Engineering animal models of dystonia

Engineering Animal Models of Dystonia

Janneth Oleas, BS,1 Fumiaki Yokoi, PhD,1 Mark P. DeAndrade, MS,1 Antonio Pisani, MD,2 and Yuqing Li, PhD1*

1Department of Neurology, College of Medicine, University of Florida, Gainesville, Florida2Department of Neuroscience, University Tor Vergata/Laboratory of Neurophysiology and Synaptic Plasticity, Fondazione Santa Lucia

Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy

ABSTRACT: Dystonia is a neurological disordercharacterized by abnormal involuntary movements thatare prolonged and often cause twisting and turning.Several genetically modified worms, fruit flies, androdents have been generated as models of genetic dys-tonias, in particular DYT1, DYT11, and DYT12 dysto-nias. Although these models do not show overtdystonic symptoms, the rodent models exhibit motordeficits in specialized behavioral tasks, such as therotarod and beam-walking tests. For example, in arodent model of DYT12 dystonia, which is generallystress triggered, motor deficits are observed only afterthe animal is stressed. Moreover, in a rodent model ofDYT1 dystonia, the motor and electrophysiological defi-cits can be rescued by trihexyphenidyl, a common anti-cholinergic medication used to treat dystonic symptomsin human patients. Biochemically, the DYT1 and DYT11animal models also share some similarities to patients,such as a reduction in striatal D2 dopamine receptor

and binding activities. In addition, conditional knockoutmouse models for DYT1 and DYT11 dystonia demon-strate that loss of the causal dystonia-related proteinsin the striatum leads to motor deficits. Interestingly, lossof the DYT1 dystonia causal protein in Purkinje cellsshows an improvement in motor performance, suggest-ing that gene therapy targeting of the cerebellum orintervention in its downstream pathways may be useful.Finally, recent studies using DYT1 dystonia worm andmouse models led to a potential novel therapeuticagent, which is currently undergoing clinical trials.These results indicate that genetic animal models arepowerful tools to elucidate the pathophysiology and tofurther develop new therapeutics for dystonia. VC 2013Movement Disorder Society

Key Words: dystonia; animal model; DYT1; DYT11;DYT12

Dystonia is a neurological disorder characterized bysustained contractions of muscles, which cause abnor-mal movement, twisting, and postures.1 Several meth-ods of classifying dystonia exist. The earliest methodof classifying dystonia was to segregate it as either pri-mary or secondary.2 In primary dystonia, the dystonicsymptoms are not caused by another condition orenvironmental factor and can be hereditary or

nonhereditary. In secondary dystonia, the dystonicsymptoms are usually the result of another condition(eg stroke, brain injury, or metabolic disease) or ofcertain medications.3 A more recent classification sys-tem has been developed that segregates dystonia basedon clinical characteristics and etiology.1 Currenthypotheses suggest that dystonia is a neurodevelop-mental disorder4 that is caused by an abnormal motorcircuitry in the cerebral cortex, basal ganglia, thala-mus, and cerebellum, which, in turn, leads to abnor-mal synaptic plasticity and altered neurotransmission5–

10 and thereby likely causes the dystonic symptoms.The animal models of dystonia can be classified into

two fundamental groups: phenotypic and genotypic.The primary goal of the phenotypic animal models isto mimic the dystonic phenotype seen in patients. Onecommon method has been to inject pharmacologicalcompounds into specific brain regions, such as the cer-ebellum or striatum, or systemically to attempt toreproduce an overt dystonic phenotype.7,11,12 Theadvantage of this approach is that it has the possibility

------------------------------------------------------------*Correspondence to: Dr. Yuqing Li, Department of Neurology, Collegeof Medicine, University of Florida, PO Box 100236, Gainesville, FL32610; [email protected]

Supported by grants from the Tyler’s Hope for a Dystonia Care, Inc., theNational Institutes of Health (NS37409, NS47466, NS47692, NS54246,NS57098, NS65273, NS72872, and NS74423), and startup funds fromthe Departments of Neurology at UAB and UF (Yuqing Li).

Relevant conflicts of interest/financial disclosures: Nothing to report.Full author roles may be found in the Acknowledgments section online.

Received: 7 December 2012; Revised: 25 May 2013; Accepted: 29May 2013

Published online in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/mds.25583

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990 Movement Disorders, Vol. 28, No. 7, 2013

of the potential identification of a brain region, cellreceptor, or signaling pathway that may contribute todystonia onset. These models are also particularly use-ful in analyzing secondary dystonia caused by braininjury or from the side effects of drugs.13 However,one must consider that the primary goal of genotypicdystonia animal models is to mimic the genetic muta-tions found in patients, which can be accomplished bytargeted mutagenesis or through insertional transgenictechniques. This type of model is particularly usefulfor analyzing the pathophysiology of genetic dystoniasby examining the normal function of the causal pro-teins and for analyzing the mutated effects. The pri-mary aim of this review is to highlight the findings ofgenotypic models of DYT1, DYT11, and DYT12 dys-tonias using worms (Caenorhabditis elegans), fruit flies(Drosophila melanogaster), and rodents to illustratetheir utility in elucidating dystonia pathophysiologyand to uncover novel therapeutics.

DYT1 Dystonia

Background

DYT1 dystonia [Online Mendelian Inheritance inMan (OMIM) identifier, 128100] is a primary, gener-alized, early onset torsion dystonia.14 Patients typicallypresent with symptoms in childhood, but they canpresent as late as 26 years of age.15,16 Typically,symptoms first present in the limbs; then, over severalyears, the symptoms in many patients become general-ized.17 DYT1 dystonia is an autosomal-dominant dis-order with a reduced and incomplete penetrance of30% to 40%. It is caused by a trinucleotide (GAG)deletion in the DYT1/TOR1A gene, which encodesthe ubiquitously expressed torsinA protein.18 Thismutation removes one of a pair of glutamic acid resi-dues from the C-terminal region of the torsinA pro-tein. This mutation is commonly referred to as eitherDYT1 DGAG or torsinADE. TorsinA is a member ofthe AAA1 superfamily (ATPases associated withdiverse cellular activities) and has been implicated in avariety of cellular processes, including chaperone-mediated protein folding and vesicular trafficking.19,20

Invertebrate Models of DYT1 Dystonia

Worms (C. elegans) and fruit flies (D. melanogaster)have a short generation time and a well defined nerv-ous system, and these properties make them very goodmodel organisms in which to study neurological disor-ders.21–23 C. elegans have three torsin-related genes:tor-1, tor-2, and ooc-5. All three genes encodeATPases that are putative proteins for the Clp/Hsp100and AAA1 superfamilies.24,25 Caldwell and colleaguesconducted in vivo assays in C. elegans to investigatethe effect of these torsin proteins on polyglutamine-induced protein aggregation.26 First, they observed

that human torsinA expression, wild-type TOR-2overexpression, or TOR-2 and OOC-5 coexpressionusing these approaches were able to reduce proteinaggregation in C. elegans. However, the expression ofmutated TOR-2 had lost its ability to reduce the proteinaggregation (Table 1). In another study, overexpressionof human torsinA or TOR-2 in C. elegans, specificallyin dopaminergic neurons, was able to protect dopami-nergic neurons from 6-hydroxydopamine (6-OHDA)-induced neuronal loss, possibly through down-regulation of the dopamine transporter.27 However,this protection was decreased when either mutant tor-sinA or mutant TOR-2 proteins were expressed.Finally, human torsinA was able to suppress endoplas-mic reticulum (ER) stress response in C. elegans, bothat baseline and in response to the protein glycosylationinhibitor tunicamycin, which induces ER stress.28 How-ever, in the presence of mutant human torsinA, baselineER stress was found to be increased.28

When human torsinADE was expressed in either themuscle or neurons of fruit flies, temperature-dependent locomotion deficits were observed.29 Inaddition, protein aggregation of mutant torsinA wasobserved at normal temperature. Fruit flies thatexpressed an 18-base pair deletion in human DYT1/TOR1A, a mutation reported in a family with earlyonset dystonia,30,31 showed similar motor deficits afterexposure to 38�C. Furthermore, abnormalities in syn-apses and larva neuromuscular junction were observedin these fruit flies.31 Fruit flies only have a single torsingene: dtorsin.32 Down-regulation of dtorsin results inincreased neuronal degeneration.33 Furthermore, anull mutation of dtorsin will result in semi-lethality,sterility, locomotion deficits, and decreased dopaminelevels in the brains of larva and adult heterozygotes.34

These invertebrate studies have been integral inidentifying the function of torsinA, such as a role inchaperone-mediated protein folding, protection ofdopaminergic neurons from neurotoxicity and ERstress, and the effects of modulating expression orintroducing mutations on behavior. These findingstaken together support the continued use of inverte-brate animal models to study DYT1 dystonia and par-ticularly the role of cellular functions.

Rodent Models of DYT1 Dystonia

In mammals, there are four genes in the torsin family:torsinA, torsinB, torsin2A, and torsin3A. The humanDYT1/TOR1A gene and the rodent Dyt1/Tor1a genes,particularly in mice and rats, are highly homologous;therefore, they are excellent model organisms.

Dyt1 DGAG Knock-in, Knockout, and KnockdownMouse Models of DYT1 Dystonia

Multiple DYT1 genotypic dystonia models have beengenerated using rodents (Table 2). The most significant

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model is the Dyt1 DGAG heterozygous knock-in (KI)line of mice, because this mouse line recapitulates thetrinucleotide deletion in Dyt1/Tor1a observed mostoften in patients with DYT1 dystonia. Similar tohumans, this deletion results in a loss of one of a pair ofglutamic acid residues in torsinA.35,36

The Dyt1 KI mouse exhibited motor deficits andabnormal gait, which, although no overt dystonia, isthought to represent a dystonia-like phenotype.35

Dyt1 KI mice also displayed a subtle anxiety-likebehavior and enhancement of cued fear memory.37

These results were similar to DYT1 dystonia mutationcarriers, who exhibited increased anxiety, verbal mem-ory retroactive interference, and higher semantic flu-ency performance.38

In addition, Dyt1 KI mice exhibited reduced releaseof striatal dopamine at baseline and after ampheta-mine stimulation.39 Furthermore, a reduction in stria-tal D2 dopamine receptor (D2R) binding wasobserved in Dyt1 KI mice,40 which was consistentwith post-mortem and in vivo positron emissiontomography (PET) imaging studies, which revealed areduction in D2R binding in human DYT1 dystoniamutation carriers.41

Finally, Dyt1 KI mice exhibited cerebellothalamo-cortical (CbTC) pathway abnormalities using PET anddiffusion tensor imaging (DTI) imaging techniques.42

This was similar to the white-matter alterationsobserved in the sensorimotor cortex43 and the superiorcerebellar peduncle44 in patients with DYT1 dystonia

and primary dystonia, suggesting alterations in CbTCtract integrity.44,45 Furthermore, the KI mice haveexhibited alterations in corticostriatal long-termdepression (LTD).40 Neurotransmission deficits alsowere reported in cell cultures from hippocampal neu-rons in Dyt1 KI mice.46,47

Dyt1 KI mice had reduced torsinA protein levels inthe brain,36,48 consistent with the accelerated degrada-tion of mutant torsinA observed in cultured cells.49,50

Therefore, to determine whether a loss-of-functionmutation of torsinA leads to DYT1 dystonia, Dyt1knockdown (KD) mice were developed. These mice hadapproximately 36% reduction of torsinA protein, andDyt1 knockout (KO) mice were similarly developed.Similar to Dyt1 KI mice, Dyt1 KD mice displayedmotor deficits, increased locomotor activity, and altera-tions of striatal dopamine metabolism.51 In contrast,Dyt1 homozygous KO,36,52 Dyt1 homozygous KI,35,36

and KO/KI double-mutant mice53 resulted in neonatallethality, suggesting that a gross loss of torsinA functionimpaired normal development in mice. To date, thesefindings are consistent with no report of human carrierswith mutations in both DYT1 alleles.

Brain Region-specific Dyt1 Conditional KnockoutMice

Brain region-specific Dyt1 conditional KO micehave been used to understand how specific brainregion or cell types contribute to the pathophysiology

TABLE 1. Invertebrate models of DYT1 dystonia

Model Important findings

C. elegans containing fluorescentpolyglutamine-induced protein aggregates

# Polyglutamine-induced protein aggregation when overexpressing human torsinA, TOR-2, and coexpression ofTOR-2 and OOC-526

TOR-2 (D368) mutant (DYT1 associated mutation) was not able to reduce protein aggregationC. elegans (dopamine-specific neuronalpromoter)

" Resistance of DA neurons to 6-OHDA by overexpression of human torsinA or worm TOR-227

# Resistance of DA neurons to 6-OHDA by expression of torsinADE, mutant TOR-2 proteins, or by coexpressionof wild-type torsinA and torsinADE

C. elegans (ges-1 intestinal promoter) # ER stress response at basal and when stress was induced by tunicamycin with wild-type torsinA expression28

" ER stress response at basal conditions with torsinADE expression and by coexpression of wild-type torsinAand torsinADE, but no significant change in response to tunicamycin

D. melanogaster Gal4/UAS system for over-expression of torsinADE in the nervoussystem and muscle

Protein aggregation by torsinADE29

Impaired locomotion after exposure to 38�CUltrastructure alterations at the neuromuscular junction in larvae

D. melanogaster Gal4/UAS system for over-expression or down-regulation of dtorsin

Down-regulation or overexpression of dtorsin caused subtle rough eye phenotypes (neurotoxicity) and abnormaleye pigment granule configuration in young flies33

Down-regulation of dtorsin caused retinal degeneration in aged fliesOverexpression of dtorsin caused protection from neural degenerationdtorsin Localizes to nuclear envelope and ER

D. melanogaster Gal4/UAS system for over-expression of torsinADE and DFY HtorA inneurons or muscles

Protein aggregation in neurons and muscles by torsinADE, but absent in DFY HtorA mutants31

Impaired locomotion after exposure to 38�C in flies expressing torsinADE and DFY HtorAUltrastructure alterations at the neuromuscular junction in larvae and synapses

D. melanogaster null mutant for dtorsin Prepulpal semi-lethality, sterility and altered locomotion behavior34

# Dopamine levels in brain (heterozygote)# GTP cyclohydrolase protein and activity (heterozygote)

6-OHDA, 6-hydroxydopamine; ER, endoplasmic reticulum; GTP, guanosine-50-triphosphate; TOR-2(D368), lacking a codon for amino acid 368; torsinADE,human mutant torsinA with a GAG deletion; DFY HtorA, human mutant torsinA with an 18-base-pair deletion.

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TABLE 2. Embryonic stem (ES) cell-based dystonia mouse models

Models Microdialysis Monoamines Locomotion RotarodBeam

WalkingGait

AnalysisNeuronal

PhenotypeAdditionalfindings

DYT1 dystonia modelsDyt1DGAG KI # DA at baseline and

by AMPH in stria-tum (�2.3 mo;male/female)39

# HVA in striatum (11mo; male)35; "HVA in striatum(10 mo)39; # DA inmidbrain (6 and10 mo; male/female)39

" Activity (6 mo;male)35; " daytimemotility (3 and 6mo; male/female)39

Not affected35 (6 mo;male/female); #motor skills (3, 6,and 10 mo; male/female)39

" Slips (6 mo; male)35;" transversal timeand slips (3 mo;(male/female)39

" Paw overlap dis-tance (6 mo;male)35; affectedgait (male/female)39

Abnormal nuclear envelopein CNS (Dyt1DGAGhomozygous KI and KOembryonic day 15,18)36;abnormal nuclear enve-lope of cerebral corticalneurons (Dyt1DGAGhomozygous KI-new-born)56; inclusions forubiquitin and torsinA inbrain stem (6 mo;male)35; enlarged andreduced TH-positive cellsin substantia nigra (3–6mo; male/female)39;shorter primary dendrites(8 mo) and reducedspine numbers in Pur-kinje cells (3–8 mo)62

Microstructural abnormalitiesin cerebellothalamocorti-cal pathway (4 mo)42; #corticostriatal LTD (3–4weeks; male)40; # D2Rbinding40; # D2R instriatum (6 mo; male)40;" synaptic vesicle recy-cling46; " glutamatemEPSC frequency47 (hip-pocampus cultured neu-rons from heterozygousand homozygousDyt1DGAG KI mice post-natal days 0–1); #torsinAin brain53; #torsinA instriatum48

Dyt1 KD NA # DOPAC in striatum(8–9 mo; male)51

" Horizontal activity (6mo; male)51

Not affected (6 mo;male/female)51

" Slips (6 mo; male)51 # Hind base (male)52 NA

Purkinje cell-specific Dyt1conditional KO

NA NA NA Not affected (�5.2–7.7 mo; male/female)53

# Slips (�5.6–8 mo;male/female)53; nodifference in slipnumbers betweenCT and Dyt1 DGAGKI/Dyt1 pKOdouble-mutantmice (�5.3–5.7mo; male/female)53

Normal gait (�5.4–7.8mo; male/female)53

Shorter primary dendrites (8mo) and reduced spinenumbers (3–8 mo) inPurkinje cells62; normalnuclear envelope in cer-ebellar cells (�2–4 mo;male/female)53

Cortex-specific Dyt1 condi-tional KO

NA Not affected in stria-tum (�8.7–11.3mo; male/female)52

" Activity (�3.8–6.5mo; male/female)52

Not affected (�3.8–6.5 mo; male/female)52

" Slips (�3.8–6.3 mo;male/female)52

# Hind base (�5.4–8mo; male)52

Normal nuclear envelope incerebral cortical neurons(8 weeks)56

Striatum-specific Dyt1 condi-tional KO

NA Not affected in stria-tum (�10–11.3mo; male/female)56

Not affected (�4–5.5mo; male/female)56

Not affected (�3.4–4.8 mo; male/female)56

" Slips (�4.6–6 mo;male/female)56

NA Normal nuclear envelope instriatal medium spinyneurons (6 weeks)56

# D2R binding in striatum(�8.8–10.5 mo)56

Cholinergic-specific Dyt1conditional KO

Normal DA at baselineand by AMPH instriatum (7 mo)58

NA Not affected (3–8 mo;male/female)58

# Latency to fall (8–11mo; male/female)58

Not affected (8–11mo; male/female)58

NA NA Normal striatal acetylcholinecontent, uptake andrelease; abnormal D2Rresponse and alteredmuscarinic receptorfunction (8–10 weeks)58

DYT11 dystonia modelsSgce KO Normal DA at baseline;

" DA release byAMPH in striatum(5 mo)96

" DA, DOPAC, HVA instriatum (�11–11.7 mo; male/female)87

" Horizontal activity(female); " verticalmovement number,movement time,activity (male/female)87

Not affected87 " Slips, impairedmotor learning(�6.5–7.5 mo;male/female)87

Not affected87 Abnormal nuclear envelopein striatum (2–5.5mo)93; abnormal nuclearenvelopes in cerebellarPurkinje cells (2–4mo)94

Myoclonus (�7.2–8 mo;male/female)87; anxiety-like behavior (�7.3–8.2mo; male)87; depression-like behaviors (�7.8–8.6mo; female)87; # D2R,normal D1R and DAT instriatum (5 mo)96

Striatum-specific Sgce con-ditional KO

NA NA Not affected (�5.2–8mo; male/female)93

# Latency to fall in tri-als 3 and 5; #total latency to fall(�5.7–8.3 mo;male/female)93

" Slips (�5.5–8 mo;male/female)93

NA Normal nuclear envelope instriatum93

No myoclonus (�6.3–9 mo;male/female).93

Purkinje cell-specific Sgceconditional KO

NA NA # Stereotypic activity,movement num-bers, movementtime (�6.2–7.5mo; male/female)94

# Latency to fall on4th trial (�7–8mo; male/female)94

Impaired motor learn-ing (�6.6–8 mo;male/female)94

NA Normal nuclear envelope incerebellar Purkinjecells94

No significant myoclonus(�8.7–10 mo; male/female)94

DYT1 and DYT11 dystonia modelDyt1 and Sgce double-

mutantNA NA # Horizontal movement

numbers comparedwith Dyt1DGAG KI(�4.4–5.3 mo;male/female)48

NA " Slips (5.5 mo; male/female)48

NA Normal nuclear envelope incortical neurons (5.5mo; male/female)48

# TorsinA in striatum (5.5mo; male/female)48

DYT12 dystonia modelHeterozygous Atp1a3 KO NA Not affected, but indi-

vidual neurotrans-mitter levelscorrelated to openfield activity levelsshowed thatstressed mice hadchanges in thedopaminergic andserotonergicsystems106

# Clockwise circling(stressed 6 mo;male/female); vol-untary activity lev-els by wheelrunning: 1 grouphyperactive, theother group hypo-active (6 mo;stressed female)106

# Latency to fall(stressed 6 mo;female)106

" Slips (stressed 6-mo; female)106

NA NA # Sensitivity to warm stimuli(stressed female)106

AMPH, amphetamine; CNS, central nervous system; CT, control littermate; DA, dopamine; DAT, dopamine transporter; DOPAC, 3,4-dihydroxyphenylaceticacid; Dyt1DGAG KI, heterozygous mutant Dyt1 gene with a GAG deletion; D1R D2R, dopamine receptor type 1 and type 2; HVA, homovanillic acid; KD,knockdown; KI, knock-in; KO, knockout; LTD, long-term depression; mo, months; NA, not analyzed; TH, tyrosine hydroxylase.



of the disease (Table 2). When the phenotypes of con-ditional KO mice match with those of Dyt1 KI mice,it is proposed that the corresponding regions or cellshave relevance to the pathophysiology. The cerebralcortex-specific Dyt1 conditional KO mice (Dyt1cKO), which were generated by crossing Emx1-cre54

and Dyt1 loxP mice, exhibited motor deficits andhyperactivity.52 The striatum-specific Dyt1 conditionalKO mice (Dyt1 sKO), which were generated by cross-ing Rgs9L-cre55 and Dyt1 loxP mice, exhibited motordeficits and reduced striatal D2R binding activity.56

However, neither cKO mice nor sKO mice had a sig-nificant alteration in striatal monoamine levels. Next,cholinergic neuron-specific Dyt1 conditional KO mice,which were generated by crossing ChAT-cre57 andDyt1 loxP mice, exhibited motor deficits. Further-more, striatal cholinergic interneurons in these micerevealed alterations in response to muscarinic receptoractivation and D2R receptor activation, but nochanges were observed in response to either g-aminobutyric acid A (GABAA) or metabotropic gluta-mate receptor activation.58 The cerebral cortex andstriatum, along with cholinergic innervation, seemedto be important components of the basal ganglia cir-cuitry. Those studies suggested that loss of torsinAfunction in these areas resulted in motor deficits, andthe findings may prove important to better under-standing the pathogenesis of dystonia.

Another aspect of this puzzle that has yet to be inves-tigated is the use of conditional knockout techniques todemonstrate the contribution of the direct and indirectpathways of the basal ganglia. Striatal medium spinyneurons (MSNs) that express D1 dopamine receptormediate the direct pathway, which is thought to beinvolved in the initiation of movement; whereas striatalMSNs that express D2 dopamine receptor mediate theindirect pathway, and it is possible that this may pre-vent unwanted movements. Therefore, the motor defi-cits in DYT1 dystonia may be mediated, at least in part,through changes in D2R function in the basal gangliacircuit. The relative contributions of presynaptic D2Ron cholinergic interneurons and postsynaptic D2R onMSNs to the pathophysiology of DYT1 dystoniaremain to be determined.

Several studies have suggested the role of the cere-bellum in the pathophysiology of dystonia. Forinstance, cerebellectomies of either dystonic (dt) ratsor tottering mutant mice were able to improve theirdystonic-like symptoms.7,59 Moreover, crossing thetottering mutant mice with pcd mutant mice, whichhave Purkinje cell-specific degeneration, were similarlyable to improve their dystonic-like symptoms.60 There-fore, Purkinje cell-specific Dyt1 conditional KO (Dyt1pKO) mice were generated by crossing Pcp2-cre61

with Dyt1 loxP mice. These mice had alterations inPurkinje cell dendritic morphology and exhibited an

improvement in motor performance compared withwild-type mice.53,62 Because Purkinje cells integratecerebellar signals and the cerebellum modulates thebasal ganglia circuits,63 loss of torsinA function inPurkinje cells may balance the abnormal basal gangliacircuits and attenuate the dystonic symptoms.

Transgenic Rodent Models of DYT1 Dystonia

Various transgenic rodent models that overex-pressed wild-type torsinA or mutant torsinADE havebeen produced using different promoters (Table 3).A line of transgenic mice that overexpressed humantorsinADE, driven by the neuron-specific enolase pro-moter, displayed self-clasping of hind limbs, hyperki-nesia, altered circling behavior, abnormal gait,brainstem pathology, and disrupted striatal dopa-mine levels.64,65 A second line of transgenic micewas generated using the human cytomegalovirus(hCMV) immediate-early promoter to drive the over-expression of human torsinADE, and these have beenreferred to as hMT mice. The hMT mice exhibitedmotor learning deficits,66 motor deficits,67 andabnormal gait. Furthermore, these mice had anincrease in dopamine turnover, altered dopaminerelease, decreased basal locomotion induced byamphetamine,68 and decreased dopamine transporterfunction.69

The hMT mice have been studied extensively usingelectrophysiological techniques. These mice exhibiteddefected LTD, enhanced long-term potentiation, anda deficit of synaptic depotentiation (SD).70 A short-ened pause response by thalamic stimulation in cho-linergic interneurons also was observed that wascaused by altered D2R function affecting the synap-tic convergence between thalamostriatal and cortico-striatal responses.71 Furthermore, hMT miceexhibited an excitatory, rather than inhibitory, stria-tal cholinergic interneuron response after dopamineD2R activation,72,73 and they also had decreasedD2R receptor levels.74 In addition, electrophysiologi-cal studies revealed that there was a critical altera-tion of striatal dopamine D2R-mediated functionboth in cholinergic interneurons and in the controlof GABAergic synaptic transmission in MSNs inhMT mice.72,75 These observations are compatiblewith current theories on the pathogenesis of dysto-nia, suggesting that both an increased propensity to“potentiation” (long-term potentiation-like) and afailure of “depression” mechanisms (LTD-like andSD-like) lead to a “loss of inhibition” in the motorsystem, which may explain, at least in part, thepathogenesis of the excess of abnormal movementsobserved in dystonic patients, although this remainsspeculative.9

A third line of transgenic mice that overexpressedhuman torsinADE in dopaminergic neurons of the

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midbrain resulted in motor deficits and altered dopa-mine release in response to cocaine. These findingssuggested that disruption of torsinA activity by over-expressing the mutant torsinA in dopaminergic neu-rons affected dopamine transmission.76

A fourth line of transgenic mice was generated usinga murine prion protein promoter, and this line of micerevealed that overexpressing wild-type or torsinADE

caused inclusion bodies predominantly in the brain-stem, nuclear envelope abnormalities, altered monoa-mine levels, and motor deficits.77 However, both thewild-type and torsinADE were expressed as fusion pro-teins with a carboxyl terminal attachment of a V5-Histag. The functional consequences of these fusion pro-teins have not been well characterized. Realizing thedeficiency of the approach, the same research groupgenerated transgenic rats expressing human torsinADE

from the human torsinA promoter. Those mutant ratsexhibited altered synaptic plasticity, motor deficits,and nuclear membrane alterations.78

Most of the transgenic rodent models showed animpairment of motor behavior. However, investigatorsshould be cautious when interpreting these results,because the observed behavioral and cellular abnor-malities may have been a result of non-physiologicaland ectopic protein expression. Furthermore, it wasdifficult to generate proper control animals for thesetransgenic mice because of differences in the transgeneinsertion site, copy number, expression level, and pat-tern of expression.

DYT11 Dystonia

Background

DYT11 dystonia (OMIM 159900) is the major sub-type of myoclonus-dystonia and is characterized bymyoclonic jerks with dystonic symptoms.79,80 In addi-tion, it is often accompanied by psychiatric symptoms,such as depression and anxiety disorders. DYT11 dys-tonia generally presents in childhood but, in someindividuals, can present in late adulthood.81 It iscaused by mutations in SGCE, which encodes thetransmembrane glycoprotein e-sarcoglycan.79 SGCE ismaternally imprinted and paternally expressed.82 Fur-thermore, it has been demonstrated that there is exclu-sive paternal expression of e-sarcoglycan in the brainsof mice83 and humans.84

Rodent Models of DYT11 Dystonia

Epsilon-sarcoglycan was first identified in mice as ahomolog of a-sarcoglycan.85,86 Two lines of Sgce KOmice have been reported. The first lacks exon 4, whichresults in a frame-shift mutation and has exhibited sig-nificant relevant phenotypes.83,87 Another mouse linewas generated that lacked exons 6 through 9, whichdid not result in a frame-shift mutation and did not

exhibit an overt phenotype.88 Below, we focus on thefindings from the first line of mice.

Sgce Heterozygous Knockout Mice

Sgce heterozygous KO mice lacking exon 4 weregenerated by crossing Sgce loxP mice83 with CMV-cremice89 (Table 2). Paternally-inherited Sgce KO micehad myoclonus and deficits in fine motor coordinationand balance, motor learning in the beam-walking test,and anxiety and depression-like behaviors.87 Themotor learning deficit was recently demonstrated inpatients with DYT11 dystonia who had impaired sac-cadic adaptation,90 and this was thought to be a formof cerebellar motor learning.91 Sgce KO mice alsoexhibited abnormal nuclear envelopes in striatal neu-rons and cerebellar Purkinje cells, suggesting thatDYT11 dystonia belongs to a growing family ofnuclear envelopathies.92–94 In addition, loss ofe-sarcoglycan did not cause a reduction of other sarco-glycan isoforms (a, d, or f), suggesting thate-sarcoglycan did not form a sarcoglycan complexsimilar to other sarcoglycans.93 Furthermore, the levelsof striatal dopamine and its metabolites in Sgce KOmice were significantly increased.87 The hyperdopami-nergic state seemed to be potentially further strength-ened by evidence that patients with DYT11 dystoniahad a reduction in striatal D2R binding.95 Similarly,Sgce KO mice exhibited reduced striatal D2R proteinlevels and an increased dopamine release after amphet-amine administration.96 In conclusion, those studiessuggested that DYT11 dystonia had functional altera-tions of the monoamine system in the striatum andthat the Sgce KO mice reasonably modeled DYT11dystonia.

Brain Region-specific Sgce Conditional KnockoutMice

Brain region-specific Sgce KO models have been gen-erated to dissect brain circuits involved in the pathoge-nesis of DYT11 dystonia. Paternally inherited,striatum-specific Sgce conditional KO (Sgce sKO) miceexhibited motor deficits but no myoclonus and alsohad normal nuclear envelopes in the striatum.93 Pater-nally inherited, cerebellar Purkinje cell-specific Sgceconditional KO (Sgce pKO) mice had motor learningdeficits but no myoclonus and a normal nuclear enve-lope in the cerebellar Purkinje cells.94 The results sug-gested that loss of e-sarcoglycan function in thestriatum and in the cerebellar Purkinje cells contrib-uted to motor deficits and motor learning deficitsobserved in the complete Sgce KO mice.87 In contrast,loss of e-sarcoglycan function in these regions alonedid not contribute to myoclonus or nuclear envelopeabnormalities, suggesting that other brain regions or acombination of these brain regions may contribute tothese phenotypes.



DYT12 Dystonia

Background

DYT12 dystonia (OMIM 128235), or rapid-onsetdystonia-parkinsonism (RDP), is characterized bysymptoms of both dystonia and parkinsonism, whichinclude resting tremor, akinesia, bradykinesia, andpostural instability.97 In DYT12 dystonia, the humanpatients’ symptoms can appear within minutes to afew days and do not remit.97–101 The symptoms canbe triggered by a physiological stressor, such as a highfever or pregnancy.98,102 DYT12 dystonia is caused bymissense mutations in the ATP1A3 gene, which enco-des the Na1, K1-ATPase a3 isoform. The a3 isoformis only expressed in neurons and cardiac cells.97,103 Itis hypothesized that loss of the a3 isoform duringstress may interfere with the normal response to physi-ological stressors. These responses have beenreferred to as stress-induced channelopathies, and thistype of finding has been observed in other diseases,

such as myasthenia gravis and chronic fatiguesyndrome.8,104

Rodent Model of DYT12 Dystonia

Na1, K1-ATPase a3 isoform-deficient mice weregenerated as a DYT12 dystonia model from gene tar-geting that resulted in aberrant splicing of the gene.105

The heterozygous Atp1a3 KO mice exhibited anincrease in locomotor activity at baseline and inresponse to methamphetamine in the open-field test.In addition, heterozygous Atp1a3 KO mice exhibitedimpaired learning in the Morris water maze anddecreased levels of the hippocampal NR1 subunit ofNMDA receptor. More important, the heterozygousAtp1a3 KO mice had no motor deficits before stressinduction; however, after an immobilized stress proto-col, female heterozygous Atp1a3 KO mice exhibitedmotor deficits in the rotarod and beam-walkingtests.106 The stressed Atp1a3 KO mice also exhibited

TABLE 3. Transgenic DYT1 dystonia rodent models

Promoter Microdialysis Monoamines Locomotion RotarodBeam

WalkingGait

AnalysisNeuronal

phenotypeAdditionalfindings

Rat neuron-specificenolase

NA # DA in striatum (micewith affected phe-notype)64; #DOPAC/DA in stria-tum (mice withunaffected andaffected pheno-type; 1.5 mo)64

Self-clasping, hyperki-nesia, circling (1.5mo; mice withaffected pheno-type)64; # rearing(3 mo)65

Not affected (3, 6, 9,and 12 mo)65

NA # Forelimb and hindlimb stride length(9 mo)65

Perinuclear inclusions for ubiquitinand TorsinA in PPN and PAG64

Transgene expression lost afterrepeated breeding65

Human cytomegalovi-rus (CMV)

Not affected DA atbaseline in stria-tum68; # DA byAMPH in striatum(6 mo; male)68; #DAT activity instriatum (6 mo;male)69

Not affected in stria-tum (6 mo;male)68; " DOPAC/DA and HVA/DA instriatum (3–4 mo;male)67

Not affected (9 mo;male)66; # basallocomotion activityand by AMPH (6mo; male)69

# Motor learning (9mo; male)66; notaffected (3–4 mo;male)67

" Transversal time andslips (3–4 mo;male)67

" Hind base width 3–4mo; male)67

No torsinA or ubiquitin inclusionsin striatum (male)66; no tor-sinA or ubiquitin inclusions; nobleb formation in nuclearenvelope (male)67

Abnormal response to D2 DAreceptor stimulation of ChIs (9mo)72; normal DAT, VMAT2,striatal DA receptors density(6 mo; female)68; impairedLTD and SD in medium spinyneurons70; " striatal acetyl-cholinesterase activity (�2mo)70; altered GABAergic func-tion in striatum (�2 mo)75; #D2R, # RGS9 in striatum (�2mo)74; # pause response bythalamic stimulation in ChIs(male)71

Murine prion NA htorsinADE mice: "DOPAC, 5-HT, and5-HIAA in brainstem; htorsinAmice: #DA, 5-HTand 5-HIAA instriatum (5 mo;male/female)77

htorsinADE mice: "activity (6 mo;male/female) 77;htorsinA mice: #activity

htorsinADE: # latencyto fall, # learning(6 mo; male/female)77; htorsinAmice: not affected

htorsinADE: notaffected (6 mo;male/female)77;htorsinA mice: "transversal time

htorsinADE: notaffected (6 mo;male/female)77;htorsinA mice: #mean stride lengthfor forelimbs andhind limbs

htorsinADE and htorsinA: torsinA orubiquitin inclusions mainly inbrainstem, nuclear envelopedisruption in brainstem andstriatum (1.5 mo; male)77

Altered motor circuits integrity bydiffusion tensor imaging incortex, striatum, cerebellum (5mo; male)77

Tor1A promoter (ratmodel)

NA NA NA # Latency to fall attraining sessions(2 mo; male)78

" Transversal time 3.5cm round beam (2mo; male)78

" Variation of steplength (hind limb;11 mo; male)78

Abnormal protein localization ofhuman torsinADE to nuclearenvelope (cortex, hippocam-pus, striatum, olfactory bulb,substantia nigra; 2 mo; male);altered nuclear envelope struc-ture in cortex and striatum (3mo; male)78

Limb grasping (11 mo; male) andclasping; impaired LTD andSD in medium spiny neurons(8–12 weeks; male)78

Human TH promoterfragment (trans-gene expression indopaminergic neu-rons of midbrain)

TH-htorsinADE: notaffected DA atbaseline and # DAby cocaine in stria-tum (male); TH-htorsinA mice: "DA release bycocaine instriatum76

TH-htorsinADE mice:normal DA andDOPAC in striatum;TH-htorsinA mice:# DA and #DOPAC in striatum(2.5 mo; female)76

TH-htorsinADE mice:Normal baselinelocomotion; TH-htorsinA mice: #baseline locomo-tion (4–10 mo;male)76

Not affected (2-mo, 4-mo, or 6-mn male/female)76

TH-htorsinADE mice: "transversal timeand slips; male/female)76

NA NA

5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, 5-hydroxytryptophan; AMPH, amphetamine; ChIs, cholinergic interneurons; DA, dopamine; DAT, dopamine trans-porter; DOPAC, 3,4-dihydroxyphenylacetic acid; D2R, dopamine receptor type 2; HVA, homovanillic acid; htorsinA, human wild-type torsinA; htorsinADE, humanmutant torsinA with a GAG deletion; LTD, long-term depression; mo, months; NA, not analyzed; PPN, pedunculopontine nucleus; PAG, periaqueductal gray;RGS9, regulator of G-protein signaling 9; SD, synaptic depotentiation; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter.

O L E A S E T A L .


alterations in their sensory response to warm stimuli,circling behavior, and the monoamine neurotransmit-ter system. Moreover, in a separate study, stressincreased the susceptibility to depression-like pheno-types in Atp1a3 KO mice.107

Dystonia Animal Models andPreclinical Drug Discovery

Currently, there is no known cure for dystonia.However, physical therapy, medications, and surgeryaim to lessen symptoms. Positive symptomatic out-comes have resulted from botulinum toxin treatmentfor focal dystonia and cranial dystonias.108 Trihexy-phenidyl, g-aminobutyric acid derivatives, and dopa-minergic agents are also used for dystonia patients.Deep brain stimulation for generalized and cervicaldystonia has been effective in select patients.109 How-ever, not all patients respond favorably to these treat-ments, likely as a result of the heterogeneity ofdystonia and the differences in underlying pathophysi-ology. Therefore, there is tremendous need for newand more effective treatments for dystonia.

Although none of the genetic dystonia rodent mod-els have exhibited overt dystonic symptoms, thebeam-walking and rotarod tests have successfullydetected motor deficits in a majority of genotypicrodent models.35,39,48,51,56,58,66,67,76–78,87,93,94,106 Twoparticular cases worth noting are the DYT12 andDYT1 dystonia mouse models. In patients withDYT12 dystonia, symptoms often are triggered by astressor, resulting in a rapid progression of dystonicsymptoms and parkinsonism. In the mouse model ofDYT12 dystonia, there are no beam-walking deficits;however, when a stress protocol is used, the deficitsemerge.106 Furthermore, the beam-walking deficits inDyt1 KI mice could be rescued using the anticholi-nergic trihexyphenidyl, a common human dystoniamedication.40 We believe these two studies providesome preliminary evidence that the beam-walking testmay be an indirect test for dystonia in genotypicrodent models, although this will need furthervalidation.

We posit that potential therapeutics aimed at treat-ing genetic dystonias possibly may rescue the beam-walking deficits observed in respective genotypic mod-els. For example, an in vivo screen using transgenicC. elegans resulted in the identification of the antibi-otic ampicillin as being able to enhance wild-type tor-sinA activity and rescuing beam-walking deficits andtorsinA protein levels in Dyt1 KI mice.110 The effectof ampicillin on patients with DYT1 dystonia is cur-rently being investigated in a clinical trial (NationalClinical Trials identifier NCT01433757). In anothercase, loss of the torsinA protein in the Purkinje cellsof the cerebellum (Dyt1 pKO) in combination with

the global Dyt1 DGAG mutation (Dyt1 KI) in miceexhibited lower numbers of slips compared with Dyt1KI mice on the beam-walking test.53 This finding indi-cated the possibility that the molecular lesions of tor-sinA in cerebellar Purkinje cells by gene therapy orintervention in the signaling pathway downstream ofthe cerebellar Purkinje cells may rescue motorsymptoms in DYT1 dystonia patients. In addition,further refinement of drugs that target the cortico-striatal pathway and its modulatory pathways hope-fully will provide new treatments for DYT1 dystoniaand other related dystonias. It is likely that other can-didate tests like beam-walking will emerge as we gaina better understanding of the animal models ofdystonia.

Improving Existing Models forDystonia and Future Directions

One approach for the development of a better ani-mal model would be gene targeting mediated by zincfinger nucleases (ZFNs), which are engineered proteinsthat bind to DNA at specific sites and producedouble-strand breaks in DNA.111 The advantage ofZFNs lies in targeting efficiency and the possibility ofcreating gene modifications from different organ-isms,111 omitting the need to produce embryonic stemcell-based chimeric animal models.112 Because ZFNtechnology can be applicable to various organ-isms,113,114 it can be useful to generate novel Dyt1 KIor Dyt1 KO rats. Rats have larger bodies and brainsizes compared with mice, and this may facilitate eas-ier in vivo electrophysiological, behavioral, and imag-ing studies.

In addition, the engineered rodent dystonia modelsdeveloped to date suffer from the lack of overt motordeficits. Future efforts should focus on developingmodels with early onset and pronounced symptoms.The process of generating such models also will pro-vide insights into novel factors that contribute to onsetand severity of symptoms. Recent studies have demon-strated that lethality in Dyt1 homozygous KO micevaried from 12 hours to 3 weeks, depending on thegenetic background.115 Therefore, it is possible thatmotor symptoms also may be influenced by geneticbackground. Also, it has been demonstrated that addi-tional gene mutations can affect the temporal onset ofmotor deficits, like in the case of mutant mice thatharbor both the Dyt1 KI mutation and the Sgce KOmutation.48 Finally, stress-induced motor deficits inthe DYT12 dystonia mouse model suggest that envi-ronmental factors also can influence the onset andseverity of the phenotype. Combinations of theseapproaches hopefully will lead to better mammalianmodels and will facilitate the development of noveltherapeutics.



Conclusion

Although not all engineered rodent models haverevealed overt dystonic symptoms, motor deficits inthese models may correspond to dystonic symptoms inhumans. In addition to behavioral similarities betweengenotypic models of dystonia and patients, biochemi-cal similarities also exist. For example, reductions instriatal D2R and/or its binding activities are commonfindings in both DYT1 and DYT11 dystonia mousemodels. Analysis of these rodent models also havedemonstrated pathophysiological changes in the corti-costriatal, thalamostriatal, and CbTC pathways, andunderstanding these changes will be critical to therapydevelopment. It is our hope that further investigationof these pathways will lead to novel therapeutics fortreating dystonia. Using a combination of invertebrateand rodent models, ampicillin, a common antibiotic,was able to rescue motor deficits in animals and is acandidate for human trials. Moreover, Dyt1 pKOmice demonstrated that molecular lesions of torsinAin cerebellar Purkinje cells placed by gene therapy or,alternatively, intervening in the signaling pathwaysdownstream of cerebellar Purkinje cells may be able torescue motor symptoms. These results indicate thatthe genetic animal models developed can be useful tofurther study the pathophysiology of dystonia and toattempt to develop novel therapeutics.

Acknowledgments: We thank Michael S. Okun, MD, for review-ing and editing this article.

References1. Albanese A, Bhatia K, Bressman SB, et al. Phenomenology and

classification of dystonia: a consensus update. Mov Disord 2013;28:863–873.

2. Fahn S, Eldridge R. Definition of dystonia and classification ofthe dystonic states. Adv Neurol 1976;14:1–5.

3. Bertram KL, Williams DR. Diagnosis of dystonic syndromes—anew eight-question approach. Nat Rev Neurol 2012;8:275–283.

4. Niethammer M, Carbon M, Argyelan M, Eidelberg D. Heredi-tary dystonia as a neurodevelopmental circuit disorder: evidencefrom neuroimaging. Neurobiol Dis 2011;42:202–209.

5. Tinazzi M, Rosso T, Fiaschi A. Role of the somatosensory sys-tem in primary dystonia. Mov Disord 2003;18:605–622.

6. Zhuang P, Li Y, Hallett M. Neuronal activity in the basal gan-glia and thalamus in patients with dystonia. Clin Neurophysiol2004;115:2542–2557.

7. Neychev VK, Fan X, Mitev VI, Hess EJ, Jinnah HA. The basalganglia and cerebellum interact in the expression of dystonicmovement. Brain 2008;131(pt 9):2499–2509.

8. Breakefield XO, Blood AJ, Li Y, Hallett M, Hanson PI,Standaert DG. The pathophysiological basis of dystonias. NatRev Neurosci 2008;9:222–234.

9. Quartarone A, Pisani A. Abnormal plasticity in dystonia: disrup-tion of synaptic homeostasis. Neurobiol Dis 2011;42:162–170.

10. Sadnicka A, Hoffland BS, Bhatia KP, van de Warrenburg BP,Edwards MJ. The cerebellum in dystonia—help or hindrance?Clin Neurophysiol 2012;123:65–70.

11. Calderon DP, Fremont R, Kraenzlin F, Khodakhah K. The neuralsubstrates of rapid-onset dystonia-parkinsonism. Nat Neurosci2011;14:357–365.

12. Ikeda K, Satake S, Onaka T, et al. Enhanced inhibitory neuro-transmission in the cerebellar cortex of the Atp1a3-deficient heter-ozygous mice. J Physiol 2013.

13. Raike RS, Jinnah HA, Hess EJ. Animal models of generalized dys-tonia. NeuroRx 2005;2:504–512.

14. Ozelius LJ, Hewett JW, Page CE, et al. The early-onset torsiondystonia gene (DYT1) encodes an ATP-binding protein. NatGenet 1997;17:40–48.

15. Bressman SB, de Leon D, Brin MF, et al. Idiopathic dystoniaamong Ashkenazi Jews: evidence for autosomal dominant inheri-tance. Ann Neurol 1989;26:612–620.

16. Fahn S. The genetics of idiopathic torsion dystonia. Int J Neurol1991;25-26:70–80.

17. Muller U. The monogenic primary dystonias. Brain 2009;132(pt8):2005–2025.

18. Ozelius LJ, Hewett JW, Page CE, et al. The gene (DYT1) forearly-onset torsion dystonia encodes a novel protein related to theClp protease/heat shock family. Adv Neurol 1998;78:93–105.

19. Burdette AJ, Churchill PF, Caldwell GA, Caldwell KA. The early-onset torsion dystonia-associated protein, torsinA, displays molec-ular chaperone activity in vitro. Cell Stress Chaperones2010;15:605–617.

20. Granata A, Watson R, Collinson LM, Schiavo G, Warner TT.The dystonia-associated protein torsinA modulates synaptic vesi-cle recycling. J Biol Chem 2008;283:7568–7579.

21. Bargmann CI. Neurobiology of the Caenorhabditis elegansgenome. Science 1998;282:2028–2033.

22. Shulman JM, Shulman LM, Weiner WJ, Feany MB. From fruit flyto bedside: translating lessons from Drosophila models of neuro-degenerative disease. Curr Opin Neurol 2003;16:443–449.

23. Bilen J, Bonini NM. Drosophila as a model for human neurodege-nerative disease. Annu Rev Genet 2005;39:153–171.

24. Consortium CeS. Genome sequence of the nematode C. elegans: aplatform for investigating biology. Science 1998;282:2012–2018.

25. Basham SE, Rose LS. The Caenorhabditis elegans polarity geneooc-5 encodes a Torsin-related protein of the AAA ATPase super-family. Development 2001;128:4645–4656.

26. Caldwell GA, Cao S, Sexton EG, Gelwix CC, Bevel JP, CaldwellKA. Suppression of polyglutamine-induced protein aggregation inCaenorhabditis elegans by torsin proteins. Hum Mol Genet 2003;12:307–319.

27. Cao S, Gelwix CC, Caldwell KA, Caldwell GA. Torsin-mediatedprotection from cellular stress in the dopaminergic neurons ofCaenorhabditis elegans. J Neurosci 2005;25:3801–3812.

28. Chen P, Burdette AJ, Porter JC, et al. The early-onset torsiondystonia-associated protein, torsinA, is a homeostatic regulator ofendoplasmic reticulum stress response. Hum Mol Genet 2010;19:3502–3515.

29. Koh YH, Rehfeld K, Ganetzky B. A Drosophila model of earlyonset torsion dystonia suggests impairment in TGF-beta signaling.Hum Mol Genet 2004;13:2019–2030.

30. Leung JC, Klein C, Friedman J, et al. Novel mutation in theTOR1A (DYT1) gene in atypical early onset dystonia and poly-morphisms in dystonia and early onset parkinsonism. Neuroge-netics 2001;3:133–143.

31. Lee DW, Seo JB, Ganetzky B, Koh YH. DeltaFY mutation inhuman torsin A [corrected] induces locomotor disability andaberrant synaptic structures in Drosophila. Mol Cells 2009;27:89–97.

32. Ozelius LJ, Page CE, Klein C, et al. The TOR1A (DYT1) genefamily and its role in early onset torsion dystonia. Genomics1999;62:377–384.

33. Muraro NI, Moffat KG. Down-regulation of torp4a, encodingthe Drosophila homologue of torsinA, results in increased neuro-nal degeneration. J Neurobiol 2006;66:1338–1353.

34. Wakabayashi-Ito N, Doherty OM, Moriyama H, et al. Dtorsin,the Drosophila ortholog of the early-onset dystonia TOR1A(DYT1), plays a novel role in dopamine metabolism [serialonline]. PLoS One 2011;6:e26183.

35. Dang MT, Yokoi F, McNaught KS, et al. Generation and charac-terization of Dyt1 DeltaGAG knock-in mouse as a model forearly-onset dystonia. Exp Neurol 2005;196:452–463.

O L E A S E T A L .


36. Goodchild RE, Kim CE, Dauer WT. Loss of the dystonia-associated protein torsinA selectively disrupts the neuronalnuclear envelope. Neuron 2005;48:923–932.

37. Yokoi F, Dang MT, Miller CA, et al. Increased c-fos expressionin the central nucleus of the amygdala and enhancement of cuedfear memory in Dyt1 DeltaGAG knock-in mice. Neurosci Res2009;65:228–235.

38. Balas M, Peretz C, Badarny S, Scott RB, Giladi N. Neuropsycho-logical profile of DYT1 dystonia. Mov Disord 2006;21:2073–2077.

39. Song CH, Fan X, Exeter CJ, Hess EJ, Jinnah HA. Functionalanalysis of dopaminergic systems in a DYT1 knock-in mousemodel of dystonia. Neurobiol Dis 2012;48:66–78.

40. Dang MT, Yokoi F, Cheetham CC, et al. An anticholinergicreverses motor control and corticostriatal LTD deficits in Dyt1DGAG knock-in mice. Behav Brain Res 2012;226:465–472.

41. Asanuma K, Ma Y, Okulski J, et al. Decreased striatal D2 recep-tor binding in non-manifesting carriers of the DYT1 dystoniamutation. Neurology 2005;64:347–349.

42. Ulug AM, Vo A, Argyelan M, et al. Cerebellothalamocorticalpathway abnormalities in torsinA DYT1 knock-in mice. ProcNatl Acad Sci U S A 2011;108:6638–6643.

43. Carbon M, Kingsley PB, Su S, et al. Microstructural white matterchanges in carriers of the DYT1 gene mutation. Ann Neurol2004;56:283–286.

44. Carbon M, Ghilardi MF, Argyelan M, Dhawan V, Bressman SB,Eidelberg D. Increased cerebellar activation during sequencelearning in DYT1 carriers: an equiperformance study. Brain2008;131(pt 1):146–154.

45. Argyelan M, Carbon M, Niethammer M, et al. Cerebellothalamo-cortical connectivity regulates penetrance in dystonia. J Neurosci2009;29:9740–9747.

46. Kakazu Y, Koh JY, Ho KW, Gonzalez-Alegre P, Harata NC. Syn-aptic vesicle recycling is enhanced by torsinA that harbors theDYT1 dystonia mutation. Synapse 2012;66:453–464.

47. Kakazu Y, Koh JY, Iwabuchi S, Gonzalez-Alegre P, Harata NC.Miniature release events of glutamate from hippocampal neuronsare influenced by the dystonia-associated protein torsinA. Synapse2012;66:807–822.

48. Yokoi F, Yang G, Li J, DeAndrade MP, Zhou T, Li Y. Earlieronset of motor deficits in mice with double mutations in Dyt1and Sgce. J Biochem 2010;148:459–466.

49. Giles LM, Chen J, Li L, Chin LS. Dystonia-associated mutationscause premature degradation of torsinA protein and cell-type-specific mislocalization to the nuclear envelope. Hum Mol Genet2008;17:2712–2722.

50. Gordon KL, Gonzalez-Alegre P. Consequences of the DYT1mutation on torsinA oligomerization and degradation. Neuro-science 2008;157:588–595.

51. Dang MT, Yokoi F, Pence MA, Li Y. Motor deficits andhyperactivity in Dyt1 knockdown mice. Neurosci Res 2006;56:470–474.

52. Yokoi F, Dang MT, Mitsui S, Li J, Li Y. Motor deficits andhyperactivity in cerebral cortex-specific Dyt1 conditional knock-out mice. J Biochem 2008;143:39–47.

53. Yokoi F, Dang MT, Li Y. Improved motor performance in Dyt1DGAG heterozygous knock-in mice by cerebellar Purkinje-cellspecific Dyt1 conditional knocking-out. Behav Brain Res 2012;230:389–398.

54. Jin XL, Guo H, Mao C, et al. Emx1-specific expression of foreigngenes using “knock-in” approach. Biochem Biophys Res Commun2000;270:978–982.

55. Dang MT, Yokoi F, Yin HH, Lovinger DM, Wang Y, Li Y. Dis-rupted motor learning and long-term synaptic plasticity in micelacking NMDAR1 in the striatum. Proc Natl Acad Sci U S A2006;103:15254–15259.

56. Yokoi F, Dang MT, Li J, Standaert DG, Li Y. Motor deficits anddecreased striatal dopamine receptor 2 binding activity in thestriatum-specific Dyt1 conditional knockout mice [serial online].PLoS One 2011;6:e24539.

57. Rossi J, Balthasar N, Olson D, et al. Melanocortin-4 receptorsexpressed by cholinergic neurons regulate energy balance and glu-cose homeostasis. Cell Metab 2011;13:195–204.

58. Sciamanna G, Hollis R, Ball C, et al. Cholinergic dysregulationproduced by selective inactivation of the dystonia-associated pro-tein torsinA. Neurobiol Dis 2012;47:416–427.

59. LeDoux MS, Lorden JF, Ervin JM. Cerebellectomy eliminates themotor syndrome of the genetically dystonic rat. Exp Neurol1993;120:302–310.

60. Campbell DB, North JB, Hess EJ. Tottering mouse motor dys-function is abolished on the Purkinje cell degeneration (pcd)mutant background. Exp Neurol 1999;160:268–278.

61. Barski JJ, Dethleffsen K, Meyer M. Cre recombinase expressionin cerebellar Purkinje cells. Genesis 2000;28(3-4):93–98.

62. Zhang L, Yokoi F, Jin YH, et al. Altered Dendritic Morphologyof Purkinje cells in Dyt1 DeltaGAG knock-in and Purkinje cell-specific Dyt1 conditional knockout mice [serial online]. PLoS One2011;6:e18357.

63. Hoshi E, Tremblay L, Feger J, Carras PL, Strick PL. The cerebel-lum communicates with the basal ganglia. Nat Neurosci 2005;8:1491–1493.

64. Shashidharan P, Sandu D, Potla U, et al. Transgenic mouse model ofearly-onset DYT1 dystonia. Hum Mol Genet 2005;14:125–133.

65. Lange N, Hamann M, Shashidharan P, Richter A. Behavioural andpharmacological examinations in a transgenic mouse model ofearly-onset torsion dystonia. Pharmacol Biochem Behav 2011;97:647–655.

66. Sharma N, Baxter MG, Petravicz J, et al. Impaired motor learn-ing in mice expressing torsinA with the DYT1 dystonia mutation.J Neurosci 2005;25:5351–5355.

67. Zhao Y, DeCuypere M, LeDoux MS. Abnormal motor functionand dopamine neurotransmission in DYT1 DeltaGAG transgenicmice. Exp Neurol 2008;210:719–730.

68. Balcioglu A, Kim MO, Sharma N, Cha JH, Breakefield XO,Standaert DG. Dopamine release is impaired in a mouse model ofDYT1 dystonia. J Neurochem 2007;102:783–788.

69. Hewett J, Johanson P, Sharma N, Standaert D, Balcioglu A. Func-tion of dopamine transporter is compromised in DYT1 transgenicanimal model in vivo. J Neurochem 2010;113:228–235.

70. Martella G, Tassone A, Sciamanna G, et al. Impairment of bidir-ectional synaptic plasticity in the striatum of a mouse model ofDYT1 dystonia: role of endogenous acetylcholine. Brain 2009;132(pt 9):2336–2349.

71. Sciamanna G, Tassone A, Mandolesi G, et al. Cholinergic dys-function alters synaptic integration between thalamostriatal andcorticostriatal inputs in DYT1 dystonia. J Neurosci 2012;32:11991–12004.

72. Pisani A, Martella G, Tscherter A, et al. Altered responses todopaminergic D2 receptor activation and N-type calcium currentsin striatal cholinergic interneurons in a mouse model of DYT1dystonia. Neurobiol Dis 2006;24:318–325.

73. Sciamanna G, Tassone A, Martella G, et al. Developmental pro-file of the aberrant dopamine D2 receptor response in striatalcholinergic interneurons in DYT1 dystonia [serial online]. PLoSOne 2011;6:e24261.

74. Napolitano F, Pasqualetti M, Usiello A, et al. Dopamine D2receptor dysfunction is rescued by adenosine A2A receptor antag-onism in a model of DYT1 dystonia. Neurobiol Dis 2010;38:434–445.

75. Sciamanna G, Bonsi P, Tassone A, et al. Impaired striatal D2receptor function leads to enhanced GABA transmission in amouse model of DYT1 dystonia. Neurobiol Dis 2009;34:133–145.

76. Page ME, Bao L, Andre P, et al. Cell-autonomous alteration ofdopaminergic transmission by wild type and mutant (DeltaE) Tor-sinA in transgenic mice. Neurobiol Dis 2010;39:318–326.

77. Grundmann K, Reischmann B, Vanhoutte G, et al. Overexpres-sion of human wildtype torsinA and human DeltaGAG torsinA ina transgenic mouse model causes phenotypic abnormalities. Neu-robiol Dis 2007;27:190–206.

78. Grundmann K, Glockle N, Martella G, et al. Generation of anovel rodent model for DYT1 dystonia. Neurobiol Dis 2012;47:61–74.

79. Zimprich A, Grabowski M, Asmus F, et al. Mutations in the geneencoding epsilon-sarcoglycan cause myoclonus-dystonia syn-drome. Nat Genet 2001;29:66–69.



80. Asmus F, Zimprich A, Tezenas Du Montcel S, et al. Myoclonus-dystonia syndrome: epsilon-sarcoglycan mutations and phenotype.Ann Neurol 2002;52:489–492.

81. Foncke EM, Gerrits MC, van Ruissen F, et al. Distal myoclonusand late onset in a large Dutch family with myoclonus-dystonia.Neurology 2006;67:1677–1680.

82. Piras G, El Kharroubi A, Kozlov S, et al. Zac1 (Lot1), a potentialtumor suppressor gene, and the gene for epsilon-sarcoglycan arematernally imprinted genes: identification by a subtractive screenof novel uniparental fibroblast lines. Mol Cell Biol 2000;20:3308–3315.

83. Yokoi F, Dang MT, Mitsui S, Li Y. Exclusive paternal expressionand novel alternatively spliced variants of epsilon-sarcoglycanmRNA in mouse brain. FEBS Lett 2005;579:4822–4828.

84. Ritz K, van Schaik BD, Jakobs ME, et al. SGCE isoform charac-terization and expression in human brain: implications formyoclonus-dystonia pathogenesis? Eur J Hum Genet 2011;19:438–444.

85. Ettinger AJ, Feng G, Sanes JR. epsilon-Sarcoglycan, a broadlyexpressed homologue of the gene mutated in limb-girdle musculardystrophy 2D. J Biol Chem 1997;272:32534–32538.

86. Ettinger AJ, Feng G, Sanes JR. epsilon-Sarcoglycan, a broadlyexpressed homologue of the gene mutated in limb-girdle musculardystrophy 2D [erratum]. J Biol Chem 1998;273:19922.

87. Yokoi F, Dang MT, Li J, Li Y. Myoclonus, motor deficits,alterations in emotional responses and monoamine metabolismin epsilon-sarcoglycan deficient mice. J Biochem 2006;140:141–146.

88. Lancioni A, Rotundo IL, Kobayashi YM, et al. Combined defi-ciency of alpha and epsilon sarcoglycan disrupts the cardiac dys-trophin complex. Hum Mol Genet 2011;20:4644–4654.

89. Schwenk F, Baron U, Rajewsky K. A cre-transgenic mouse strainfor the ubiquitous deletion of loxP-flanked gene segments includ-ing deletion in germ cells. Nucleic Acids Res 1995;23:5080–5081.

90. Hubsch C, Vidailhet M, Rivaud-Pechoux S, et al. Impaired sacca-dic adaptation in DYT11 dystonia. J Neurol Neurosurg Psychia-try 2011;82:1103–1106.

91. Prsa M, Thier P. The role of the cerebellum in saccadic adapta-tion as a window into neural mechanisms of motor learning. EurJ Neurosci 2011;33:2114–2128.

92. Nagano A, Arahata K. Nuclear envelope proteins and associateddiseases. Curr Opin Neurol 2000;13:533–539.

93. Yokoi F, Dang MT, Zhou T, Li Y. Abnormal nuclear envelopesin the striatum and motor deficits in DYT11 myoclonus-dystoniamouse models. Hum Mol Genet 2012;21:916–925.

94. Yokoi F, Dang MT, Yang G, et al. Abnormal nuclear envelope inthe cerebellar Purkinje cells and impaired motor learning inDYT11 myoclonus-dystonia mouse models. Behav Brain Res2012;227:12–20.

95. Beukers RJ, Booij J, Weisscher N, Zijlstra F, van Amelsvoort TA,Tijssen MA. Reduced striatal D2 receptor binding in myoclonus-dystonia. Eur J Nucl Med Mol Imaging 2009;36:269–274.

96. Zhang L, Yokoi F, Parsons DS, Standaert DG, Li Y. Alteration ofstriatal dopaminergic neurotransmission in a mouse model of DYT11myoclonus-dystonia [serial online]. PLoS One 2012;7:e33669.

97. Brashear A, Dobyns WB, de Carvalho Aguiar P, et al. The pheno-typic spectrum of rapid-onset dystonia-parkinsonism (RDP) andmutations in the ATP1A3 gene. Brain 2007;130(pt 3):828–835.

98. Dobyns WB, Ozelius LJ, Kramer PL, et al. Rapid-onset dystonia-parkinsonism. Neurology 1993;43:2596–2602.

99. Linazasoro G, Indakoetxea B, Ruiz J, Van Blercom N, Lasa A.Possible sporadic rapid-onset dystonia-parkinsonism. Mov Disord2002;17:608–609.

100. Pittock SJ, Joyce C, O’Keane V, et al. Rapid-onset dystonia-par-kinsonism: a clinical and genetic analysis of a new kindred. Neu-rology 2000;55:991–995.

101. Zaremba J, Mierzewska H, Lysiak Z, Kramer P, Ozelius LJ,Brashear A. Rapid-onset dystonia-parkinsonism: a fourth familyconsistent with linkage to chromosome 19q13. Mov Disord2004;19:1506–1510.

102. Brashear A, DeLeon D, Bressman SB, Thyagarajan D, FarlowMR, Dobyns WB. Rapid-onset dystonia-parkinsonism in a secondfamily. Neurology 1997;48:1066–1069.

103. de Carvalho Aguiar P, Sweadner KJ, Penniston JT, et al. Muta-tions in the Na1/K1-ATPase alpha3 gene ATP1A3 are associatedwith rapid-onset dystonia parkinsonism. Neuron 2004;43:169–175.

104. Cannon SC. Pathomechanisms in channelopathies of skeletal mus-cle and brain. Annu Rev Neurosci 2006;29:387–415.

105. Moseley AE, Williams MT, Schaefer TL, et al. Deficiency inNa,K-ATPase alpha isoform genes alters spatial learning, motoractivity, and anxiety in mice. J Neurosci 2007;27:616–626.

106. DeAndrade MP, Yokoi F, van Groen T, Lingrel JB, Li Y. Charac-terization of Atp1a3 mutant mice as a model of rapid-onset dys-tonia with parkinsonism. Behav Brain Res 2011;216:659–665.

107. Kirshenbaum GS, Saltzman K, Rose B, Petersen J, Vilsen B,Roder JC. Decreased neuronal Na1, K1-ATPase activity inAtp1a3 heterozygous mice increases susceptibility to depression-like endophenotypes by chronic variable stress. Genes BrainBehav 2011;10:542–550.

108. Karp BI. Botulinum toxin physiology in focal hand and cranialdystonia. Toxins (Basel) 2012;4:1404–1414.

109. Yu H, Neimat JS. The treatment of movement disorders by deepbrain stimulation. Neurotherapeutics 2008;5:26–36.

110. Cao S, Hewett JW, Yokoi F, et al. Chemical enhancement of tor-sinA function in cell and animal models of torsion dystonia. DisModel Mech 2010;3(5-6):386–396.

111. Carroll D. Genome engineering with zinc-finger nucleases. Genet-ics 2011;188:773–782.

112. Yan Z, Sun X, Engelhardt JF. Progress and prospects: techniquesfor site-directed mutagenesis in animal models. Gene Ther 2009;16:581–588.

113. Maeder ML, Thibodeau-Beganny S, Osiak A, et al. Rapid "open-source" engineering of customized zinc-finger nucleases for highlyefficient gene modification. Mol Cell 2008;31:294–301.

114. Reyon D, Kirkpatrick JR, Sander JD, et al. ZFNGenome: a com-prehensive resource for locating zinc finger nuclease target sites inmodel organisms [serial online]. BMC Genomics 2011;12:83.

115. Tanabe LM, Martin C, Dauer WT. Genetic background modu-lates the phenotype of a mouse model of DYT1 dystonia [serialonline]. PLoS One 2012;7:e32245.

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Engineering animal models of dystonia