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Maintenance of Susceptibility to Neurodegeneration Following Intrastriatal Injections of Quinolinic Acid in a New Transgenic Mouse Model of Huntington's Disease
Experimental Neurology 175, 297–300 (2002) doi:10.1006/exnr.2002.7885, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Maintenance of Susceptibility to Neurodegeneration Following Intrastriatal Injections of Quinolinic Acid in a New Transgenic Mouse Model of Huntington’s Disease Å. Peterse´n,* K. Chase,† Z. Puschban,* M. DiFiglia,‡ P. Brundin,* and N. Aronin† *Section for Neuronal Survival, Wallenberg Neuroscience Center, BMCAIO, Lund University, 221 84 Lund, Sweden; †Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts; and ‡Department of Neurology,Massachusetts General Hospital, Boston, Massachusetts Received October 24, 2001; accepted January 22, 2002
A transgenic mouse model of Huntington’s disease (R6/1 and R6/2 lines) expressing exon 1 of the HD gene with 115-150 CAG repeats resisted striatal damage following injection of quinolinic acid and other neurotoxins. We examined whether excitotoxin resistance characterizes mice with mutant huntingtin transgenes. In a new transgenic mouse with 3 kb of mutant human huntingtin cDNA with 18, 46, or 100 CAG repeats, we found no change in susceptibility to intrastriatal injections of the excitotoxin quinolinic acid, compared to wild-type littermates. The new transgenic mice were injected with the same dose of quinolinic acid (30 nmol) as had been the R6 mice. Our findings highlight the importance of studying pathogenetic mechanisms in different transgenic models of a disease. © 2002 Elsevier Science (USA) Key Words: excitotoxicity; striatum; Huntington’s disease.
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms, together with marked cell death in the striatum and cortex (14). It is caused by an expansion of a CAG repeat in exon 1 of the huntingtin gene (8). The pathogenetic mechanisms resulting from this mutation are not known. Excitotoxicity has been proposed to contribute to neuronal death in HD, because injections of excitotoxins into the striatum cause a pattern of cell loss similar to that observed in HD (for review see 2). In contrast, the R6 lines of transgenic HD mice, which express only exon 1 of mutant huntingtin (11), are unresponsive to excitotoxic effects of intrastriatal injections of N-methyl-Daspartate (NMDA), quinolinic acid (QA, an NMDA receptor agonist), and malonate, a mitochondrial toxin which kills cells partially through an NMDA-receptor-
dependent mechanism (4 – 6). We therefore tested the hypothesis that mutant huntingtin may render striatal neurons insensitive to excitotoxicity in transgenic HD mice (tgHD). These transgenic models of HD (9) have a substantially longer human huntingtin cDNA (3221 bases) compared to R6 lines (less than 200 bases) and express 18 (tgCT18), 46 (tgHD46), or 100 (tgHD100) CAG repeats. These transgenic HD mice develop striatal and cortical dendritic and nuclear changes reminiscent of those found in HD (9). The changes include nuclear inclusions, diffuse nuclear presence of huntingtin, and dysmorphic dendrites. The mice used in this study were heterozygous for the HD gene, expressing 18, 46, or 100 CAG repeats (one base change at position 28 CAG) or wild-type (WT) littermates. The mice were divided into two age groups, 4 –7 months old and older than 7 months. Under anesthesia [Avertin (2,2,2-tribomoethanol/2-methyl-2 butanol), 0.23 ml per 10 g body wt, Aldrich, St. Louis, MO], 30 nmol of QA (Sigma, St. Louis, MO) or vehicle (phosphate-buffered saline) was stereotaxically injected into the striatum as reported previously (4). The mice were perfused with 4% paraformaldehyde 4 days after the intrastriatal injection and the striatum was sectioned coronally on a sliding microtome at 40 m thickness. Every fourth section was processed for Fluorojade (FJ, Histo Chem, Jefferson, AR), as previously described (4, 13). FJ identifies damaged or dying neurons (13). After intrastriatal injection of excitotoxin, we previously observed a correlation between number of FJ-stained striatal cells and the number of apoptotic terminaltransferase-mediated deoxyuridine triphosphate– biotin nick end-labeled (TUNEL) striatal cells, loss of DARPP-32 immunostained, and loss of cresyl-violetstained neurons (4). Adjacent sections were stained with cresyl violet (ICN Biomedicals Inc., Aurora, OH) for assessment of residual cell numbers. All morpho-
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logical analyses were performed on blind-coded slides using an Olympus CAST-Grid system (Olympus Danmark A/S, Albertslund, Denmark). Both the striatal lesion volume (defined by presence of FJ-stained cells) and the total striatal volume were delineated in the FJ-stained sections. The volume was then calculated, taking into account the frequency of sections (1: 4) and their thickness (0.04 mm), according to the Cavalieri principle (i.e. total volume ⫽ total sampled area ⫻ 4 ⫻ 0.04; (3)). In the same sections, the peristriatal cortical volume (neocortical regions surrounding the head of the caudate-putamen and bordering onto the corpus callosum) was measured. The number of FJ-positive cells and the total number of neurons stained with cresyl violet in the striatum were also counted using stereological principles (3). Every fourth section throughout the entire striatum was sampled for each staining. All data were analyzed using analysis of variance (ANOVA) with genotype (WT and tgHD), repeat length (18, 46, and 100 CAG repeats), and age (4 –7 months; 8⫹ months) as factors. Cell death due to intrastriatal injections of QA was assessed in FJ-stained sections (Figs. 1A and 1B). There were no significant differences between the different mice regarding the numbers of FJ-positive striatal cells (Fig. 1C) [three-factor ANOVA, repeat length F(2, 67) ⫽ 2.83, n.s.; genotype F(1, 67) ⫽ 0.73, n.s.; age F(1, 67) ⫽ 0.82, n.s.]. There were also no differences between the groups regarding lesion volumes [Fig. 1D, repeat length F(2, 67) ⫽ 4.12, n.s.; genotype F(1, 67) ⫽ 0.38, n.s.; age F(1, 67) ⫽ 0.32, n.s.]. The possibility that there was a prior difference in striatal or cortical volumes between wild-type and transgenic mice before injection of QA was also assessed in all mice in the noninjected hemisphere (Table 1). There were no significant differences in striatal volumes [three-factor ANOVA, repeat length F(2, 67) ⫽ 2.91 n.s.; genotype F(1, 67) ⫽ 0.03, n.s.; age F(1, 67) ⫽ 1.33, n.s.] or in peristriatal volumes [three-factor ANOVA: repeat length F(2, 65) ⫽ 1.51, n.s.; genotype F(1, 67) ⫽ 0.07, n.s.; age F(1, 67) ⫽ 1.51, n.s.] among the different mice. The total number of remaining striatal neurons on the intact side was assessed in cresyl violet-stained sections; there were no differences among the different types of mice (data not shown). Although the tgHD and R6 transgenic mice share expansions of CAG repeats in a huntingtin cDNA background, the tgHD and R6 mice differ in genetic design and behavioral phenotype. Consideration must be given to differences in strain, physiology, and neuronal morphology to discern differences in excitotoxic responses. R6 mice are bred on a CBA/C57Bl6 background and the present tgHD mice on a SJL/C57Bl6 background (9, 11). However, resistance to excitotoxins in the R6 mice is unlikely to result from strain traits, because each of the background strains is highly sus-
ceptible to QA-induced striatal damage (6). Differences in transgene designs might well account for changes in cellular responsiveness to QA excitotoxicity. The R6 transgene uses a portion of the human huntingtin gene promoter, whereas the tgHD has a neuron-specific enolase promoter. Transcription efficiencies probably differ. The tgHD and R6 mice express different lengths of the HD gene and the CAG repeats; the R6 mice contain exon 1 with 115–150 CAG repeats and tgHD mice express a 3-kb fragment of the gene with 18 (control length), 46, or 100 CAG repeats. The huntingtin protein context presents a fundamental difference in transgene effect. Transgenic mice with expanded CAG repeats in exon 1 of the huntingtin gene have many more nuclear inclusions, an earlier onset (1–3 months) and shorter life span (1 month), compared to mice with similar CAG repeat lengths in a larger huntingtin gene (7, 9, 12). Our results therefore indicate that transgenics with CAG repeat expansions, within a large huntingtin context, maintain excitotoxic responsiveness. Neurophysiologically, R6/2 mice might be able to withstand excitotoxic stimulation. Medium-sized spiny striatal neurons in brain slices from symptomatic R6/2 mice exhibit fivefold increases in basal cytoplasmic calcium levels and better capacity to handle calcium overload following QA (6). Medium spiny neurons in R6/2 mice displayed depolarized resting membrane potentials and increased input resistances (10). In contrast, the tgHD100 mice have a normal resting membrane potential (9). Furthermore, application of NMDA increases peak current densities and intracellular Ca 2⫹ levels significantly higher than in wild-type control mice (9). It is possible that R6 mice and tgHD100 mice respond to NMDA receptor activation (tgHD might have increased responsiveness), but that R6 lines are better able to handle the increased Ca 2⫹ load. The tgHD mice have morphological features similar to those found in HD. Neuronal changes in the striatum, including nuclear inclusions and frequent dysmorphic dendrites, predicted the onset and severity of motor impairment (9). Spine density in medium spiny neurons was not clearly altered (unpublished data). Medium spiny neurons from symptomatic R6/2 mice have decreased dendritic spine densities and smaller dendritic fields compared to presymptomatic R6/2 mice (10). These differences raise the possibility that, with a low spine density, the medium spiny neurons in R6/2 mice could have fewer NMDA receptors. Excitotoxicity has been suggested to play a role in HD (2), although it is by no means proven. Earlier work shows that application of NMDA to tgHD100 neurons in tissue slice leads to increased intracellular calcium levels compared to WT neurons (9), but we observed no increased cell death in vivo. Therefore, it is possible that tgHD100 neurons develop an enhanced capacity to acutely handle increases in cytosolic calcium, but even-
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FIG. 1. Photomicrographs of the FJ-stained striatum in WT (A) and tgHD100 (B) mice of 7 months of age (bar 1 mm). There was no difference in the number of FJ-positive neurons (C) or in lesion volumes (D) between the different mice.
tually succumb to its detrimental effect on cellular homeostasis. The current study underscores the importance of carefully comparing different transgenic mouse models of the same human disease. It shows that effects of expressing a polyglutamine expansion differ dramatically depending on the full protein context in which it is expressed. Significantly, in transgenic mice exhibiting neuropathology similar to HD, the response to QA administration persists. In this setting, the blunted
response to QA found in R6 lines might offer a window to examine protection against excitotoxicity. Thus, R6 mice which only express around 3% of the full length of huntingtin are resistant to damage after intrastriatal QA injections (4, 6), whereas the tgHD mice of the present study which express approximately 33% of the full length of huntingtin retain sensitivity to QA excitotoxicity. Different responsiveness to excitotoxicity among various HD mouse models depends on neuronal conse-
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TABLE 1
4.
Assessment of Striatal and Peristriatal Cortical Volumes Expressed as Means ⫾ SEM Type of mouse
N
Striatum (mm 3)
Cortex (mm 3)
WT 18 Q (4–7m) tg 18 Q (4–7m) WT 18 Q (8m⫹) tg 18 Q (8m⫹) WT 46 Q (4–7m) tg 46 Q (4–7m) WT 46 Q (8m⫹) tg 46 Q (8m⫹) WT 100 Q (4–7m) tg 100 Q (4–7m) WT 100 Q (8m⫹) tg 100 Q (8m⫹)
5 5 2 4 8 6 4 12 8 12 6 7
6.8 ⫾ 0.2 6.5 ⫾ 0.3 6.8 ⫾ 0.4 6.0 ⫾ 0.2 6.0 ⫾ 0.2 5.6 ⫾ 0.3 6.3 ⫾ 0.2 6.3 ⫾ 0.1 5.9 ⫾ 0.2 6.6 ⫾ 0.2 6.2 ⫾ 0.3 6.9 ⫾ 0.3
12.1 ⫾ 0.6 10.9 ⫾ 0.2 11.5 ⫾ 0.1 11.2 ⫾ 0.5 11.0 ⫾ 0.4 10.4 ⫾ 0.6 11.4 ⫾ 0.4 11.7 ⫾ 0.3 11.0 ⫾ 0.4 11.9 ⫾ 0.2 11.5 ⫾ 0.2 11.9 ⫾ 0.3
quences of the expression of the aberrant gene. Comparison of morphological and electrophysiological properties of the neurons in the various mouse models may permit dissection of possible mechanisms of excitotoxicity in HD.
5.
6.
7.
8.
9.
ACKNOWLEDGMENTS We thank Britt Lindberg and Bengt Mattsson for excellent technical assistance. This work is supported by grants from Anders Wall Foundation (Å.P.); Swedish Research Council (P.B.); National Institutes of Health NS 38194 (N.A.), NS 16367, and NS 35711 (M.D.); DOD USAMRMC 9829059 (N.A.); the Huntington’s Disease Society of America (M.D.); and the Hereditary Disease Foundation (N.A., M.D.). We also thank the NIH Diabetes and Endocrine Research Center at UMMS, DK32520, for its support of the Peptide Core and the Transgenic Core. Note added in proof. A recently published study (15) has demonstrated an increased sensitivity to excitotoxin-induced damage in the striatum of YAC72 transgenic HD mice.
REFERENCES Davies, S. W., M. Turmaine, B. A. Cozens, M. DiFiglia, A. H. Sharp, C. A. Ross, E. Scherzinger, E. E. Wanker, L. Mangiarini, and G. P. Bates. 1997. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90: 537–548. 2. DiFiglia, M. 1990. Excitotoxic injury of the neostriatum: A model for Huntington’s disease. Trends Neurosci. 13: 286 –289. 3. Gundersen, H. J. G., P. Bagger, B. F. Bendsten, S. M. Evans, L. Korbu, N. Marcussen, A. Moller, K. Nielsen, J. R. Nyengaard, B. Pakkenberg, F. B. Sorensen, A. Vesterby, and M. J. West. 1988. The new stereological tools: Dissector, fractionator, nucleator and point-sample intercepts and their use in pathological research and diagnosis. Acta Pathol. Microbiol. Immunol. Scand. 96: 857– 881.
10.
11.
12.
1.
13.
14. 15.
Hansson, O., Å. Peterse´ n, M. Leist, P. Nicotera, R. F. Castilho, and P. Brundin. 1999. Transgenic mice expressing a Huntington’s disease mutation are resistant to quinolinic acid-induced striatal excitotoxicity. Proc. Natl. Acad. Sci. USA 96: 8727– 8732. Hansson, O., R. F. Castilho, L. Korhonen, D. Lindholm, G. P. Bates, and P. Brundin. 2001. Partial resistance to malonateinduced striatal cell death in transgenic mouse models of Huntington’s disease is dependent on age and CAG repeat length. J. Neurochem. 78: 694 –703. Hansson, O., E. Guatteo, N. B. Mercuri, G. Bernardi, X.-J. Li, R. Castilho, and P. Brundin. 2001. Resistance to NMDA toxicity correlates with appearance of nuclear inclusions, behavioral deficits and with changes in calcium homeostasis in mice transgenic for exon 1 of the Huntington gene. Eur. J. Neurosci. 14: 1492–1504. Hodgson, J. G., N. Agopyan, C. A. Gutekunst, B. R. Leavitt, F. LePiane, R. Singaraja, D. J. Smith, N. Bissada, K. McCutcheon, J. Nasir, L. Jamot, X. J. Li, M. E. Stevens, E. Rosemond, J. C. Roder, A. G. Philips, E. M. Rubin, S. M. Hersch, and M. R. Hayden. 1999. A YAC mouse model for Huntington’s disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 23: 181–192. Huntington’s Disease Collaborative Research Group. 1993. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 72: 971–983. Laforet, G. A., E. Sapp, K. Chase, C. McIntyre, F. M. Boyce, M. Campbell, B. A. Cadigan, L. Warzecki, D. A. Tagle, H. Reddy, C. Cepeda, C. R. Calvert, E. S. Jokel, G. J. Klapstein, M. A. Ariano, M. S. Levine, M. DiFiglia, and N. Aronin. 2001. Changes in cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model of Huntington’s disease. J. Neurosci. 21: 9112–9123. Levine, M. S., G. J. Klapstein, A. Koppel, E. Gruen, C. Cepeda, M. E. Vargas, E. S. Jokel, E. M. Carpenter, H. Zanjani, R. S. Hurst, A. Efstratiadis, S. Zeitlin, and M.–F. Chesselet. 1999. Enhanced sensitivity to N-methyl-D-aspartate receptor activation in transgenic and knockin mouse models of Huntington’s disease. J. Neurosci. Res. 58: 515–532. Mangiarini, L., K. Sathasivam, M. Seller, B. Cozens, A. Harper, C. Hetherington, M. Lawton, Y. Trottier, H. Lehrach, S. W. Davies, and G. P. Bates. 1996. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87: 493–506. Reddy, P. H., M. Williams, V. Charles, L. Garrett, L. PikeBuchanan, W. O. Whetsell, Jr., G. Miller, and D. A. Tagle. 1998. Behavioural abnormalities and selective neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA. Nature Genet. 20: 198 –202. Schmued, L. C., C. Albertson, and W. Slikker. 1997. FluoroJade: A novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res. 751: 37– 46. Vonsattel, J. P., and M. DiFiglia. 1998. Huntington’s disease. J. Neuropathol. Exp. Neurol. 57: 369 –384. Zeron, M. M., O. Hansson, N. Chen, C. L. Wellington, B. R. Leavitt, P. Brundin, M. R. Hayden, and L. A. Raymond. 2002. Increased sensitivity to N-Methyl-D-Aspartate Receptor-mediated excitotoxicity in a mouse model of Huntington’s disease. Neuron 33: 849 – 860.
BRIEF COMMUNICATION Maintenance of Susceptibility to Neurodegeneration Following Intrastriatal Injections of Quinolinic Acid in a New Transgenic Mouse Model of Huntington’s Disease Å. Peterse´n,* K. Chase,† Z. Puschban,* M. DiFiglia,‡ P. Brundin,* and N. Aronin† *Section for Neuronal Survival, Wallenberg Neuroscience Center, BMCAIO, Lund University, 221 84 Lund, Sweden; †Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts; and ‡Department of Neurology,Massachusetts General Hospital, Boston, Massachusetts Received October 24, 2001; accepted January 22, 2002
A transgenic mouse model of Huntington’s disease (R6/1 and R6/2 lines) expressing exon 1 of the HD gene with 115-150 CAG repeats resisted striatal damage following injection of quinolinic acid and other neurotoxins. We examined whether excitotoxin resistance characterizes mice with mutant huntingtin transgenes. In a new transgenic mouse with 3 kb of mutant human huntingtin cDNA with 18, 46, or 100 CAG repeats, we found no change in susceptibility to intrastriatal injections of the excitotoxin quinolinic acid, compared to wild-type littermates. The new transgenic mice were injected with the same dose of quinolinic acid (30 nmol) as had been the R6 mice. Our findings highlight the importance of studying pathogenetic mechanisms in different transgenic models of a disease. © 2002 Elsevier Science (USA) Key Words: excitotoxicity; striatum; Huntington’s disease.
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms, together with marked cell death in the striatum and cortex (14). It is caused by an expansion of a CAG repeat in exon 1 of the huntingtin gene (8). The pathogenetic mechanisms resulting from this mutation are not known. Excitotoxicity has been proposed to contribute to neuronal death in HD, because injections of excitotoxins into the striatum cause a pattern of cell loss similar to that observed in HD (for review see 2). In contrast, the R6 lines of transgenic HD mice, which express only exon 1 of mutant huntingtin (11), are unresponsive to excitotoxic effects of intrastriatal injections of N-methyl-Daspartate (NMDA), quinolinic acid (QA, an NMDA receptor agonist), and malonate, a mitochondrial toxin which kills cells partially through an NMDA-receptor-
dependent mechanism (4 – 6). We therefore tested the hypothesis that mutant huntingtin may render striatal neurons insensitive to excitotoxicity in transgenic HD mice (tgHD). These transgenic models of HD (9) have a substantially longer human huntingtin cDNA (3221 bases) compared to R6 lines (less than 200 bases) and express 18 (tgCT18), 46 (tgHD46), or 100 (tgHD100) CAG repeats. These transgenic HD mice develop striatal and cortical dendritic and nuclear changes reminiscent of those found in HD (9). The changes include nuclear inclusions, diffuse nuclear presence of huntingtin, and dysmorphic dendrites. The mice used in this study were heterozygous for the HD gene, expressing 18, 46, or 100 CAG repeats (one base change at position 28 CAG) or wild-type (WT) littermates. The mice were divided into two age groups, 4 –7 months old and older than 7 months. Under anesthesia [Avertin (2,2,2-tribomoethanol/2-methyl-2 butanol), 0.23 ml per 10 g body wt, Aldrich, St. Louis, MO], 30 nmol of QA (Sigma, St. Louis, MO) or vehicle (phosphate-buffered saline) was stereotaxically injected into the striatum as reported previously (4). The mice were perfused with 4% paraformaldehyde 4 days after the intrastriatal injection and the striatum was sectioned coronally on a sliding microtome at 40 m thickness. Every fourth section was processed for Fluorojade (FJ, Histo Chem, Jefferson, AR), as previously described (4, 13). FJ identifies damaged or dying neurons (13). After intrastriatal injection of excitotoxin, we previously observed a correlation between number of FJ-stained striatal cells and the number of apoptotic terminaltransferase-mediated deoxyuridine triphosphate– biotin nick end-labeled (TUNEL) striatal cells, loss of DARPP-32 immunostained, and loss of cresyl-violetstained neurons (4). Adjacent sections were stained with cresyl violet (ICN Biomedicals Inc., Aurora, OH) for assessment of residual cell numbers. All morpho-
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0014-4886/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
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logical analyses were performed on blind-coded slides using an Olympus CAST-Grid system (Olympus Danmark A/S, Albertslund, Denmark). Both the striatal lesion volume (defined by presence of FJ-stained cells) and the total striatal volume were delineated in the FJ-stained sections. The volume was then calculated, taking into account the frequency of sections (1: 4) and their thickness (0.04 mm), according to the Cavalieri principle (i.e. total volume ⫽ total sampled area ⫻ 4 ⫻ 0.04; (3)). In the same sections, the peristriatal cortical volume (neocortical regions surrounding the head of the caudate-putamen and bordering onto the corpus callosum) was measured. The number of FJ-positive cells and the total number of neurons stained with cresyl violet in the striatum were also counted using stereological principles (3). Every fourth section throughout the entire striatum was sampled for each staining. All data were analyzed using analysis of variance (ANOVA) with genotype (WT and tgHD), repeat length (18, 46, and 100 CAG repeats), and age (4 –7 months; 8⫹ months) as factors. Cell death due to intrastriatal injections of QA was assessed in FJ-stained sections (Figs. 1A and 1B). There were no significant differences between the different mice regarding the numbers of FJ-positive striatal cells (Fig. 1C) [three-factor ANOVA, repeat length F(2, 67) ⫽ 2.83, n.s.; genotype F(1, 67) ⫽ 0.73, n.s.; age F(1, 67) ⫽ 0.82, n.s.]. There were also no differences between the groups regarding lesion volumes [Fig. 1D, repeat length F(2, 67) ⫽ 4.12, n.s.; genotype F(1, 67) ⫽ 0.38, n.s.; age F(1, 67) ⫽ 0.32, n.s.]. The possibility that there was a prior difference in striatal or cortical volumes between wild-type and transgenic mice before injection of QA was also assessed in all mice in the noninjected hemisphere (Table 1). There were no significant differences in striatal volumes [three-factor ANOVA, repeat length F(2, 67) ⫽ 2.91 n.s.; genotype F(1, 67) ⫽ 0.03, n.s.; age F(1, 67) ⫽ 1.33, n.s.] or in peristriatal volumes [three-factor ANOVA: repeat length F(2, 65) ⫽ 1.51, n.s.; genotype F(1, 67) ⫽ 0.07, n.s.; age F(1, 67) ⫽ 1.51, n.s.] among the different mice. The total number of remaining striatal neurons on the intact side was assessed in cresyl violet-stained sections; there were no differences among the different types of mice (data not shown). Although the tgHD and R6 transgenic mice share expansions of CAG repeats in a huntingtin cDNA background, the tgHD and R6 mice differ in genetic design and behavioral phenotype. Consideration must be given to differences in strain, physiology, and neuronal morphology to discern differences in excitotoxic responses. R6 mice are bred on a CBA/C57Bl6 background and the present tgHD mice on a SJL/C57Bl6 background (9, 11). However, resistance to excitotoxins in the R6 mice is unlikely to result from strain traits, because each of the background strains is highly sus-
ceptible to QA-induced striatal damage (6). Differences in transgene designs might well account for changes in cellular responsiveness to QA excitotoxicity. The R6 transgene uses a portion of the human huntingtin gene promoter, whereas the tgHD has a neuron-specific enolase promoter. Transcription efficiencies probably differ. The tgHD and R6 mice express different lengths of the HD gene and the CAG repeats; the R6 mice contain exon 1 with 115–150 CAG repeats and tgHD mice express a 3-kb fragment of the gene with 18 (control length), 46, or 100 CAG repeats. The huntingtin protein context presents a fundamental difference in transgene effect. Transgenic mice with expanded CAG repeats in exon 1 of the huntingtin gene have many more nuclear inclusions, an earlier onset (1–3 months) and shorter life span (1 month), compared to mice with similar CAG repeat lengths in a larger huntingtin gene (7, 9, 12). Our results therefore indicate that transgenics with CAG repeat expansions, within a large huntingtin context, maintain excitotoxic responsiveness. Neurophysiologically, R6/2 mice might be able to withstand excitotoxic stimulation. Medium-sized spiny striatal neurons in brain slices from symptomatic R6/2 mice exhibit fivefold increases in basal cytoplasmic calcium levels and better capacity to handle calcium overload following QA (6). Medium spiny neurons in R6/2 mice displayed depolarized resting membrane potentials and increased input resistances (10). In contrast, the tgHD100 mice have a normal resting membrane potential (9). Furthermore, application of NMDA increases peak current densities and intracellular Ca 2⫹ levels significantly higher than in wild-type control mice (9). It is possible that R6 mice and tgHD100 mice respond to NMDA receptor activation (tgHD might have increased responsiveness), but that R6 lines are better able to handle the increased Ca 2⫹ load. The tgHD mice have morphological features similar to those found in HD. Neuronal changes in the striatum, including nuclear inclusions and frequent dysmorphic dendrites, predicted the onset and severity of motor impairment (9). Spine density in medium spiny neurons was not clearly altered (unpublished data). Medium spiny neurons from symptomatic R6/2 mice have decreased dendritic spine densities and smaller dendritic fields compared to presymptomatic R6/2 mice (10). These differences raise the possibility that, with a low spine density, the medium spiny neurons in R6/2 mice could have fewer NMDA receptors. Excitotoxicity has been suggested to play a role in HD (2), although it is by no means proven. Earlier work shows that application of NMDA to tgHD100 neurons in tissue slice leads to increased intracellular calcium levels compared to WT neurons (9), but we observed no increased cell death in vivo. Therefore, it is possible that tgHD100 neurons develop an enhanced capacity to acutely handle increases in cytosolic calcium, but even-
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FIG. 1. Photomicrographs of the FJ-stained striatum in WT (A) and tgHD100 (B) mice of 7 months of age (bar 1 mm). There was no difference in the number of FJ-positive neurons (C) or in lesion volumes (D) between the different mice.
tually succumb to its detrimental effect on cellular homeostasis. The current study underscores the importance of carefully comparing different transgenic mouse models of the same human disease. It shows that effects of expressing a polyglutamine expansion differ dramatically depending on the full protein context in which it is expressed. Significantly, in transgenic mice exhibiting neuropathology similar to HD, the response to QA administration persists. In this setting, the blunted
response to QA found in R6 lines might offer a window to examine protection against excitotoxicity. Thus, R6 mice which only express around 3% of the full length of huntingtin are resistant to damage after intrastriatal QA injections (4, 6), whereas the tgHD mice of the present study which express approximately 33% of the full length of huntingtin retain sensitivity to QA excitotoxicity. Different responsiveness to excitotoxicity among various HD mouse models depends on neuronal conse-
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TABLE 1
4.
Assessment of Striatal and Peristriatal Cortical Volumes Expressed as Means ⫾ SEM Type of mouse
N
Striatum (mm 3)
Cortex (mm 3)
WT 18 Q (4–7m) tg 18 Q (4–7m) WT 18 Q (8m⫹) tg 18 Q (8m⫹) WT 46 Q (4–7m) tg 46 Q (4–7m) WT 46 Q (8m⫹) tg 46 Q (8m⫹) WT 100 Q (4–7m) tg 100 Q (4–7m) WT 100 Q (8m⫹) tg 100 Q (8m⫹)
5 5 2 4 8 6 4 12 8 12 6 7
6.8 ⫾ 0.2 6.5 ⫾ 0.3 6.8 ⫾ 0.4 6.0 ⫾ 0.2 6.0 ⫾ 0.2 5.6 ⫾ 0.3 6.3 ⫾ 0.2 6.3 ⫾ 0.1 5.9 ⫾ 0.2 6.6 ⫾ 0.2 6.2 ⫾ 0.3 6.9 ⫾ 0.3
12.1 ⫾ 0.6 10.9 ⫾ 0.2 11.5 ⫾ 0.1 11.2 ⫾ 0.5 11.0 ⫾ 0.4 10.4 ⫾ 0.6 11.4 ⫾ 0.4 11.7 ⫾ 0.3 11.0 ⫾ 0.4 11.9 ⫾ 0.2 11.5 ⫾ 0.2 11.9 ⫾ 0.3
quences of the expression of the aberrant gene. Comparison of morphological and electrophysiological properties of the neurons in the various mouse models may permit dissection of possible mechanisms of excitotoxicity in HD.
5.
6.
7.
8.
9.
ACKNOWLEDGMENTS We thank Britt Lindberg and Bengt Mattsson for excellent technical assistance. This work is supported by grants from Anders Wall Foundation (Å.P.); Swedish Research Council (P.B.); National Institutes of Health NS 38194 (N.A.), NS 16367, and NS 35711 (M.D.); DOD USAMRMC 9829059 (N.A.); the Huntington’s Disease Society of America (M.D.); and the Hereditary Disease Foundation (N.A., M.D.). We also thank the NIH Diabetes and Endocrine Research Center at UMMS, DK32520, for its support of the Peptide Core and the Transgenic Core. Note added in proof. A recently published study (15) has demonstrated an increased sensitivity to excitotoxin-induced damage in the striatum of YAC72 transgenic HD mice.
REFERENCES Davies, S. W., M. Turmaine, B. A. Cozens, M. DiFiglia, A. H. Sharp, C. A. Ross, E. Scherzinger, E. E. Wanker, L. Mangiarini, and G. P. Bates. 1997. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90: 537–548. 2. DiFiglia, M. 1990. Excitotoxic injury of the neostriatum: A model for Huntington’s disease. Trends Neurosci. 13: 286 –289. 3. Gundersen, H. J. G., P. Bagger, B. F. Bendsten, S. M. Evans, L. Korbu, N. Marcussen, A. Moller, K. Nielsen, J. R. Nyengaard, B. Pakkenberg, F. B. Sorensen, A. Vesterby, and M. J. West. 1988. The new stereological tools: Dissector, fractionator, nucleator and point-sample intercepts and their use in pathological research and diagnosis. Acta Pathol. Microbiol. Immunol. Scand. 96: 857– 881.
10.
11.
12.
1.
13.
14. 15.
Hansson, O., Å. Peterse´ n, M. Leist, P. Nicotera, R. F. Castilho, and P. Brundin. 1999. Transgenic mice expressing a Huntington’s disease mutation are resistant to quinolinic acid-induced striatal excitotoxicity. Proc. Natl. Acad. Sci. USA 96: 8727– 8732. Hansson, O., R. F. Castilho, L. Korhonen, D. Lindholm, G. P. Bates, and P. Brundin. 2001. Partial resistance to malonateinduced striatal cell death in transgenic mouse models of Huntington’s disease is dependent on age and CAG repeat length. J. Neurochem. 78: 694 –703. Hansson, O., E. Guatteo, N. B. Mercuri, G. Bernardi, X.-J. Li, R. Castilho, and P. Brundin. 2001. Resistance to NMDA toxicity correlates with appearance of nuclear inclusions, behavioral deficits and with changes in calcium homeostasis in mice transgenic for exon 1 of the Huntington gene. Eur. J. Neurosci. 14: 1492–1504. Hodgson, J. G., N. Agopyan, C. A. Gutekunst, B. R. Leavitt, F. LePiane, R. Singaraja, D. J. Smith, N. Bissada, K. McCutcheon, J. Nasir, L. Jamot, X. J. Li, M. E. Stevens, E. Rosemond, J. C. Roder, A. G. Philips, E. M. Rubin, S. M. Hersch, and M. R. Hayden. 1999. A YAC mouse model for Huntington’s disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 23: 181–192. Huntington’s Disease Collaborative Research Group. 1993. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 72: 971–983. Laforet, G. A., E. Sapp, K. Chase, C. McIntyre, F. M. Boyce, M. Campbell, B. A. Cadigan, L. Warzecki, D. A. Tagle, H. Reddy, C. Cepeda, C. R. Calvert, E. S. Jokel, G. J. Klapstein, M. A. Ariano, M. S. Levine, M. DiFiglia, and N. Aronin. 2001. Changes in cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model of Huntington’s disease. J. Neurosci. 21: 9112–9123. Levine, M. S., G. J. Klapstein, A. Koppel, E. Gruen, C. Cepeda, M. E. Vargas, E. S. Jokel, E. M. Carpenter, H. Zanjani, R. S. Hurst, A. Efstratiadis, S. Zeitlin, and M.–F. Chesselet. 1999. Enhanced sensitivity to N-methyl-D-aspartate receptor activation in transgenic and knockin mouse models of Huntington’s disease. J. Neurosci. Res. 58: 515–532. Mangiarini, L., K. Sathasivam, M. Seller, B. Cozens, A. Harper, C. Hetherington, M. Lawton, Y. Trottier, H. Lehrach, S. W. Davies, and G. P. Bates. 1996. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87: 493–506. Reddy, P. H., M. Williams, V. Charles, L. Garrett, L. PikeBuchanan, W. O. Whetsell, Jr., G. Miller, and D. A. Tagle. 1998. Behavioural abnormalities and selective neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA. Nature Genet. 20: 198 –202. Schmued, L. C., C. Albertson, and W. Slikker. 1997. FluoroJade: A novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res. 751: 37– 46. Vonsattel, J. P., and M. DiFiglia. 1998. Huntington’s disease. J. Neuropathol. Exp. Neurol. 57: 369 –384. Zeron, M. M., O. Hansson, N. Chen, C. L. Wellington, B. R. Leavitt, P. Brundin, M. R. Hayden, and L. A. Raymond. 2002. Increased sensitivity to N-Methyl-D-Aspartate Receptor-mediated excitotoxicity in a mouse model of Huntington’s disease. Neuron 33: 849 – 860.