International Journal of
Molecular Sciences
Article
A2A Receptor Dysregulation in Dystonia DYT1
Knock-Out Mice
Vincenza D’Angelo 1,† , Mauro Giorgi 2,† , Emanuela Paldino 3 , Silvia Cardarelli 2 , Francesca R. Fusco 3 ,
Ilaria Saverioni 2 , Roberto Sorge 1 , Giuseppina Martella 1,3 , Stefano Biagioni 2 , Nicola B. Mercuri 1 ,
Antonio Pisani 4,5 and Giuseppe Sancesario 1, *
1
2
3
4
5
*
†
Citation: D’Angelo, V.; Giorgi, M.;
Paldino, E.; Cardarelli, S.; Fusco, F.R.;
Saverioni, I.; Sorge, R.; Martella, G.;
Biagioni, S.; Mercuri, N.B.; et al. A2A
Receptor Dysregulation in Dystonia
DYT1 Knock-Out Mice. Int. J. Mol.
Sci. 2021, 22, 2691. https://doi.org/
10.3390/ijms22052691
Academic Editor: José L. Lanciego
Received: 14 February 2021
Accepted: 1 March 2021
Published: 7 March 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
Department of Systems Medicine, Tor Vergata University of Rome, 00133 Rome, Italy;
dangelo@med.uniroma2.it (V.D.); sorge@uniroma2.it (R.S.); martella@med.uniroma2.it (G.M.);
mercurin@med.uniroma2.it (N.B.M.)
Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome,
00185 Rome, Italy; mauro.giorgi@uniroma1.it (M.G.); silvia.cardarelli@uniroma1.it (S.C.);
ilaria.saverioni@gmail.com (I.S.); stefano.biagioni@uniroma1.it (S.B.)
IRCCS Santa Lucia Foundation, 00179 Rome, Italy; e.paldino@hsantalucia.it (E.P.);
f.fusco@hsantalucia.it (F.R.F.)
IRCCS Mondino Foundation, 27100 Pavia, Italy; pisani@uniroma2.it
Department of Brain and Behavioral Sciences, University of Pavia, 27100 Pavia, Italy
Correspondence: sancesario@med.uniroma2.it; Tel.: +39-33-8146-6058
These authors to be considered equally first author.
Abstract: We aimed to investigate A2A receptors in the basal ganglia of a DYT1 mouse model of
dystonia. A2A was studied in control Tor1a+/+ and Tor1a+/− knock-out mice. A2A expression was
assessed by anti-A2A antibody immunofluorescence and Western blotting. The co-localization of A2A
was studied in striatal cholinergic interneurons identified by anti-choline-acetyltransferase (ChAT)
antibody. A2A mRNA and cyclic adenosine monophosphate (cAMP) contents were also assessed. In
Tor1a+/+, Western blotting detected an A2A 45 kDa band, which was stronger in the striatum and the
globus pallidus than in the entopeduncular nucleus. Moreover, in Tor1a+/+, immunofluorescence
showed A2A roundish aggregates, 0.3–0.4 µm in diameter, denser in the neuropil of the striatum
and the globus pallidus than in the entopeduncular nucleus. In Tor1a+/−, A2A Western blotting
expression and immunofluorescence aggregates appeared either increased in the striatum and the
globus pallidus, or reduced in the entopeduncular nucleus. Moreover, in Tor1a+/−, A2A aggregates
appeared increased in number on ChAT positive interneurons compared to Tor1a+/+. Finally, in
Tor1a+/−, an increased content of cAMP signal was detected in the striatum, while significant levels
of A2A mRNA were neo-expressed in the globus pallidus. In Tor1a+/−, opposite changes of A2A
receptors’ expression in the striatal-pallidal complex and the entopeduncular nucleus suggest that
the pathophysiology of dystonia is critically dependent on a composite functional imbalance of the
indirect over the direct pathway in basal ganglia.
Keywords: A2A; cAMP; A2A mRNA; dystonia; DYT1; basal ganglia; D2
published maps and institutional affiliations.
1. Introduction
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
The clinical features of dystonia are relatively well-defined, produced by the ill-timed
activation of agonist-antagonist muscles, and presenting either as focal or generalized.
Conversely, the pathological mechanisms underlying such a heterogeneous group of
movement disorders remain elusive [1–3]. Unlike symptomatic dystonia secondary to
histological damages affecting basal ganglia, no neuropathologic correlates are detectable
at microscopic levels for primary dystonia [4,5].
A common form of primary early onset generalized dystonia is caused by 3 bp deletion
(GAG) in the coding region of the TOR1A (DYT1) gene, which results in a defective protein
called torsinA, whose role in dystonia pathology is unclear [6]. In animal models for
Int. J. Mol. Sci. 2021, 22, 2691. https://doi.org/10.3390/ijms22052691
https://www.mdpi.com/journal/ijms
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DYT1 dystonia, multiple lines of evidence revealed the impairment of dopamine receptor
type 2 (D2 receptor), with D2 downregulation, sparse D2 synapses, reduced coupling
between the D2 receptor and its cognate G proteins, as well as the loss of D2 dependent
electrophysiological inhibition and severely altered synaptic plasticity in medium spiny
neurons and cholinergic interneurons in the striatum [7–18]. Therefore, the striatum and
D2 receptors have been considered central in the cellular pathomechanism underlying
DYT1 dystonia [15].
Noteworthy, the deficit in D2 receptor-mediated transmission can be counteracted by
A2A receptor pharmacological antagonism in the striatum of a genetic mouse model of
DYT1, and also of DYT11 and DYT25 dystonia, suggesting that an imbalance in D2/A2A
functions may represent a convergent pathogenetic mechanism [11,19,20]. Moreover, a
peculiarity of D2 receptors is their selective co-localization with A2A receptors in striatal
cholinergic interneurons [21,22], and mainly in the sub-population of striatal medium spiny
GABAergic neurons containing enkephalin, which are known to lead the indirect striatalpallidal pathway [23,24]. D2 and A2A receptors in enkephalin neurons form complex
heteromers and exert reciprocal antagonistic interaction, inhibiting or stimulating the
second messenger cyclic adenosine monophosphate (cAMP) synthesis, respectively [25,26].
Therefore, besides the dysfunction of D2 receptors, A2A receptors may be of interest
in dystonia pathophysiology for several reasons. In this paper, we aimed to investigate
whether the D2 receptor dysfunction is coupled with any change in the A2A receptor
expression in the basal ganglia of a DYT1 mouse model. We report that opposite to the
downregulation of D2 receptors in the striatum, an up-regulation of the A2A receptors
occurs in the striatal-pallidal complex, whereas A2A down-regulation occurs in the entopeduncular nucleus of a DYT1 dystonia mouse model.
2. Results
2.1. Expression of A2A Receptors
We first quantified the presence of A2A receptors by Western blot on proteins extracted
from the striatum, globus pallidus and entopeduncular nucleus of Tor1a+/+ and Tor1a+/−.
A single specific band was detected in all areas of the basal ganglia approximately at 45
kDa, which corresponds to the migration level of A2A receptor monomers (Supplementary
Materials Figure S1). In Tor1a+/+, a higher A2A expression was detected in the striatum
that was not significantly different from the globus pallidus, whereas the lowest A2A
expression was detected in the entopeduncular nucleus (p < 0.001). Moreover, Western
blotting analysis revealed a significant increase in A2A receptor levels in the striatum
(n = 11, df = 1, F = 5.189, p = 0.034) and globus pallidus (n = 9, df = 1, F = 6.791, p = 0.020),
but a significant decrease in A2A expression in the entopeduncular nucleus (n = 8, df = 1,
F = 7.629, p = 0.016) of mutant Tor1a+/− compared to control Tor1a+/+ mice (Figure 1).
2.2. Cyclic AMP Levels
We then investigated whether the changes in A2A expression in the basal ganglia of
Tor1a+/− mice consistently affected second messenger cAMP content in tissue extracts.
Actually, the cAMP level was significantly increased in the striatum (df = 1, F = 4.828,
p < 0.05), but was unchanged in the globus pallidus (df = 1, F = 0.151, p > 0.5) and in the
entopeduncular nucleus (df = 1, F = 0.403, p > 0.5) of Tor1a+/− mice (n = 7) compared with
Tor1a+/+ (n = 7) (Figure 2).
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−
β
Figure 1. Expression of A2A in basal ganglia of Tor1a+/+ and Tor1a+/− mice. (A) Densitometric
analysis optical density (O.D.) of A2A-immunostained bands in correspondent areas of caudate–
putamen (CP), globus pallidus (GP), and entopeduncular nucleus (EN) of Tor1a+/+ and Tor1a+/
− −.
−
ff
Results were expressed as the mean ± SD of the values obtained in each group. One-way ANOVA,
** p < 0.020. (B) Representative immunoblots of A2A content in correspondent area of CP, GP and EN
β
content was detected
of Tor1a+/+ and Tor1a+/− mice. As an internal reference standard, the β-actin
in each lane of the same blots to correct for protein loading.
−
ff
Figure 2. Cyclic adenosine monophosphate (cAMP) level in tissue homogenates of caudate–putamen
(CP), globus pallidus (GP), and entopeduncular nucleus (EN) in control Tor1a+/+ and in mutant
−
Tor1a+/− mice.
Values, expressed as pmol of cAMP/ mg protein, are represented as mean ± SD.
Significant increase in cAMP content was detected in the CP of Tor1a+/− versus Tor1a+/+. One-way
ANOVA, * p < 0.05.
2.3. Expression of A2A mRNA
Real-time PCR amplification of A2A mRNA was performed in the two groups of
animals to investigate A2A changes at the transcription level in basal ganglia. The primers,
−
−
Int. J. Mol. Sci. 2021, 22, 2691
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which amplified a specific mRNA fragment (140 bp), have shown that the A2A mRNA
expression level in the caudate–putamen of Tor1a+/+ (n = 6) is much higher than in the
globus pallidus (n = 8) (Figure 3A), whereas in the entopeduncular nucleus its expression
is under the detection level (data not shown). The pattern and relative intensity of A2A
mRNA expression detected in the caudate–putamen of Tor1a+/
− mice (n = 8) were similar
−
compared to the caudate–putamen of Tor1a+/+ mice (Figure 3B). Surprisingly, a significant
increase in A2A mRNA expression was detected in the globus pallidus of Tor1a+/−mice
(n = 8) compared
to Tor1a+/+ mice (Figure 3C).
−
Figure 3. (A)—Comparative real-time PCR of A2A mRNA in caudate–putamen (CP) and globus
pallidus (GP) of Tor1a+/+ control mice (n = 6, df = 1, F = 71.172, p = 0.001). (B)—The expression
− of Tor1a+/− mice
of A2A mRNA in the CP of Tor1a+/+ mice is compared to the levels in the CP
(n = 8, df = 1, F = 0.311, p = 0.586). (C)—The expression of A2A mRNA in the GP of Tor1a+/+ mice is
−
compared to the levels in the GP of Tor1a+/− mice (n = 7, df = 1, F = 4.743, p = 0.048). Results were
expressed as the mean ± SD of values obtained from each group. Data in (A–C) were analyzed with
one-way ANOVA followed by Bonferroni’s correction for multiple data. * p < 0.05, *** p < 0.001
2.4. A2A Immunofluorescence in Confocal Microscopy
To allow a precise morphological definition of the A2A receptor aggregates on the
striatal-pallidal complex in control and mutant mice, we performed a fluorescence staining, followed by detection with confocal microscopy. A better understanding of A2A
receptors’ subcellular distribution came out in images acquired using 63× oil immersion
objective (1.4 numerical aperture) with an additional digital zoom factor (1×–1.5×–2×).
The immuno-fluorescent signal appeared extremely specific without background staining,
showing A2A positive small grains with elliptical or roundish shape about 0.3–0.4 µm
in diameter, isolated or contiguous, composing irregular clusters of size variable in the
different areas of the basal ganglia of Tor1a+/+ mice (Figure 4A,C,E).
Noteworthy, in Tor1a+/+ mice, A2A small grains appeared to be diffusely covering
the neuronal compartments of the striatum, globus pallidus and entopeduncular nucleus,
uniformly distributed in the neuropil, whereas grains were rare and almost absent on
the cell nuclei and in striatal axonal bundles (Figure 4A,C,E). Moreover, among the basal
ganglia regions, the number of A2A receptors per microscopic field was much higher in
the striatum and globus pallidus than in the entopeduncular nucleus.
In Tor1a+/− mice, A2A receptor grains appeared either increased in number in
the striatum and globus pallidus, or reduced in number in the entopeduncular nucleus
(Figure 4B,D,F), compared with control Tor1a+/+ (Figure 4A,C,E). Moreover, the distribution pattern of A2A positive fluorescent grains was clearly different in the basal ganglia of
Tor1a+/− mice, wherein A2A receptor positive grains appeared as tiny spots uniformly
distributed in the neuropil, but also clustered on neuronal bodies of the striatum and
globus pallidus (Figure 4B,D), unlike in Tor1a+/+ mice (Figure 4A,C).
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Figure 4. Representative immuno-fluorescence microphotographs of high magnification (63× oil
immersion objective plus zoom factor 1×–1.5×–2.5×) confocal laser scanning microscopy, showing
the roundish morphology of A2A positive tiny spots and their distributions in the striatum (A,B),
globus pallidus (C,D), and entopeduncular nucleus (E,F) of control Tor1a+/+ (A,C,E), and of mutant
− mice. A2A receptor labeling is visualized in red-Cy3 fluorescence, while cell nuclei
Tor1a+/− (B,D,F)
′
are visualized by 4′ ,6–diamidino–2–phenylindole (DAPI) fluorescence in blue. White arrows in B
and D point to clustering of A2A grains around neuronal bodies. Scale Bar in F = 5 µm.
Densitometric analysis confirmed a higher density of the number of A2A receptors
per microscopic field in the striatum (df = 1, F = 14.039, p = 0.013) and in the globus pallidus
(df = 1, F = 22.859, p = 0.003), and their lower density in the entopeduncular nucleus (df = 1,
F = 9.031, p = 0.024) of Tor1a+/− (n = 4), compared with correspondent basal ganglia areas
of the Tor1a+/+ mice (n = 4) (Figure 5).
While the numbers of A2A receptors were either increased in the striatal-pallidal
complex or decreased
in the entopeduncular nucleus, their size appeared smaller in all the
−
basal ganglia structures of TOR1a+/−. A representative number of A2A positive grains
were randomly selected from the basal ganglia of control and mutant mice: the mean
perimeters per A2A positive grain in TOR1a+/+ versus TOR1a+/− were, respectively,
(two tailed
T test): in the striatum, 2.06 ± 0.19 > 1.62 ± 0.08 SEM µm (n = 13, t = 2.077 df = 24,
−
p = 0.04); in the globus pallidus, 2.342 ± 0.14 > 1.378 ± 0.06 SEM µm (n = 15, t = 6.088 df = 28,
p = 0.0001); and in the entopeduncular nucleus, 4.938 ± 0.39 > 1.61 ± 0.08 SEM µm (n = 14,
t = 8.238, df = 26, p = 0.0001). However, the fluorescence intensity of the A2A positive
grains was comparable in the correspondent areas of TOR1a+/+ versus TOR1a+/− (data
not shown).
2.5. Colocalization of A2A in Cholinergic Neurons
Finally, we evaluated whether any change also occurs in the expression of A2A
receptors in the striatal choline-acetyltransferase (ChAT) positive cholinergic interneurons
of mutant Tor1a+/− mice. Double-labeling fluorescence was obtained, with ChAT reactive
large neuronal bodies and thick primary dendrites marked in red (Figure 6B,E), and A2A
receptor positive grains marked in green in the neuropil (Figure 6A,D). In the merged
ChAT/A2A images, A2A receptors appeared as yellow grains uniformly diffused on
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ChAT neuronal bodies and thick dendrites (Figure 6C,F), but absent in the white matter
bundles appearing as black holes in the microscopic field (Figure 6A,C,D,F), confirming the
specificity of A2A receptor staining on neurons. Compared to Tor1a+/+ mice (Figure 6C),
in Tor1a+/− the A2A receptor grains (Figure 6F) appeared markedly more numerous on
the surface of ChAT neurons (yellow grains) and in the surrounding neuropil (green grains).
Figure 5. Comparative densitometric analysis of the number (n◦ ) of A2A positive grains per microscopic field in caudate–putamen (CP), globus pallidus (GP), and entopeduncular nucleus (EN) of
− − mice. One-way ANOVA, ** p < 0.02.
control Tor1a+/+ and of mutant Tor1a+/
−
−
Figure 6. Localization of A2A receptors in cholinergic striatal interneurons. Representative confocal
laser scanning microscopy images double-labeled with antibody for anti A2A receptors and with
antibody anti-ChAT for cholinergic interneurons. A2A receptors are visualized in green-cy2 fluorescence (A,D); ChAT positive neurons are visualized in red-cy3 fluorescence (B,E); merged images
in C, F with A2A positive grains visualized in yellow on ChAT-positive interneurons, and in green
in the neuropil. Cell nuclei are visualized by DAPI fluorescence in blue. (A–C) Tor1a+/+ mice;
(D–F) Tor1a+/− mice. Scale bar in F = 50 µm.
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The overlapping areas of A2A receptors on striatal ChAT neuronal bodies were
36.37 + 2.92 SEM µm2 (neuronal bodies n = 12 from 3 mice) in Tor1a+/+ mice, and
47.44 + 2.98 SEM µm2 (neuronal bodies n = 13 from 3 mice) in Tor1a+/− mice (unpaired
two-tailed T test, t = 2.645, df = 23, p = 0.014).
3. Discussion
In our study, different techniques demonstrate specific changes in A2A receptors
in the basal ganglia circuits of Tor1a+/− mice, with increased or decreased expression,
respectively, in the striatal-pallidal complex and entopeduncular nucleus. We briefly
discuss the limits of our morpho-chemical study, and the possible relevance of A2A changes
to the pathophysiology of dystonia in this DYT1 animal model.
The high specificity of the polyclonal antibody was demonstrated in the Western blot
analysis, with a selective band corresponding to the 45 kDa migration level of A2A receptor
monomers [27], and by the well-defined immunofluorescent reaction product, configuring
the sharp morphology of A2A receptor aggregates without surrounding diffuse background
staining. Moreover, our Western blot analysis and immunohistochemical method were
extremely sensitive, also detecting the low level of A2A receptors in the entopeduncular
nucleus of control Tor1a+/+ mice, and even their decrease in this nucleus, contemporary
to the opposite changes of A2A expression in the contiguous striatal-pallidal complex on
the same tissue section of Tor1a+/− mice.
3.1. Morphological Characteristics of A2A Receptor Aggregates
In previous studies with electron microscopy, the A2A labeling in the striatum was
most commonly localized in dendrites and dendritic spines, in fewer amounts in axon
terminals and in very low levels in neuronal soma and glia, whereas in the external
globus pallidus (GP), the A2A labeling was mainly presynaptic [28,29]. In our study, the
immunofluorescence A2A aggregates appeared arranged in a homogenous spot, with a
diameter between 0.3 and 0.4 µm, which approaches the dimensions and morphology of
synaptic complexes [30]. However, we cannot define the subcellular localization of the
A2A aggregates at synaptic and extra synaptic levels since we cannot detect anything but
the fluorescent A2A signals, losing the surrounding subcellular structures configuring
the synaptic complex as detected in electron microscopy. Instead, the advantage of the
immuno-fluorescence technique and confocal microscopy with high power objective is that
they can allow at a time detection of A2A aggregates, approaching synaptic size at cellular
level, as well as their distribution in the tissue in a relatively large microscopic field.
3.2. Distribution of A2A Aggregates
The high distribution of adenosine A2A receptors in the striatum and globus pallidus
of control Tor1a+/+ mice and their selective increase in the same areas of Tor1a+/− mice
suggest a major role of A2A within the indirect pathway for basal ganglia physiology
and dystonia pathophysiology. However, it is worth noting that besides the selective
postsynaptic localization of A2A receptors on striatal medium spiny enkephalin neurons
in the indirect pathway [25], a morphological segregation of A2A receptors has been
demonstrated at presynaptic levels in glutamatergic terminals that make synapses on
striatal medium spiny neurons of the direct pathway, where they exert a selective facilitator
modulation of cortico-striatal neurotransmission [31]. Further studies should investigate
the localization of A2A receptors at synaptic and extra-synaptic levels in dystonic mice,
clarifying the distinct changes of A2A receptors in basal ganglia sub-regions.
Our immuno-fluorescence technique and confocal microscopy with high power objective demonstrate the increased distribution density of A2A in Tor1a+/− mice in the
neuropil, likely targeting dendrites and spines in the striatum and globus pallidus, as
well as on the soma of the striatal projecting neurons, striatal cholinergic interneurons,
and neurons in the globus pallidus. The widespread increase in A2A receptors in the
striatal-pallidal complex may have functional relevance, affecting acetylcholine release in
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the striatum and regulating neuronal excitability and plasticity on cortico-striatal glutamatergic terminals, on GABAergic projecting neurons, and on GABAergic terminals in the
globus pallidus [21,25,31,32].
Moreover, the light A2A expression generally observed in the entopeduncular nucleus of Tor1a+/+ mice [33] and its significant decrease in Tor1a+/− mice, detected with
different techniques, cannot be considered functionally irrelevant, and this suggests that
A2A receptors in the direct pathway could also be involved in basal ganglia function and
in dystonia pathophysiology. We hypothesize that the increased expression of A2A in
the striatum and in the globus pallidus is likely involved in increased inhibitory GABA
input at striatal-pallidal synapses demonstrated in globus pallidus in DYT1 dystonia [32],
disinhibiting the subthalamic nucleus and its excitatory glutamatergic input to the entopeduncular nucleus [34]. In this scenario, the reduced A2A expression in the entopeduncular
nucleus may be viewed, at least in part, as an adaptive mechanism to regulate increased
glutamatergic input from the subthalamic nucleus to basal ganglia output. Future studies
should investigate the elaborated role of pre- and postsynaptic A2A receptors in the glutamatergic synapses of the direct- and indirect pathway neurons, respectively, on GABAergic
medium spiny neurons’ soma and dendrites in the striatum, and of their terminals in the
entopeduncular nucleus and globus pallidus in the pathophysiology of dystonia. While
the numbers of A2A positive dots were either increased or decreased, respectively, in the
striatal-pallidal complex and entopeduncular nucleus of Tor1a+/− mice, at the same time,
the size of A2A positive dots was significantly decreased in all three structures. Such a
decrease in the size of A2A receptor aggregates could be associated with the contemporary
decrease in the D2 synapses [18], and the consequent deficiency in A2A/D2 colocalization
and the formation of heteromers, at least in the striatum. The functional relevance of such
A2A microstructural change should be checked in the direct and indirect pathway since, in
general, the efficiency of neuronal connectivity is directly related to the synapses’ size [35].
Further studies should investigate whether the opposite changes in the numbers of D2
and A2A receptors in dystonic mice could also compromise the formation of D2/A2A
etheromers, changing their interaction not only quantitatively but also qualitatively.
3.3. Changes in Second Messenger cAMP
To verify whether the tissue levels of cAMP are influenced by the changes in A2A
expression, we evaluated the basal content of cAMP in tissue homogenates of the different
basal ganglia nuclei from Tor1a+/+ and Tor1a+/− knock-out mice. Surprisingly, we found
significantly increased cAMP levels only in the striatum of Tor1a+/−, whereas in the
globus pallidus and in the entopeduncular nucleus, cAMP levels were similar in Tor1a+/+
and in Tor1a+/− mice. The cAMP synthesis in striatal neurons is stimulated by dopamine
D1 receptors, which are significantly decreased in DYT1 transgenic mice [36], and by A2A
receptors, which are significantly increased in Tor1a+/− mice (present work), and so likely
determining the increased striatal cAMP levels in the Tor1a+/− striatum.
To explain the discrepancy between the higher levels of cAMP in the Tor1a+/−
striatum and the unchanged cAMP levels in the globus pallidus and entopeduncular
nucleus, we should consider the interference of other related factors in addition to the
increased or decreased stimulating action of the A2A receptors. We can hypothesize that
the A2A receptor dependent cAMP synthesis is out of the inhibitory control of the deficient
D2 receptors, but the D2 receptors appear to be functionally impaired both in the striatum
as well as in the globus pallidus [8,32].
In a previous work, we reported that PDE10A-dependent cAMP hydrolyzing activity
as well as total PDE activity are unaffected in the striatum of a mutant mouse model of DYT1
dystonia, wherein at the same time the PDE10A-dependent cAMP hydrolyzing activity
was increased in the globus pallidus and decreased in the entopeduncular nucleus [37].
Therefore, we could hypothesize that in the mutant DYT1 striatum, an insufficient total PDE
activity cannot timely catabolize the A2A dependent increased cAMP synthesis, whereas
the equilibrium between cAMP synthesis and catabolism is preserved in the globus pallidus
Int. J. Mol. Sci. 2021, 22, 2691
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and in the entopeduncular nucleus by a contemporary increase or decrease in PDE activity,
respectively. The reasons for the discrepant compensatory PDE activity in front of A2Adependent cAMP synthesis in the striatum of mutant DYT1 mice are unknown. We can
just speculate on the different cellular distributions of the PDE10A isoform in different
striatal neurons.
Indeed, PDE10A is selectively expressed in medium spiny neurons, but not in striatal
interneurons, and in particular it is absent in cholinergic interneurons [38,39]. A2A receptors on cholinergic interneurons play a crucial role in striatal network function, activating
the release of acetylcholine in the striatum, and striatal cholinergic dysfunction has been
shown to have a widespread role in the pathophysiology of dystonia [21,40]. Therefore, we
can hypothesize that the increased levels of cAMP in the striatum may reflect, at least in
part, the A2A-dependent cAMP synthesis in cholinergic interneurons, wherein cAMP is
not hydrolysable by the absent PDE10A, nor adequately hydrolyzed by another unknown
phosphodiesterase isoform.
Anyway, the relevant impact of cAMP on the membrane excitability of striatal mediumsized spiny neurons and cholinergic interneurons has been widely described [41–43]. Our
observation of a chronic elevation in neuronal cAMP in the striatum of Tor1a+/− can
open a new window to investigate the role of cAMP changes in the severely altered
synaptic plasticity of medium spiny neurons and cholinergic interneurons in dystonia
mouse models [8,11–16,40,44].
3.4. Changes in A2A mRNA
Finally, to evaluate whether the changes in A2A expression in Tor1a+/− mice are
regulated at translational or post-translational levels, we used the sensitive real-time PCR
technique analysis of A2A mRNA in basal ganglia. According to previous studies, the
A2A mRNA can be found only on the striatum in cell bodies of medium spiny striatal
neurons and occasionally in dendrites, coding for the synthesis of A2A receptors, which
would be transported to the terminals of striatal-pallidal efferent neurons till the globus
pallidus [45]. Within the basal ganglia, we detected significant levels of A2A mRNA only
in the striatum of Tor1a+/+ that were similar to A2A mRNA levels in the striatum of
Tor1a+/−, suggesting that the increased A2A expression in the Tor1a+/− striatum can
be regulated at post-translational levels. However, the striatal results of A2A mRNA
should be kept with caution since in tissue homogenates we cannot distinguish eventual
differentiated A2A mRNA levels in different classes of striatal neurons either to the direct or
indirect pathway, obtaining on the whole apparently unchanged striatal A2A mRNA levels.
Instead, a surprising A2A mRNA significant increase was observed in the globus
pallidus of Tor1a+/−, whereas A2A mRNA was detected just in a very low amount in the
globus pallidus of Tor1a+/+. Therefore, in Tor1a+/− it is likely that the globus pallidus is
coding for its own A2A receptors, and such A2A neo-expression may change, at least in
part, its dependent role from the striatal A2A inputs. The A2A mRNA changes in the basal
ganglia of Tor1a+/− appear to be specific, since in Parkinson’s disease A2A mRNA appears
either decreased in the striatum or unchanged in the globus pallidus [46]. Future studies
should clarify the cell types expressing A2A mRNA in the globus pallidus of Tor1a+/−
mice, and the function of their intrinsic A2A receptors.
4. Materials and Methods
4.1. Animals
C57BL/6 Tor1a+/− knock-out mice, which mimic the loss of function of the DYT1
dystonia Tor1a mutation [47], were bred at Santa Lucia Foundation Animal Facility. Control
Tor1a+/+, and Tor1a+/− knock-out mice were kept under an artificial day-night cycle,
with free access to food and water. Male control and mutant mice were sacrificed at
the age of 5–6 months. DNA was isolated and amplified from 1 to 2 mm tail fragments
with the Extract-N-Amp Tissue polymerase chain reaction (PCR) kit (XNAT2 kit; SigmaAldrich Merck Life Science, Milan, Italy), and genotyping was performed as previously
Int. J. Mol. Sci. 2021, 22, 2691
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reported [17]. All the efforts were made to minimize the number of animals utilized and
their suffering. Treatment and handling of mice were carried out in compliance with both
the European Council and Italian guidelines (2010/63EU, 20 October 2010; D.L. 26/2014,
4 March 2014; 86/609/EEC, 24 November 1986; D.L. 116/1992—27 January 1992), according
to experimental protocols approved by the Animal Ethics Committee of the University of
Rome Tor Vergata (D.M. 153/2001-A, 9 April 2001, and 43/2002-A, 4 May 2002) and by the
IRCCS Santa Lucia Foundation Animal Care and Use Committee (D.M.9/2006-A, 2006),
and authorized by the Italian Ministry of Health (authorization 223/2017-PR, May 2017).
For biochemical studies, the animals were killed by cervical dislocation, and the brains
were removed rapidly and placed on an ice-cold plate. Thick brain sections were cut with
an Oxford vibratome, and the caudate–putamen, globus pallidus and entopeduncular
nucleus were dissected out rapidly from both hemispheres under a stereomicroscope, and
promptly frozen in liquid nitrogen and stored at −80 ◦ C [48].
For morphological studies, the animals were deeply anesthetized with tiletamine/
zolazepam (80 mg/kg) and xylazine (10 mg/kg) and perfused trans-cardially with
1% heparin in 50 mL 0.1 M sodium phosphate buffer (PBS), and with 250 mL 4%
para-formaldehyde in 0.1 M in PBS (pH 7.4). The brains were removed immediately
and post-fixed in the same fixative solution overnight at 4 ◦ C; then they were equilibrated with 30% sucrose overnight, and finally frozen in liquid nitrogen and stored at
−80 ◦ C [48].
4.2. Quantitative Analysis of A2A Protein
The quantitative analysis of A2A expressions in basal ganglia was assessed by Western blotting. Tissues were lysated in 20 mM Tris-HCl buffer pH 7.2 containing 0.2 mM
EGTA, 5 mM MgCl2, 0.1% v/v Triton X-100, 5 mM β–mercaptoethanol, 1 mM PMSF and
2% v/v antiprotease cocktail (Sigma–Aldrich Merck Life Science, Milan, Italy). Thirty
micrograms of proteins were loaded on a 9% SDS polyacrylamide gel and subjected to
electrophoresis under a reducing condition. The proteins were then transferred to a nitrocellulose membrane (Bio–Rad, Milan, Italy). The blots were incubated overnight at
4 ◦ C with a rabbit polyclonal anti–A2A receptor antibody (1:1000; BML–SA654, Enzo Life
Sciences, Farmingdale, NY, USA), or mouse anti-β-actin (1:10,000; Sigma-Aldrich Merck
Life Science, Milan, Italy) as a reference standard. A2A receptors-reactive bands were
revealed by horseradish peroxidase-conjugated secondary antibodies (1:10,000, Jackson
Immunoresearch, Cambridgeshire, UK), incubated in a lumi-light-enhanced chemiluminescence substrate (Bio-Rad, Milan, Italy), and exposed to Chemidoc (Bio–Rad, Milan, Italy).
Densitometric analysis of scanned blots was performed using the NIH ImageJ version l.29
program (NIH, Bethesda, MD, USA).
4.3. cAMP Measurement
The frozen samples were immediately submerged in 0.1 M HCl, and rapidly homogenized. The homogenates were centrifuged at 13,000× g for 30 min at room temperature
and the supernatants were acetylated, according to the instructions of the commercial kit
manufacturer (Direct EIA kit, Enzo Life Sciences, Farmingdale, NY, USA). The kit uses a
polyclonal antibody to cAMP, which in a competitive manner binds the cyclic nucleotides
in the sample, or cyclic nucleotides conjugated with alkaline phosphatase molecules added
to the incubation medium. The samples were incubated for 2 h at room temperature, and
thereafter the substrate p-nitrophenyl phosphate (pNPP) was added for 1 h to reveal the
residual alkaline phosphatase activity in the medium, which generates a yellow product,
readable on a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Fisher
Scientific, Monza, Italy) at 405 nm. The intensity of the yellow color in the medium is
inversely proportional to the concentration of cyclic nucleotides in the samples and in
comparative standards.
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4.4. RNA Extraction and Real–Time PCR
Total RNA was prepared using the TRI Reagent (Sigma-Aldrich Merck Life Science,
Milan, Italy), according to the manufacturer’s instructions, and quantified with a NanoDrop
1000 spectrophotometer (Thermo Fisher Scientific, Monza, Italy). One microgram of RNA
was reverse-transcribed using the QuantiTect reverse transcription kit (Qiagen, Milan, Italy).
Real-time PCR was performed on reverse-transcription products with the PowerUp™
SYBR™ Green Master Mix in the QuantStudio 3 Real-Time PCR System (Thermo Fisher
Scientific, Monza, Italy). Thermal cycling conditions consisted of an initial denaturation
step at 95 ◦ C for 2 min, followed by 40 cycles at 95 ◦ C for 15 s, 60 ◦ C for 15 s, and
72 ◦ C for 1 min. The threshold cycle (Ct) (defined as the fractional PCR cycle number at
which fluorescence reaches 10 times the baseline SD) was used for comparison analysis.
The 2−∆∆Ct method was used to evaluate the relative expression ratio for A2A receptors
compared with the internal control gene β–actin. The following sequences of primers
were used: A2A receptors (a) forward 5′ –TCTTCTTCGCCTGCTTTGTCC–3′ , (b) reverse 5′ –
GCCCTCATACCCGTCACCA–3′ ; β-actin (a) forward 5′ –GCGCAAGTACTCTGTGTGGA–
3′ , (b) reverse 5′ –AAGGGTGTAAAACGCAGCT–3′ .
4.5. A2A Immunofluorescence in Confocal Microscopy
Coronal brain sections (40 µm thick) were cut with a freezing microtome. Tissue
sections were then incubated for 1 h at room temperature in 10% donkey serum solution in
PBS 0.25%–Triton X-100 (PBS-Tx). The sections were incubated with the primary rabbit
anti-A2A antibody (BML–SA654, Enzo Life Sciences, Farmingdale, NY, USA) 1:200, 3 days
at 4 ◦ C; thereafter, the sections were incubated with cyanine 3 (cy3) conjugated secondary
antibody (Jackson Immuno Research, Cambridgeshire, UK) 1:200, 2 h at room temperature.
For negative controls, representative sections were processed with the omission of the
primary or of the secondary antibody. Cell nuclei were detected with a blue-fluorescent
DNA stain by 4′ ,6–diamidino–2–phenylindole (DAPI) (D9542, Sigma-Aldrich Merck Life
Science, Milan, Italy). After washout, tissue sections were mounted on plus polarized glass
slides with Vectashield mounting medium (Super Frost Plus, Thermo Fisher Scientific,
Monza, Italy) and cover-slipped.
Fluorescent images were acquired with a LSM700 Zeiss confocal laser scanning microscope (Zeiss, Oberkochen, Germany), with a 5×, a 20× objective, or a 63× oil immersion
lens (1.4 numerical aperture) with an additional digital zoom factor (1×–1.5×–2×) under
no saturation conditions. Single-section images (1024 × 1024) or z-stack projections in the
z-dimension (z-spacing, 1 µm) were collected. Z-stack images were acquired to analyze the
whole neuronal soma, which spans multiple confocal planes. The confocal pinhole was
kept at 1, the gain and the offset were adjusted to prevent saturation of the brightest signal
and sequential scanning for each channel was performed. The confocal settings, including
laser power, photomultiplier gain, and offset, were kept constant for each marker.
A negative staining was obtained omitting the primary or the secondary antibody.
The 5× and 20× objectives were used to define areas of interest in the dorsolateral striatum,
globus pallidus, and entopeduncular nucleus; the distribution of A2A receptors was first
acquired using 20× objective with an additional digital zoom factor (1×–1.5×–2×), and
thereafter 63× oil immersion objective (1.4 numerical aperture).
For quantitative analysis of the number of A2A positive spots per microscopic field
and of their perimeters, images were collected from at least 3–4 slices per animal, processed
simultaneously from each basal ganglia nucleus (n ≥ 4 mice/genotype), and exported for
analysis with ImageJ software (NIH, Bethesda, MD, USA). Software background subtraction was utilized to reduce noise.
4.6. A2A Localization in Striatal Cholinergic Interneurons
A double immunofluorescent staining for choline-acetyltransferase (ChAT) and A2A
was used to evaluate the colocalization of A2A receptors in striatal cholinergic interneurons.
Brain sections were incubated with goat anti-ChAT (Nova biological, CA, USA) and rabbit
Int. J. Mol. Sci. 2021, 22, 2691
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anti-A2A receptors (BML–SA654, Enzo Life Sciences, Farmingdale, NY, USA). Both primary
antibodies were used at a 1:200 dilution, in 0.1 M PBSS containing 0.3% Triton X–100 for
72 h at 4 ◦ C. Sections were then rinsed three times for 5 min at room temperature and
subsequently incubated with cyanine 2 (Cy2) and cyanine 3 (cy3) conjugated secondary
antibodies (Jackson Immuno Research, Cambridgeshire, UK) for 2 h at room temperature
at 1:200 dilution in a 0.1 M PB solution containing 0.3% Triton X–100. For negative controls,
representative sections were processed with the omission of the primary or of the secondary
antibodies. Cell nuclei were detected with a blue-fluorescent DNA stain DAPI (D9542,
Sigma-Aldrich Merck Life Science, Milan, Italy). Subsequently, sections were rinsed in PBS,
mounted on plus polarized glass slides with Vectashield mounting medium (Super Frost
Plus, Thermo Fisher Scientific, Monza, Italy) and cover-slipped.
The sections were preliminary examined under an epi-illumination fluorescence
microscope (Zeiss Axioskop 2, Oberkochen, Germany). Confocal laser scanner microscopy
(Zeiss LSM800, Oberkochen, Germany) was used to acquire immunofluorescent images as
reported in the previous subsection.
Immunofluorescence intensity and colocalization analysis were evaluated by using
the Java image processing and plugin analysis program included in Fiji ImageJ (NIH,
Bethesda, MD, USA). To quantify the density of the A2A specific marker on a defined area,
the “overlapping signal” of the A2A signal on ChAT neuronal bodies was calculated.
4.7. Experimental Design and Statistical Analysis
Biological samples and microscopic fields were randomly selected from the basal
ganglia areas of the two experimental groups. All data were initially entered into an
Excel database (Microsoft, Redmond, WA, USA) and the analysis was performed using the
Statistical Package for the Social Sciences Windows, version 15.0 (SPSS, Chicago, IL, USA).
Descriptive statistics consisted of the mean ± standard deviation (SD) for parameters with
gaussian distributions (after confirmation with histograms and the Kolgomorov–Smirnov
test), or of the mean ± standard error mean (SEM). Comparisons were performed in compliance with data characteristics either with the Paired Samples Test, or ANOVA one-way
or ANOVA two factors and multiple comparisons by Bonferroni test. A p value of < 0.05
was considered statistically significant.
5. Conclusions
The increased expression of A2A receptors in the striatal-pallidal complex of Tor1a+/−
knock-out mice may suggest a gaining of function of such receptors, overwhelming the D2
loss of function in the indirect pathway; moreover, the associated decrease in A2A expression in the entopeduncular nucleus may be functionally significative. Future studies should
clarify the relevance of A2A changes in the direct and indirect pathway in dysregulating
the basal ganglia network in dystonia pathophysiology.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006
7/22/5/2691/s1. Figure S1—Image of Western blot analysis of A2A receptors in brain tissue.
Author Contributions: V.D. and M.G. designed research, performed research, and were involved in
drafting the manuscript; E.P., S.C. and I.S. performed research; R.S. statistically analyzed data; F.R.F.
and A.P. interpreted data and critically revised the manuscript; S.B. resources; S.B., G.M., N.B.M.
critically revised the manuscript; G.S. conceived research, interpreted data and wrote the paper. All
authors have read and approved the final version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and of the European Council (2010/63EU, 20 October 2010), approved by
the Institutional Ethics Committee of University of Rome Tor Vergata (43/2002-A, 4 May 2002), and
of the IRCCS Santa Lucia Foundation Animal Care and Use Committee (D.M.9/2006-A, 2006), and
authorized by the Italian Ministry of Health (authorization 223/2017-PR, May 2017).
Int. J. Mol. Sci. 2021, 22, 2691
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Informed Consent Statement: Not applicable.
Data Availability Statement: Data and methods used in the research have been presented in sufficient detail in the paper. Additional data can be requested to the corresponding author.
Acknowledgments: We kindly acknowledge Graziano Bonelli for excellent technical assistance, and
James Lynch for style reviewing.
Conflicts of Interest: The authors declare no competing financial interests.
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