the mutant
gene since the striatum is particularly vulnerable to the
local and systemic effects of inhibitors of mitochondrial
function [40-42) and to the toxic effects of excitatory
amino acids C433.
The mechanism by which the expansion of CAG
repeats in the IT15 gene ultimately causes the HD
phenotype is yet unknown. It is apparent from our
results that the presence of expanded trinucleotide repeats does not alter the hybridization signal in the striatum in presymptomatic H D, and that the remaining
striatal neurons in more advanced cases of HD express
normal levels of IT15 mRNA. Similarly, previous studies have shown that preproenkephalin mRNA levels
in remaining striatal neurons of HD are unchanged
[44],suggesting that there is no general disturbance of
transcriptional activity in the population of susceptible
striatal neurons. The conclusion that the HD mutation
does not result in changes in IT15 mRNA levels is in
line with the presence of IT15 mRNA in lymphoblast
lines from homozygotes for the HD mutation [3]. In
this respect, HD differs from another disorder associated with trinucleotide repeats, i.e., fragile X syndrome
[45],where the presence of a massively expanded triplet repeat in the 5’ untranslated region results in a
repression of transcription of the FMR-1 gene {46].
HD is also unlike myotonic dystrophy where the consequences of the trinucleotide repeat expansion in the
3’ untranslated region seems to be a change in the
steady-state levels of the resultant mRNA [47, 48).
This does not exclude, however, the possibility that
the expanded trinucleotide repeat may have an effect
Landwehrmeyer et al: Huntington’s Disease Gene
Fig 8. Comparison of IT15 mRNA expression in striatum (A,
B ) and lateral globus paallidus (C, DI of Huntington’s disease
( H D ) (B, D ) and control (A. C) at the cellular level. Note the
similar labeling of niediurn sized neuronal profiles in H D and
control striatum and the lozu neuronal density in H D striatum.
Nerironal density in lateral globus pallidus in constrast is increased in H D brain. Bar = 20 pm.
at the R N A level. For example, the mutant IT15
mRNA may lead to a novel localization of the IT15
mRNA within certain cellular compartments. It is also
possible that the presence of extended repeats within
the IT15 mRNA may alter its interactions with proteins, resulting in an altered splicing pattern or an altered efficiency of translation. These possibilities require additional investigations.
Alternatively, the IT1 5 mutation may have its dominant effect at the D N A or the protein level. Although
transcription of IT15 itself appears to be unaffected
by the expansion of CAG repeats, the presence of an
extended polyglutamine stretch, a common motif for
transcription factors [49], may alter regulation of transcription of other genes in its vicinity. In another disor-
228 Annals of Neurology
Vol 37
N o 2 February 1995
der associated with an amplification of trinucleotide
repeats, spinobulbar muscular atrophy (SBMA), expansion of CAG repeats occurs in the 5’ coding region of
the androgen receptor [50] (a ligand-activated, DNAbinding, transcriptional regulatory protein), resulting in
a polyglutamine stretch similar to that predicted in
HD. The polyglutamine-expanded androgen receptor
subnormally transactivates an androgen-responsive reporter gene 1511. It is not known whether IT15 (or
fragments of IT15) acts as a transregulatory protein but
an expanded polyglutamine stretch in its N-terminal
domain could result in a modified transcriptional regulatory property 1471. Immunohistochemical studies
have shown a cytoplasmatic (unpublished observations)
and a cytoplasmatic and nuclear localization of the IT15
protein [52] and are equivocal with respect to a possible function of the IT15 protein as a transcription factor. Last, the HD mutation may confer a new property
on the IT15 protein that ultimately results in neuronal
death. It is of interest that none of the protein sequences deposited in the Swiss protein data base contain a region of more than 38 reiterated glutamines,
suggesting that organisms do not tolerate proteins with
very long stretches of glutamine { 5 3 ] .
Two other neurodegenerative disorders that bear
some resemblance to the clinical and neuropathological
aspects of H D have recently been found to be associated with expansion of CAG repeats, i.e., spinocerebellar ataxia type 1 (SCA 1)1541 and dentatorubropallidoluysian atrophy (DRPLA) 155, 56}. Similar to HD, the
disease phenotypes are associated with more than 40
CAG repeats {54-56’1; longer repeat lengths are associated with paternal transmission and correlate with age
of onset of symptoms and a phenotype suggestive
of more extensive involvement of neuronal systems
(e.g., myoclonic epilepsy in DRPLA). Like the IT15
transcript in HD, the DRPLA gene transcript is widely
expressed including apparently normal cerebral regions
{ 5 5 } . In DRPLA there appears to be somatic variation
in the size of the enlarged allele 155}, a finding that
has been reported in HD brains as well 1571 (but compare { 581). Whether somatic variations in repeat length
contribute to the pattern of neuronal loss characteristic
for HD is unclear at the present time, however. Although it might be difficult in some patients with prominent choreic movements, personality changes, and
mild cerebellar signs to clinically distinguish DRPLA
from HD 1591, the neuropathological findings in these
two diseases are clearly different.
These disorders illustrate that despite the similarities
of trinucleotide repeat-associated neurodegenerative
diseases (prominent involvement of brain, slow degenerative process, anticipation, association of more severe
phenotype with larger expansions), the mechanisms by
which the respective trinucleotide expansion in the different genes lead to neuronal loss are likely to be different. A more complete understanding of the mechanisms that lead to the HD phenotype will have to focus
on interactions of the mutant IT15 gene and protein
with various factors in the neurons in which it is expressed.
Supported by USPHS grants NS 16367 to J. G., M. D., and J. V.
(Huntington’s Disease Center Without Walls) and PHSMH 31862
(Brain Tissue Resource Center) to J. V. and E. B., grants from
Bristol-Myers Squibb (J.G.), AGO8856 and N S 19613 to A. B. Y.
and J. B. P., K11 HD 00983 to L. S. D. IV, and DFG La 70212-1
to G. B. L.
We express our appreciation to all the H D patients and their families
who so unfailingly gave of themselves to assist with this project. We
also thank Lisa Kanaley, Iris DeQuiroz, and Nelson Marsol for help
in providing brain tissue and David G. Standaert MD, PhD, for
stimulating and helpful discussions.
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