Brain Research Bulletin 69 (2006) 306–310
Normal sensitivity to excitotoxicity in a transgenic Huntington’s disease rat C. Winkler a , J.M.A.C. Gil b,c , I.M. Ara´ujo b,d , O. Rieß e , ˚ Peters´en b,∗ T. Skripuletz f , S. von H¨orsten f , A. b
a Department of Neurology, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany Neuronal Survival Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Sciences, Lund University, BMC A10, SE-221 84 Lund, Sweden c Center for Neurosciences and Cell Biology and Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal d Center for Neurosciences and Cell Biology and Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal e Department of Medical Genetics, University of T¨ ubingen, Calwerstr. 7, 72076 T¨ubingen, Germany f Department of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany
Received 3 July 2005; received in revised form 15 December 2005; accepted 4 January 2006 Available online 20 January 2006
Abstract Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by a CAG repeat expansion in the HD gene. Excitotoxic cell damage by excessive stimulation of glutamate receptors has been hypothesized to contribute to the pathogenesis of HD. Transgenic mouse models of HD have shown variable sensitivity to excitotoxicity. The models differ in the genetic background, the type and length of the promoter driving the transgene expression, the CAG repeat length and/or the HD gene construct length. Furthermore, one has to differentiate whether transgenic or knock-in models have been used. All these factors may be involved in determining the responsiveness to an excitotoxic insult. Here, we explored the responsiveness to excitotoxic damage using a transgenic HD rat model carrying 22% of the rat HD gene which is driven by the rat HD promoter and which harbors 51 CAG repeats. 3 and 18 months old transgenic HD rats and their wild-type littermates received unilateral intrastriatal injections of the glutamate analogue quinolinic acid. Lesion size was assessed 7 days later using the degenerative stain Fluoro-Jade and by immunohistochemistry for the neuronal protein NeuN. No difference in susceptibility to excitotoxicity was found between the groups. Our study supports mouse data showing maintained susceptibility to excitotoxicity with the expression of around 25% of the full HD gene. Differences in sensitivity to excitotoxicity between genetic animal models of HD may be dependent on the length of the expressed HD gene although additional factors are also likely to be important. © 2006 Elsevier Inc. All rights reserved. Keywords: HD animal models; Quinolinic acid; Fluoro-Jade
1. Introduction Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in the HD gene [4]. Affected individuals suffer from personality changes, motor deficits, weight loss, and dementia before they die 15–20 years after disease onset. No effective treatment is available today. The neuropathology is characterized by loss of neurons, primarily in the neostriatum and the cerebral cortex. Although the pathogenesis remains unknown, involvement of
Abbreviations: ANOVA, analysis of variance; FJ, Fluoro-Jade; HD, Huntington’s disease; NeuN, neuronal nuclei; NMDA, N-methyl-d-aspartate; PBS, phosphate buffered saline; QA, quinolinic acid ∗ Corresponding author. Tel.: +46 46 222 0525; fax: +46 46 222 0531. ˚ Peters´en). E-mail address:
[email protected] (A. 0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2006.01.003
excitotoxicity, i.e. the excessive stimulation of glutamate receptors, has been suggested to play a role [1–3,10]. Biochemical and cell biological data further support this hypothesis by defining a direct involvement of huntingtin in the glutamate/NMDA signaling pathway [14,15,18]. Since the generation of genetically modified mouse models of HD, the role of excitotoxicity for the presentation of the HD phenotype has been investigated in at least six different transgenic lines. In these studies, HD mice and their wild-type littermates have received intrastriatal injections of quinolinic acid (QA), an N-methyl-d-aspartate (NMDA) receptor agonist, that is known to cause excitotoxic damage. Conflicting results ranging from resistance to excitotoxicity to increased sensitivity to the excitotoxic insult have been obtained in HD mouse models [6–8,11,13,18]. Differences in the genetic background of the particular mouse strain, the promoter driving the transgene con-
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struct, the length of the HD gene, and the number of CAG repeats have been discussed to explain these results [11]. To further elucidate whether HD animal models have an altered sensitivity to excitotoxicity, we decided to examine it in a recently generated HD rat. These rats carry a truncated huntingtin cDNA fragment with 51 CAG repeats under the control of the native rat huntingtin promoter, with the gene product corresponding to 22% of the HD gene [16]. The model is on a Sprague–Dawley genetic background. These animals display HD-like symptoms such as cognitive impairments and progressive motor dysfunction as well as neuropathological characteristics such as neuronal intranuclear inclusions and enlarged lateral ventricles [16]. Previous studies in the R6 and the YAC mouse models have shown that excitotoxic susceptibility may develop over time [18,6]. We therefore used both young and aged HD rats (3 and 18 months of age), and examined their responsiveness to intrastriatal injections of QA using similar analyses as employed in previous mouse studies. 2. Materials and methods
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degenerating neurons [12], as previously described [7]. A further series was processed for immunohistochemistry for the neuron-specific nuclear protein NeuN (Chemicon, Temecula, CA, USA). Briefly, free-floating sections were quenched in 3% hydrogen peroxide/10% methanol in 0.1 M PBS for 15 min. After rinsing in 0.1 M PBS, sections were incubated in a blocking solution containing 5% normal horse serum and 0.25% Triton X-100 in 0.1 M PBS. They were then incubated for 48 h at 4 ◦ C with a primary mouse anti-NeuN antibody (1:1000) in the blocking solution. After rinsing, sections were incubated for 1 h at room temperature with a secondary biotinylated horse anti-mouse antibody (1:200; Dakopatts, Copenhagen, Denmark) followed by another rinse. The bound antibodies were then visualized by an avidin–biotin–peroxidase complex system (Vectastain ABC Elite Kit, Vector Labs, Burlingame, CA, USA) using 3,3-diaminobenzidine as the chromogen. 2.2.2. Stereology All morphological analyses were performed on blind-coded slides using an Olympus CAST-Grid system (Olympus Danmark A/S, Albertslund, Denmark). The striatal lesion area (defined by the presence of FJ-labelled cells or the lack of NeuN positive cells) was delineated in sections processed for either FJ-staining or NeuN immunohistochemistry. The total striatal area at the non-injected side was delineated in NeuN stained sections. The striatal lesion volume and the total striatal volume were then calculated, taking into account the frequency of sections (1:8) and their thickness (30 m), according to the Cavalieri principle [5].
2.1. Animals
2.3. Statistical analyses
Transgenic HD rats with 51 CAG repeats [16], and their wild-type littermates were used in this study. The transgenic HD rat expresses 727 amino acids of the HD gene (cDNA position 324–2321 corresponding to 22% of full length), which are under the control of 886 bp of the rat huntingtin promotor (position 900 to 15). The genetic background of the transgenic HD rat is Sprague–Dawley strain (outbred) derived. Animals are presently in F14 of inbreeding and for the experiments F10 animals were used. Due to this fact, susceptibility to NMDA receptor-mediated toxicity should be close to that of Sprague–Dawley rats, but animals can now be considered as inbreds. The rats were housed under a 12-h light:12-h dark cycle with food and water ad libitum. Rats of 3- (N; wild-type rats = 8, transgenic HD rats = 10) and 12- (N; wild-type rats = 6, transgenic HD rats = 9) months of age were used in this study. Rats at 3 months of age displayed signs of reduced anxiety in a social interaction test of anxiety and in the elevated plus maze test of anxiety. Furthermore these animals display ophistotonus-like head movements, while other classical signs of the HD-like phenotype, such motor dysfunction and cognitive decline develop at 9–12 months of age (Nguyen et al., 2005, unpublished data). At 18 months of age, the transgenic rats showed a reduction of anxiety-like behaviors, a cognitive decline in a spatial learning task and progressive impairments of hind- and forelimb coordination [16]. All research and animal care procedures were approved by the District Government, Hannover, Germany, and performed according to international guidelines for the use of laboratory animals.
For group comparisons, data were subjected to a two-factor analysis of variance (ANOVA) using the Statview 5.4 Package (Abacus concepts, Berkeley, CA, USA). Statistical significance was set at P < 0.05. Data are presented as means ± standard error of the mean (S.E.M.).
2.2. Surgery Quinolinic acid (QA; Sigma, St. Louis, MO, USA) was dissolved in 0.1 M phosphate buffered saline (PBS, pH 7.4). Under isoflurane anesthesia, the rats received a single unilateral intrastriatal injection of 80 nmol of QA in 1 l at the following stereotactic coordinates using a 5 l Hamilton syringe: 1.2 mm rostral to bregma, 2.7 mm right of the midline, and 4.5 mm ventral to the dural surface, with the incision bar set at zero. The injection rate was 0.2 l/min, and the cannula was left in place for an additional 3 min before slowly retracting it. 2.2.1. Histology Rats were perfused with 4% paraformaldehyde 7 days after the surgery. The brains were postfixed over night in the same solution and dehydrated in 20% sucrose/0.1 M PBS. Serial coronal sections were cut on a freezing microtome at 30 m thickness. One set of series was processed for the Nissl stain cresyl violet (ICN Biomedicals Inc., Aurora, OH, USA). Another series of sections was processed for Fluoro-Jade (FJ; Histo-Chem, Jefferson, AR, USA), that stains
3. Results The size of the striatal lesion induced by the unilateral injection of 80 nmol of QA in transgenic and wild-type rats of 3 or 18 months of age was analyzed 7 days post-surgery in sections stained for FJ (Fig. 1A–C) or processed for NeuN immunohistochemistry (Fig. 1D–F). Areas positive for FJ were immunonegative for NeuN, indicating loss of the neuronal phenotype. Within the FJ-positive areas vast numbers of degenerating cells exhibiting dense nuclei were observed as confirmed by the Nissl stain cresyl violet (data not shown). In wild-type littermates the striatal lesion volume amounted to approximately 43% of the total striatal volume. Thus, it would have been possible to observe both an increased or a decreased sensitivity of QA in the transgenic HD rats. However, there was no significant difference between the FJ-stained lesion volumes of the different genotypes (two factor ANOVA, F(1, 29) = 0.12, P n.s.), although a small difference between the rats of 3 and 18 months of age was detected (two factor ANOVA, F(1, 29) = 5.73, P = 0.02) but no interaction between genotype and age (two factor ANOVA, F(1, 29) = 0.63, P n.s.) (Figs. 1A–C and 2A). Similarly, no difference between the lesions volumes assessed in NeuN processed sections were found between the two genotypes (two factor ANOVA, F(1, 29) = 0.10, P n.s.), the two age groups (two factor ANOVA, F(1, 29) = 0.25, P n.s.) nor any interaction between age and genotype (two factor ANOVA, F(1, 29) = 0.01, P n.s.) (Figs. 1D–F and 2B). We were then interested in studying whether the striatum was atrophied prior to the lesion. We examined the total striatal volume at the non-injected side in sections processed for NeuN immunohistochemistry (Fig. 2C). The
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Fig. 1. Striatal quinolinic acid-induced lesions assessed with Fluoro-Jade staining and NeuN immunohistochemistry. Rats were subjected to an intrastriatal injection of 1 l of quinolinic acid (80 nmol) and the lesion volumes were assessed 7 days post-surgery. Representative photographs showing QA-induced striatal lesion in 18-month-old wild-type (A) and transgenic (B) HD rats using FJ-staining. Higher magnification of FJ staining in a wild-type rat (C). Representative photographs showing the adjacent section processed for NeuN immunohistochemistry in 18-month-old wild-type (D) and transgenic (E) HD rats. Higher magnification of NeuN immunohistochemistry in a wild-type rat (F). Scale bar in (A): 1 mm, scale bar in (C): 140 m.
data was analyzed using a two-factor ANOVA and revealed no difference in the total striatal volume between genotypes (F(1, 29) = 1.37, P n.s.), age groups (F(1, 29) = 3.75, P n.s.) nor any interaction between genotype and age (F(1, 29) = 0.59, P n.s). 4. Discussion In HD, there is a selective neuronal loss in the cerebral cortex and the striatum, despite ubiquitous expression of the mutant huntingtin [17]. Interestingly, the affected regions are all densely innervated by glutamatergic input. Furthermore, intrastriatal injections of glutamate analogues give rise to an HD-like neuropathology [1]. These factors led to the hypothesis of excitotoxicity being involved in the disease. With the production of genetically modified animal models for HD, an interest arose to study whether they would have an altered susceptibility to excitotoxicity. In this study, young and old transgenic HD rats [16] did not display an altered sensitivity to excitotoxicity. The intrastriatal QA injection into wild-type littermates induced a lesion that corresponded to approximately 43% of the intact striatal volume in the injected side 7 days after surgery. Thus,
this lesion in the control animals was of intermediate size and would have allowed us to show either reduced or increased sensitivity to QA injected into the transgenic HD rats. However, the same susceptibility to excitotoxicity as observed in control animals was also found in transgenic HD rats. Since there was no apparent atrophy of the striatum in the transgenic HD rats at 3 or 18 months of age, the lack of difference in QA-induced lesion volumes between the genotypes could not be explained by different striatal volumes prior to the lesion. Results ranging from reduced to increased sensitivity to excitotoxicity have been found in the various rodent models for HD. Although the animals vary in many parameters such as strain, transgene construct, promotor, CAG repeat length, and expression levels, the length of the expressed HD gene may be involved in determining the response to excitotoxicity. The HD rat expresses 22% of the HD gene and displayed no altered susceptibility to excitotoxicity. This result is in agreement with a previous study where we have shown a maintained excitotoxic response in transgenic mice expressing around 30% of the full HD gene [11]. This result was independent of CAG repeat length and age of the mice. However, in the R6 line of trans-
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subunit NR2B, which is specifically enriched in HD-sensitive medium spiny striatal neurons [9]. It is indeed apparent that there are many differences between the mouse models, but what they arise from is not fully understood. However, the data from the studies in HD mouse models together with our result in the HD rat may indicate that the length of the expressed HD gene could be important when studying cellular responses to neurotoxins in animal models of this disorder. In conclusion, this study shows that transgenic HD rats maintain their sensitivity to excitotoxicity as compared to their wild-type littermates. Acknowledgements We are grateful to Bengt Mattsson for excellent technical ˚ assistance and Tim Karl for his help during experiments. A.P. is supported by the Swedish Brain Foundation. J.G. has a fellowship from the Foundation for Science and Technology (FCT, Portugal; SFRH/BD/6068/2001). I.M.A. is supported by a short term fellowship from the Federation of European Biochemical Societies (FEBS). O.R. and S.vH. are supported by grants from the High Q Foundation. References
Fig. 2. Maintained sensitivity to excitotoxicity in HD rats. The striatal lesion volume following an injection of 80 nmol QA did not differ between 3- and 18month-old transgenic HD rats, or their wild-type littermates (N; 3-month-old wild-type rats = 8, transgenic rats = 10; 18-month-old wild-type rats = 6, transgenic rats = 9). The lesion volumes were assessed in sections stained with FJ (A) and processed for NeuN immunohistochemistry (B). There was no difference in the total striatal volumes between the genotypes of the different ages as assessed in sections processed for NeuN immunohistochemistry (C).
genic HD mice that expresses around 3% of the HD gene with 117–150 CAG repeats, a progressive appearance of a resistance to excitotoxicity has been found [6,7]. Similarly, in other mouse models that also express only a few percent of the full HD gene, the N171-82 Q mouse [8] and the recently generated short stop mouse expressing around 120 CAG repeats [13], protection from QA-induced toxicity has been reported. Upregulation of protective pathways has been suggested to account for the reduced sensitivity to an excitotoxic insult in the N171-82Q mice [8]. In contrast, in the YAC HD mice expressing 72 CAG repeats in the full length HD gene, a progressive increased sensitivity to excitotoxicity has been reported [18]. As has been shown in neurons derived from these mice, this phenomenon may be due to the potentiation of currents through the NMDA receptor
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