Motor Deficits and Decreased Striatal Dopamine Receptor 2 Binding Activity in the Striatum-Specific Dyt1 Conditional Kno...

MISSING IMAGE

Material Information

Title:
Motor Deficits and Decreased Striatal Dopamine Receptor 2 Binding Activity in the Striatum-Specific Dyt1 Conditional Knockout Mice
Physical Description:
Serial
Creator:
Yokoi, Fumiaki
Dang, Mai Tu
Li, Jianyong
Standaert, David G.
Li, Yuquing
Publisher:
PLoS ONE
Publication Date:

Subjects

Genre:
serial   ( sobekcm )

Notes

Abstract:
DYT1 early-onset generalized dystonia is a hyperkinetic movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Recently, significant progress has been made in studying pathophysiology of DYT1 dystonia using targeted mouse models. Dyt1 DGAG heterozygous knock-in (KI) and Dyt1 knock-down (KD) mice exhibit motor deficits and alterations of striatal dopamine metabolisms, while Dyt1 knockout (KO) and Dyt1 DGAG homozygous KI mice show abnormal nuclear envelopes and neonatal lethality. However, it has not been clear whether motor deficits and striatal abnormality are caused by Dyt1 mutation in the striatum itself or the end results of abnormal signals from other brain regions. To identify the brain region that contributes to these phenotypes, we made a striatum-specific Dyt1 conditional knockout (Dyt1 sKO) mouse. Dyt1 sKO mice exhibited motor deficits and reduced striatal dopamine receptor 2 (D2R) binding activity, whereas they did not exhibit significant alteration of striatal monoamine contents. Furthermore, we also found normal nuclear envelope structure in striatal medium spiny neurons (MSNs) of an adult Dyt1 sKO mouse and cerebral cortical neurons in cerebral cortex-specific Dyt1 conditional knockout (Dyt1 cKO) mice. The results suggest that the loss of striatal torsinA alone is sufficient to produce motor deficits, and that this effect may be mediated, at least in part, through changes in D2R function in the basal ganglia circuit.
Citation/Reference:
Yokoi F, Dang MT, Li J, Standaert DG, Li Y (2011) Motor Deficits and Decreased Striatal Dopamine Receptor 2 Binding Activity in the Striatum-Specific Dyt1 Conditional Knockout Mice. PLoS ONE 6(9): e24539. doi:10.1371/journal.pone.0024539
Funding:
This work was supported by National Institutes of Health grants (NS37409, NS47466, NS47692, NS54246, NS57098, NS65273, NS72782, and NS74423) and startup funds from the Lucille P. Markey Charitable Trust and Beckman Institute (UIUC), Department of Neurology (UAB), and Tyler’s Hope for a Dystonia Cure, Inc. (UF). Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Record Information

Source Institution:
University of Florida Institutional Repository
Holding Location:
University of Florida
Rights Management:
All rights reserved by the source institution and holding location.
System ID:
AA00005007:00001


This item is only available as the following downloads:


Full Text


OPEN a ACCESS Freely available online


Motor Deficits and Decreased Striatal Dopamine

Receptor 2 Binding Activity in the Striatum-Specific Dytl

Conditional Knockout Mice

Fumiaki Yokoi', Mai Tu Dang2, Jianyong Li3, David G. Standaert4, Yuqing Li'*
1 Department of Neurology, College of Medicine, University of Florida, Gainesville, Florida, United States of America, 2The Children's Hospital of Philadelphia,
Philadelphia, Pennsylvania, United States of America, 3 Department of Biochemistry, Virginia Tech, Blacksburg, Virginia, United States of America, 4 Department of
Neurology, School of Medicine, Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, Alabama, United States
of America


Abstract
DYT1 early-onset generalized dystonia is a hyperkinetic movement disorder caused by mutations in DYTI (TOR1A), which
codes for torsinA. Recently, significant progress has been made in studying pathophysiology of DYT1 dystonia using
targeted mouse models. Dytl AGAG heterozygous knock-in (KI) and Dytl knock-down (KD) mice exhibit motor deficits and
alterations of striatal dopamine metabolisms, while Dytl knockout (KO) and Dytl AGAG homozygous KI mice show
abnormal nuclear envelopes and neonatal lethality. However, it has not been clear whether motor deficits and striatal
abnormality are caused by Dytl mutation in the striatum itself or the end results of abnormal signals from other brain
regions. To identify the brain region that contributes to these phenotypes, we made a striatum-specific Dytl conditional
knockout (Dytl sKO) mouse. Dytl sKO mice exhibited motor deficits and reduced striatal dopamine receptor 2 (D2R) binding
activity, whereas they did not exhibit significant alteration of striatal monoamine contents. Furthermore, we also found
normal nuclear envelope structure in striatal medium spiny neurons (MSNs) of an adult Dytl sKO mouse and cerebral
cortical neurons in cerebral cortex-specific Dytl conditional knockout (Dytl cKO) mice. The results suggest that the loss of
striatal torsinA alone is sufficient to produce motor deficits, and that this effect may be mediated, at least in part, through
changes in D2R function in the basal ganglia circuit.

Citation: Yokoi F, Dang MT, Li J, Standaert DG, Li Y (2011) Motor Deficits and Decreased Striatal Dopamine Receptor 2 Binding Activity in the Striatum-Specific
Dyt1 Conditional Knockout Mice. PLoS ONE 6(9): e24539. doi:10.1371/journal.pone.0024539
Editor: Izumi Sugihara, Tokyo Medical and Dental University, Japan
Received May 4, 2011; Accepted August 12, 2011; Published September 12, 2011
Copyright: 2011 Yokoi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants (NS37409, NS47466, NS47692, NS54246, NS57098, NS65273, NS72782, and NS74423)
and startup funds from the Lucille P. Markey Charitable Trust and Beckman Institute (UIUC), Department of Neurology (UAB), and Tyler's Hope for a Dystonia Cure,
Inc. (UF). Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
E-mail: yuqing.li@neurology.ufl.edu


Introduction

Dystonia is a movement disorder that is exhibited by
involuntary, repetitive, sustained muscle contractions or abnormal
postures [1]. Dystonia is classified into two groups, primary and
secondary dystonia. Primary dystonia develops spontaneously in
the absence of any apparent cause or associated disease.
Secondary dystonia is caused by other diseases, such as Parkinson's
or Huntington's diseases, brain injury, or drug side effects. Genetic
dystonia belongs to the primary dystonia group and is classified
into more than 20 types, hll..J.. 1 less than half of them have
known gene mutations [2]. DYT1 early-onset generalized torsion
dystonia is an inherited movement disorder and is caused by
mutations in DYT1 (TOR1A) coding for torsinA with about 30%
penetrance [3]. TorsinA is a member of AAA+ family of ATPases
and may work in t.itfi kin,; of polytopic membrane proteins and
protein processing in the secretary pathway [4,5]. ATPase activity
[6,7] and molecular chaperon activity of torsinA [8] were also
reported in vitro. Nearly all DYT1 dystonia patients have a 3 base-
pairs deletion (AGAG) in DYT1 in one allele, corresponding to a
loss of a glutamic acid residue in the C-terminal region of torsinA


PLoS ONE www.plosone.org


[3], while an 18 bp deletion mutation [9] and an Arg288Gln
missense mutation [10] have been reported. A frame shift
mutation caused by 4 bp-deletion coding for the C-terminal
region of torsinA was also reported in a possible myoclonus-
dystonia patient [11]. Dyt1 AGAG heterozygous KI male mice
exhibit reduction of striatal torsinA and motor deficits [12-14].
Dyt1 KD male mice and Dyt1 cKO mice also exhibit motor
deficits, suggesting loss of torsinA function may contribute to
motor deficits in these Dyt1 mutant mice [15,16]. Ampicillin-
injected Dytl AGAG heterozygous KI male mice express normal
level of striatal torsinA and exhibit normal motor performance,
suggesting recovery of torsinA level in the striatum may rescue the
motor deficits in Dyt1 AGAG heterozygous KI male mice [13].
Alil.....1. DYT1 dystonia patients do not respond to levodopa
treatment in most cases, there are several reports suggesting
functional alterations in the striatal dopaminergic system in DYT1
dystonia. A postmortem dopamine (DA) content analysis in a
DYT1 dystonia patient brain suggested a reduced DA in rostral
portions of the putamen and caudate nucleus [17]. Another
postmortem study reported a reduced dopamine receptor binding
in the striatum and increased striatal 3,4-dihydroxyphenylacetic


September 2011 1 Volume 6 1 Issue 9 | e24539


*0*PLoS one






Striatum-Specific Dytl Conditional KO Mice


acid (DOPAC)/DA ratio in DYT1 dystonia patients [18]. A
positron emission tomography study suggested that striatal D2R
availability is reduced in both manifesting and non-manifesting
DYT1 mutation carriers [19]. Dytl AGAG heterozygous KI male
mice exhibit reduced homovanillic acid (HVA) level in the
striatum [12] and Dytl KD mice show decreased level of striatal
DOPAC [15]. Moreover a transgenic mouse model overexpress-
ing human mutant torsinA derived by human cytomegalovirus
(CMV) immediate early promoter exhibit a reduction of D2R in
the striatum [20]. These reports suggested that functional
alterations of the striatal dopaminergic system may contribute to
the ]. ii.... .. of DYT1 dystonia, while its precise functional
role has not been well defined. For example, it is not known
whether motor deficits and alterations of the striatal dopaminergic
system are caused by the mutation in the striatum itself or end
results of abnormal signals from other brain regions.
Abnormal nuclear envelopes have been reported in transfected
cells over-expressing the mutant forms of torsinA [21-23], Dytl
KO mice and Dytl AGAG homozygous KI mice exhibiting
neonatal lethality [24]. Both transgenic mice overexpressing
human WT torsinA and mutant torsinA using murine prion
promoter exhibit abnormal nuclear envelopes and abnormal
motor performance [25]. It was suggested that neuron-specific
nuclear envelope abnormality in Dytl AGAG homozygous KI
mice is caused by malfunction of torsinA with incomplete
compensation by torsinB which is weakly expressed in neurons
[26]. However, such abnormality has not been found in Dytl
AGAG heterozygous KI mouse or DYT1 dystonia patient brains,
casting doubts about its role in the. 1 1.... ,,. ;. of DYT1 dystonia.
In the present study, we hypothesized that a selective loss of
torsinA function in the striatum may affect motor performance in
mice. We used cre-loxP technology [27] applied to mouse gene
recombination [28] to selectively inactivate Dytl in the striatum
using a I. ^ .- line that has restricted recombination in the
striatum [29]. Dytl sKO mice were made and their motor
performance, dopaminergic system, and the role of torsinA in
maintaining the nuclear envelope structures were analyzed. Since
abnormal nuclear envelope was reported in cerebral cortical
neurons in Dytl KO and Dytl AGAG homozygous KI mice, we
further analyzed nuclear envelopes in cerebral cortical neurons of
Dytl cKO mice [16].

Results

Making of Dytl sKO mice
K. ^ .- and Dytl loxP double heterozygous mice were prepared
by crossing Dytl loxP mice [16] and i. mice [29]. Dyt1 sKO
mice were prepared by crossing I. ^ .- Dytl loxP double
heterozygous mice and Dytl loxP mice (Fig. 1A). Genotyping was
performed by multiplex PCR with tail DNA (Fig. 1B). Dytl sKO
mice were born according to Mendelian ratio and developed to
adult. The striatum-specific deletion ofDytl exons 3 and 4 in Dytl
sKO mice was confirmed by PCR using DNA isolated from each
brain region (Fig. 1C).

No overt abnormal postures and normal locomotion in
Dytl sKO mice
When suspended from the tail, both Dytl sKO and Control
littermate (CT) mice showed normal splaying of hindpaws and had
no observable hindpaw extension or truncal arching. All mice
exhibited strong righting reflexes when tipped on their side. The
results suggest that Dytl sKO mice had no overt abnormal
postures.


1 2 3 4 5
Dytl IoxP 0 _-


, Rgs9-cre


Dytl sKO

(striatum)


- 4-


1 2 3 4 5 6


cre --
IoxP -b
WT "





0





(bp) CT sKO
700 -
600 -
500 -
4-.A
400 -

300 -


Figure 1. Making of Dytl sKO mice. (A) The breeding strategy to
generate Dytl sKO mice. Dytl loxP mice [16] and Rgs9-cre mice [29]
were prepared as described earlier. Rgs9-cre Dytl loxP double
heterozygous mice were prepared by crossing Dytl loxP mice and
Rgs9-cre mice. Dytl sKO mice were prepared by crossing Rgs9-cre Dytl
loxP double heterozygous mice and Dytl loxP mice. The primer sites to
amplify Dytl exons 3 and 4-deleted locus were shown by an arrow pair
under the map. (B) A representative image of the multiplex PCR-based
genotyping. Top bands are PCR products of cre. Middle bands are PCR
products of Dytl loxP locus. The bottom bands are PCR products of Dytl
WT locus. Lanes 1,3, 5: Dytl loxP homozygous mice; Lanes 2, 7: Rgs9-cre
Dytl loxP double heterozygous mice; Lanes 4: Dytl sKO mouse; Lane 6:
Dytl loxP heterozygous mouse. (C) Tissue-specific deletion of Dytl
exons 3 and 4 in Dyt1sKO mice was confirmed by PCR using DNA
isolated from each brain region. The deletion was detected only in the
striatum of Dyt1sKO mouse as predicted (A). CT: control littermate
mouse.
doi:10.1371/journal.pone.0024539.g001


Since Dytl AGAG heterozygous KI male mice [12] and Dytl
KD male mice [15] exhibit moderate hyperactivities, and Dytl
cKO mice exhibit prominent hyperactivities in the open-field test
[16], spontaneous activities in Dytl sKO mice were assessed in the


September 2011 1 Volume 6 1 Issue 9 | e24539


= = 4


~, ~ PLoS ONE I www.plosone.org






Striatum-Specific Dytl Conditional KO Mice


open-field apparatus and compared to those in CT mice. Dytl
sKO mice did not exhibit significant differences in comparison to
CT mice in either horizontal locomotion (Fig. 2A; horizontal
activity, p = 0.55; B; total distance, p = 0.83; C; movement number,
p=0.54; D; movement time, p=0.91) or vertical locomotion
(Fig. 2E; vertical movement number, p = 0.87). There was no
significant difference in stereotypic activity (Fig. 2F; p = 0.45),
stereotypic movement time (Fig. 2G; p = 0.47), clockwise or anti-
clockwise revolution between Dytl sKO mice and CT mice
(Fig. 2H; p = 0.90 and p = 0.62, respectively). The results suggest
that loss of torsinA in the striatum alone does not affect
spontaneous locomotion.

Significant motor deficits of hindpaws in Dytl sKO mice
Motor performance was assessed by the accelerated rotarod and
beam-walking tests. Each mouse was put on the accelerated
rotarod and the latency to fall was measured. Since mice can hold
onto the rotarod with four paws, the latency to fall is an indicator
of total motor performance and shorter latency indicates motor
deficits. Dyt1 sKO mice did not show significant difference in
latency to fall (Fig. 3A; p = ( I suggesting no motor symptoms in
total motor performance with four paws. We further analyzed the
motor coordination and balance by the beam-walking test. Mice
were trained to transverse a medium square beam for two days.
The trained mice were tested twice on four different beams and
total numbers of hindpaw slips were analyzed. Dyt1 sKO mice
showed 144% more slip numbers in the beam-walking test (Fig. 3B;
p = 11 11 suggesting motor deficits in hindpaws.
In the accelerated rotarod test, Dytl sKO mice did not exhibit
overt motor deficits with four paws. The result is consistent with
other DYT1 dystonia mouse models. Dyt1 AGAG heterozygous


.4 4,000

S3,000
E 2,000

g 0 1,000

01
CT sKO


E




gC




30
S25
S20
15

25
10
0


2,000
2-1
1,500

"i 1,000

500

0
CT sKO


CT sKO


CT sKO


CT sKO


Figure 3. Motor performances in Dytl sKO mice and CT mice.
(A) Latency to fall in the accelerated rotarod test. Dytl sKO mice did not
exhibit significant difference in the latency to fall, suggesting no motor
deficits with four paws. (B) Beam-walking performances in Dytl sKO
mice and CT mice. The data in the CT mice were normalized to zero.
Dytl sKO mice showed significant increased slips numbers in beam-
walking test, suggesting motor deficits in hindpaws. Vertical bars
represent means standard errors. *p<0.05.
doi:10.1371/journal.pone.0024539.g003


KI mice [12], Dyt1 KD mice [15] or Dytl cKO mice [16] do not
exhibit significant motor deficits either in the accelerated rotarod
tests. On the other hand, Dytl sKO mice, Dytl cKO mice, Dyt1
AGAG heterozygous KI male mice and Dyt1 KD male mice


D


S300
250 8 300,
g 200 ,
E150 200,
EE 100 1
S 50,
0 0

CT sKO


200

150

E 100

50

0 0


CT sKO


CTsKO
CT sKO


15 C3 CT
1= m sKO



0

T sO C V
CT sKO CWREV ACWREV


Figure 2. Locomotion of Dytl sKO mice and CT mice in the open field test. Spontaneous activities in Dytl sKO mice were assessed in the
open-field apparatus and compared to those in CT mice. Dytl sKO mice did not exhibit significant differences in comparison to CT mice in horizontal
locomotion (A, horizontal activity; B, total distance; C, movement number; D, movement time) or vertical locomotion (E, vertical movement number).
There was no significant difference in stereotypic activity (F), stereotypic movement time (G), clockwise (H, CWREV) or anti-clockwise revolution (H,
ACWREV). Vertical bars represent means standard errors.
doi:1 0.1371/journal.pone.0024539.g002


SPLoS ONE I www.plosone.org 3 September 2011 1 Volume 6 1 Issue 9 | e24539






Striatum-Specific Dytl Conditional KO Mice


exhibit motor deficits in the beam-walking test. Since dystonic
symptoms commonly start from the legs in DYT1 dystonia
patients, the beam-walking test may be one of the most
appropriate behavior tests to detect the early motor symptoms in
DYTldystonia mouse models.

Decreased radioligand binding to D2R in the striatal
membrane fractions from Dytl sKO mice
We measured radioligand binding activity of [3H] YM-09151-2
to D2R in the striatal membrane fractions from three Dytl sKO
and three CT mice. To see the overall distribution of data,
representative saturation binding curves of [3H] YM-09151-2
were drawn using the transformed composite data of mean values
obtained for the three Dytl sKO (open circles) and three CT (solid
circles) mice (Fig. 4A). The fit lines in the representative Scatchard
plot were also created by least square means from the transformed
composite data of the mean values (Fig. 4B; Dytl sKO,

A


CQ
800


C? 600.
50 0

o 400


3- 200


0 0
200



B


2 6000

E 5000

4000

a 3000
LL
0 2000

o 1000

0


).0 0.2 0.4 0.6 0.8 1.0
Free (nM)


O CT
0 sKO


0 200 400 600 800

Bound (fmol/mg)

Figure 4. Radioligand binding activity to D2R in the striatal
membrane fractions. (A) A representative saturation binding curve of
[3H] YM-09151-2 to the striatal membranes fractions from three Dytl
sKO (open circles) and three CT (solid circles) mice. The transformed
composite data of mean values obtained for the three mice in each
genotype are plotted. (B) A representative Scatchard transformation of
the data. The fit lines were created by least square means from the
transformed composite data of mean values.
doi:10.1371/journal.pone.0024539.g004


r = 0.9788; CT, r = 0.9585) as described [30]. To analyze the
statistical significance of B,, and ]K, between Dytl sKO and CT
mice, B,, and K1 of each mouse were also individually analyzed in
Scatchard plot and those of each genotype were compared by
Student's t test as described [31]. The B,, value for [3H] YM-
09151-2 binding, reflecting levels of radioligand binding to D2R,
was ;_i... ,1 lower in Dytl sKO mice in comparison to CT
mice (means standard errors; Dytl sKO, 541 28 fmol/mg,
n = 3; CT, 793 46 fmol/mg, n = 3; p = 0.009). Receptor affinity
for the ligand as indicated by Kb, however, was unchanged in Dytl
sKO mice (Dytl sKO, 149- 46 pM, n= 3; CT, 135 12 pM,
n= 3; p= 0.79).

Normal monoamine contents in the striatum of Dytl sKO
mice
To determine whether the motor deficits accompany any
alterations of striatal DA metabolism, we measured concentrations
of DA, DOPAC and HVA in the striatum. Unlike Dytl AGAG
heterozygous KI mice or Dytl KD mice, the levels of DA, DOPAC
or HVA and the ratios of DOPAC or HVA to DA in the Dytl
sKO mice were not ;_..i. ,11 different from those in CT mice
(Table 1); suggesting loss of torsinA function in the striatum alone
does not affect monoamine contents in the striatum.

Normal nuclear envelopes of neurons in Dytl sKO and
Dytl cKO mice
To analyze whether loss of torsinA in adult neurons affects
nuclear envelope structures, we examined the nuclear envelope
structures of striatal MSNs in a Dytl sKO mouse using a
transmission electron microscopy. We first examined 11 striatal
MSNs in a CT mouse and found no abnormal nuclear envelope as
expected (Fig. 5A, B). Alil.... 1, we examined 29 striatal MSNs of
a Dytl sKO mouse, we could not find any blebbing or other
nuclear envelope abnormalities among them (Fig. 5C, D). Since
abnormal nuclear envelope was reported in cerebral cortical
neurons of Dytl KO and Dytl AGAG homozygous KI mice but
not in their striatal neurons [24], we further analyzed cerebral
cortical neurons in Dytl cKO mice to determine whether loss of
torsinA in cerebral cortical neurons contributes to abnormal
envelope in adult mice. All..-. 1, we examined 50 cerebral
cortical neurons of two CT mice (Fig. 5E, F) and 84 cerebral
cortical neurons of two Dytl cKO mice (Fig. 5G, H), we could not
find any blebbing or other nuclear envelope abnormalities among
them, either. Since Dytl cKO mice exhibit motor deficits [16],
these results suggest that abnormal nuclear envelope structure is
not a cause of motor impairment in Dytl cKO or sKO mice. On
the other hand, we confirmed abnormal nuclear envelopes in the


Table 1. DA and its metabolites in the striatum.


Content or ratio CT
DA 8.6512.36


DOPAC
HVA
DOPAC/DA
HVA/DA


0.8210.12
1.3710.14
2.58+0.21
3.19+0.23


Dytl sKO
9.2612.29
0.7610.11
1.3610.13
2.51 0.20
3.18+0.22


The values of neurochemical are shown as mean + standard errors (in ng/mg of
wet tissue). The turnovers of metabolites are shown as the ratio of the
neurochemicals after natural log transformation to obtain a normal distribution.
CT: control littermate mice; Dytl sKO: striatum-specific Dytl conditional
knockout mice.
doi:10.1371/journal.pone.0024539.t001


September 2011 1 Volume 6 1 Issue 9 | e24539


,.* ', PLoS ONE I www.plosone.org







Striatum-Specific Dytl Conditional KO Mice


:~~~,~,~,~,~,~,~,~,~,~,~,~.:rr..:s:
~yL~I,,.,,,~ii~
~x;. .

~~ r.;~7
~" :~J;u
.s


Figure 5. Normal nuclear envelope structure of the striatal
MSNs in Dyt sKO and CT mice, and cerebral cortical neurons in
Dyt1 cKO and CT mice. Representative electron microscope images
of the nuclei of the striatal MSNs in CT (A) and Dytl sKO mice (C).
Enlarged images of CT (B) and Dytl sKO (D) mice clearly show normal
nuclear envelopes in MSNs. Representative electron microscope images
of the nuclei of the cerebral cortical neurons in CT (E) and Dytl cKO (G)
mice. Enlarged images of CT (F) and Dytl cKO (H) mice also show
normal nuclear envelopes in cerebral cortical neurons. Nucleus (N) and
nuclear envelope (arrow) are shown in each figure. No abnormal
nuclear envelopes of neurons were detected in Dytl sKO or Dytl cKO
mice as well as CT mice. Magnification in A, C, E, G: 5,000x.
doi:10.1371/journal.pone.0024539.gOO5



cerebral cortical neurons of our newborn Dyt1 AGAG homozy-
gous KI mouse [12] used as a positive control (Fig. 6A-C).


Discussion

All..... 1, DTTI is expressed in multiple brain regions, motor
symptoms are prominent in DYT1 dystonia patients. Reduced


PLoS ONE | www.plosone.org


D2R binding activity and alteration of striatal monoamine
metabolism were reported in DYT1 dystonia patients [17-19].
Dyt1 AGAG heterozygous KI male and Dyt1 KD male mice
exhibit motor deficits. Dyt1 AGAG heterozygous KI male mice
exhibit reduced HVA in the striatum [12] and Dyt1 KD mice
exhibit reduced striatal DOPAC [15]. Furthermore, a transgenic
mouse model overexpressing human mutant torsinA derived by
human CMV immediate early promoter show reduction of D2R
in the striatum and impaired LTD which is rescued by
adenosine A2A receptor antagonism [20]. In the same mouse
model, altered responses to D2R activation and N-type calcium
currents in striatal cholinergic interneurons were reported [32].
Moreover anticholinergic drugs are quite effective in clinical
practice [1]. To determine whether the alterations of the
dopaminergic system are cell-autonomous, we made Dyt1 sKO
mice and analyzed their motor performance and dopaminergic
system. Dyt1 sKO mice exhibited motor deficits and reduced
striatal D2R binding activity, whereas they did not exhibit
significant alteration of striatal monoamine contents. MSNs in
the indirect pathway and cholinergic interneurons express D2R
and contribute to synaptic plasticity in the striatum [33,34].
Reduction of D2R may affect signal transduction pathways in
these neurons and affect basal ganglia circuit. Since the loss of
D2R is known to cause motor deficits in mice [31], the present
results suggest that the loss of striatal torsinA affects signal
transduction pathway 1l...._. D2R in the basal ganglia circuit
and exhibit motor deficits.
Several studies suggest a reduction of striatal D2R binding
activity in DYT1 dystonia patients. A postmortem study reported
a trend of [3H] YM-09151-2 binding reduction in the striatum
[18]. Another positron emission tomography study also suggested
that the striatal D2R availability was reduced in both manifesting
and non-manifesting DYTI mutation carriers [19]. However, the
mechanism of the striatal D2R binding activity reduction has not
been known. In the present study, we made Dyt1 sKO mice and
analyzed their D2R binding activity. B,,, value for [3H] YM-
09151-2 binding activity to D2R was ;_..; ,11 lower in Dyt1
sKO mice in comparison to CT mice, while the receptor affinity to
the ligand as indicated by Kd, was comparable between the
genotypes. The results suggest that the number of functional D2R
may be reduced in the striatal membrane fractions, while the
affinity of the ligand itself is not largely altered. The results also
suggest that reduced striatal D2R binding activity is not an end
result caused by alterations of striatal monoamines, but it is caused
by loss of torsinA function in the striatum itself. It was suggested
that torsinA contributes to protein processing in the secretary
pathway [5]. Over-expression of WT torsinA in vitro also inhibit
t.it ff kin;i of polytopic membrane proteins [4], suggesting proper
level of torsinA protein is critical for normal protein maturation
and t.itffi kin,. Deficient t.itffi kin,, of D2R caused by loss of
torsinA in the striatum may underlie decreased D2R-binding in
the striatal membrane fractions.
The results suggest that the loss of torsinA function in the
striatum itself contributes to the ]..1d....1, ;.1.._ of DYT1
dystonia. Loss of striatal torsinA may affect signal transduction
pathway 1l...._. D2R in the basal ganglia circuit and exhibit
motor deficits. This is consistent with the clinical observation that
deep brain stimulation targeting GPi in the basal ganglia circuits is
an effective surgical therapy for DYT1 dystonia [35]. Interestingly,
Dyt1 cKO mice also exhibit similar motor deficits without
alteration of monoamine contents in the striatum [16]. Collec-
tively, our results suggest that loss of torsinA function in the
corticostriatal pathway may affect the basal ganglia circuits and
contribute to motor impairment without alteration of DA


September 2011 1 Volume 6 1 Issue 9 | e24539






Striatum-Specific Dytl Conditional KO Mice


U~ i

VF
A. N


Figure 6. Abnormal nuclear envelope structure of the cerebral cortical neurons in a newborn Dytl AGAG homozygous KI mouse. (A)
A representative electron microscope image of the cerebral cortical neurons of a newborn Dytl AGAG homozygous KI mouse. This section has two
abnormal nuclear envelope structures (arrows) on the left and right parts of the image. Enlarged images of the left (B) and the right (C) parts of the
nuclear envelope clearly show abnormal nuclear envelope structures in the cerebral cortical neuron. Nucleus (N) and abnormal nuclear envelope
structure (arrow) are shown in each figure. Magnification in A: 6,000x.
doi:10.1371/journal.pone.0024539.g006


metabolism itself. Further studies focusing on the corticostriatal
pathway will elucidate details of the molecular mechanism of this
disease.
A postmortem study reported increased striatal DOPAC/DA
ratio in DYT1 dystonia patients [18]. Dyt1 AGAG heterozygous
KI male mice exhibit decreased HVA [12], while Dytl KD mice
exhibited decreased DOPAC [15] in their striata. However, the
mechanism of the striatal monoamine alterations has not been
clear. In the present study, Dytl sKO mice did not exhibit
alteration of DA, DOPAC or HVA contents in the striatum,
suggesting that loss of torsinA function in the striatum itself may
not affect monoamine contents in the striatum. The results also
suggest that dopaminergic tone in the striatum may have a
limited role in the 1 i,1..._. 1. of DYT1 dystonia because Dytl
sKO mice exhibited motor deficit without alteration of striatal
monoamine contents. The results are consistent with the clinical
observation that levodopa is not effective for DYT1 patients. The
significant alterations in the striatal DA metabolites found in Dyt1
AGAG heterozygous KI male mice and Dyt1 KD mice may be
caused by loss of torsinA function in dopaminergic neurons
derived from the substantial nigra. Generating substantial nigra-
specific Dytl conditional KO mice in the future may further allow
us to determine the origin of the alterations of DA metabolism
observed in Dyt1 AGAG heterozygous KI male mice and Dyt1
KD mice.
Abnormal nuclear envelopes have been reported in transfected
cells over-expressing the mutant forms of torsinA [21-23].
Abnormal nuclear envelopes were also reported in Dyt1 KO
mice and Dyt1 AGAG homozygous KI mice [24]. However,
abnormal nuclear envelopes were not found in Dyt1 AGAG
heterozygous KI mice which exhibit motor deficits, casting
doubts about its role in the 1 i,1..._. ,. ; of DYT1 dystonia. To
analyze whether the motor deficits in Dytl sKO and cKO mice
are caused by abnormal nuclear envelopes, we examined the
nuclear envelopes of striatal MSNs or cortical neurons. However,
we could not find any blebbing or other nuclear envelope
abnormalities in both Dyt1 sKO and cKO mice, suggesting that
nuclear envelope abnormality may not play any role in motor
impairment seen in these mice. Since abnormal envelopes in vivo
are detected only in neonatal Dyt1 AGAG homozygous KI mice
and Dyt1 KO mice, that exhibit neonatal lethality, the abnormal
nuclear envelope may be an indicator of neuronal cell death in
these dying mice.



PLoS ONE | www.plosone.org


Materials and Methods

Making of the striatum-specific Dytl conditional
knockout mice
Dyt1 loxP mice [16] and I. mice [29] were prepared as
described earlier. Genotyping for Dyt1 sKO and CT mice was
performed by multiplex PCR using tail DNA with F (5'-
ATTCAAAAATGTTGTCATAGCCAGG-3') and T (5'-CTA-
CAGTGACCTGAATCATGTGGC-3') primer sets for Dyt1 loxP
locus [16], and creA (5'-ATCTCCGGTATTGAAACTC-
CAGCGC-3') and cre6 (5'-CACTCATGGAAAATAGCGATC-
3') primer sets for cre locus [36]. To confirm the striatum-specific
deletion of Dyt1 exons 3 and 4, the olfactory bulb, striatum,
cerebral cortex, cerebellum, and brainstem were dissected from
Dyt1 loxP and Dyt1 sKO mouse brains. The tissues were digested
with lysis buffer [100 mM Tris-Cl (pH 8.5), 5 mM EDTA*2Na,
0.2% SDS, 200 mM NaCI, 1 mM CaC12, 0.1 mg/ml Proteinase
K (Invitrogen)] at 55C overnight. Each DNA was isolated by
adding equal volume of isopropanol and then washing with 70%
ethanol. The deletion of exons 3 and 4 was confirmed by PCR
using F and Tcko2 (5'-CCATAGCTGGACCTGCAATTAAG-
3') primers as described earlier [16,37]. A group consisted of 11
Dyt1 sKO mice (7 males and 4 females) and 13 CT mice (6 males
and 7 females) was used for the behavior tests. A pair of Dyt sKO
and CT mice was used to prepare brain sections for transmission
electron microscopy analysis. Since onset of DYT1 dystonia is
usually childhood or adolescence, we used adult mice in this study.
All experiments were performed by investigators blind to the
genotypes. Mice were housed under a 12 hours-light and
12 hours-dark cycle with access to food and water ad libitum. All
experiments were carried out in compliance with the USPHS
Guide for Care and Use of Laboratory Animals and approved by
IACUC of University of Alabama at Birmingham with Animal
Protocol Number 091008198.

Preparation of Dytl cKO mice and a Dytl AGAG
homozygous KI mouse
Two pairs of adult Dyt1 cKO and CT mice were prepared and
genotyped as described earlier [16]. A neonatal Dyt1 AGAG
homozygous KI mouse was prepared by crossing Dyt1 AGAG
heterozygous KI mice and genotyped as described earlier [12,38].
These mice were used for transmission electron microscopy
analysis.


September 2011 1 Volume 6 1 Issue 9 | e24539






Striatum-Specific Dytl Conditional KO Mice


Behavioral semi-quantitative assessments of motor
disorders
Behavioral semi-quantitative assessments of motor disorders
were performed as described earlier [12,39]. Mouse from 96 to
139-days old was placed on a table and assessments of hindpaw
clasping, hindpaw dystonia, truncal dystonia and balance
adjustments to a postural challenge were performed. The hindpaw
clasping was assessed as hindpaw movements for postural
adjustment and attempt to straighten up while the mouse was
suspended by the mid-tail. The hindpaw dystonia was assessed as
the increased spacing between the limbs, poor limb coordination,
crouching posture and impairment of gait. Truncal dystonia was
assessed as the flexed posture. Postural challenge was performed
by flipping the mouse onto its back and the ease of righting was
noted.

Accelerated rotarod test
The motor performance was assessed with Economex acceler-
ating rotarod (Columbus Instruments) as described earlier [12].
The apparatus started at an initial speed of 4 rpm. Rod speed was
gradually accelerated at a rate of 0.2 rpm/s. The latency to fall
was measured with a cutoff time of 2 min. Mice from 103 to 146-
days old were tested for three trials on each day for 2 days. The
trials within the same day were performed at approximately
1 hour intervals.

Open-field test
The open-field test was performed under light condition as
described earlier [40,41]. Spontaneous activities of individual mice
from 124 to 167 days old were recorded by infrared light beam
sensors in a 41x41x31 cm acryl case for 15 min at 1 min
intervals using DigiPro software (AccuScan Instruments).

Beam-walking test
The beam-walking test was performed as described earlier
[12,15,41,42]. Briefly, the beam-walking test was performed
within the last 8 hours of the light period after acclimation to a
sound-attenuated testing room for 1 hour. The mice from 141 to
184 days old were trained to transverse a medium square beam
(14 mm wide) in three consecutive trials each day for 2 days and
tested twice each on the medium square beam and a medium
round beam (17 mm diameter) on the third day. The mice were
then tested twice each on a small round beam (10 mm diameter)
and a small square beam (7 mm wide) on the fourth day. Their
hind paw slips on each side during transverse on the 80 (,-1... ,. 1,
beams were counted.

Radioligand binding assay to D2R in the striatal
membrane fractions
Radioligand binding assay to the striatal D2R was performed by
using [3H] YM-09151-2 based on a method reported earlier [43].
The striata were dissected from three Dytl sKO mice and three
CT mice of 268-321 days old (average 284 days old) and
homogenized in nine volumes of ice-cold 50 mM Tris*C1, 8 mM
MgC12, 5 mM EDTA*2Na, pH 7.1. Each homogenate was
centrifuged at 18,000x g for 20 min at 4'C and the pellet was
suspended in the same buffer and stored at -80'C. The protein
concentration of the membrane preparation was determined by
Bradford assay using bovine serum albumin as a standard. An
aliquot of the homogenate was suspended in 200 pl1 of binding
buffer [50 mM Tris-C1, 120 mM NaCI, 5 mM KC1, 5 mM
MgC12, 1.5 mM CaC12, 1 mM EDTA*2Na, 10 pM pargyline
hydrochloride '_ .... \.iii. I. and 0.1% ascorbic acid, pH 7.4]


PLoS ONE www.plosone.org


with 0.03-0.96 nM [3H] YM-09151-2 (2.64TBq/mmol, Perkin
Elmer). The reaction mixture was incubated at 25'C for 40 min,
and then rapidly filtered under vacuum 1l....1. glass microfibre
GF/B filter (Whatman). The filter was then washed four times
with 4 ml of the ice-cold binding buffer without the isotope and
then dried. The radioactivity of the filters was measured in 6 ml of
ScintiSafe Econol (Fisher Scientific) by a Beckman liquid
scintillation counter. Non-specific binding of [3H] YM-09151-2
was measured in presence of 30 gM (S)-(-)-sulpiride ...
Aldrich). The experiments were performed in duplicate.

HPLC analysis
Striata were dissected from the brains of 13 Dyt1 sKO and 13
CT mice from 302 to 345 days old and homogenized in ice-cold
0.2 N perchloric acid. The homogenate were centrifuged for
15 min at 15,000 x g at 4'C to remove debris. Twenty microliters
of the supernatant representing 2 mg of tissue, was the applied to a
C18C reverse phase HPLC column (Varian) connected to an ESA
model 5200A electrochemical detector. DA, DOPAC and HVA
were analyzed using a running buffer of 50 mM potassium
phosphate buffer with 0.5 mM octyl sulfate ',1..... and 8%
acetonitrile as described earlier [12,15,41]. DA, DOPAC, and
HVA were separated at 0.8 ml/min and quantified by comparing
to the standard reagents S'- i-

Transmission electron microscopy analysis
Brain sections for transmission electron microscopy were
prepared as described earlier [14]. Dyt1 sKO and their CT mice
(n = 1 each, 6 weeks of age), Dyt1 cKO mice and their CT mice
(n= 2 each, 8 weeks of age), and a newborn Dyt1 AGAG
homozygous KI mouse were perfused with chilled 0.1 M
phosphate-buffered saline (pH 7.4) followed by Karnovsky's
fixative in phosphate buffered 2% glutaraldehyde and 2.5%
paraformaldehyde. The brains were dissected out and left in
Karnovsky's fixative overnight. The tissue was then trimmed and
washed in cacodylate buffer with no further additives. Microwave
fixation was used with the secondary 2% osmium tetroxide
fixative, followed by the addition of 3% potassium ferricyanide for
30 minutes. Af. i I 1;,i_ ;11 water, saturated uranyl acetate was
added for en bloc staining. The tissue was dehydrated in a series of
increasing concentrations of ethanol starting at 50%. Acetonitrile
was used as the transition fluid between ethanol and the epoxy.
Infiltration series was done with an epoxy mixture using the epon
substitute Lx112. The resulting blocks were polymerized at 90C
overnight, trimmed with a razor blade, and ultrathin sectioned
with diamond knives. Sections were then stained with uranyl
acetate and lead citrate, and the nuclear envelopes in the striata of
Dyt1 sKO and their CT mice, and cerebral cortices of Dyt1 cKO
mice, their CT mice and a Dyt1 AGAG homozygous KI mouse
were examined or photographed with a Hitachi H600 transmis-
sion electron microscope.

Statistics
Data in the open-field test, latency to fall in the accelerated
rotarod, and HPLC were analyzed by ANOVA mixed model with
SAS/STAT Analyst program (Version 9.1.3; SAS institute Inc.
NC) as described earlier [12,16,41]. The turnovers of metabolites
were analyzed as the ratio of the neurochemicals after natural log
transformation to obtain a normal distribution. Slips numbers of
hindpaws in beam-walking test were analyzed by logistic
regression (GENMOD) with Poisson distribution using GEE
model in the software. Sex, age and body weight were input as
variables. Vertical movement numbers in the open-field test,
latency to fall in the accelerated rotarod test, and slips numbers in


September 2011 1 Volume 6 1 Issue 9 | e24539







Striatum-Specific Dytl Conditional KO Mice


the beam-walking test were analyzed after natural log transfor-
mation to obtain a normal distribution. The data in CT mice were
normalized to zero. Radioligand binding data were analyzed by
Student's t test. B,, and Kd of each mouse were individually
calculated in Scatchard plot and those of each genotype were
compared. Significance was assigned at p<0.05.


References

1. Breakefield XO, Blood AJ, Li Y, Hallett M, Hanson PI, et al. (2008) The
pathophysiological basis of dystonias. Nat Rev Neurosci 9: 222-234.
2. Muller U (2009) The monogenic primary dystonias. Brain 132: 2005-2025.
3. Ozelius LJ, HewettJW, Page CE, Bressman SB, Kramer PL, et al. (1997) The
early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein. Nat
Genet 17: 40-48.
4. Torres GE, Sweeney AL, Beaulieu JM, Shashidharan P, Caron MG (2004)
Effect of torsinA on membrane proteins reveals a loss of function and a
dominant-negative phenotype of the dystonia-associated DeltaE-torsinA mutant.
Proc Natl Acad Sci U S A 101: 1565015655.
5. HewettJW, Tannous B, Niland BP, Nery FC, Zeng J, et al. (2007) Mutant
torsinA interferes with protein processing through the secretary pathway in
DYT1 dystonia cells. Proc Nad Acad Sci U S A 104: 7271-7276.
6. Kustedjo K, Deechongkit S, Kelly JW, Cravatt BF (2003) Recombinant
expression, purification, and comparative characterization of torsinA and its
torsion dystonia-associated variant Delta E-torsinA. Biochemistry 42:
15333-15341.
7. Konakova M, Pulst SM (2005) Dystonia-associated forms of torsinA are deficient
in ATPase activity. J Mol Neurosci 25: 105117.
8. Burdette AJ, Churchill PF, Caldwell GA, Caldwell KA (2010) The early-onset
torsion dystonia-associated protein, torsinA, displays molecular chaperone
activity in vitro. Cell Stress Chaperones 15: 605617.
9. Leung JC, Klein C, Friedman J, Vieregge P, Jacobs H, et al. (2001) Novel
mutation in the TOR1A (DYT1) gene in atypical early onset dystonia and
polymorphisms in dystonia and early onset parkinsonism. Neurogenetics 3:
133 143.
10. Zirn B, Grundmann K, Huppke P, PuthenparampilJ, Wolburg H, et al. (2008)
Novel TOR1A mutation p.Arg288Gln in early-onset dystonia (DYTI).J Neurol
Neurosurg Psychiatry 79: 1327 1330.
11. Ritz KGM, Foncke EM, van Ruissen F, van der Linden C, Vergouwen MD,
Bloem BR, Vandenberghe W, Crols R, SpeelmanJD, Baas F, Tijssen MA (2009)
Myoclonus-dystonia: clinical and genetic evaluatio... [J Neurol Neurosurg
Psychiatry. 2009] PubMed result. J Neurol Neurosurg Psychiatry 80: 653658.
12. Dang MT, Yokoi F, McNaught KS, Jengelley TA, Jackson T, et al. (2005)
Generation and Characterization of Dytl deltaGAG Knock-in Mouse as a
Model for Early-Onset Dystonia. Exp Neurol 196: 452463.
13. Cao S, Hewett JW, Yokoi F, Lu J, Buckley AC, et al. (2010) Chemical
enhancement of torsinA function in cell and animal models of torsion dystonia.
Dis Model Mech 3: 386 396.
14. Yokoi F, Yang G, LiJ, Deandrade MP, Zhou T, et al. (2010) Earlier onset of
motor deficits in mice with double mutations in Dytl and Sgce.J Biochem 148:
459-466.
15. Dang MT, Yokoi F, Pence MA, Li Y (2006) Motor deficits and hyperactivity in
Dytl knockdown mice. Neurosci Res 56: 470-474.
16. Yokoi F, Dang MT, Mitsui S, LiJ, Li Y (2008) Motor deficits and hyperactivity
in cerebral cortex-specific Dytl conditional knockout mice. J Biochem 143:
39-47.
17. Furukawa Y, Hornykiewicz O, Fahn S, Kish SJ (2000) Striatal dopamine in
early-onset primary torsion dystonia with the DYT1 mutation. Neurology 54:
1193-1195.
18. Augood SJ, Hollingsworth Z, Albers DS, Yang L, Leung JC, et al. (2002)
Dopamine transmission in DYT1 dystonia: a biochemical and autoradiogra-
phical study. Neurology 59: 445-448.
19. Asanuma K, Ma Y, OkulskiJ, Dhawan V, Chaly T, et al. (2005) Decreased
striatal D2 receptor binding in non-manifesting carriers of the DYT1 dystonia
mutation. Neurology 64: 347-349.
20. Napolitano F, Pasqualetti M, Usiello A, Santini E, Pacini G, et al. (2010)
Dopamine D2 receptor dysfunction is rescued by adenosine A2A receptor
antagonism in a model of DYT1 dystonia. Neurobiol Dis 38: 434-445.
21. Naismith TV, Heuser JE, Breakefield XO, Hanson PI (2004) TorsinA in the
nuclear envelope. Proc Nad Acad Sci U S A 101: 7612-7617.
22. Gonzalez-Alegre P, Paulson HL (2004) Aberrant cellular behavior of mutant
torsinA implicates nuclear envelope dysfunction in DYT1 dystonia. J Neurosci
24: 25932601.





PLoS ONE | www.plosone.org


Acknowledgments

We thank Lisa Foster, Andrea M\L C ,ii. .h and their staff for animal care,
and Lou Ann Miller, Miki Jinno and Mark P. DeAndrade for their
technical assistance.


Author Contributions

Conceived and designed the experiments: FY MTD DGS YL. Performed
the experiments: FY MTDJL YL. Analyzed the data: FY YL. Contributed
reagents/materials/analysis tools: JL DGS YL. Wrote the paper: FY DGS
YL.




23. Goodchild RE, Dauer WT (2004) Mislocalization to the nuclear envelope: an
effect of the dystonia-causing torsinA mutation. Proc Natl Acad Sci U S A 101:
847-852.
24. Goodchild RE, Kim CE, Dauer WT (2005) Loss of the dystonia-associated
protein torsinA selectively disrupts the neuronal nuclear envelope. Neuron 48:
923-932.
25. Grundmann K, Reischmann B, Vanhoutte G, Hubener J, Teismann P, et al.
(2007) Overexpression of human wildtype torsinA and human DeltaGAG
torsinA in a transgenic mouse model causes phenotypic abnormalities.
Neurobiol Dis 27: 190-206.
26. Kim CE, Perez A, Perkins G, Ellisman MH, Dauer WT (2010) A molecular
mechanism underlying the neural-specific defect in torsinA mutant mice. Proc
Nad Acad Sci U S A 107: 9861-9866.
27. Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian
cells by the Cre recombinase of bacteriophage P1. Proc Nad Acad Sci U S A 85:
5166-5170.
28. Schwenk F, Baron U, Rajewsky K (1995) A cre-transgenic mouse strain for the
ubiquitous deletion of loxP-flanked gene segments including deletion in germ
cells. Nucleic Acids Res 23: 5080-5081.
29. Dang MT, Yokoi F, Yin HH, Lovinger DM, Wang Y, et al. (2006) Disrupted
motor learning and long-term synaptic plasticity in mice lacking NMDAR1 in
the striatum. Proc Nad Acad Sci U S A 103: 1525415259.
30. McBride WJ, Chernet E, Dyr W, Lumeng L, Li TK (1993) Densities of
dopamine D2 receptors are reduced in CNS regions of alcohol-preferring P rats.
Alcohol 10: 387 390.
31. Wang Y, Xu R, Sasaoka T, Tonegawa S, Kung MP, et al. (2000) Dopamine D2
long receptor-deficient mice display alterations in striatum-dependent functions.
J Neurosci 20: 8305-8314.
32. Pisani A, Martella G, Tscherter A, Bonsi P, Sharma N, et al. (2006) Altered
responses to dopaminergic D2 receptor activation and N-type calcium currents
in striatal cholinergic interneurons in a mouse model of DYT1 dystonia.
Neurobiol Dis 24: 318 325.
33. Wang Z, Kai L, Day M, RonesiJ, Yin HH, et al. (2006) Dopaminergic control
of corticostriatal long-term synaptic depression in medium spiny neurons is
mediated by cholinergic interneurons. Neuron 50: 443 452.
34. Shen W, Flajolet M, Greengard P, Surmeier DJ (2008) Dichotomous
dopaminergic control of striatal synaptic plasticity. Science 321: 848 851.
35. Vidailhet M, Vercueil L, HouetoJL, KIystkowiak P, Lagrange C, et al. (2007)
Bilateral, pallidal, deep-brain stimulation in primary generalised dystonia: a
prospective 3 year follow-up study. Lancet Neurol 6: 223 229.
36. Campos VE, Du M, Li Y (2004) Increased seizure susceptibility and cortical
malformation in beta-catenin mutant mice. Biochem Biophys Res Commun
320: 606-614.
37. Zhang L, Yokoi F,Jin YH, Deandrade MP, Hashimoto K, et al. (2011) Altered
Dendritic Morphology of Purkinje cells in Dytl DeltaGAG Knock-In and
Purkinje Cell-Specific Dytl Conditional Knockout Mice. PLoS One 6: e18357.
38. Yokoi F, Dang MT, Miller CA, Marshall AG, Campbell SL, et al. (2009)
Increased c-fos expression in the central nucleus of the amygdala and
enhancement of cued fear memory in Dytl DeltaGAG knock-in mice. Neurosci
Res 65: 228-235.
39. Fernagut P, Diguet E, Stefanova N, Biran M, Wenning G, et al. (2002) Subacute
systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in
adult C57B1/6 mice: behavioral and histopathological characterisation.
Neuroscience 114: 1005.
40. Cao BJ, Li Y (2002) Reduced anxiety- and depression-like behaviors in Emxl
homozygous mutant mice. Brain Res 937: 32-40.
41. Yokoi F, Dang MT, LiJ, Li Y (2006) Myoclonus, motor deficits, alterations in
emotional responses and monoamine metabolism in epsilon-sarcoglycan
deficient mice. J Biochem 140: 141-146.
42. DeAndrade MP, Yokoi F, van Groen T, Lingrel JB, Li Y (2011) Character-
ization of Atpla3 mutant mice as a model of rapid-onset dystonia with
parkinsonism. Behav Brain Res 216: 659-665.
43. Terai M, Hidaka K, Nakamura Y (1989) Comparison of [3H]YM-09151-2 with
[3H]spiperone and [3H]raclopride for dopamine d-2 receptor binding to rat
striatum. EurJ Pharmacol 173: 177-182.


September 2011 1 Volume 6 1 Issue 9 | e24539




Full Text

PAGE 1

MotorDeficitsandDecreasedStriatalDopamine Receptor2BindingActivityintheStriatum-SpecificDyt1ConditionalKnockoutMiceFumiakiYokoi1,MaiTuDang2,JianyongLi3,DavidG.Standaert4,YuqingLi1*1 DepartmentofNeurology,CollegeofMedicine,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica, 2 TheChildren’sHospitalofPhiladelphia, Philadelphia,Pennsylvania,UnitedStatesofAmerica, 3 DepartmentofBiochemistry,VirginiaTech,Blacksburg,Virginia,UnitedStatesofAmerica, 4 Departmentof Neurology,SchoolofMedicine,CenterforNeurodegenerationandExperimentalTherapeutics,UniversityofAlabamaatBirmingham,Birmingham,Ala bama,UnitedStates ofAmericaAbstractDYT1early-onsetgeneralizeddystoniaisahyperkineticmovementdisordercausedbymutationsin DYT1 ( TOR1A ),which codesfortorsinA.Recently,significantprogresshasbeenmadeinstudyingpathophysiologyofDYT1dystoniausing targetedmousemodels. Dyt1 D GAGheterozygousknock-in(KI)and Dyt1 knock-down(KD)miceexhibitmotordeficitsand alterationsofstriataldopaminemetabolisms,while Dyt1 knockout(KO)and Dyt1 D GAGhomozygousKImiceshow abnormalnuclearenvelopesandneonatallethality.However,ithasnotbeenclearwhethermotordeficitsandstriatal abnormalityarecausedby Dyt1 mutationinthestriatumitselfortheendresultsofabnormalsignalsfromotherbrain regions.Toidentifythebrainregionthatcontributestothesephenotypes,wemadeastriatum-specific Dyt1 conditional knockout( Dyt1 sKO)mouse. Dyt1 sKOmiceexhibitedmotordeficitsandreducedstriataldopaminereceptor2(D2R)binding activity,whereastheydidnotexhibitsignificantalterationofstriatalmonoaminecontents.Furthermore,wealsofound normalnuclearenvelopestructureinstriatalmediumspinyneurons(MSNs)ofanadult Dyt1 sKOmouseandcerebral corticalneuronsincerebralcortex-specific Dyt1 conditionalknockout( Dyt1 cKO)mice.Theresultssuggestthatthelossof striataltorsinAaloneissufficienttoproducemotordeficits,andthatthiseffectmaybemediated,atleastinpart,through changesinD2Rfunctioninthebasalgangliacircuit.Citation: YokoiF,DangMT,LiJ,StandaertDG,LiY(2011)MotorDeficitsandDecreasedStriatalDopamineReceptor2BindingActivityintheStriatum-Specific Dyt1 ConditionalKnockoutMice.PLoSONE6(9):e24539.doi:10.1371/journal.pone.0024539 Editor: IzumiSugihara,TokyoMedicalandDentalUniversity,Japan Received May4,2011; Accepted August12,2011; Published September12,2011 Copyright: 2011Yokoietal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisworkwassupportedbyNationalInstitutesofHealthgrants(NS37409,NS47466,NS47692,NS54246,NS57098,NS65273,NS72782,andNS74423) andstartupfundsfromtheLucilleP.MarkeyCharitableTrustandBeckmanInstitute(UIUC),DepartmentofNeurology(UAB),andTyler’sHopeforaDyst oniaCure, Inc.(UF).PublicationofthisarticlewasfundedinpartbytheUniversityofFloridaOpen-AccessPublishingFund.Thefundershadnoroleinstudydes ign,data collectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:yuqing.li@neurology.ufl.eduIntroductionDystoniaisamovementdisorderthatisexhibitedby involuntary,repetitive,sustainedmusclecontractionsorabnormal postures[1].Dystoniaisclassifiedintotwogroups,primaryand secondarydystonia.Primarydystoniadevelopsspontaneouslyin theabsenceofanyapparentcauseorassociateddisease. Secondarydystoniaiscausedbyotherdiseases,suchasParkinson’s orHuntington’sdiseases,braininjury,ordrugsideeffects.Genetic dystoniabelongstotheprimarydystoniagroupandisclassified intomorethan20types,althoughlessthanhalfofthemhave knowngenemutations[2].DYT1early-onsetgeneralizedtorsion dystoniaisaninheritedmovementdisorderandiscausedby mutationsin DYT1 ( TOR1A )codingfortorsinAwithabout30% penetrance[3].TorsinAisamemberofAAA + familyofATPases andmayworkintraffickingofpolytopicmembraneproteinsand proteinprocessinginthesecretorypathway[4,5].ATPaseactivity [6,7]andmolecularchaperonactivityoftorsinA[8]werealso reported invitro .NearlyallDYT1dystoniapatientshavea3basepairsdeletion( D GAG)in DYT1 inoneallele,correspondingtoa lossofaglutamicacidresidueintheC-terminalregionoftorsinA [3],whilean18bpdeletionmutation[9]andanArg288Gln missensemutation[10]havebeenreported.Aframeshift mutationcausedby4bp-deletioncodingfortheC-terminal regionoftorsinAwasalsoreportedinapossiblemyoclonusdystoniapatient[11]. Dyt1 D GAGheterozygousKImalemice exhibitreductionofstriataltorsinAandmotordeficits[12–14]. Dyt1 KDmalemiceand Dyt1 cKOmicealsoexhibitmotor deficits,suggestinglossoftorsinAfunctionmaycontributeto motordeficitsinthese Dyt1 mutantmice[15,16].Ampicillininjected Dyt1 D GAGheterozygousKImalemiceexpressnormal levelofstriataltorsinAandexhibitnormalmotorperformance, suggestingrecoveryoftorsinAlevelinthestriatummayrescuethe motordeficitsin Dyt1 D GAGheterozygousKImalemice[13]. AlthoughDYT1dystoniapatientsdonotrespondtolevodopa treatmentinmostcases,thereareseveralreportssuggesting functionalalterationsinthestriataldopaminergicsysteminDYT1 dystonia.Apostmortemdopamine(DA)contentanalysisina DYT1dystoniapatientbrainsuggestedareducedDAinrostral portionsoftheputamenandcaudatenucleus[17].Another postmortemstudyreportedareduceddopaminereceptorbinding inthestriatumandincreasedstriatal3,4-dihydroxyphenylacetic PLoSONE|www.plosone.org1September2011|Volume6|Issue9|e24539

PAGE 2

acid(DOPAC)/DAratioinDYT1dystoniapatients[18].A positronemissiontomographystudysuggestedthatstriatalD2R availabilityisreducedinbothmanifestingandnon-manifesting DYT1 mutationcarriers[19]. Dyt1 D GAGheterozygousKImale miceexhibitreducedhomovanillicacid(HVA)levelinthe striatum[12]and Dyt1 KDmiceshowdecreasedlevelofstriatal DOPAC[15].MoreoveratransgenicmousemodeloverexpressinghumanmutanttorsinAderivedbyhumancytomegalovirus (CMV)immediateearlypromoterexhibitareductionofD2Rin thestriatum[20].Thesereportssuggestedthatfunctional alterationsofthestriataldopaminergicsystemmaycontributeto thepathogenesisofDYT1dystonia,whileitsprecisefunctional rolehasnotbeenwelldefined.Forexample,itisnotknown whethermotordeficitsandalterationsofthestriataldopaminergic systemarecausedbythemutationinthestriatumitselforend resultsofabnormalsignalsfromotherbrainregions. Abnormalnuclearenvelopeshavebeenreportedintransfected cellsover-expressingthemutantformsoftorsinA[21–23], Dyt1 KOmiceand Dyt1 D GAGhomozygousKImiceexhibiting neonatallethality[24].Bothtransgenicmiceoverexpressing humanWTtorsinAandmutanttorsinAusingmurineprion promoterexhibitabnormalnuclearenvelopesandabnormal motorperformance[25].Itwassuggestedthatneuron-specific nuclearenvelopeabnormalityin Dyt1 D GAGhomozygousKI miceiscausedbymalfunctionoftorsinAwithincomplete compensationbytorsinBwhichisweaklyexpressedinneurons [26].However,suchabnormalityhasnotbeenfoundin Dyt1 D GAGheterozygousKImouseorDYT1dystoniapatientbrains, castingdoubtsaboutitsroleinthepathogenesisofDYT1dystonia. Inthepresentstudy,wehypothesizedthataselectivelossof torsinAfunctioninthestriatummayaffectmotorperformancein mice.Weused cre-loxP technology[27]appliedtomousegene recombination[28]toselectivelyinactivate Dyt1 inthestriatum usinga Rgs9-cre linethathasrestrictedrecombinationinthe striatum[29]. Dyt1 sKOmiceweremadeandtheirmotor performance,dopaminergicsystem,andtheroleoftorsinAin maintainingthenuclearenvelopestructureswereanalyzed.Since abnormalnuclearenvelopewasreportedincerebralcortical neuronsin Dyt1 KOand Dyt1 D GAGhomozygousKImice,we furtheranalyzednuclearenvelopesincerebralcorticalneuronsof Dyt1 cKOmice[16].Results Makingof Dyt1 sKOmiceRgs9-cre and Dyt1loxP doubleheterozygousmicewereprepared bycrossing Dyt1loxP mice[16]and Rgs9-cre mice[29]. Dyt1 sKO micewerepreparedbycrossing Rgs9-creDyt1loxP double heterozygousmiceand Dyt1loxP mice(Fig.1A).Genotypingwas performedbymultiplexPCRwithtailDNA(Fig.1B). Dyt1 sKO micewerebornaccordingtoMendelianratioanddevelopedto adult.Thestriatum-specificdeletionof Dyt1 exons3and4in Dyt1 sKOmicewasconfirmedbyPCRusingDNAisolatedfromeach brainregion(Fig.1C).Noovertabnormalposturesandnormallocomotionin Dyt1 sKOmiceWhensuspendedfromthetail,both Dyt1 sKOandControl littermate(CT)miceshowednormalsplayingofhindpawsandhad noobservablehindpawextensionortruncalarching.Allmice exhibitedstrongrightingreflexeswhentippedontheirside.The resultssuggestthat Dyt1 sKOmicehadnoovertabnormal postures. Since Dyt1 D GAGheterozygousKImalemice[12]and Dyt1 KDmalemice[15]exhibitmoderatehyperactivities,and Dyt1 cKOmiceexhibitprominenthyperactivitiesintheopen-fieldtest [16],spontaneousactivitiesin Dyt1 sKOmicewereassessedinthe Figure1.MakingofDyt1sKOmice. (A)Thebreedingstrategyto generate Dyt1 sKOmice. Dyt1loxP mice[16]and Rgs9-cre mice[29] werepreparedasdescribedearlier. Rgs9-creDyt1loxP double heterozygousmicewerepreparedbycrossing Dyt1loxP miceand Rgs9-cre mice. Dyt1 sKOmicewerepreparedbycrossing Rgs9-creDyt1 loxP doubleheterozygousmiceand Dyt1loxP mice.Theprimersitesto amplify Dyt1 exons3and4-deletedlocuswereshownbyanarrowpair underthemap.(B)ArepresentativeimageofthemultiplexPCR-based genotyping.TopbandsarePCRproductsof cre .MiddlebandsarePCR productsof Dyt1loxP locus.ThebottombandsarePCRproductsof Dyt1 WTlocus.Lanes1,3,5: Dyt1loxP homozygousmice;Lanes2,7: Rgs9-cre Dyt1loxP doubleheterozygousmice;Lanes4: Dyt1 sKOmouse;Lane6: Dyt1loxP heterozygousmouse.(C)Tissue-specificdeletionof Dyt1 exons3and4in Dyt1 sKOmicewasconfirmedbyPCRusingDNA isolatedfromeachbrainregion.Thedeletionwasdetectedonlyinthe striatumof Dyt1 sKOmouseaspredicted( D ).CT:controllittermate mouse. doi:10.1371/journal.pone.0024539.g001 Striatum-Specific Dyt1 ConditionalKOMice PLoSONE|www.plosone.org2September2011|Volume6|Issue9|e24539

PAGE 3

open-fieldapparatusandcomparedtothoseinCTmice. Dyt1 sKOmicedidnotexhibitsignificantdifferencesincomparisonto CTmiceineitherhorizontallocomotion(Fig.2A;horizontal activity, p =0.55;B;totaldistance, p =0.83;C;movementnumber, p =0.54;D;movementtime, p =0.91)orverticallocomotion (Fig.2E;verticalmovementnumber, p =0.87).Therewasno significantdifferenceinstereotypicactivity(Fig.2F; p =0.45), stereotypicmovementtime(Fig.2G; p =0.47),clockwiseoranticlockwiserevolutionbetween Dyt1 sKOmiceandCTmice (Fig.2H; p =0.90and p =0.62,respectively).Theresultssuggest thatlossoftorsinAinthestriatumalonedoesnotaffect spontaneouslocomotion.Significantmotordeficitsofhindpawsin Dyt1 sKOmiceMotorperformancewasassessedbytheacceleratedrotarodand beam-walkingtests.Eachmousewasputontheaccelerated rotarodandthelatencytofallwasmeasured.Sincemicecanhold ontotherotarodwithfourpaws,thelatencytofallisanindicator oftotalmotorperformanceandshorterlatencyindicatesmotor deficits. Dyt1 sKOmicedidnotshowsignificantdifferencein latencytofall(Fig.3A; p =0.48),suggestingnomotorsymptomsin totalmotorperformancewithfourpaws.Wefurtheranalyzedthe motorcoordinationandbalancebythebeam-walkingtest.Mice weretrainedtotransverseamediumsquarebeamfortwodays. Thetrainedmiceweretestedtwiceonfourdifferentbeamsand totalnumbersofhindpawslipswereanalyzed. Dyt1 sKOmice showed144%moreslipnumbersinthebeam-walkingtest(Fig.3B; p =0.043),suggestingmotordeficitsinhindpaws. Intheacceleratedrotarodtest, Dyt1 sKOmicedidnotexhibit overtmotordeficitswithfourpaws.Theresultisconsistentwith otherDYT1dystoniamousemodels. Dyt1 D GAGheterozygous KImice[12], Dyt1 KDmice[15]or Dyt1 cKOmice[16]donot exhibitsignificantmotordeficitseitherintheacceleratedrotarod tests.Ontheotherhand, Dyt1 sKOmice, Dyt1 cKOmice, Dyt1 D GAGheterozygousKImalemiceand Dyt1 KDmalemice Figure2.LocomotionofDyt1sKOmiceandCTmiceintheopenfieldtest. Spontaneousactivitiesin Dyt1 sKOmicewereassessedinthe open-fieldapparatusandcomparedtothoseinCTmice. Dyt1 sKOmicedidnotexhibitsignificantdifferencesincomparisontoCTmiceinhorizontal locomotion(A,horizontalactivity;B,totaldistance;C,movementnumber;D,movementtime)orverticallocomotion(E,verticalmovementnumber). Therewasnosignificantdifferenceinstereotypicactivity(F),stereotypicmovementtime(G),clockwise(H,CWREV)oranti-clockwiserevolution (H, ACWREV).Verticalbarsrepresentmeans 6 standarderrors. doi:10.1371/journal.pone.0024539.g002 Figure3.MotorperformancesinDyt1sKOmiceandCTmice. (A)Latencytofallintheacceleratedrotarodtest. Dyt1 sKOmicedidnot exhibitsignificantdifferenceinthelatencytofall,suggestingnomotor deficitswithfourpaws.(B)Beam-walkingperformancesin Dyt1 sKO miceandCTmice.ThedataintheCTmicewerenormalizedtozero. Dyt1 sKOmiceshowedsignificantincreasedslipsnumbersinbeamwalkingtest,suggestingmotordeficitsinhindpaws.Verticalbars representmeans 6 standarderrors.* p 0.05. doi:10.1371/journal.pone.0024539.g003 Striatum-Specific Dyt1 ConditionalKOMice PLoSONE|www.plosone.org3September2011|Volume6|Issue9|e24539

PAGE 4

exhibitmotordeficitsinthebeam-walkingtest.Sincedystonic symptomscommonlystartfromthelegsinDYT1dystonia patients,thebeam-walkingtestmaybeoneofthemost appropriatebehaviorteststodetecttheearlymotorsymptomsin DYT1dystoniamousemodels.DecreasedradioligandbindingtoD2Rinthestriatal membranefractionsfrom Dyt1 sKOmiceWemeasuredradioligandbindingactivityof[3H]YM-09151-2 toD2Rinthestriatalmembranefractionsfromthree Dyt1 sKO andthreeCTmice.Toseetheoveralldistributionofdata, representativesaturationbindingcurvesof[3H]YM-09151-2 weredrawnusingthetransformedcompositedataofmeanvalues obtainedforthethree Dyt1 sKO(opencircles)andthreeCT(solid circles)mice(Fig.4A).ThefitlinesintherepresentativeScatchard plotwerealsocreatedbyleastsquaremeansfromthetransformed compositedataofthemeanvalues(Fig.4B; Dyt1 sKO, r2=0.9788;CT,r2=0.9585)asdescribed[30].Toanalyzethe statisticalsignificanceof Bmaxand Kd,between Dyt1 sKOandCT mice, Bmaxand Kdofeachmousewerealsoindividuallyanalyzedin Scatchardplotandthoseofeachgenotypewerecomparedby Student’s t testasdescribed[31].The Bmaxvaluefor[3H]YM09151-2binding,reflectinglevelsofradioligandbindingtoD2R, wassignificantlylowerin Dyt1 sKOmiceincomparisontoCT mice(means 6 standarderrors; Dyt1 sKO,541 6 28fmol/mg, n=3;CT,793 6 46fmol/mg,n=3; p =0.009).Receptoraffinity fortheligandasindicatedby Kd,however,wasunchangedin Dyt1 sKOmice( Dyt1 sKO,149 6 46pM,n=3;CT,135 6 12pM, n=3; p =0.79).Normalmonoaminecontentsinthestriatumof Dyt1 sKO miceTodeterminewhetherthemotordeficitsaccompanyany alterationsofstriatalDAmetabolism,wemeasuredconcentrations ofDA,DOPACandHVAinthestriatum.Unlike Dyt1 D GAG heterozygousKImiceor Dyt1 KDmice,thelevelsofDA,DOPAC orHVAandtheratiosofDOPACorHVAtoDAinthe Dyt1 sKOmicewerenotsignificantlydifferentfromthoseinCTmice (Table1);suggestinglossoftorsinAfunctioninthestriatumalone doesnotaffectmonoaminecontentsinthestriatum.Normalnuclearenvelopesofneuronsin Dyt1 sKOand Dyt1 cKOmiceToanalyzewhetherlossoftorsinAinadultneuronsaffects nuclearenvelopestructures,weexaminedthenuclearenvelope structuresofstriatalMSNsina Dyt1 sKOmouseusinga transmissionelectronmicroscopy.Wefirstexamined11striatal MSNsinaCTmouseandfoundnoabnormalnuclearenvelopeas expected(Fig.5A,B).Althoughweexamined29striatalMSNsof a Dyt1 sKOmouse,wecouldnotfindanyblebbingorother nuclearenvelopeabnormalitiesamongthem(Fig.5C,D).Since abnormalnuclearenvelopewasreportedincerebralcortical neuronsof Dyt1 KOand Dyt1 D GAGhomozygousKImicebut notintheirstriatalneurons[24],wefurtheranalyzedcerebral corticalneuronsin Dyt1 cKOmicetodeterminewhetherlossof torsinAincerebralcorticalneuronscontributestoabnormal envelopeinadultmice.Althoughweexamined50cerebral corticalneuronsoftwoCTmice(Fig.5E,F)and84cerebral corticalneuronsoftwo Dyt1 cKOmice(Fig.5G,H),wecouldnot findanyblebbingorothernuclearenvelopeabnormalitiesamong them,either.Since Dyt1 cKOmiceexhibitmotordeficits[16], theseresultssuggestthatabnormalnuclearenvelopestructureis notacauseofmotorimpairmentin Dyt1 cKOorsKOmice.On theotherhand,weconfirmedabnormalnuclearenvelopesinthe Figure4.RadioligandbindingactivitytoD2Rinthestriatal membranefractions. (A)Arepresentativesaturationbindingcurveof [3H]YM-09151-2tothestriatalmembranesfractionsfromthree Dyt1 sKO(opencircles)andthreeCT(solidcircles)mice.Thetransformed compositedataofmeanvaluesobtainedforthethreemiceineach genotypeareplotted.(B)ArepresentativeScatchardtransformationof thedata.Thefitlineswerecreatedbyleastsquaremeansfromthe transformedcompositedataofmeanvalues. doi:10.1371/journal.pone.0024539.g004 Table1. DAanditsmetabolitesinthestriatum.ContentorratioCTDyt1sKOpDA 8.65 6 2.369.26 6 2.290.86 DOPAC 0.82 6 0.120.76 6 0.110.70 HVA 1.37 6 0.141.36 6 0.130.98 DOPAC/DA 2.58 6 0.212.51 6 0.200.82 HVA/DA 3.19 6 0.233.18 6 0.220.98 Thevaluesofneurochemicalareshownasmean 6 standarderrors(inng/mgof wettissue).Theturnoversofmetabolitesareshownastheratioofthe neurochemicalsafternaturallogtransformationtoobtainanormaldistribution. CT:controllittermatemice; Dyt1 sKO:striatum-specific Dyt1 conditional knockoutmice. doi:10.1371/journal.pone.0024539.t001 Striatum-Specific Dyt1 ConditionalKOMice PLoSONE|www.plosone.org4September2011|Volume6|Issue9|e24539

PAGE 5

cerebralcorticalneuronsofournewborn Dyt1 D GAGhomozygousKImouse[12]usedasapositivecontrol(Fig.6A–C).DiscussionAlthough DYT1 isexpressedinmultiplebrainregions,motor symptomsareprominentinDYT1dystoniapatients.Reduced D2Rbindingactivityandalterationofstriatalmonoamine metabolismwerereportedinDYT1dystoniapatients[17–19]. Dyt1 D GAGheterozygousKImaleand Dyt1 KDmalemice exhibitmotordeficits. Dyt1 D GAGheterozygousKImalemice exhibitreducedHVAinthestriatum[12]and Dyt1 KDmice exhibitreducedstriatalDOPAC[15].Furthermore,atransgenic mousemodeloverexpressinghumanmutanttorsinAderivedby humanCMVimmediateearlypromotershowreductionofD2R inthestriatumandimpairedLTDwhichisrescuedby adenosineA2Areceptorantagonism[20].Inthesamemouse model,alteredresponsestoD2RactivationandN-typecalcium currentsinstriatalcholinergicinterneuronswerereported[32]. Moreoveranticholinergicdrugsarequiteeffectiveinclinical practice[1].Todeterminewhetherthealterationsofthe dopaminergicsystemarecell-autonomous,wemade Dyt1 sKO miceandanalyzedtheirmotorperformanceanddopaminergic system. Dyt1 sKOmiceexhibitedmotordeficitsandreduced striatalD2Rbindingactivity,whereastheydidnotexhibit significantalterationofstriatalmonoaminecontents.MSNsin theindirectpathwayandcholinergicinterneuronsexpressD2R andcontributetosynapticplasticityinthestriatum[33,34]. ReductionofD2Rmayaffectsignaltransductionpathwaysin theseneuronsandaffectbasalgangliacircuit.Sincethelossof D2Risknowntocausemotordeficitsinmice[31],thepresent resultssuggestthatthelossofstriataltorsinAaffectssignal transductionpathwaythroughD2Rinthebasalgangliacircuit andexhibitmotordeficits. SeveralstudiessuggestareductionofstriatalD2Rbinding activityinDYT1dystoniapatients.Apostmortemstudyreported atrendof[3H]YM-09151-2bindingreductioninthestriatum [18].Anotherpositronemissiontomographystudyalsosuggested thatthestriatalD2Ravailabilitywasreducedinbothmanifesting andnon-manifesting DYT1 mutationcarriers[19].However,the mechanismofthestriatalD2Rbindingactivityreductionhasnot beenknown.Inthepresentstudy,wemade Dyt1 sKOmiceand analyzedtheirD2Rbindingactivity. Bmaxvaluefor[3H]YM09151-2bindingactivitytoD2Rwassignificantlylowerin Dyt1 sKOmiceincomparisontoCTmice,whilethereceptoraffinityto theligandasindicatedby Kd,wascomparablebetweenthe genotypes.TheresultssuggestthatthenumberoffunctionalD2R maybereducedinthestriatalmembranefractions,whilethe affinityoftheliganditselfisnotlargelyaltered.Theresultsalso suggestthatreducedstriatalD2Rbindingactivityisnotanend resultcausedbyalterationsofstriatalmonoamines,butitiscaused bylossoftorsinAfunctioninthestriatumitself.Itwassuggested thattorsinAcontributestoproteinprocessinginthesecretory pathway[5].Over-expressionofWTtorsinA invitro alsoinhibit traffickingofpolytopicmembraneproteins[4],suggestingproper leveloftorsinAproteiniscriticalfornormalproteinmaturation andtrafficking.DeficienttraffickingofD2Rcausedbylossof torsinAinthestriatummayunderliedecreasedD2R-bindingin thestriatalmembranefractions. TheresultssuggestthatthelossoftorsinAfunctioninthe striatumitselfcontributestothepathophysiologyofDYT1 dystonia.LossofstriataltorsinAmayaffectsignaltransduction pathwaythroughD2Rinthebasalgangliacircuitandexhibit motordeficits.Thisisconsistentwiththeclinicalobservationthat deepbrainstimulationtargetingGPiinthebasalgangliacircuitsis aneffectivesurgicaltherapyforDYT1dystonia[35].Interestingly, Dyt1 cKOmicealsoexhibitsimilarmotordeficitswithout alterationofmonoaminecontentsinthestriatum[16].Collectively,ourresultssuggestthatlossoftorsinAfunctioninthe corticostriatalpathwaymayaffectthebasalgangliacircuitsand contributetomotorimpairmentwithoutalterationofDA Figure5.Normalnuclearenvelopestructureofthestriatal MSNsinDyt1sKOandCTmice,andcerebralcorticalneuronsinDyt1cKOandCTmice. Representativeelectronmicroscopeimages ofthenucleiofthestriatalMSNsinCT(A)and Dyt1 sKOmice(C). EnlargedimagesofCT(B)and Dyt1 sKO(D)miceclearlyshownormal nuclearenvelopesinMSNs.Representativeelectronmicroscopeimages ofthenucleiofthecerebralcorticalneuronsinCT(E)and Dyt1 cKO(G) mice.EnlargedimagesofCT(F)and Dyt1 cKO(H)micealsoshow normalnuclearenvelopesincerebralcorticalneurons.Nucleus(N)and nuclearenvelope(arrow)areshownineachfigure.Noabnormal nuclearenvelopesofneuronsweredetectedin Dyt1 sKOor Dyt1 cKO miceaswellasCTmice.MagnificationinA,C,E,G:5,000 6 doi:10.1371/journal.pone.0024539.g005 Striatum-Specific Dyt1 ConditionalKOMice PLoSONE|www.plosone.org5September2011|Volume6|Issue9|e24539

PAGE 6

metabolismitself.Furtherstudiesfocusingonthecorticostriatal pathwaywillelucidatedetailsofthemolecularmechanismofthis disease. ApostmortemstudyreportedincreasedstriatalDOPAC/DA ratioinDYT1dystoniapatients[18]. Dyt1 D GAGheterozygous KImalemiceexhibitdecreasedHVA[12],while Dyt1 KDmice exhibiteddecreasedDOPAC[15]intheirstriata.However,the mechanismofthestriatalmonoaminealterationshasnotbeen clear.Inthepresentstudy, Dyt1 sKOmicedidnotexhibit alterationofDA,DOPACorHVAcontentsinthestriatum, suggestingthatlossoftorsinAfunctioninthestriatumitselfmay notaffectmonoaminecontentsinthestriatum.Theresultsalso suggestthatdopaminergictoneinthestriatummayhavea limitedroleinthepathogenesisofDYT1dystoniabecause Dyt1 sKOmiceexhibitedmotordeficitwithoutalterationofstriatal monoaminecontents.Theresultsareconsistentwiththeclinical observationthatlevodopaisnoteffectiveforDYT1patients.The significantalterationsinthestriatalDAmetabolitesfoundin Dyt1 D GAGheterozygousKImalemiceand Dyt1 KDmicemaybe causedbylossoftorsinAfunctionindopaminergicneurons derivedfromthesubstantianigra.Generatingsubstantianigraspecific Dyt1 conditionalKOmiceinthefuturemayfurtherallow ustodeterminetheoriginofthealterationsofDAmetabolism observedin Dyt1 D GAGheterozygousKImalemiceand Dyt1 KDmice. Abnormalnuclearenvelopeshavebeenreportedintransfected cellsover-expressingthemutantformsoftorsinA[21–23]. Abnormalnuclearenvelopeswerealsoreportedin Dyt1 KO miceand Dyt1 D GAGhomozygousKImice[24].However, abnormalnuclearenvelopeswerenotfoundin Dyt1 D GAG heterozygousKImicewhichexhibitmotordeficits,casting doubtsaboutitsroleinthepathogenesisofDYT1dystonia.To analyzewhetherthemotordeficitsin Dyt1 sKOandcKOmice arecausedbyabnormalnuclearenvelopes,weexaminedthe nuclearenvelopesofstriatalMSNsorcorticalneurons.However, wecouldnotfindanyblebbingorothernuclearenvelope abnormalitiesinboth Dyt1 sKOandcKOmice,suggestingthat nuclearenvelopeabnormalitymaynotplayanyroleinmotor impairmentseeninthesemice.Sinceabnormalenvelopes invivo aredetectedonlyinneonatal Dyt1 D GAGhomozygousKImice and Dyt1 KOmice,thatexhibitneonatallethality,theabnormal nuclearenvelopemaybeanindicatorofneuronalcelldeathin thesedyingmice.MaterialsandMethods Makingofthestriatum-specific Dyt1 conditional knockoutmiceDyt1loxP mice[16]and Rgs9-cre mice[29]werepreparedas describedearlier.Genotypingfor Dyt1 sKOandCTmicewas performedbymultiplexPCRusingtailDNAwithF(5 9 ATTCAAAAATGTTGTCATAGCCAGG-3 9 )andT(5 9 -CTACAGTGACCTGAATCATGTGGC-3 9 )primersetsfor Dyt1loxP locus[16],andcreA(5 9 -ATCTCCGGTATTGAAACTCCAGCGC-3 9 )andcre6(5 9 -CACTCATGGAAAATAGCGATC3 9 )primersetsfor cre locus[36].Toconfirmthestriatum-specific deletionof Dyt1 exons3and4,theolfactorybulb,striatum, cerebralcortex,cerebellum,andbrainstemweredissectedfrom Dyt1loxP and Dyt1 sKOmousebrains.Thetissuesweredigested withlysisbuffer[100mMTris N Cl(pH8.5),5mMEDTA N 2Na, 0.2%SDS,200mMNaCl,1mMCaCl2,0.1mg/mlProteinase K(Invitrogen)]at55 u Covernight.EachDNAwasisolatedby addingequalvolumeofisopropanolandthenwashingwith70% ethanol.Thedeletionofexons3and4wasconfirmedbyPCR usingFandTcko2(5 9 -CCATAGCTGGACCTGCAATTAAG3 9 )primersasdescribedearlier[16,37].Agroupconsistedof11 Dyt1 sKOmice(7malesand4females)and13CTmice(6males and7females)wasusedforthebehaviortests.Apairof Dyt1 sKO andCTmicewasusedtopreparebrainsectionsfortransmission electronmicroscopyanalysis.SinceonsetofDYT1dystoniais usuallychildhoodoradolescence,weusedadultmiceinthisstudy. Allexperimentswereperformedbyinvestigatorsblindtothe genotypes.Micewerehousedundera12hours-lightand 12hours-darkcyclewithaccesstofoodandwater adlibitum .All experimentswerecarriedoutincompliancewiththeUSPHS GuideforCareandUseofLaboratoryAnimalsandapprovedby IACUCofUniversityofAlabamaatBirminghamwithAnimal ProtocolNumber091008198.Preparationof Dyt1 cKOmiceanda Dyt1 D GAG homozygousKImouseTwopairsofadult Dyt1 cKOandCTmicewerepreparedand genotypedasdescribedearlier[16].Aneonatal Dyt1 D GAG homozygousKImousewaspreparedbycrossing Dyt1 D GAG heterozygousKImiceandgenotypedasdescribedearlier[12,38]. Thesemicewereusedfortransmissionelectronmicroscopy analysis. Figure6.AbnormalnuclearenvelopestructureofthecerebralcorticalneuronsinanewbornDyt1D GAGhomozygousKImouse. (A) Arepresentativeelectronmicroscopeimageofthecerebralcorticalneuronsofanewborn Dyt1 D GAGhomozygousKImouse.Thissectionhastwo abnormalnuclearenvelopestructures(arrows)ontheleftandrightpartsoftheimage.Enlargedimagesoftheleft(B)andtheright(C)partsofthe nuclearenvelopeclearlyshowabnormalnuclearenvelopestructuresinthecerebralcorticalneuron.Nucleus(N)andabnormalnuclearenvelope structure(arrow)areshownineachfigure.MagnificationinA:6,000 6 doi:10.1371/journal.pone.0024539.g006 Striatum-Specific Dyt1 ConditionalKOMice PLoSONE|www.plosone.org6September2011|Volume6|Issue9|e24539

PAGE 7

Behavioralsemi-quantitativeassessmentsofmotor disordersBehavioralsemi-quantitativeassessmentsofmotordisorders wereperformedasdescribedearlier[12,39].Mousefrom96to 139-daysoldwasplacedonatableandassessmentsofhindpaw clasping,hindpawdystonia,truncaldystoniaandbalance adjustmentstoaposturalchallengewereperformed.Thehindpaw claspingwasassessedashindpawmovementsforpostural adjustmentandattempttostraightenupwhilethemousewas suspendedbythemid-tail.Thehindpawdystoniawasassessedas theincreasedspacingbetweenthelimbs,poorlimbcoordination, crouchingpostureandimpairmentofgait.Truncaldystoniawas assessedastheflexedposture.Posturalchallengewasperformed byflippingthemouseontoitsbackandtheeaseofrightingwas noted.AcceleratedrotarodtestThemotorperformancewasassessedwithEconomexacceleratingrotarod(ColumbusInstruments)asdescribedearlier[12]. Theapparatusstartedataninitialspeedof4rpm.Rodspeedwas graduallyacceleratedatarateof0.2rpm/s.Thelatencytofall wasmeasuredwithacutofftimeof2min.Micefrom103to146daysoldweretestedforthreetrialsoneachdayfor2days.The trialswithinthesamedaywereperformedatapproximately 1hourintervals.Open-fieldtestTheopen-fieldtestwasperformedunderlightconditionas describedearlier[40,41].Spontaneousactivitiesofindividualmice from124to167daysoldwererecordedbyinfraredlightbeam sensorsina41 6 41 6 31cmacrylcasefor15minat1min intervalsusingDigiProsoftware(AccuScanInstruments).Beam-walkingtestThebeam-walkingtestwasperformedasdescribedearlier [12,15,41,42].Briefly,thebeam-walkingtestwasperformed withinthelast8hoursofthelightperiodafteracclimationtoa sound-attenuatedtestingroomfor1hour.Themicefrom141to 184daysoldweretrainedtotransverseamediumsquarebeam (14mmwide)inthreeconsecutivetrialseachdayfor2daysand testedtwiceeachonthemediumsquarebeamandamedium roundbeam(17mmdiameter)onthethirdday.Themicewere thentestedtwiceeachonasmallroundbeam(10mmdiameter) andasmallsquarebeam(7mmwide)onthefourthday.Their hindpawslipsoneachsideduringtransverseonthe80cm-length beamswerecounted.RadioligandbindingassaytoD2Rinthestriatal membranefractionsRadioligandbindingassaytothestriatalD2Rwasperformedby using[3H]YM-09151-2basedonamethodreportedearlier[43]. Thestriataweredissectedfromthree Dyt1 sKOmiceandthree CTmiceof268–321daysold(average284daysold)and homogenizedinninevolumesofice-cold50mMTris N Cl,8mM MgCl2,5mMEDTA N 2Na,pH7.1.Eachhomogenatewas centrifugedat18,000 6 gfor20minat4 u Candthepelletwas suspendedinthesamebufferandstoredat 2 80 u C.Theprotein concentrationofthemembranepreparationwasdeterminedby Bradfordassayusingbovineserumalbuminasastandard.An aliquotofthehomogenatewassuspendedin200mlofbinding buffer[50mMTris N Cl,120mMNaCl,5mMKCl,5mM MgCl2,1.5mMCaCl2,1mMEDTA N 2Na,10mMpargyline hydrochloride(Sigma-Aldrich)and0.1%ascorbicacid,pH7.4] with0.03–0.96nM[3H]YM-09151-2(2.64TBq/mmol,Perkin Elmer).Thereactionmixturewasincubatedat25 u Cfor40min, andthenrapidlyfilteredundervacuumthroughglassmicrofibre GF/Bfilter(Whatman).Thefilterwasthenwashedfourtimes with4mloftheice-coldbindingbufferwithouttheisotopeand thendried.Theradioactivityofthefilterswasmeasuredin6mlof ScintiSafeEcono1(FisherScientific)byaBeckmanliquid scintillationcounter.Non-specificbindingof[3H]YM-09151-2 wasmeasuredinpresenceof30mM(S)-(-)-sulpiride(SigmaAldrich).Theexperimentswereperformedinduplicate.HPLCanalysisStriataweredissectedfromthebrainsof13 Dyt1 sKOand13 CTmicefrom302to345daysoldandhomogenizedinice-cold 0.2Nperchloricacid.Thehomogenatewerecentrifugedfor 15minat15,000 6 gat4 u Ctoremovedebris.Twentymicroliters ofthesupernatantrepresenting2mgoftissue,wastheappliedtoa C18CreversephaseHPLCcolumn(Varian)connectedtoanESA model5200Aelectrochemicaldetector.DA,DOPACandHVA wereanalyzedusingarunningbufferof50mMpotassium phosphatebufferwith0.5mMoctylsulfate(Sigma)and8% acetonitrileasdescribedearlier[12,15,41].DA,DOPAC,and HVAwereseparatedat0.8ml/minandquantifiedbycomparing tothestandardreagents(Sigma).TransmissionelectronmicroscopyanalysisBrainsectionsfortransmissionelectronmicroscopywere preparedasdescribedearlier[14]. Dyt1 sKOandtheirCTmice (n=1each,6weeksofage), Dyt1 cKOmiceandtheirCTmice (n=2each,8weeksofage),andanewborn Dyt1 D GAG homozygousKImousewereperfusedwithchilled0.1M phosphate-bufferedsaline(pH7.4)followedbyKarnovsky’s fixativeinphosphatebuffered2%glutaraldehydeand2.5% paraformaldehyde.Thebrainsweredissectedoutandleftin Karnovsky’sfixativeovernight.Thetissuewasthentrimmedand washedincacodylatebufferwithnofurtheradditives.Microwave fixationwasusedwiththesecondary2%osmiumtetroxide fixative,followedbytheadditionof3%potassiumferricyanidefor 30minutes.Afterwashingwithwater,saturateduranylacetatewas addedforenblocstaining.Thetissuewasdehydratedinaseriesof increasingconcentrationsofethanolstartingat50%.Acetonitrile wasusedasthetransitionfluidbetweenethanolandtheepoxy. Infiltrationserieswasdonewithanepoxymixtureusingtheepon substituteLx112.Theresultingblockswerepolymerizedat90 u C overnight,trimmedwitharazorblade,andultrathinsectioned withdiamondknives.Sectionswerethenstainedwithuranyl acetateandleadcitrate,andthenuclearenvelopesinthestriataof Dyt1 sKOandtheirCTmice,andcerebralcorticesof Dyt1 cKO mice,theirCTmiceanda Dyt1 D GAGhomozygousKImouse wereexaminedorphotographedwithaHitachiH600transmissionelectronmicroscope.StatisticsDataintheopen-fieldtest,latencytofallintheaccelerated rotarod,andHPLCwereanalyzedbyANOVAmixedmodelwith SAS/STATAnalystprogram(Version9.1.3;SASinstituteInc. NC)asdescribedearlier[12,16,41].Theturnoversofmetabolites wereanalyzedastheratiooftheneurochemicalsafternaturallog transformationtoobtainanormaldistribution.Slipsnumbersof hindpawsinbeam-walkingtestwereanalyzedbylogistic regression(GENMOD)withPoissondistributionusingGEE modelinthesoftware.Sex,ageandbodyweightwereinputas variables.Verticalmovementnumbersintheopen-fieldtest, latencytofallintheacceleratedrotarodtest,andslipsnumbersinStriatum-Specific Dyt1 ConditionalKOMice PLoSONE|www.plosone.org7September2011|Volume6|Issue9|e24539

PAGE 8

thebeam-walkingtestwereanalyzedafternaturallogtransformationtoobtainanormaldistribution.ThedatainCTmicewere normalizedtozero.Radioligandbindingdatawereanalyzedby Student’s t test. Bmaxand Kdofeachmousewereindividually calculatedinScatchardplotandthoseofeachgenotypewere compared.Significancewasassignedat p 0.05.AcknowledgmentsWethankLisaFoster,AndreaMcCulloughandtheirstaffforanimalcare, andLouAnnMiller,MikiJinnoandMarkP.DeAndradefortheir technicalassistance.AuthorContributionsConceivedanddesignedtheexperiments:FYMTDDGSYL.Performed theexperiments:FYMTDJLYL.Analyzedthedata:FYYL.Contributed reagents/materials/analysistools:JLDGSYL.Wrotethepaper:FYDGS YL.References1.BreakefieldXO,BloodAJ,LiY,HallettM,HansonPI,etal.(2008)The pathophysiologicalbasisofdystonias.NatRevNeurosci9:222–234. 2.MullerU(2009)Themonogenicprimarydystonias.Brain132:2005–2025. 3.OzeliusLJ,HewettJW,PageCE,BressmanSB,KramerPL,etal.(1997)The early-onsettorsiondystoniagene(DYT1)encodesanATP-bindingprotein.Nat Genet17:40–48. 4.TorresGE,SweeneyAL,BeaulieuJM,ShashidharanP,CaronMG(2004) EffectoftorsinAonmembraneproteinsrevealsalossoffunctionanda dominant-negativephenotypeofthedystonia-associatedDeltaE-torsinAmutant. ProcNatlAcadSciUSA101:15650–15655. 5.HewettJW,TannousB,NilandBP,NeryFC,ZengJ,etal.(2007)Mutant torsinAinterfereswithproteinprocessingthroughthesecretorypathwayin DYT1dystoniacells.ProcNatlAcadSciUSA104:7271–7276. 6.KustedjoK,DeechongkitS,KellyJW,CravattBF(2003)Recombinant expression,purification,andcomparativecharacterizationoftorsinAandits torsiondystonia-associatedvariantDeltaE-torsinA.Biochemistry42: 15333–15341. 7.KonakovaM,PulstSM(2005)Dystonia-associatedformsoftorsinAaredeficient inATPaseactivity.JMolNeurosci25:105–117. 8.BurdetteAJ,ChurchillPF,CaldwellGA,CaldwellKA(2010)Theearly-onset torsiondystonia-associatedprotein,torsinA,displaysmolecularchaperone activityinvitro.CellStressChaperones15:605–617. 9.LeungJC,KleinC,FriedmanJ,ViereggeP,JacobsH,etal.(2001)Novel mutationintheTOR1A(DYT1)geneinatypicalearlyonsetdystoniaand polymorphismsindystoniaandearlyonsetparkinsonism.Neurogenetics3: 133–143. 10.ZirnB,GrundmannK,HuppkeP,PuthenparampilJ,WolburgH,etal.(2008) NovelTOR1Amutationp.Arg288Glninearly-onsetdystonia(DYT1).JNeurol NeurosurgPsychiatry79:1327–1330. 11.RitzKGM,FonckeEM,vanRuissenF,vanderLindenC,VergouwenMD, BloemBR,VandenbergheW,CrolsR,SpeelmanJD,BaasF,TijssenMA(2009) Myoclonus-dystonia:clinicalandgeneticevaluatio…[JNeurolNeurosurg Psychiatry.2009]-PubMedresult.JNeurolNeurosurgPsychiatry80:653–658. 12.DangMT,YokoiF,McNaughtKS,JengelleyTA,JacksonT,etal.(2005) GenerationandCharacterizationofDyt1deltaGAGKnock-inMouseasa ModelforEarly-OnsetDystonia.ExpNeurol196:452–463. 13.CaoS,HewettJW,YokoiF,LuJ,BuckleyAC,etal.(2010)Chemical enhancementoftorsinAfunctionincellandanimalmodelsoftorsiondystonia. DisModelMech3:386–396. 14.YokoiF,YangG,LiJ,DeandradeMP,ZhouT,etal.(2010)Earlieronsetof motordeficitsinmicewithdoublemutationsinDyt1andSgce.JBiochem148: 459–466. 15.DangMT,YokoiF,PenceMA,LiY(2006)Motordeficitsandhyperactivityin Dyt1knockdownmice.NeurosciRes56:470–474. 16.YokoiF,DangMT,MitsuiS,LiJ,LiY(2008)Motordeficitsandhyperactivity incerebralcortex-specificDyt1conditionalknockoutmice.JBiochem143: 39–47. 17.FurukawaY,HornykiewiczO,FahnS,KishSJ(2000)Striataldopaminein early-onsetprimarytorsiondystoniawiththeDYT1mutation.Neurology54: 1193–1195. 18.AugoodSJ,HollingsworthZ,AlbersDS,YangL,LeungJC,etal.(2002) DopaminetransmissioninDYT1dystonia:abiochemicalandautoradiographicalstudy.Neurology59:445–448. 19.AsanumaK,MaY,OkulskiJ,DhawanV,ChalyT,etal.(2005)Decreased striatalD2receptorbindinginnon-manifestingcarriersoftheDYT1dystonia mutation.Neurology64:347–349. 20.NapolitanoF,PasqualettiM,UsielloA,SantiniE,PaciniG,etal.(2010) DopamineD2receptordysfunctionisrescuedbyadenosineA2Areceptor antagonisminamodelofDYT1dystonia.NeurobiolDis38:434–445. 21.NaismithTV,HeuserJE,BreakefieldXO,HansonPI(2004)TorsinAinthe nuclearenvelope.ProcNatlAcadSciUSA101:7612–7617. 22.Gonzalez-AlegreP,PaulsonHL(2004)Aberrantcellularbehaviorofmutant torsinAimplicatesnuclearenvelopedysfunctioninDYT1dystonia.JNeurosci 24:2593–2601. 23.GoodchildRE,DauerWT(2004)Mislocalizationtothenuclearenvelope:an effectofthedystonia-causingtorsinAmutation.ProcNatlAcadSciUSA101: 847–852. 24.GoodchildRE,KimCE,DauerWT(2005)Lossofthedystonia-associated proteintorsinAselectivelydisruptstheneuronalnuclearenvelope.Neuron48: 923–932. 25.GrundmannK,ReischmannB,VanhoutteG,HubenerJ,TeismannP,etal. (2007)OverexpressionofhumanwildtypetorsinAandhumanDeltaGAG torsinAinatransgenicmousemodelcausesphenotypicabnormalities. NeurobiolDis27:190–206. 26.KimCE,PerezA,PerkinsG,EllismanMH,DauerWT(2010)Amolecular mechanismunderlyingtheneural-specificdefectintorsinAmutantmice.Proc NatlAcadSciUSA107:9861–9866. 27.SauerB,HendersonN(1988)Site-specificDNArecombinationinmammalian cellsbytheCrerecombinaseofbacteriophageP1.ProcNatlAcadSciUSA85: 5166–5170. 28.SchwenkF,BaronU,RajewskyK(1995)Acre-transgenicmousestrainforthe ubiquitousdeletionofloxP-flankedgenesegmentsincludingdeletioningerm cells.NucleicAcidsRes23:5080–5081. 29.DangMT,YokoiF,YinHH,LovingerDM,WangY,etal.(2006)Disrupted motorlearningandlong-termsynapticplasticityinmicelackingNMDAR1in thestriatum.ProcNatlAcadSciUSA103:15254–15259. 30.McBrideWJ,ChernetE,DyrW,LumengL,LiTK(1993)Densitiesof dopamineD2receptorsarereducedinCNSregionsofalcohol-preferringPrats. Alcohol10:387–390. 31.WangY,XuR,SasaokaT,TonegawaS,KungMP,etal.(2000)DopamineD2 longreceptor-deficientmicedisplayalterationsinstriatum-dependentfunctions. JNeurosci20:8305–8314. 32.PisaniA,MartellaG,TscherterA,BonsiP,SharmaN,etal.(2006)Altered responsestodopaminergicD2receptoractivationandN-typecalciumcurrents instriatalcholinergicinterneuronsinamousemodelofDYT1dystonia. NeurobiolDis24:318–325. 33.WangZ,KaiL,DayM,RonesiJ,YinHH,etal.(2006)Dopaminergiccontrol ofcorticostriatallong-termsynapticdepressioninmediumspinyneuronsis mediatedbycholinergicinterneurons.Neuron50:443–452. 34.ShenW,FlajoletM,GreengardP,SurmeierDJ(2008)Dichotomous dopaminergiccontrolofstriatalsynapticplasticity.Science321:848–851. 35.VidailhetM,VercueilL,HouetoJL,KrystkowiakP,LagrangeC,etal.(2007) Bilateral,pallidal,deep-brainstimulationinprimarygeneraliseddystonia:a prospective3yearfollow-upstudy.LancetNeurol6:223–229. 36.CamposVE,DuM,LiY(2004)Increasedseizuresusceptibilityandcortical malformationinbeta-cateninmutantmice.BiochemBiophysResCommun320:606–614. 37.ZhangL,YokoiF,JinYH,DeandradeMP,HashimotoK,etal.(2011)Altered DendriticMorphologyofPurkinjecellsinDyt1DeltaGAGKnock-Inand PurkinjeCell-SpecificDyt1ConditionalKnockoutMice.PLoSOne6:e18357. 38.YokoiF,DangMT,MillerCA,MarshallAG,CampbellSL,etal.(2009) Increasedc-fosexpressioninthecentralnucleusoftheamygdalaand enhancementofcuedfearmemoryinDyt1DeltaGAGknock-inmice.Neurosci Res65:228–235. 39.FernagutP,DiguetE,StefanovaN,BiranM,WenningG,etal.(2002)Subacute systemic3-nitropropionicacidintoxicationinducesadistinctmotordisorderin adultC57Bl/6mice:behaviouralandhistopathologicalcharacterisation. Neuroscience114:1005. 40.CaoBJ,LiY(2002)Reducedanxiety-anddepression-likebehaviorsinEmx1 homozygousmutantmice.BrainRes937:32–40. 41.YokoiF,DangMT,LiJ,LiY(2006)Myoclonus,motordeficits,alterationsin emotionalresponsesandmonoaminemetabolisminepsilon-sarcoglycan deficientmice.JBiochem140:141–146. 42.DeAndradeMP,YokoiF,vanGroenT,LingrelJB,LiY(2011)CharacterizationofAtp1a3mutantmiceasamodelofrapid-onsetdystoniawith parkinsonism.BehavBrainRes216:659–665. 43.TeraiM,HidakaK,NakamuraY(1989)Comparisonof[3H]YM-09151-2with [3H]spiperoneand[3H]raclopridefordopamined-2receptorbindingtorat striatum.EurJPharmacol173:177–182.Striatum-Specific Dyt1 ConditionalKOMice PLoSONE|www.plosone.org8September2011|Volume6|Issue9|e24539