Generation of a novel rodent model for DYT1 dystonia

Neurobiology of Disease 47 (2012) 61–74

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Generation of a novel rodent model for DYT1 dystonia Kathrin Grundmann a,⁎, 1, Nicola Glöckle a, 1, Giuseppina Martella b, c, Giuseppe Sciamanna b, c, Till-Karsten Hauser d, Libo Yu a, Salvador Castaneda e, Bernd Pichler e, Birgit Fehrenbacher f, Martin Schaller f, Brigitte Nuscher g, h, Christian Haass g, h, Jasmin Hettich a, Zhenyu Yue i, Huu Phuc Nguyen a, Antonio Pisani b, c, Olaf Riess a, Thomas Ott a a

Dept. of Medical Genetics, University of Tuebingen, Germany Dept. of Neuroscience, University “Tor Vergata”, Rome, Italy c Fondazione Santa Lucia I.R.C.C.S., Rome, Italy d Dept. of Neuroradiology, University of Tuebingen, Germany e Department of Preclinical imaging and Radiopharmacy, University Hospital of Tuebingen, Germany f Dept. of Dermatology, University of Tuebingen, Germany g German Center for Neurodegenerative Diseases, Ludwig Maximilian University Munich, Germany h Adolf-Butenandt-Institute, Biochemistry, Ludwig Maximilian University, Germany i Department of Neurology & Neuroscience Friedman Brain Institute Mount Sinai School of Medicine New York, NY 10029 b

a r t i c l e

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Article history: Received 28 November 2011 Revised 13 March 2012 Accepted 17 March 2012 Available online 26 March 2012 Keywords: Transgenic rat model TorsinA DYT1 dystonia Movement disorder

a b s t r a c t A mutation in the coding region of the Tor1A gene, resulting in a deletion of a glutamic acid residue in the torsinA protein (ΔETorA), is the major cause of the inherited autosomal-dominant early onset torsion dystonia (DYT1). The pathophysiological consequences of this amino acid loss are still not understood. Currently available animal models for DYT1 dystonia provided important insights into the disease; however, they differ with respect to key features of torsinA associated pathology. We developed transgenic rat models harboring the full length human mutant and wildtype Tor1A gene. A complex phenotyping approach including classical behavioral tests, electrophysiology and neuropathology revealed a progressive neurological phenotype in ΔETorA expressing rats. Furthermore, we were able to replicate key pathological features of torsinA associated pathology in a second species, such as nuclear envelope pathology, behavioral abnormalities and plasticity changes. We therefore suggest that this rat model represents an appropriate new model suitable to further investigate the pathophysiology of ΔETorA and to test for therapeutic approaches. © 2012 Elsevier Inc. All rights reserved.

Introduction DYT1 dystonia is an autosomal-dominantly inherited movement disorder. Most cases are caused by a 3 base pair (GAG) deletion in the coding region of the Tor1A gene (DYT1/TOR1A) (Ozelius et al., 1997). DYT1 dystonia manifests predominantly in childhood as Abbreviations: CV, coefficient of variation; MSN, medium spiny motor neurons; EPSP, excitatory postsynaptic potentials; LTP, long term potentiation; LTD, long term depression; SD, synaptic depotentiation; LFS, low frequency stimulation; RMP, resting membrane potential; DA, dopamine; DOPAC, 3,4-dihydroxy-phenylacetic acid; D1, dopamine receptor type 1; D2, dopamine receptor type 2; AMPA, α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid; ADC, apparent diffusion coefficient; DWI, diffusion weight imaging; RD, radial diffusivity; FA, fractional anisotropy; ROI, region of interest approach; HFS, high frequency stimulation; MC, motor cortex; SWM, subcortical white matter; FOV, field of view; AIR, automated image reconstruction. ⁎ Corresponding author at: Dept. of Medical Genetics, University of Tuebingen, Calwer Str. 7, 72076 Tuebingen, Germany. Fax: + 49 7071 295228. E-mail address: [email protected] (K. Grundmann). 1 These authors contributed equally to this work. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2012.03.024

twisting movements and abnormal postures induced by involuntary muscle contractions. The protein torsinA belongs to the AAA + (ATPase associated with different cellular activities) protein family. Studies in recent years have implicated torsinA in cellular response to stress (Hewett et al., 2003; Kuner et al., 2004), in neurite outgrowth (Ferrari-Toninelli et al., 2004; Hewett et al., 2006), synaptic plasticity (Martella et al., 2009) and dopaminergic transmission (Augood et al., 2002, 2003; Marsden et al., 1985; Torres et al., 2004). Despite the increasing knowledge about the normal function of the protein, the relationship between the impaired function of mutant torsinA protein and the development of DYT1 dystonia is still unclear. Findings from cell biological studies, neurochemical analyses, functional imaging and electrophysiological studies in mouse models and in humans indicate that dystonic movements are the result of dysfunction of CNS motor systems involving selective regions implicated in movement control (Martella et al., 2009; Niethammer et al., 2011; Tanabe et al., 2009). These disease characteristics require the investigation of the pathophysiology of the torsinA mutation in an

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in vivo setting. Accordingly, a variety of animal models for DYT1 dystonia have been generated including transgenic and gene targeted mouse models (Dang et al., 2005; Goodchild et al., 2005; Grundmann et al., 2007; Sharma et al., 2005; Shashidharan et al., 2005). These mouse models display important characteristics of the human disease. However, the phenotype of the existing mouse models appears to be inconsistent most likely due to differences in genetic backgrounds, the promoters chosen to drive the expression of the transgene but also to the varying degree of characterization. Collectively, it is difficult to dissect at this stage which phenotypic features are related to torsinA dysfunction and which are side effects of the models (Grundmann et al., 2007; Sharma et al., 2005; Shashidharan et al., 2005). One limitation of the existing mouse models overexpressing the wildtype and mutant torsinA is the use of foreign promoters to drive the expression of the torsinA protein (Grundmann et al., 2007; Sharma et al., 2005; Shashidharan et al., 2005). Although the mouse models for DYT1 dystonia provided important insights into disease mechanisms there is still need for an animal model belonging to a different species. Replication of pathological features observed in preceding mouse models might provide further evidence that these features are direct results of an impact of the mutant protein and not related to the model system expressing the mutant protein. This motivated us to generate a transgenic rat model overexpressing the human mutant and wildtype torsinA protein. We used a 16.25 kbps human genomic fragment containing the full length human Tor1A gene including promoter and regulatory regions with the intention to express the human torsinA protein under the control of the endogenous human promoter and its regulatory elements. This model enabled us to investigate which of the so far reported pathological characteristics caused by the mutant torsinA protein in cell culture or in mouse models can be replicated in vivo in a second rodent species. Materials and methods Generation of DYT1 transgenic rats The 16.25 kbps full length human DYT1 alleles including upstream and downstream flanking regions were inserted into a pUC19 vector. Following linearization and purification the transgenes were used to generate transgenic rats by microinjection of the DNA construct into the pronucleus of rat zygotes. Transgenic rats were kept on Sprague–Dawley background and bred as hemizygotes using standard procedures. Genotypes of offspring were determined by PCR and sequencing of DNA extracted from ear biopsies. Primer pair 5′-TAAAAATGTGTATCCGAGTGGAAAT (forward) and 5′-AAGGACTGAGTGTTGTTTCTTTTC (reverse) amplified a 230 bp segment within the coding sequence of exon 5 under the following conditions: 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, 40 cycles. Only lines that showed stable germline transmission of the transgenes were used for further analyses. To estimate the relative copy number of the transgene primers specific for human torsinA were designed (5′-GAAAACCCTGTCCTTACCCA (forward) and 5′-CCCTACTCACTGTTCTTGTTATTGAA (reverse)). Equal amounts of genomic DNA from the various lines were amplified by real time PCR on a Roche Lightcycler 480. As a calibrator the cycle threshold-value from line three, the weakest expressing line, was chosen. The relative copy number of transgenes was calculated with an adaptation of 2 − ΔΔCT method from Livak and Schmittgen (2001). Western blot analyses and immunohistochemistry Western blot analyses and immunohistochemistry were performed as described previously (Grundmann et al., 2007) using torsinA-specific monoclonal antibody D-M2A8 (dilution: 1:200; Cell

Signaling Technology, Inc.) (Hewett et al., 2004). Immunolabeling was visualized by using biotin-coupled antibodies (Vector Laboratories, Burlingame, CA). Immunofluorescence For double immunofluorescence staining brain sections were incubated at 4 °C overnight simultaneously with anti-laminA/C (1:100) (Cell signaling) and D-M2A8 antibody (1:50) (Cell signaling), respectively. After subsequent washes, sections were incubated with the appropriate secondary antibodies conjugated with Fluorescein Avidin DCS (Vector Laboratories Inc., Burlingame, USA) (20 μg/ml; 2 h; RT) and with Texas Red® Avidin DCS (Vector Laboratories Inc., Burlingame, USA) (20 μg/ml; 2 h; RT). After washing, sections were mounted with Vectashield mounting medium (Vector Laboratories Inc., Burlingame, USA). Sections were analyzed using an Axiofluor microscope and imaging software (Carl Zeiss, Oberkochen, Germany). Transmission electron microscopy Brains of two transgenic male rats of each genotype and two nontransgenic littermates at the age of 2 months were dissected, immersed in Karnovsky's fixative and stored at 4 °C. Brain tissues were washed in 0.1 M sodium cacodylate and postfixed in 2% osmium tetroxide in 0.2 M sodium cacodylate. Tissues were dehydrated in a graded series of alcohol and finally embedded in glycide ether and cut using an ultra microtome (Ultracut, Reichert, Vienna, Austria). Ultra-thin sections (30 nm) were mounted on copper grids and analyzed using a Zeiss LIBRA 120 transmission electron microscope (Carl Zeiss, Oberkochen, Germany). Behavioral studies All behavioral studies were performed at the age of 2, 5, 8 and 11 months and were performed by a trained observer blind to the genetic status. A cohort of 16 male ΔETorA rats, 16 male WtTorA rats as well as 16 male nontransgenic littermates were analyzed by means of beam walking and rotarod. In order to check for general health, motor functions and neurological reflexes this cohort was examined according to the “modified SHIRPA” protocol (Masuya et al., 2005) (http://www.brc. riken.go.jp/lab/gsc/mouse/AboutUs/shirpalist.htm) including hind limb clasping and limb grasping. For hind limb clasping, each rat was picked up by its tail and suspended for 10 s to observe clasping of the hind limbs. In order to classify this phenotype the score developed by Korenova et al. (2009) was used. For limb grasping animals were hung by their tail. Animals get 1 point if they are able to catch the hind-limbs and 0 points if not. A second cohort of 15 male rats of each genotype and 15 nontransgenic littermates was analyzed using the PhenoMaster system (TSE, Bad Homburg). The following parameters were measured: drinking and feeding, activity (ambulatory, fine movement, rearing) and metabolic performance (O2 consumption, CO2 production, respiratory exchange rate, energy expenditure). A detailed description of these experiments is given in the Supplementary online material (see Supplementary methods for details). Rotarod and beam walking The rotarod test was performed using an accelerating rotarod (Hugo Basile, Collegeville PA, USA). Three training sessions were followed by two test sessions. During training each rat was placed on the rotarod at a constant speed for a maximum of 120 s. The number of falls from the rotarod was recorded. On testing days (days 4 and 5), we switched to the accelerating mode of the rotarod, with acceleration from 4 to 40 rpm over a 4-min period. The time each animal

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Fig. 1. Transgene construct and torsinA expression in transgenic rats. (A) The schematic diagram shows the full length human torsinA including upstream and downstream flanking regions resulting in a 16.25 kbp fragment which was inserted into a pUC19 vector via NdeI and BamHI. (B) Relative copy number calculation: Equal amounts of genomic DNA were amplified by means of realtime PCR. Expression levels of torsinA protein in the various lines were calculated by immunoblot analysis using monoclonal antibody D-M2A8 and normalization to β-actin signals. The weakest expressing line 3 was chosen as an internal calibrator for copy number estimation. (C) Western blot analysis of tissues from different brain regions of the rat lines with the highest expression level of each genotype at the age of 2 months demonstrating a 37 kDa product representing transgene expression in the cortex, cerebellum, brainstem, striatum and bulbus olfactorius. β-actin was used a loading control.

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was able to maintain its balance on the rod was measured. The same cohort of rats was tested by beam walking test. Three different sizes and shapes of the traversing segment (3 cm × 3 cm square crosssectioned, 2 cm × 4 cm rectangular cross-sectioned and 3.5 cm round cross-sectioned) were provided. One training session was followed by four test sessions. At training day 1 rats were made to traverse the square crosssection of 3 cm × 3 cm twice. On test days rats were made to traverse each segment once starting with the 3 cm × 3 cm square cross-sectioned beam, followed by the 2 cm × 4 cm rectangular cross-sectioned and 3.5 cm round cross-sectioned beam. Beam traversal time was recorded.

after obtaining a stable LTP. Whole-cell patch-clamp recordings were performed from MSNs visualized using IR-DIC videomicroscopy in the dorsal striatum, as described (Sciamanna et al., 2009). Cells were clamped at the holding potential (HP) of −60 mV, to measure spontaneous excitatory postsynaptic currents (sEPSCs). Data were acquired with pCLAMP 10.2 (Axon Instruments) and analyzed off-line with MiniAnalysis 5.1 (Synaptosoft) software. Drugs (Alomone Labs, Israel; Sigma, Italy; Tocris Cookson, UK) were applied by dissolving them to the final concentration and by switching the perfusion from control saline to drug-containing saline after a three ways tap had been turned on. Statistical analysis

Footprint analysis Gait abnormalities were detected by means of foot print analysis performed once at the age of 11 months in the original cohort of 16 male ΔETorA rats and controls. Fore- and hind paws were dipped in blue and red non-toxic paint, respectively. Rats were placed on a sheet of paper and were permitted to walk thus allowing their footprints to leave a paw print pattern on the white paper. Four parameters were measured: stride length, hind- and forepaw base width and the overlap between fore- and hind paws. For each parameter three measurements were taken (in cm). Paw prints at the beginning and at the end as well as runs in which rats made stops, turns or obvious decelerations were excluded from the analysis. We determined the coefficient for variation (CV) as indicator for regularity. CV was calculated from the equation: 100× standard deviation/mean (Amende et al., 2005; Hausdorff et al., 1998).

Statistical analysis was carried out using Prism statistical software package (GraphPad Software, Inc., San Diego, CA, USA) and SPSS software (SPSS, Chicago, IL, USA). All results are expressed as means ± standard error. Results are reported significant if p ≤ 0.05. For multiple comparisons of behavioral data one way ANOVA was used. Genetic status (ΔETorA, WtTorA, nontransgenic) was treated as between-subjects factor. Data subjected to one way ANOVA were analyzed by Bonferroni posttest when normal (Gaussian) distribution was determined. Otherwise Mann–Whitney U-test was used. Student's t-tests were employed for analysis of foot print data. Statistical analysis of the data of automated home cage testing is provided in the Supplementary data. Results

Electrophysiology from striatal and nigral neurons

Generation of transgenic rats

Animal experiments were carried out in accord with Internal Institutional Review Committee, EU directive and Italian rules (86/609/ EEC; D.Lvo 116/1992, 63/2100 EU, 153/2001A-IHM and 5/2010 UV). Transgenic rats (8–12 weeks old) were sacrificed under ether anesthesia and the brain removed from the skull. Parasagittal slices (300 μm thick) were prepared as previously described (Ding et al., 2008; Martella et al., 2009). Intracellular current-clamp recordings were performed using sharp microelectrodes filled with 2 M KCl (40–60 MΩ), as described (Martella et al., 2009) (see Supplementary methods for details). Corticostriatal glutamatergic excitatory postsynaptic potentials (EPSPs) were evoked in striatal medium spiny neurons (MSNs) in the presence of bicuculline (10 μM). A bipolar electrode was placed in cortical layer V. For paired-pulse facilitation, two stimuli were delivered with an interstimulus interval of 50 ms, and measuring the ratio of EPSP2, divided by EPSP1. For high-frequency stimulation (HFS, three trains of 3 s duration, 100 Hz frequency, 20 s apart), stimulus intensity was raised to reach threshold level. After HFS delivery, the amplitude of EPSPs was plotted over time as percentage of the control pre-HFS EPSP amplitude (15 min before HFS). To induce striatal long-term potentiation (LTP), Mg 2 + was omitted from the bathing medium (Calabresi et al., 1992). Synaptic depotentiation (SD) was induced by a low-frequency stimulation (LFS) protocol (2 Hz, 10 min), applied

Transgenic rats were generated by standard pronuclear injection of purified transgenic 16.25 kbps constructs containing full length human Tor1A gene including the potential promoter and all intronic regions (Fig. 1A). Two constructs, WtTorA and the plasmid containing the mutant Tor1A gene, were used for the generation of rat lines overexpressing human ΔETorA and human WtTorA, respectively. As genetic background, Sprague Dawley outbred rats were used. Initial Western blot analysis of whole brain homogenates revealed that the majority of the founder lines robustly expressed the mutant protein. We focused the further analyses on three lines of each genotype presenting the highest levels of the transgene, respectively (ΔETorA: lines 3, 4 and 8, WtTorA: lines 10, 11 and 15). Real-Time Quantitative PCR analyses of the genomic DNA revealed that transgenic rats showed a varying number of tandem integrates of the transgene. The copy number correlated well with the protein expression determined by Western blotting. Overall, lines expressing the human WtTorA showed a lower expression level compared to lines expressing human ΔETorA (Fig. 1B). To assess torsinA expression in different brain regions, we performed Western blot analysis using D-M2A8 antibody which showed that all lines independent from the genotype express torsinA at high levels in the cortex and striatum. Intermediate expression levels of the human torsinA protein were seen in the cerebellum and low

Fig. 2. torsinA protein is expressed in neural tissues. (1) Staining with antibody D-M2A8 reveals expression in different neural tissues of ΔETorA rats demonstrated here at the age of 2 months. Strong expression is detected in cortex and hippocampus. Moderate expression is seen in the cerebellum and the striatum and low expression was detected in substantia nigra, pons, brain stem and olfactory bulb. (CA1: CA1 field of the hippocampus; CA2: CA2 field of the hippocampus; CA3: CA3 field of the hippocampus; CPu: Caudate putamen; GrDG: granular layer of dentate gyrus; Cn: Cerebellum; py: pyramidal cell layer of the hippocampus; LPi: lamina pyramidalis interna of the cortex; LPe: Lamina pyramidalis externa of the cortex, Sn: Substantia nigra). (2) D-M2A8 immunostaining of brain regions of WtTorA rats revealed a diffuse cytoplasmatic staining of torsinA in cells of the cortex (A–C) whereas the immunoreactive product is particularly enriched in the nuclear envelope in ΔETorA rats as demonstrated here for the cortex (D–F), hippocampus (G–I), and the striatum (J–L), respectively (CA1: CA1 field of the hippocampus; CA2: CA2 field of the hippocampus; CA3: CA3 field of the hippocampus; py: pyramidal cell layer of the hippocampus; scale bar in A,D,G,J: 200 μm, in B,E,H,K: 10 μm and in C,F,I,L: 5 μm). (3) The aberrant protein distribution of human ΔEtorsinA in the nuclear envelope observed in the striatum of ΔETorA rats (Fig. A) was seen in various brain regions expressing the human mutant protein including regions with intermediate and low ΔEtorsinA expression levels including bulbus olfactorius and substantia nigra (panels B, D). Cells in different brainstem regions, in the cerebellum and in pons were unremarkable (panels C,D,F). Scale bar of lower magnification: 200 μm, scale bar of higher magnification 10 μm.

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Fig. 3. Alteration of the nuclear membrane in neurons of ΔETorA rats. (1) Double-label immunohistochemistry for torsinA and lamin A/C in ΔETorA rats and WtTorA rats (A). Examples of torsinA-immunofluorescence (green) (B, F) and lamin A/C-immunofluorescence (red) (C,D,G) are shown from cells of the striatum. TorsinA was enriched in the NE in ΔETorA rats, whereas a homogenous cytoplasmatic distribution was observed in WtTorA rats (E,F). Staining with lamin A/C antibody reveals a discontinuous pattern of the NE structure (C), which was not seen in WtTorA rats (G) or in nontransgenic controls (D). (2) Electron microscopical analyses of the nuclear envelope of neurons of the striatum of ΔETorA rats revealed a severally altered ultrastructure of the NE membrane. Stretches of the nuclear envelope were characterized by an almost complete convergence of the two membranes resulting in the phenomenon that even at higher magnification the nuclear envelope was not detectable any more. These regions were interspersed with regions with an abnormal wide space of the nuclear outer and inner membranes ((2)C and D). Nontransgenic rats presented nuclei with a regular perinuclear space which was comparable to what was seen in non-transgenic littermates as demonstrated for striatal cells ((2) A and B). (Scale bar: each 0.5 μm).

expression levels in brainstem and olfactory bulb. While expression levels of the transgene in different brain regions did only slightly vary between the lines of each genotype, WtTorA rats presented a more homogenous expression of the protein in the brain regions analyzed for torsinA expression (Fig. 1C). A hematological screen including a complete blood count, differential blood count and measurement of blood sugar was performed at the age of two months in three rats of all six lines and was found to be normal (data not shown). Neuropathology of ΔETorA rats The regional expression of the transgene in brain was further analyzed by immunohistochemistry using monoclonal antibody D-M2A8 (Hewett et al., 2004). Immunohistochemistry was performed at the age of 2, 5, 8 and 11 months corresponding to the behavioral studies. Immunoreactivity in ΔETorA rats was most prominent in cerebral cortex (lamina pyramidalis externa, lamina pyramidalis interna), and in the hippocampus. In the hippocampal formation immunoreactivity was present in the granule cell layer and in the pyramidal cell layer

throughout all subfields of the Ammon's horn as well as dentate gyrus. Expression of the transgene was highest in the CA1 and CA2 region and in dentate gyrus, whereas the CA3 subfield was only moderately stained. Moderate levels of the transgene were also detected in the cerebellum and in the striatum, whereas low levels were identified in the brain stem, pons, substantia nigra and in the olfactory bulb as shown for a rat overexpressing ΔETorA of line 4 at the age of 2 months (Fig. 2 (1)). The expression pattern at later ages (5, 8 and 11 months) did not differ from the pattern of torsinA expression at the age of 2 months. Again, WtTorA rats showed a more homogenous expression pattern in the different brain regions as demonstrated by Western blot analysis, however, the overall expression level was lower (data not shown). Higher magnification revealed that WtTorA rats displayed a diffuse cytoplasmic staining of torsinA which was more intense compared to nontransgenic rats but similar in distribution (Figs. 2 (2)A–C), whereas ΔETorA rats presented with a shift of the subcellular localization of the transgene from the cytoplasm into the nuclear membrane in different regions of the brain as demonstrated here for the cortex, the

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hippocampus and the striatum (Figs. 2 (2)D–L). This aberrant protein distribution was seen not only in various regions expressing human ΔETorA including brain regions with high expression levels of the human mutant protein such as cortex, hippocampus and striatum (Figs. 2 (2)A–C, Fig. 2 (3)A) but also in regions with intermediate and low ΔE torsinA expression levels including olfactory bulb and substantia nigra (Figs. 2 (3)B, D). Cells in different brainstem regions, in the cerebellum and in pons were unremarkable (Figs. 2 (3)C, D, F). We then analyzed the subcellular localization of torsinA in different neuronal populations by immunofluorescence (Fig. 3 (1)). Again we were able to confirm a predominant concentration of the mutated protein in the nuclear membrane of the ΔETorA rats (Fig. 3 (1)B) which was not seen in the WtTorA rats (Fig. 3 (1)F) and in nontransgenic controls (Fig. 3 (1)D). Most interestingly, staining with both anti-lamin A/C antibody and D-M2A8 antibody reveals a discontinuous staining of the nuclear envelope (NE) structure (Figs. 3 (1)C, B). The integrity of the NE was further analyzed by electron microscopy in neurons of the striatum and the cortex of ΔETorA rats at the age of 3 months (Fig. 3(2)). WtTorA rats presented nuclei with a regular perinuclear space which was comparable to what was seen in nontransgenic littermates as demonstrated for striatal cells (Figs. 3 (2)A and B). In contrast, in ΔETorA rats we detected nuclei with a severely disturbed ultrastructure of the nuclear envelope. Stretches of the NE were characterized by an almost complete convergence of the two membranes. These regions were interspersed with regions with an abnormal wide space of the nuclear outer and inner membranes (Figs. 3 (2)C and D). This was seen in almost all cells in the striatum and in the cortex (data not shown).

Behavioral phenotyping We systematically studied the motor and behavioral phenotype of line 4 overexpressing ΔETorA and line 11 overexpressing WtTorA, which express relatively high levels of the transgene, respectively.

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Inspection of general health by means of a modified SHIRPA protocol (Masuya et al., 2005) did not reveal significant differences between nontransgenic rats and transgenic animals at any time point. Both, the ΔETorA rats and the WtTorA rats, exhibited normal fertility. The rats did not display an obvious phenotype on routine inspection. Notably, one way ANOVA revealed that the rats differed in body weight (2 months: F2,45 = 5.36, p b 0.01; 5 months: F2,45 = 7.35, p b 0.01; 8 months: F2,45 = 24.78, p b 0.001; 11 months: F2,45 = 40.88, p b 0.001): ΔETorA rats showed a significantly lower body weight compared to controls (2 months: p b 0.05; 5 months: p b 0.005; 8 months: p b 0.001; 11 months: p b 0.001). Analysis of variance also showed a difference in body temperature between groups (2 months: F2,43 = 4,66, p b0.05; 5 months: F2,45 = 9.45, p b 0.001; 8 months: F2.45 = 9.91, p b 0.001; 11 months: F2,45 = 5.69, p b 0.01). ΔETorA rats showed a significantly lower body temperature compared to the control littermates which were already detectable at the age of 2 months (2 months: p b 0.05; 5 months: p b 0.001; 8 months: p b 0.001). At 11 months the ΔETorA rats had a significantly lower body temperature compared to WtTorA rats (pb 0.01; Supplementary Fig. 1). Rotarod and beam walking The performance on the rotarod of the ΔETorA rats was not significantly different from that of WtTorA rats and controls except for one time point (8 months; F2,41 = 3.37, p b 0.05), when the ΔETorA rats performed better when compared to the control group (Fig. 4A; p b 0.05). Performance of ΔETorA rats at this time point was indeed moderately inversely correlated with their weight (p b 0.05, r = −0.32, Supplementary Fig. 2) meaning that lighter animals performed better. When introducing body weight into variance analysis as random effect the three groups showed no significant differences. However, as evident from the analysis of the training session, there were significant group differences (day 1: F2,141 = 3.21, p b 0.05; day 2: F2,185 = 5.55, p b 0.01; day 3: F2,184 = 3.86, p b 0.01). The ΔETorA rats started at a markedly lower level, falling off the rotarod significantly

Fig. 4. Motor phenotype of ΔETorA rats. (A) Rotarod analysis revealed no significant changes in ΔETorA rats (line 4) compared to WtTorA rats (line 11) and controls (wt). The better performance at the age of 8 months is most likely related to the lower weight of the animals. Inclusion of body weight into analysis of variance as random effect does not reveal significant differences between the three groups. (B) Analysis of training sessions demonstrated a worse performance of ΔETorA rats compared to the other groups at the first session. ΔETorA rats started at a markedly lower level, falling off the rotarod significantly more often compared to nontransgenic animals (day 1: p b 0.05; day 2: p b 0.001; day 3: p b 0.01). (C) Impairments in the beam walking task were detected at the age of two months and were restricted to the 3.5 cm round beam (p b 0.05).

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Fig. 5. Neurological phenotype of ΔETorA rats. Clasping behavioral: Tail suspension test indicates a clasping-like behavioral in ΔETorA rats at different degrees (A,B), which was not seen in WtTorA rats (C) and nontransgenic littermates (D). Limb grasping test: An increasing number of animals showed abnormalities in the limb grasping tests starting at the age of 5 months reaching significance at the age of 11 months (p b 0.05) (E). Footprint analysis of nontransgenic mice showed a normal gait with a clear overlap of front (red) and hind limb steps (blue). ΔETorA rats exhibited irregularities in their gait pattern with respect to the step length. Coefficient of variation was determined to quantify variations in step length (F), which was significant for the hind limbs (p b 0.05) but could be observed in tendency also for the front limbs (p = 0.065) (G).

more often compared to nontransgenic animals (day 1: p b 0.05; day 2: p b 0.001; day 3: p b 0.01) (Fig. 4B). The impairment of acquisition of a certain motor task at first trial was also seen for the beam walking test. At the age of 2 months, ΔETorA rats needed significantly more time to traverse the most challenging beam (3.5 cm round beam; F2,45 = 3.26, p b 0.05, Fig. 4C) compared to the control group (pb 0.05). At all following time points, ΔETorA rats did not significantly differ from their littermates or even showed a tendency to perform better (Fig. 4C).

The neurological examination as part of the modified SHIRPA protocol (Masuya et al., 2005) revealed that the integration of the full length mutant torsinA into the rat was able to induce deficits regarding

different motor responses which mainly affect the hind limbs. At the age of 5 months, some ΔETorA rats displayed clasping (Figs. 5A und B). This phenotype was progressing as evident from Tables 1a and 1b. Not only the number of animals affected increased over time but also the intensity of the clasping mirrored by the higher score they got according to the classification of Korenova et al. (2009). At the age of 11 months all rats overexpressing ΔETorA showed clasping of varying degrees (Table 1b). Abnormalities were also detected in the limb grasping test (F2,45 = 3.22, p b 0.05). ΔETorA rats showed a lower score which was evident already at the age of 5 months. Discrepancy increased over time and became significant at the age of 11 months (p b 0.05) (Fig. 5E). Slight neurological impairments could give rise to small foot placement deficits. We therefore added footprint analysis to the original

Table 1a Hind limb clasping in transgenic rats at the age of 8 months.

Table 1b Hind limb clasping in transgenic rats at the age of 11 months.

ΔETorA-transgenic rats present with motor reflex deficits

8 Months

ΔETorA rats

WtTorA rats

Nontransgenic rats

11 Months

ΔETorA Rats

WtTorA Rats

Nontransgenic rats

Hind limbs clasped or crossed with toes flexed Hind limbs almost in contact, toes spread Hind limbs extended with spreading toes

7

–

–

9

–

–

8

2

1

7

1

1

1

14

15

Hind limbs clasped or crossed with toes flexed Hind limbs almost in contact, toes spread Hind limbs extended with spreading toes

–

15

15

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phenotyping protocol at the age of 11 months to evaluate the regularity and precision of the ΔETorA rat's gait. We did not observe significant differences between the different rat lines for stride length, overlap, fore- and hind paws width (data not shown). However, the raw data indicated a greater variance in step length in ΔETorA rats compared to nontransgenic littermates (Fig. 5F). To quantify the stride-to-stride variations, we determined the coefficient of variation (CV) (Amende et al., 2005; Hausdorff et al., 1998). The variation of step length was significant for the hind limbs (p b 0.05), but could be seen in tendency for the fore limbs (p = 0.065, Fig. 5G). Collectively, these findings indicate that ΔETorA rats developed a progressive neurological phenotype which affected predominantly the hind limbs and seemed to

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subsequently impair the performance on the beam walk and the regularity of gait. Automated home caging analysis revealed development of late onset glucose intolerance Analysis of activity parameters in automated home cages (including rearing, ambulatory movements during dark and light phase) did not reveal major abnormalities between both transgenic lines and controls. Analysis of metabolic parameters, however, disclosed a strong increase of energy expenditure and water consumption in ΔETorA rats (p b 0.001 at 11 months; Supplementary Figs. 3A,B). We

Fig. 6. Impairment of striatal LTD in ΔETorA rats. A) Left. Summary plots showing that HFS of corticostriatal glutamatergic fibers induces an LTD in MSNs from both NT and WtTorA rat slices. Conversely, HFS failed to cause any synaptic change in MSNs from ΔETorA rats. Right. The superimposed traces show representative EPSPs recorded before (pre) and 25 min after (post) HFS in NT, WtTorA and ΔETorA rats. B) Pre-incubation with either amphetamine or L-dopa (100 μM, 25 min) was not able to restore LTD in ΔETorA rats. C) Similarly, quinpirole (10 μM, 20 min) treatment was unable to restore LTD in ΔETorA rats. Conversely, pirenzepine (100 nM 25 min) preincubation was able to fully restore LTD in mutant rats. Each data point represents the mean ± SEM.

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then analyzed urine and blood sugar levels which were found to be increased in ΔETorA rats of line 4. Obviously the rats of line 4 developed late-onset diabetes mellitus which seemed to be specific for this line. Rats of other ΔETorA lines (lines 3 and 8) as well as rats overexpressing WtTorA did not show abnormalities in glucose levels at any time point. Notably, ΔETorA rats with abnormal glucose levels did not differ from animals with normal glucose levels in all parameters detected by Shirpa protocol and all classical behavioral tests compared to non-diabetes animals of the same transgenic line (Supplementary Fig. 4). In particular, ΔETorA rats with and without increased blood sugar levels did not differ with respect to clasping behavioral (Supplementary Table 1). We therefore excluded abnormalities in the blood glucose being related to the observed phenotype in ΔETorA rats. Analysis of striatal synaptic plasticity revealed impaired long-term plasticity at corticostriatal synapses A first set of experiments was performed from nigral neurons. In dopaminergic neurons recorded from ΔETorA rats, the response both to dopamine and D2 autoreceptors was not significantly different from both WtTorA transgenic and nontransgenic rats (see Supplementary results), confirming previous evidence obtained from transgenic mice (Martella et al., 2009; Napolitano et al., 2010). In both intracellular and whole-cell recordings, MSNs were silent at rest; tonic action potential discharge was elicited by depolarizing current pulses, while strong inward rectification was observed upon injection of hyperpolarizing current. The intrinsic membrane properties, such as resting membrane potential (RMP), input resistance, action potential amplitude and duration, delay to spike threshold, and firing frequency from MSNs of nontransgenic rats (n = 15) did not differ from those recorded from WtTorA (n = 12) or ΔETorA MSNs (n = 19; p > 0.05 ANOVA among groups; Supplementary Table 2). To look for possible alterations in glutamate release probability, we measured spontaneous excitatory postsynaptic currents (sEPSCs). A combination of NMDA and AMPA glutamate receptor antagonists, MK801 (30 μM) and CNQX (10 μM) fully blocked spontaneous synaptic events. On average, the amplitude and frequency of sEPSCs recorded from NT MSNs (n = 15) ranged between 8 and 40 pA, and 0.6 and 7 Hz, respectively and did not differ from WtTorA (n = 16) and ΔETorA (n = 24) MSNs (p > 0.05 ANOVA followed by Tukey post hoc test; data not shown). To analyze short-term plasticity, EPSPs were evoked by cortical stimulation. EPSPs recorded from NT rat MSNs had mean amplitude (20.23 ± 4.5 mV; n = 23) similar to that measured from WtTorA (21.98 ± 5.08 mV; n = 20) or ΔETorA (23.33 ± 5.8 mV; n = 31) rats (p > 0.05 ANOVA followed by post-hoc Tukey test; data not shown). In addition, no significant difference in paired-pulse ratio (PPR), considered a reliable indicator of transmitter release probability, were found between neurons from NT (n = 16; EPSP2/EPSP1 ratio 1.43 ± 0.5), WtTorA (n = 18; EPSP2/ EPSP1 ratio 1.38 ± 0.4) and ΔETorA rats (n = 24; EPSP2/EPSP1 ratio 1.36 ± 0.7) (data not shown; p > 0.05; ANOVA followed by post-hoc Tukey test). High frequency stimulation (HFS) of glutamatergic afferents induced a robust LTD in MSNs from NT rats, similar to that reported previously in rat brain slices (Calabresi et al., 1994) (Fig. 6A; 57.9 ± 4.66% of control EPSP amplitude, measured 25 min post-HFS; n = 12, t-test p b 0.001). Similarly, in MSNs from WtTorA rats, HFS caused a LTD that was not different from that measured in controls (Fig. 6A; 48.03 ± 7.66% of control EPSP amplitude; n = 15, Mann–Whitney p b 0.001). In slices from ΔETorA, however, HFS was unable to determine any long-lasting change in synaptic efficacy (Fig. 6A; 100.94 ± 7% of control EPSP amplitude; n = 18; t-test p > 0.05). To verify whether the impairment of LTD could be determined by a reduced availability of endogenous dopamine, slices were pretreated with either a dopamine (DA) releaser, amphetamine, or a dopamine precursor, L-dopa. However, pretreatment with both amphetamine (100 μM, 20 min), or L-dopa

(100 μM, 35 min) was unable to restore LTD in MSNs of ΔETorA rats (Fig. 6B; amphetamine: 93.72 ± 6.11% of control EPSP amplitude; Ldopa: 89.50 ± 3.68% of control EPSP amplitude; n = 5 for each group; t-test p > 0.05). To address the involvement of DA D2 receptors, slices were treated with the D2 receptor agonist quinpirole. In the presence of quinpirole (10 μM), however, HFS failed to restore LTD (Fig. 6C; quinpirole: 103.45 ± 9% of control EPSP amplitude; n = 13; p > 0.05 Mann– Whitney). Previous evidence obtained in mice revealed the crucial involvement of M1 muscarinic receptors in the bidirectional regulation of corticostriatal synaptic plasticity induction (Bonsi et al., 2008). To verify whether M1 receptors were involved in the loss of LTD in ΔETorA rats, as previously reported in mice overexpressing mutant torsinA (Martella et al., 2009), slices were bathed with the M1 muscarinic antagonist pirenzepine (100 nM, 25 min). Under this experimental condition, HFS induced a LTD of normal amplitude (Fig. 6C; 43.48± 4.9% of control EPSP amplitude; n = 17; p b 0.01 Mann–Whitney). The LTP induction protocol elicited an LTP of similar amplitude in MSNs from NT as well as from WtTorA and ΔETorA rats (Fig. 7A; NT: 153.36 ± 4.6% of control EPSP amplitude, n = 10, t-test p b 0.0001; WtTorA: 156.77 ± 8.2% control EPSP amplitude, n = 10, t-test p b 0.0001; ΔETorA: 151.16 ± 5.6% control EPSP amplitude, n = 13, p b 0.001 Mann–Whitney test). Once striatal LTP is stabilized, a lowfrequency stimulation (LFS) protocol reverses synaptic strength to resting levels, a plastic phenomenon defined synaptic depotentiation (SD) (Rioult-Pedotti et al., 2007). Consistent with recent evidence obtained in mice (Martella et al., 2009), LFS induced a SD in NT and WtTorA MSNs (Fig. 7A; NT: 93.110 ± 3.68% of control EPSP amplitude, n = 10, p b 0.0001 t-test; WtTorA: 88.68 ± 5.11% of control EPSP amplitude, n = 10, p b 0.0001 t-test). However, LFS failed to depotentiate corticostriatal synapses in MSNs from ΔETorA rats (Fig. 7A; 154.12 ± 5.77% of control EPSP amplitude, n = 12, t-test p > 0.05). Both LTP and SD have been shown to require the integrity of dopaminergic signaling to be expressed (Picconi et al., 2003). To address this issue, rescue experiments were performed with either amphetamine or L-dopa. Both treatments, however, were unable to restore SD in MSNs from mutant rats (Fig. 7B; amphetamine: 150.51 ± 6.21% of control EPSP amplitude, n = 8, t-test p > 0.05; L-dopa: 158.22 ± 5.7% of control EPSP amplitude, n = 8, t-test p > 0.05). In addition, in another set of recordings, slices were bathed with D1- or D2-like dopamine receptor agonists, either alone or in combination. Co-application of both D1 and D2 receptor agonists, SKF 379268 (10 μM) and quinpirole (10 μM), failed to restore SD in MSNs from transgenic rats (Fig. 7C; 156.51 ± 4.58% of control EPSP amplitude, n = 8, t-test p > 0.05). In line with the hypothesis of a pivotal role of M1 muscarinic receptors in corticostriatal bidirectional synaptic plasticity (Martella et al., 2009; Picconi et al., 2003) we tested M1 receptor antagonist in rats overexpressing mutant torsinA. Indeed, pirenzepine (100 nM) fully restored SD in MSNs from ΔETorA (Fig. 7C; 91.26 ± 3.15%, n = 12, t-test p b 0.001). Discussion To elucidate pathogenic mechanisms in DYT1 dystonia induced by the expression of the human torsinA protein, we developed and characterized a rat model of DYT1 dystonia containing the full length human DYT1 gene including the promoter region, intronic and regulatory regions. Neuropathological characterization of the torsinA overexpressing rat lines The immunohistochemical analyses revealed that the approach of integrating the human full length Tor1a gene leads to a pattern of expression of the human torsinA protein similar to what has been described for the human brain, however at clearly different levels. The different expression levels are most likely explained with differences

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Fig. 7. Impairment of SD in ΔETorA rats. A) Left. After magnesium-dependent NMDA receptor blockade relief, HFS causes a similar LTP in NT, WtTorA and ΔETorA rats. After LTP induction, low frequency stimulation (LFS) is able to cause an SD in both NT and WtTorA, but not in ΔETorA rats. Right. Representative traces of both pre- and post-HFS, and pre- and post-LFS EPSPs recorded in all the genotypes examined. B) Either L-dopa or amphetamine (both 100 μM) applied after LTP induction and 15 min before LFS are not able to rescue SD. C) Similarly, co-application of quinpirole plus SKF − 38393 (both 10 μM) failed to restore SD. C) Conversely, pirenzepine (100 nM), applied after LTP induction and 15 min before LFS, was able to restore SD in ΔETorA rats. Each data point represents the mean ± SEM of at least six independent observations.

in the copy number of the transcript, since protein levels clearly corresponded to the copy number. Noteworthy, the expression pattern of the two genotypes differs slightly from each other with a more homogenous expression of the torsinA protein in the different brain regions in lines expressing the wildtype protein compared to lines expressing the mutant protein. This cannot be explained by an impact of the integration site since the pattern of expression is almost identical in the three lines of each genotype, but might be due to interaction with the rat genome. We have to assert that the expression pattern of torsinA protein observed in human brain is not accurately replicated and is not consistent in the different genotypes. Previous studies using YACs and BACs showed that the expression of large DNA transgenes can accurately reflect the pattern of transcription and translation of the endogenous gene and has a high probability of conferring accurate tissue- and cell-specific transgene expression (Frazer et al., 1995; Giraldo and Montoliu, 2001; Zuo et al., 1999). While the use of the full length DYT1 locus might avoid artifacts caused by transgene expression in cells and tissues usually not

expressing torsinA, the integration of a ~16 kb large DNA construct does obviously not rule out interactions with the rat genome or effects of the integration site in this model. We further detected a severely altered ultrastructure of the NE by means of immunofluorescence and electron microscopy in various brain regions of ΔETorA rats. Conflicting data have been presented for the NE pathology in existing mouse models for DYT1 dystonia (Dang et al., 2005; Goodchild et al., 2005; Grundmann et al., 2007; Sharma et al., 2005). NE abnormalities have been observed in homozygous TorA knockin and knockout mouse models with reduced or even absent levels of torsinA protein (Goodchild et al., 2005) as well as in torsinA overexpressing mice, but not in all (Grundmann et al., 2007; Sharma et al., 2005; Shashidharan et al., 2005). Thus it has been hypothesized that the abnormal accumulation of the torsinA protein and the disrupted NE ultrastructure are side effects of nonphysiological levels of the protein, in particular since the NE pathology has not been observed in heterozygous mouse models carrying the GAG deletion (Dang et al., 2005; Goodchild et al., 2005; Saunders et

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al., 2002). However, there is increasing evidence from in vitro studies that the NE is a site of action of torsinA (Gonzalez-Alegre and Paulson, 2004; Goodchild and Dauer, 2004, 2005; Naismith et al., 2004). Furthermore, accumulation of torsinA in the NE was observed in fibroblasts of a human mutation carrier with physiological expression levels of torsinA (Goodchild and Dauer, 2004), whereas NE pathology has never been described in the human brain, probably due to the sparse brains available and their respective fixation methods, which might hamper the reliable analysis of NE ultrastructure (see Standaert, 2011 for review). The inconsistent findings in mouse models and the lack of evidence for NE abnormalities in human brains make it extremely difficult to dissect whether the abnormal accumulation of the protein in the NE is an artifact of the model system caused by non-physiological levels of the protein or whether this is a robust feature of torsinA dysfunction caused by the mutation. We cannot exclude the impact of high expression levels on this phenotype in the rat model. However, by demonstrating the occurrence of the NE pathology in a second species we lend support to the hypothesis that the NE pathology is a significant phenotype of ΔETorA. It is possible that physiological expression levels of the mutant protein in the human mutation carrier or in the heterozygous torsinA knockin mouse models cause subtle dysfunctions of torsinA in the NE which only leads to a visible phenomenon when higher levels of the protein are present (Cookson and Clarimon, 2005). Why the enrichment of ΔETorA to the NE takes place in certain neuron population and how the disruption of the NE ultrastructure is related to disease pathogenesis has not irrevocably been dissected. Inclusion formation was not observed in this model in contrast to preceding transgenic mouse models (Dang et al., 2005; Grundmann et al., 2007) which might be due to the lower expression level of the human transgene in this model or due to the antibodies used to detect torsinA expression. The motor phenotype of the transgenic rat model The motor behavioral characterization not only revealed a phenotype which replicated some disease characteristics observed in human mutation carriers and in preceding animal models for DYT1 dystonia but also disclosed new aspects of this phenotype so far not reported for the disease. In particular, we were able to detect a motor phenotype of increasing severity. The abnormal motor responses showed a highly variable age of onset but were fully penetrant. At the end of the observation period these abnormal motor responses appeared to affect motor functions such as regularity of gait and beam walk performance, although the latter did not reach significance. Further investigations have to clarify whether this phenotype further generalizes to other body parts and finally reach a steady state level as seen in humans. However, the progressive motor phenotype with young onset and generalization until young adulthood observed in human mutation carriers is not replicated in this model. Mouse models for DYT1 dystonia reported so far showed subtle signs in classical motor function tests, but no sign of dystonic movements or neurological abnormalities (Dang et al., 2005; Grundmann et al., 2007; Page et al., 2010; Sharma et al., 2005; Yokoi et al., 2007; Zhao et al., 2008). This transgenic rat model displays abnormal reflex responses beyond the motor phenotype. The abnormalities in ΔETorA rats disclosed by means of the SHIRPA protocol were not dystonic in nature, since the abnormal reflexes, hind limb clasping and the limb grasping reflex, are unspecific signs occurring in several conditions including Huntington's disease or spinocerebellar ataxia (Chou et al., 2008; Korenova et al., 2009; McManamny et al., 2002; Schilling et al., 1999). However, the abnormal motor reflex response appears to be a robust expression of torsinA dysfunction in this model

which can be detected easily and might therefore be well suited as disease marker for therapeutic studies. In keeping with findings not only in existing mouse models (Grundmann et al., 2007; Sharma et al., 2005) but also in human mutation carriers (Ghilardi et al., 2003) we were able to detect impairments of the acquisition of a complex motor task such as rotarod or beam walking in ΔETorA rats. However, whereas human mutation carriers (Ghilardi et al., 2003) as well as transgenic mouse models (Grundmann et al., 2007; Sharma et al., 2005) started at the same base line but displayed a slower learning curve compared to controls the rat model reported in this study started at a significantly lower level. In contrast to preceding mouse models for DYT1 dystonia the learning curve itself was not impaired in ΔETorA rats (Fig. 4B). They learned even better and finally were able to perform with the same accuracy as their littermates, proving that motor coordination and balance is not impaired in general during the observed time period. Once the motor skill is mastered, it was retained for the whole observation period indicating that the long memorization of motor skills is also not impaired. The fact that this behavioral was noted for different motor tests suggests that it was not the type of task (beam walking or rotarod) which caused the impairments but the novelty of the task. The systematic screening for physiological abnormalities and activity performed repeatedly by the automated home cage analysis did not reveal broader abnormalities but disclosed a disturbed glucose metabolism in older rats. This impaired glucose tolerance was detected exclusively in line 4. Whether the late onset glucose intolerance is a result of the integration site or due to a mutation which occurred spontaneously in this line, has to be further investigated. We further found a lower body weight and a lower body temperature of ΔETorA rats. The significance of these findings is unclear since this is not reported for preceding animal models for DYT1 dystonia or in human mutation carriers and the molecular basis is not yet understood. A higher glucose level as the underlying reason for the delayed weight gain could be excluded, since ΔETorA rats with and without diabetes did not significantly differ with respect to their weight (Supplementary Fig. 4C). We also excluded a higher activity level of the ΔETorA rats as reason for the delayed weight gain, since in contrast to other animal models for DYT1 dystonia (Grundmann et al., 2007; Sharma et al., 2005), the ΔETorA rats exhibited a normal level of activity over the whole period.

Long-term changes in synaptic efficacy at striatal synapses might be the underlying cause for the motor phenotype We were able to demonstrate severe abnormalities of plasticity in the striatum. The striatal neurons in ΔETorA rats lack LTD and SD, both of them could be restored by using M1 receptor antagonists. Most notably, we were able to replicate the findings in transgenic mice overexpressing the mutant protein (Martella et al., 2009; Sharma et al., 2005). Thus, the impairment of both LTD and SD demonstrate that a “loss of inhibition” represents a common trait feature of both mouse and rat models of DYT1 dystonia. Yet, similarly to what has been observed in transgenic mice, antagonists at M1 muscarinic receptors were able to restore a physiological plasticity, further supporting the idea of a central role of cholinergic neurotransmission in DYT1 dystonia. We were not able to disclose corresponding changes for dopamine levels and its metabolites which have been reported in previous DYT1 dystonia mouse models (Grundmann et al., 2007; Sharma et al., 2005; Zhao et al., 2008) as well as in human brains (Asanuma et al., 2005; Augood et al., 2002). Preliminary data using HPLC analysis in a relatively small number of rats (n = 5 rats for each genotype and controls) did not indicate that the dopaminergic system is affected by the integration of the human mutant and wildtype transgene into the rat genome (unpublished data).

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On the other hand, the NE pathology observed in striatal neurons in ΔETorA rats has not been observed in mouse models overexpressing ΔEtorsinA (Sharma et al., 2005). Thus, it is still not resolved how the severe alteration of striatal synaptic plasticity is related to abnormalities observed on subcellular level including NE pathology and the behavioral abnormalities observed in this model or the abnormalities in dopamine neurotransmission seen in mouse models overexpressing ΔEtorsinA (Sharma et al., 2005). Further analyses have to clarify whether there is a potential relationship between the observed neuropathological and plasticity changes and the motor phenotype or whether torsinA dysfunction affects unrelated pathways.

Salvador Castaneda and Prof Dr. B Pichler were supported by the Werner–Siemens foundation.

Conclusion

Supplementary data to this article can be found online at doi:10. 1016/j.nbd.2012.03.024.

In this study we present a transgenic rat model for DYT1 dystonia and thus a second species besides the existing mouse models overexpressing the human torsinA. We could show that the expression of the human transgene under its own promoter leads to a spatial expression pattern of torsinA in the rat brain similar to what has been observed in the human brain but does not accurately replicate the human situation. We were further able to generate a slightly progressive and fully penetrant motor phenotype which is an important aspect considering that the existing mouse models for DYT1 dystonia show only subtle motor abnormalities. Most importantly, we were able to replicate certain aspects of torsinA related pathology in a second species, namely the NE pathology and alterations in synaptic plasticity in the striatum. The confirmation of these features in a second rodent species might indicate that these phenotypes are associated with torsinA dysfunction and not specific for the model system under investigation, although at present we cannot rule out an effect determined by protein overexpression. However, the phenotype observed in this model has to be interpreted with caution, since the torsinA protein is overexpressed in both genotypic models. Thus we cannot rule out for any of the observed phenotypes in this model that they are a result of nonphysiological high expression levels, effects of the integration site and interactions with the rat genome. Furthermore, the genotypic lines chosen for in depth investigation differ in their expression levels with the line WtTorA line showing lower expression levels compared to the ΔETorA line. The low expression level of the human wildtype torsinA protein in the WtTorA rats might explain why we did not detect phenotypic abnormalities, although it has been reported that overexpression of the wildtype torsinA protein can be detrimental for neuronal cells (Grundmann et al., 2007; Page et al., 2010). In conclusion we report here the development of a transgenic rat model for DYT1 dystonia. This animal model is an important complement to the existing mouse models due to the difference in genetic background and species. This is the only model for DYT1 dystonia harboring the whole Tor1A gene thus providing the expression of the human torsinA protein under the endogenous promoter and cis regulatory elements. Eventually, since this rat model replicates key features of torsinA associated pathology observed in vivo and in vitro, this animal model might represent a novel in vivo paradigm to study the pathophysiology of DYT1 dystonia. Funding This study was supported by the Dystonia Medical Research Foundation to KG and AP; and by COST Action grant BM1101. Prof. Dr. Med. Martin Schaller was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 773, project Z2). Dr. Zhenyu Yue was supported by Bachmann–Strauss Dystonia & Parkinson Foundation and NIH R01NS060809.

Acknowledgment We wish to thank Esteban Portal for statistical analysis of behavioral data and Natalja Funk for her critical advice, technical support and discussions. We thank Ratstream for providing the cages for automated home cage analysis and TSE for the development of the cages. Appendix A. Supplementary data

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