Altered Dendritic Morphology of Purkinje cells in Dyt1
DGAG Knock-In and Purkinje Cell-Specific Dyt1
Conditional Knockout Mice
Lin Zhang1, Fumiaki Yokoi1, Yuan-Hu Jin1, Mark P. DeAndrade1, Kenji Hashimoto2, David G. Standaert1,
1Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, School of Medicine, University of Alabama at Birmingham, Birmingham,
Alabama, United States of America, 2Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan
Background: DYT1 early-onset generalized dystonia is a neurological movement disorder characterized by involuntary
muscle contractions. It is caused by a trinucleotide deletion of a GAG (DGAG) in the DYT1 (TOR1A) gene encoding torsinA;
the mouse homolog of this gene is Dyt1 (Tor1a). Although structural and functional alterations in the cerebellum have been
reported in DYT1 dystonia, neuronal morphology has not been examined in vivo.
Methodology/Principal Findings: In this study, we examined the morphology of the cerebellum in Dyt1 DGAG knock-in (KI)
mice. Golgi staining of the cerebellum revealed a reduction in the length of primary dendrites and a decrease in the number
of spines on the distal dendrites of Purkinje cells. To determine if this phenomenon was cell autonomous and mediated by a
loss of torsinA function in Purkinje cells, we created a knockout of the Dyt1 gene only in Purkinje cells of mice. We found the
Purkinje-cell specific Dyt1 conditional knockout (Dyt1 pKO) mice have similar alterations in Purkinje cell morphology, with
shortened primary dendrites and decreased spines on the distal dendrites.
Conclusion/Significance: These results suggest that the torsinA is important for the proper development of the cerebellum
and a loss of this function in the Purkinje cells results in an alteration in dendritic structure.
Citation: Zhang L, Yokoi F, Jin Y-H, DeAndrade MP, Hashimoto K, et al. (2011) Altered Dendritic Morphology of Purkinje cells in Dyt1 DGAG Knock-In and Purkinje
Cell-Specific Dyt1 Conditional Knockout Mice. PLoS ONE 6(3): e18357. doi:10.1371/journal.pone.0018357
Editor: Takeo Yoshikawa, Rikagaku Kenkyu ¯sho Brain Science Institute, Japan
Received January 29, 2011; Accepted February 28, 2011; Published March 29, 2011
Copyright: ? 2011 Zhang 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, NS72872, and NS74423),
the Department of Neurology (UAB), and Tyler’s Hope for a Dystonia Cure, Inc. 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: firstname.lastname@example.org
Dystonia is a neurological syndrome characterized by involun-
tary contractions of both agonist and antagonist muscles of
affected regions that cause twisting and abnormal movements or
postures . DYT1 dystonia is a genetically determined form of
generalized early-onset dystonia, with an age of onset between
childhood and adolescence. Symptoms usually first affect the lower
limbs and eventually progress to the entire body . DYT1
dystonia is caused by a trinucleotide deletion of a GAG (DGAG)
codon in the DYT1 (TOR1A) gene, which results in the loss of a
glutamic acid residue in the C-terminal region of the torsinA
protein . We previously generated Dyt1 DGAG knock-in (KI)
mice, a mouse model of DYT1 dystonia, showed impairments of
motor coordination and balance in the beam-walking test and
hyperactivity in the open-field test . The function of torsinA is
largely unknown, but it is a member of the AAA+ ATPase
superfamily and is believed to have a chaperone-like function
Biochemical and cellular studies show that torsinA localizes to
the endoplasmic reticulum , protects against oxidative stress,
and prevents protein aggregate formation [5,7,8,9]. In situ
hybridization studies have also revealed that torsinA mRNA is
highly expressed in the dopaminergic neurons of the substantia
nigra pars compacta, granule and pyramidal neurons of the
hippocampus, Purkinje and dentate nucleus neurons of the
cerebellum, and cholinergic neurons of the neostriatum in humans
[11,12]. Furthermore, ultrastructural studies of the striatum of
humans and macaques have revealed an association of torsinA
immunostaining with small vesicles within axons and presynaptic
terminals forming symmetric synapses .
Growing evidence suggests that the structural and/or functional
abnormalities in the cerebellum could be involved in the
pathogenesis of dystonia. Brain imaging studies have revealed
structural grey matter changes in the cerebellum of patients with
upper limb dystonia , cervical dystonia, and blepharospasm
[15,16]. Increased activation of the cerebellum in the patients with
DYT1 dystonia carriers and alterations in the olivo-cerebellar
pathway of patients with primary focal dystonia have been
reported . Furthermore, there are several reports showing that
trauma to the cerebellum or cerebellar atrophy can cause dystonia
[18,19]. In a genetically dystonic rat that harbors a mutation in the
PLoS ONE | www.plosone.org1 March 2011 | Volume 6 | Issue 3 | e18357
gene caytaxin, cerebellectomy eliminates the motor symptoms and
rescues the juvenile lethality [20,21]. Electronic lesions of dorsal
portions of the lateral vestibular nuclei (dLV), which receive input
from the Purkinje cells, are associated with the greatest
improvement in this rat . The Purkinje cells send the major
inhibitory signal from the cerebellum to the deep cerebellar nuclei,
which is mediated by the neurotransmitter c-aminobutyric acid
(GABA). Pharmacological disruption of the cerebellar signaling is
also shown to induce dystonia in mice . Lastly, the tottering
mouse, which has a recessive mutation of a calcium channel gene,
shows ataxia and paroxysmal dystonia, but this phenotype can be
eliminated by surgical removal of the cerebellum or introduction
into a Purkinje cell-specific degenerative background [24,25].
Despite these findings, few studies have sought to examine the
potential role of the Purkinje cells in the pathogenesis of DYT1
dystonia and other dystonias. In this study, we examined the
morphology of the cerebellum in Dyt1 DGAG knock-in (KI) mice.
Golgi staining of the cerebellum of KI mice revealed a reduction in
the length of primary large dendrites and a decrease in the number
of spines on the distal dendrites of Purkinje cells. Since it has been
reported that the DGAG mutation causes a reduction of torsinA in
the striatum and the entire brain [26,27,28], we sought to
determine if this phenomenon was mediated by a loss of function
of torsinA in Purkinje cells by creating a knockout of the Dyt1 gene
only in Purkinje cells (Dyt1 pKO) of mice. We previously reported
the making of Dyt1 loxP mice . In the present study, we
produced Dyt1 pKO mice by crossing the Dyt1 loxP mice with
Pcp2-cre mice, which restricts lox-mediated recombination to
Purkinje cells . We found that the Purkinje cells in the Dyt1
pKO mice have a similar morphology to that of the KI mice, with
shortened primary large dendrites and decreased spines on the
Golgi staining of Purkinje cells in KI mice
To examine the morphological structures of the Purkinje cells in
KI mice, we used Golgi staining of cerebellar sections. First, the
sizes of the Purkinje cell soma were measured and no significant
difference was found between the KI and control (CT) mice
(means 6 standard errors; CT: 10062.22%; KI: 99.1961.35%;
p.0.05, Figure 1A–1B). However, the length of the large primary
dendrite in the KI mice was approximately 20% shorter than
those in the control mice (CT: 10067.84%; KI: 80.1562.95%;
p,0.01, Figure 1C). Furthermore, the number of spines in the
quaternary dendrite branch of KI mice was approximately 27%
percent less than those in control mice (CT: 10061.9%; KI:
73.1162.17%; p,0.01, Figure 1D–1E). These results suggest the
important role of torsinA in the dendritic structure and
morphology of Purkinje cells.
Generation of the Purkinje-cell specific Dyt1 conditional
knockout (Dyt1 pKO) mice
To examine whether the morphological alterations in the KI
mice was caused by a loss of torsinA function in Purkinje cells, we
generated a Purkinje cell-specific knockout of the Dyt1. Dyt1 loxP
mice  were crossed with a line of mice with the cre recombinase
gene driven by the promoter of Pcp2, a Purkinje cell-specific gene
. Mice double heterozygous for both the Dyt1 loxP and Pcp2-cre
were then crossed with Dyt1 loxP homozygous mice to derive Dyt1
pKO mice and their control littermates (Figure 2A). Genotyping
for Dyt1 pKO and control littermates was performed by multiplex
PCR (Figure 2B). We confirmed Purkinje cell specific knockout of
Dyt1 by in situ hybridization. TorsinA mRNA was highly expressed
in the Purkinje cells in control littermates (Figure 2C.1). In
contrast, torsinA mRNA was not detected in Purkinje cells in Dyt1
pKO mice (Figure 2C.2), suggesting Dyt1 was specifically knocked
out in the Purkinje cells.
Golgi staining of Purkinje cells in Dyt1 pKO mice
To examine whether the Dyt1 pKO mice recapitulate the
dendritic morphology of the KI mice, we performed Golgi staining
on cerebellar sections from Dyt1 pKO mice at approximately 8
months old. Similar to the KI mice, no significant difference in the
size of the Purkinje cell soma in Dyt1 pKO mice compared to
control mice was observed (CT: 10063.20%; Dyt1 pKO:
94.2762.82%; p.0.05, Figure 3A–3B). Furthermore, the length
of the large primary dendrite in the Dyt1 pKO mice was
approximately 37% shorter than those in control mice (CT:
10065.65%; Dyt1 pKO 63.3661.73%; p,0.001, Figure 3C).
Lastly, the number of spines in the quaternary dendrite branch of
the Dyt1 pKO mice was approximately 33% less than those in
control mice (CT: 10063.45%; Dyt1 pKO: 67.3662.2%; p,0.01,
Figure 3D–3E). These results suggest that torsinA plays an
important role in the Purkinje cell dendritic development.
To examine whether the morphological alteration in Purkinje
cells was developmentally regulated, we analyzed the Purkinje cells
in Dyt1 pKO mice at 2–3 months old. No significant difference in
the size of the Purkinje cell soma in Dyt1 pKO mice was observed
compared to control mice (CT: 10063.71%; Dyt1 pKO:
98.0162.52%; p.0.05). There was also no significant difference
in the length of the large primary dendrite in Dyt1 pKO mice
compared to control mice (CT: 10063.81%; Dyt1 pKO
90.6863.37%; p.0.05). However, the number of spines in the
quaternary dendrite branch of the Purkinje cells was reduced in
Dyt1 pKO mice compared to control mice (CT: 10060.55%; Dyt1
pKO: 93.5860.85%; p,0.001). The results suggest that the spine
numbers reduced in advance of the reduction of the length of the
large primary dendrite by the loss of torsinA function in the
First, we examined the morphology of Purkinje cells in the
cerebellum in KI mice. We found that the primary large dendrites
of the Purkinje cells in KI mice were significantly shorter than that
of wild type mice and there was a marked reduction in the number
of spines. Next, to determine if this morphological change was
mediated by a cell-autonomous effect of loss of torsinA function,
we generated a line of mice in which torsinA was conditionally
knocked out only in Purkinje cells. The Dyt1 pKO mice showed a
similar decrease in the length of primary large dendrites and a
reduction in the number of spines. The results also suggest that the
spine numbers reduced in advance of the reduction of the length of
the large primary dendrite by the loss of torsinA function in the
Purkinje cells. These results suggest that torsinA plays an
important role in the development of the cerebellum, and that a
loss of this function in the Purkinje cells results in a cell
autonomous effect leading to an alteration in dendritic structure.
If the same alterations are present in patients with DTY1 dystonia,
this change in synaptic associations between the parallel fibers and
the Purkinje cells may contribute to the pathogenesis of the
Growing evidence suggests that structural and functional
abnormalities in the brain could be involved in the pathogenesis
of dystonia [14,15,16,31]. However, neuronal morphology of the
cerebellum has not been examined in vivo. A recent report suggests
that DYT1 dystonia is a neurodevelopmental disorder involving
Morphology of Purkinje Cells in Dyt1 Mutants
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the cortico-striatal-pallido-thalamocortical and the cerebellar-
thalamo-cortical pathways . Anatomical studies have pro-
posed an interaction between the cerebellum and the basal
ganglia through a disynaptic pathway originating in the
cerebellum and projecting to the striatum via the thalamus
. A deficiency in this connectivity was identified in patients
with DYT1 human carriers . Additionally, rats that have
undergone a hemicerebellectomy were found to have a complete
loss of striatal long-term depression (LTD) , which was also
compromised in mutant human torsinA transgenic mice .
Since the Purkinje cells are the sole output from the cerebellum,
the abnormal function of Purkinje cells, as represented by our
results, may be responsible for the change in striatal LTD in
mutant torsinA animals.
Figure 1. Purkinje cells in KI mice. (A) A representative Purkinje cell trace (left). A representative Purkinje cell from CT and KI mice at 406
magnification (right). (B) There was no significant difference in size of the Purkinje cell soma between CT and KI mice. (C) However, the large primary
dendrite of the Purkinje cells in the KI mice was significantly shorter than those in CT mice. (D) Next, the quaternary dendrite branch of CT and KI mice
was examined at 1006magnification. (E) The number of spines on the quaternary dendrite branch in the KI mice was significantly reduced compared
to CT mice. Scale bars in Panel A represent 10 mm. Scale bars in panel D represent 1 mm. Bars in Panels B, C and E represent means with standard
errors. ** p,0.01.
Morphology of Purkinje Cells in Dyt1 Mutants
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Figure 2. Generation of Dyt1 pKO mice. (A) Schematic diagram of the generation of the Dyt1 pKO mice. Filled boxes represent exons. Filled
triangles indicate loxP sites. Open triangles indicate the FRT sites that were incorporated to remove the neo cassette. Dyt1 loxP mice were crossed
with Pcp2-cre mice to obtain double heterozygotes. The double heterozygotes were crossed with Dyt1 loxP homozygotes to obtain Dyt1 pKO mice.
The primer sites for genotyping of Dyt1 locus were shown by an arrow pairs. The short bar under exon 5 is the site of probe used for in situ
hybridization. Dyt1 exons 3 and 4 were removed in Purkinje cells of Dyt1 pKO mice. (B) An agarose gel showing the various PCR products that were
used to genotype mice. The top band indicates the presence of the Pcp2-cre locus, the middle band represents the Dyt1 loxP locus, and the bottom
band represents the Dyt1 wild-type locus. Lanes 4: Dyt1 loxP homozygous mice. Lanes 2, 6: Dyt1 loxP heterozygous mice. Lanes 1, 3, 5: Dyt1 pKO mice.
(C) In situ hybridization was used to confirm the Purkinje cell-specific knockout of the Dyt1 gene. CT (C.1) and Dyt1 pKO (C.2) mice. GL: granule cell
layer; PC: Purkinje cell layer; ML: molecular layer. Scale bar represents 100 mm in C.2.
Morphology of Purkinje Cells in Dyt1 Mutants
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The Purkinje cells play an important role in motor coordination
and motor learning by integrating two types of excitatory inputs:
climbing fibers and parallel fibers. Climbing fibers originate from
the inferior olivary nucleus and convey the motor signals to the
parallel fibers. Parallel fibers are the T-shaped axons of cerebellar
granule cells, and convey the sensory and motor information
carried through the pontocerebellar and spinocerebellar mossy
fiber pathways. It is also known that the parallel fiber-Purkinje cell
(PF-PC) synapse plays an important role in the adaptive learning
process . Glutamate receptor d2 subunit (GluRd2) is selectively
expressed in the Purkinje cells of the cerebellum . Impairment
of motor coordination, Purkinje cell synapse formation, and
cerebellar LTD was reported in GluRd2 mutant mice .
GluRd2 mutant mice were unable to stabilize PF-PC synapses and
resulted in a reduction in the number of PF-PC synapses along
with impaired CF synapse elimination . We previously
reported motor deficits in KI mice  and in this study found
morphological alterations of Purkinje cells in KI mice. The results
suggest that functional alterations of the cerebellum may associate
with the pathogenesis of DYT1 dystonia.
Furthermore, torsinA is known to interact with kinesin 1 , a
motor protein involved in cellular transport and the cytoskeleton
Figure 3. Purkinje cells in Dyt1 pKO mice. (A) A representative Purkinje cell from control and Dyt1 pKO mice, produced at 406magnification. (B)
The size of the Purkinje cell soma in Dyt1 pKO mice was not significantly different than those of CT mice. (C) The large primary dendrite of the
Purkinje cells in the Dyt1 pKO mice was significantly shorter than those in CT mice. (D) A representative quaternary dendrite branch of CT and Dyt1
pKO mice was examined at 1006magnification. (E) The number of spines on the quaternary dendrite branch in the Dyt1 pKO mice were significantly
reduced compared to those of CT mice. Scale bars in Panel A represent 10 mm. Scale bars in Panel D represent 1 mm. Bars in Panels B, C and E
represent means with standard errors. ** p,0.01, *** p,0.001.
Morphology of Purkinje Cells in Dyt1 Mutants
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of the cell. In vitro studies have shown that suppression of kinesin
leads to decreased neurite extension in hippocampal neurons
. Furthermore, in human neuroblastoma cells, it was shown
that overexpression of mutant torsinA also leads to decreased
neurite extension . A decrease in neurite extension possibly
through kinesin could explain the decrease in primary dendritic
length. In addition to the shortened primary dendrite length,
however, the KI mice also showed a decrease in spine number.
This decrease in neurite extension would not explain the decrease
in number of spines. Recent reports have shown that overex-
pression of mutant human torsinA in Drosophila results in altered
synaptic morphology at the neuromuscular junction . It is,
therefore, likely that this decrease in spine number results in
altered synaptic plasticity, possibly leading to decreased parallel
Lastly, we have created a novel Purkinje cell-specific knockout
of Dyt1 to compare its morphology to the KI and wild-type mice.
Several genetic studies suggest that a loss-of-function of torsinA
contributes to the pathology of dystonia [28,29,45]. We have
found the Dyt1 pKO mice replicates the KI dendritic morphology
of Purkinje cells, in that they have decreased primary dendrite
length and decreased spine number. These findings suggest that
the DGAG mutation in the KI mice results in a loss of function of
torsinA and provide further evidence of the important role of
torsinA in the cerebellum.
In conclusion, these results add to the growing body of
evidence of the importance of the cerebellum in the pathogenesis
of dystonia and this being the first reported morphological
alteration in the cerebellum. Furthermore, these results suggest
that torsinA plays an important role in the regulations of
dendritic length and spine number in the cerebellum. Finally, the
DGAG mutation in the Dyt1 may result in a loss of function of
torsinA in the Purkinje cells. These findings will further the
understanding of the pathophysiology that underlies not only
DYT1 dystonia but also possibly other neurological movement
Materials and Methods
All experiments were carried out in compliance with the
USPHS Guide for Care and Use of Laboratory Animals and
approved by the Institutional Animal Care and Use Committee
at the University of Alabama at Birmingham with Animal
Protocol Number 10008918. All experiments were performed by
investigators blind to the genotype of the mice. Dyt1 loxP mice
were generated as previously described . To generate the
Purkinje cell-specific knockout, Dyt1 loxP mice were first crossed
with Pcp2-cre mice . The double heterozygous mice were
then crossed with Dyt1 loxP homozygous mice to derive Dyt1
pKO mice and their control littermates. Genotyping for Dyt1
pKO and control littermates was performed by multiplex PCR
using F (59-ATTCAAAAATGTTGTCATAGCCAGG-39) and
T (59-CTACAGTGACCTGAATCATGTGGC-39) primer sets
 for Dyt1 loxP and creA (59-ATCTCCGGTATTGAAA-
CTCCAGCGC-39) and cre6 (59-CACTCATGGAAAATAGC-
GATC-39) primer sets for cre . KI mice were prepared and
genotyped as previously described . Mice were housed under
a 12-h-light/dark cycle with ad libitum access to food and water.
In Situ Hybridization
Purkinje-cell specific knockout of torsinA was confirmed by in
situ hybridization. To prepare the Digoxigenin (DIG) -labeled
probe, a DNA fragment corresponding to 39-UTR of Dyt1 was
amplified by PCR with a primer sets of Dyt1insitu2 (59-
CACCAAGCTGGACTACTACCTGGA-39) and Dyt1insitu3 (59-
GAAAGCTTCTTATAGTATTAAAACC-39) and Dyt1 DNA
plasmid template. The amplified PCR fragment was then ligated
into a pGEM-T Easy vector (Promega). Next, the construct was
transformed in E. coli JM109 competent cells (Promega) and single
colonies were isolated. An appropriate clone that had the DNA
fragment in the correct direction was confirmed by PCR using T7
and Dyt1insitu3 primer sets. The plasmid DNA was purified and
cut with the restricted enzyme NcoI. The DNA fragment was
purified and dissolved in diethyl pyrocarbonate (DEPC)-treated
water. DIG-labeled probe for Dyt1 was prepared by using
digoxingenin RNA labeling kit with the SP6 promoter (Roche
Applied Science, Indianapolis, IN). In situ hybridization to sagittal
sections of the cerebellum was performed as previously described
Adult KI mice (CT: n=4; KI: n=7, approximately 8 months
old), Dyt1 pKO mice (CT: n=3; Dyt1 pKO: n=3, approximately
8 months old), and another batch of Dyt1 pKO mice (CT: n=4;
Dyt1 pKO: n=4, 2–3 months old) were used in this experiment.
After mice were deeply anesthetized with pentobarbital (1 ml/kg,
intraperitoneally), the cerebellum was quickly removed and
prepared for Golgi staining using the FD Rapid Golgi Stain Kit
(FD NeuroTechnologies, Ellicot City, MD). After staining, the
cerebellum was frozen with dry ice and sectioned parasagittally
(150 mm) using a sliding microtome (Histoslide 2000, Reichert-
Jung). The sections were then mounted on slide, dehydrated with
xylene and then cover-slipped with permount (Fisher Scientific).
Images of individual Purkinje cells were captured using a Nikon
ECLIPSE E800M microscope with a 406 Plan Fluor objective
lens. The size of each Purkinje cell soma and the length of the
large primary dendrite (5 to 16 cells from each mouse) were
measured using ImageJ software (NIH, Ver. 1.42 g). Quaternary
dendrite branches from the soma as shown in Figure 1A were
chosen at random and the spines were counted. The numbers of
spines located on randomly selected quaternary dendrite branches
(5 to 16 cells from each mouse), 10 mm in length, were counted
manually using a 406and 1006Plan Fluor objective lens and a
106 CFIUW ocular lens. The size of Purkinje cell soma, the
length of the large primary dendrite, and the spine numbers were
measured by an investigator blind to the genotypes and the data
were analyzed by another investigator.
The area (mm2) of each Purkinje cell soma and the length of the
large primary dendrite (mm) were measured and the data were
normalized to control mice and expressed as percentage. The
number of spines were counted and normalized to that of control
mice. All results were analyzed using Student’s t-test. Significance
was assigned by a P-value less than 0.05.
We thank Alena Samal and Miki Jinno for their technical assistance, and
Andrea McCullough, Kevin Feng, and their staff for animal care.
Conceived and designed the experiments: YL LZ. Performed the
experiments: LZ FY YHJ. Analyzed the data: LZ MPD. Contributed
reagents/materials/analysis tools: LZ FY YHJ YL. Wrote the paper: LZ
KH DGS YL.
Morphology of Purkinje Cells in Dyt1 Mutants
PLoS ONE | www.plosone.org6 March 2011 | Volume 6 | Issue 3 | e18357
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Morphology of Purkinje Cells in Dyt1 Mutants
PLoS ONE | www.plosone.org7 March 2011 | Volume 6 | Issue 3 | e18357