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Dopamine D1 and D2 receptors in progressive supranuclear palsy An autoradiographic study.

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Dopamine D1 and D2
Receptors in Progressive
Supranuclear Palsy: An
Autoradiographic -Study
J. Pascual, MD,"t J. Berciano, MD,S B. Grijalba, MD,*
E. del Olmo, MD," A. M. Gontdet, MD,"
J. Figols, MD,S and A. Pazos, MD"
Dopamine D1 and D2 receptors were studied in brain
tissue sections from a typical patient with progressive
supranuclear palsy and in 7 age-matched brains. The
density of D1 receptors in the caudate-putamen and
frontal cortex of the patient was within control limits.
By contrast, the density of nigral D1 receptors and striatal D2 receptors was dramatically reduced in the patient
as compared to the control brains. This work shows
again that the loss of striatal D2 receptors is the most
plausible explanation for the poor response to dopaminergic drugs in patients with progressive supranuclear
palsy. While the loss of nigral D1 receptors can be explained by the loss of nigral neurons, it seems that neurons bearing striatal D1 receptors are spared in progressive supranuclear palsy. The clinical effects of selective
D l agonists are worth testing in this devastating disorder.
Pascual J, Berciano J, Grijalba B, del Olmo E,
Gontdet AM, Figols J, Pazos A. Dopamine D1
and D2 receptors in progressive supranuclear
palsy: an autoradiographic study.
Ann Neurol 1992;32:703-707
the absence of a response to dopaminergic replacement
in PSP seems to be the concomitant degeneration of
neurons containing dopamine receptors in the striatum. In fact, Bokobta and Ruberg and coworkers
reported a reduction of about 50% in 3H-spiperonebinding sites in the caudate-putamen by using membrane homogenates from PSP brains 14, 57.
To the best of our knowledge, dopamine D1 receptors have not been examined in PSP. We report here
on dopamine D1 and D2 receptor densities in a typical PSP patient, using autoradiographic techniques to
study postmortem brain sections.
Patient Report
This man was first seen in our hospital in 1983 when he
was 64 years old. His medical history was unremarkable. He
described an unsteady gait, blurred vision not corrected by
glasses, and subtle mental changes. On examination he was
mentally slowed, apathetic, and moderately depressed. In addition, lack of voluntary vertical gaze with normal reflex
movements, slurred speech, and a rigid-akinetic syndrome
were observed. Computed tomography (CT) showed slight
brain atrophy. Despite clorimipramine and increasing doses
of levodopa plus carbidopa, his symptoms progressed and
2 years later axial rigidity with neck extension, dysphagia,
hyperreflexia, complete loss of ocular voluntary movements,
and urinary incontinence ensued. CT, performed 3 years
after the symptoms began, disclosed progression of the brain
atrophy, especially marked in the midbrain. The patient was
maintained on levodopa (up to 1 gm daily) plus carbidopa
and amitriptyline or trihexyphenidyl with no apparent benefit. H e died suddenly in 1986 while sleeping.
Methods
Progressive supranuclear palsy (PSP) is a devastating
neurodegenerative disorder characterized by loss of
voluntary vertical gaze, dysarthria, subcortical dementia, and dystonic extension of the neck, as well as a
rigid-akinetic syndrome 111. Although in its earlier
stages PSP can be misdiagnosed as Parkinson's disease
(PD), the absence of tremor and the rest of the clinical
picture usually allow a correct diagnosis 121. Another
important clue for the diagnosis of PSP is the very
poor response to levodopa or dopaminergic agonists
131. The nigrostriatal neurons appear to degenerate in
patients with PSP as in those with PD. The reason for
From the "Department of Physiology and Pharmacology, Unit of
Pharmacology, tDepamnent of Medicine, Service of Neurology, and
Section of Neuropathology, University Hospital "Marques de Valdecilla," and Faculty of Medicine, University of Cantabria, Santander,
Spain.
Received Feb 14, 1992, and in revised form Apr 1 and 27. Accepted
for publication May 3, 1992.
Address correspondence to Dr Pascual, Service of Neurology, University Hospital "Marqu6s de Valdecdla," 39008 Santander, Spain.
At autopsy, the brain was promptly removed, one half being
fixed in 10% formalin for neuropathological examination.
The other half was cut into blocks, quickly frozen and stored
at - 70°C until it was used in binding assays.
Autoradiographic labeling of dopamine D 1 and D2 receptors was performed in several brain regions of the PSP patient (age, 67 years; postmortem delay, 24 hours) as well as
in 7 male subjects with no history of neuropsychiatric disease
(average age & standard deviation [SD], 69 5 years; postmortem delay & SD, 17 ? 7 hours).
For labeling of dopamine D1 receptors and as previously
reported 163, we incubated 10-p.m-thick tissue sections with
1 nM 3H-SCH 23390 in 50 mM Tris-hydrochloric acid
(HCl), 120 mM sodium chloride (NaCl), 5 mM potassium
chloride (KCl), 2 mM calcium chloride (CaCI,), and 1 mM
magnesium chloride (MgCI,) @H 7.4) for 75 minutes at
room temperature. Adjacent sections were incubated in the
same medium in the presence of 10 pM dopamine to determine the nonspecific binding. After incubation, sections were
washed for 5 minutes in fresh cold buffer.
Labeling of D2 receptors was carried out incubating 10pm-thick caudate-putamen sections for 1 hour in the described Tris-HC1 buffer containing 1 pM ketanserin (to prevent labeling of 5-HT, receptors) and 0.4 nM 3H-spiperone
(23.3 Cilmmol). Nonspecific binding was defined by 10 pM
*
Copyright 0 1992 by the American Neurological Association 703
Fig 1 . The zona compacta ofthe substantia nigra showing loss
of neurons, gliosis, scattered melanin pigment (arrow), and ballooned neurons. (Hematoxylin-eoszn, x 600.) The insert illustrates a pigmented neuron with neurofibrilla~degeneration.
(Hematoxylin-eosin, X 1,500.)
haloperidol. Incubation was terminated by rinsing sections
twice for 5 minutes in cold Tris-HCI buffer [7, 83.
After washing, for both receptors, sections were quickly
dried in a cold-air stream. Autoradiograms were generated
by apposing the slide-mounted tissue sections to tritiumsensitive films (3H-Ultrofilm, Leica, Nussloch, Germany) at
4°C for 7 weeks in the case of D 1 receptors and for 10
weeks in the case of D2 receptors. The autoradiograms
were analyzed and quantified by using a computer-assisted
microdensitometer (Microm IP, Microm Espaiia, Barcelona,
Spain). Appropriate standards (Amersham [Buckinghamshire, UKf tritiated microscales), exposed together with the
tissues, allowed the transformation of densitometric readings
into receptor densities (fmollmg of protein) [9].
l), periaqueductal gray matter, and locus ceruleus. In
these brain regions as well as in others, mainly the
substantia innominata, dentate nucleus of the cerebellum, and red nucleus, we observed relevant but variably intense gliosis and neuronal loss, with central
chromatolysis or granulovacuolar degeneration in the
remaining neurons. Slight to moderate gliosis with neither definitive neuronal loss nor neurofibrillary degeneration was observed in the caudate-putamen.
Neurochemical Studies
These results appear in the Table and are illustrated in
Figures 2 and 3. The density of dopamine D1 receptors
Density (fmollmg ofprotein) of Dopamine Dl and 0 2
Receptors i n the Progressive Supranuclear Pa& (PSP) Brain as
Compared to Control Brains
D1
Results
Neuropathological Studies
Macroscopic examination showed generalized brain
atrophy most prominent in the midbrain, with an
enlarged sylvian aqueduct and third ventricle. Microscopic findings were diagnostic for PSP [lo]. In short,
prominent neurofibrillary tangles were seen in the globus pallidus, subthalamic nucleus, substantia nigra (Fig
704 Annals of Neurology
D2
Brain Area
Control
PSP
Control
PSP
Caudate
Putamen
Substantianigra
Frontal cortex
317 +275 +151 +109
325
339
73
122
324 f 58
429 f 69
< 30"
< 20"
78
69
40
* 14
~
"Measurementsobtained at three different striatal levels in two separate experiments.
Vol 32 No 5 November 1992
D
C
Fig 2. Autoradiographs of D l dopamine receptors as labeled
with 3HSCH 23390 in posterior striatum and midbrain sections from control subjects (A, C ) and the patient with pmgressive supranuclear palsy (PSP) {B, D). Note that in PSP striatum (B), Dl receptor densities do not dqfw from control levels
(A) in the putamen (P). In adition, D l receptor densities are
marked4 reduced in the substantia nigra (SN) of this PSP patient (0)as compared to a control {C).Bar = 2 mm.
in the caudate-putamen and the frontal cortex of the
patient was within normal limits as compared to the
control group. By contrast, D1 receptors were clearly
reduced in the substantia nigra of the patient with PSP
in comparison to the control subjects.
The density of dopamine D2 receptors was almost
negligible in the PSP striatum as compared to control
levels. In the human brain, D2 receptors are concentrated over the striatum [7}, but their very low density
(< 30-40 fmol/mg of protein) normally in the substantia nigra and neocortex does not allow comparison between the patient and control subjects.
Brief Communication: Pascual et al: D1 and D2 Dopamine Receptors in PSP
705
B
A
Pig 3. Autoradiographs of dopamine 0 2 receptors as labeled
with 3H-spiperone in anterior striatum sections from control (A)
and patient (B) brains. Note that in the patient, 0 2 receptor
densities in the caudute (C)-putamen(P) are negiglible, as compared to the control. Bar = 2 mm.
Discussion
This is the first report studying dopamine D1 and D2
receptors in PSP by using quantitative autoradiography, thus allowing analysis of the density of these receptors in discrete brain areas. Our study confirms and
extends previous findings of a reduction in dopamine
D2 receptors in the caudate-putamen of PSP patients
14, 51. In fact, the PSP patient studied here exhibited
an almost complete loss of these receptors in both the
caudate and the putamen. Dopamine D2 receptors are
thought to be located both on dopaminergic nigrostriatal terminals and, perhaps preferably, on large and medium spiny striatal neurons, thus being preserved in
pure presynaptic parkinsonian syndromes (8, 11, 121.
In our patient, and as is typical in PSP, nigral and
striatal degeneration was neuropathologically confirmed, thus correlating with the neurochemical data.
Furthermore, D2 downregulation secondary to dopaminergic treatments might partly explain the loss of
D2 receptors.
Intriguingly, D1 receptors are preserved in the PSP
strianun. In the human species, the preservation of D1
receptors in PD, despite the marked nigrostriatal degeneration taking place in this degenerative disorder,
strongly suggests a postsynaptic localization of dopamine D1 receptors [12-151. The preservation of dopa-
mine D1 receptors in this patient with PSP, in spite of
the important nigral degeneration, also weighs against
many D1 receptors being present on nigrostriatal terminals. Recently, it was reported that the neuronal degeneration occurring in PSP striatum is not homogeneous but selective for the large striatal neurons [lGl
where D2 and muscarinic receptors are believed to be
located [5}. Thus, D1 receptors could be located on
one of the several kinds of intrinsic striatal neurons
that do not degenerate in this condition. Finally, in
theory, as was recently proposed for P-adrenoceptors
[17}, dopamine D1 receptors could potentially be located on glial cells, which are known to proliferate in
PSP striatum as a result of the striatal neuronal loss.
However, lesion models in experimental animals do
not lend weight to this hypothesis 1181.
Although the loss of nigral D1 receptors in this PSP
patient could be explained by the observed neuronal
nigral degeneration, the preservation of nigral D1 receptors in patients with PD 112-151 may indicate that
the loss of D1 nigral receptors in PSP could be secondary to the degeneration of nigral terminals of gammaaminobutyric acid (GABA)ergic or substance P-containing neurons projecting from the striatum (181.
Therefore, our receptor studies in a typical patient with
PSP show again that the loss of D2 receptors is the
most plausible explanation for the absence of response
to dopaminergic drugs. However, the preservation of
“postsynaptic” dopamine D 1 receptors indicates that
the clinical effects of agonist drugs acting selectively
on D1 receptors are worth testing in PSP.
706 Annals of Neurology Vol 32 No 5 November 1992
a hi^ work was supported by grant 852184 from Comisibn Asesora
de Investigacion Cientifica y Tecnica (A. P. and J. B.).
We are indebted to the neurologists Drs Felix Fernhdez and M. Jose
reviewedwho
Sedano,
the also
manuscript.
managed this patient. Mr John Hawkins stylistically
1. Steele JC, Richardson JC, Olzewski J. Progressive supranuclear
References
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17. Waeber C, Rig0 M, Chinaglia C, et al. Beta-adrenergic subtypes
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SeVeritv Of X-Linked
Recessive Bulbospinal
Neuronopathy Correlates
with Size of the Tandem
CAG Repeat in Androgen
Keceptor Gene
Manabu Doyu, MD,*'F Gen Sobue, MD,* Eiichiro Mukai,
MD,S Teruhiko Kachi, MD,§ Takeshi Yasuda, MD,"
Terunori Mitsuma, MD,* and Akira Takahashi, MD'F
The genetic mutation of X-linked recessive bulbospinal
neuronopathy is amplification of a polymorphic tandem
CAG repeat in the androgen receptor gene. We studied
this CAG repeat in 26 Japanese patients from 2 1families
with X-linked recessive bulbospinal neuronopathy. The
number of CAG repeats was significantly correlated
with the age at onset of limb muscular weakness (r =
-0.596, p < 0.001) and age-adjusted scored disability (r
= 0 . 4 4 6 , ~< 0.03). The length of the CAG repeat therefore seem to be a determinant factor of clinical severity.
Doyu M, Sobue G, Mukai E, Kachi T, Yasuda T,
Mitsuma T, Takahashi A. Severity of X-linked
recessive bulbospinal neuronopathy correlates with
size of the tandem CAG repeat in androgen
receptor gene. Ann Neurol 1992;32:707-710
X-linked recessive bulbospinal neuronopathy (XBSNP) is an adult form of hereditary motor neuronopathy: The age at onset and severity of muscular weakness and wasting vary among patients El-41, as do the
associated endocrine features 12, 3, 51.
Androgen receptor (AR) gene mutations show an
increased number of a polymorphic tandem CAG repeats in the coding region, which is a specific abnormality in the X-BSNP gene C61. The number of CAG
repeats is highly variable in different patients 161.
We have analyzed the number of CAG repeats in
26 Japanese patients with X-BSNP, and related this to
the severity of muscle wasting and age at onset.
From the *Division of Neurology, Fourth Department of Internal
Medicine, Aichi Medical University, Nagakute, Aichi; tDepartment
of Neurology, Nagoya University School of Medicine, Nagoya; SDepartment of Neurology, National Nagoya Hospital, Nagoya;
5De artment of Neurology, National Chubu Hospital, Obu;
and Department of Neurology, Nagoya Daini Red Cross Hospital,
K
Nagoya, Japan.
Received Mar 30, 1992, and in revised form May 20. Accepted for
publication May 22, 1992.
Address correspondence to Dr Sobue, Division of Neurology,
Fourth Department of Internal Medicine, Aichi Medical University,
Nagakute, Aichi 480-11, Japan.
Copyright 0 1992 by the American Neurological Association 707
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