Human Molecular Genetics, 2001, Vol. 10, No. 22 2515-2523
© 2001 Oxford University Press
The HD mutation causes progressive lethal neurological disease in mice expressing reduced levels of huntingtin
1Skirball Institute of Biomolecular Medicine and 2Howard Hughes Medical Institute, 3Departments of Radiology and Pathology and 4Departments of Cell Biology and Physiology and Neuroscience, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA and 5Molecular Neurogenetics Unit, Massachusetts General Hospital East, Charlestown, MA 02129, USA
Received June 11, 2001; Revised and Accepted August 27, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntingtin is an essential protein that with mutant polyglutamine tracts initiates dominant striatal neurodegeneration in Huntington’s disease (HD). To assess the consequences of mutant protein when huntingtin is limiting, we have studied three lines of compound heterozygous mice in which both copies of the HD gene homolog (Hdh) were altered, resulting in greatly reduced levels of huntingtin with a normal human polyglutamine length (Q20) and/or an expanded disease-associated segment (Q111): HdhneoQ20/HdhneoQ20, HdhneoQ20/Hdhnull and HdhneoQ20/HdhneoQ111. All surviving mice in each of the three lines were small from birth, and had variable movement abnormalities. Magnetic resonance micro-imaging and histological evaluation showed enlarged ventricles in
50% of the HdhneoQ20/HdhneoQ111 and HdhneoQ20/Hdhnull mice, revealing a developmental defect that does not worsen with age. Only HdhneoQ20/HdhneoQ111 mice exhibited a rapidly progressive movement disorder that, in the absence of striatal pathology, begins with hind-limb clasping during tail suspension and tail stiffness during walking by 3–4 months of age, and then progresses to paralysis of the limbs and tail, hypokinesis and premature death, usually by 12 months of age. Thus, dramatically reduced huntingtin levels fail to support normal development in mice, resulting in reduced body size, movement abnormalities and a variable increase in ventricle volume. On this sensitized background, mutant huntingtin causes a rapid neurological disease, distinct from the HD-pathogenic process. These results raise the possibility that therapeutic elimination of huntingtin in HD patients could lead to unintended neurological, as well as developmental side-effects. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington’s disease (HD) is a dominant neurodegenerative disorder resulting from CAG expansions in the HD gene extending a polyglutamine segment in one copy of the
350 kDa protein, huntingtin (1). HD is associated primarily with selective degeneration of striatal neurons and onset of choreiform movements, psychiatric disorders and cognitive impairment (2). Genetic data suggest a gain-of-function mechanism, in which the length of the expanded glutamine stretch is the primary determinant of disease severity (1), although the mechanism by which the expanded glutamine tracts in huntingtin cause a dominant disease is not clear. Insertion of long CAG expansions into the mouse HD homolog (Hdh), extending the 7-glutamine tract of the mouse protein, reveals that mutant huntingtin does not significantly impair normal mouse development or postnatal life (3–6). Rather, at the advanced age of 2 years, heterozygous and homozygous mutant mice expressing expanded glutamine tracts (CAG knock-in mice) exhibit detectable neuronal atrophy and reactive-gliosis in the striatum and display a mild shuffling gait (7; V.C. Wheeler, C.-A. Gutekunst, V. Vrbanac, L.-A. Lebel, G. Schilling, S. Hersch, R.M. Friedlander, J.F. Gusella, J.-P. Vonsattel, D. Borchelt and M.E. MacDonald, submitted for publication). Overt disease is presaged by striatal-specific correlates; nuclear-huntingtin, intranuclear inclusions and insoluble-amyloid (5–7), suggesting that in mouse and man, mutant huntingtin initiates a time-dependent disease process to which striatal neurons are particularly vulnerable.
In contrast, circumventing the requirement for huntingtin during early embryonic development (3,8,9), conditional Hdh deletion in forebrain neurons at late embryonic or early neonatal stages produced adult mice with a rapidly progressive neurodegenerative phenotype (10). This finding showed that huntingtin function is essential to neurons, suggesting that severely decreased huntingtin as a result of the disease process could contribute to HD pathogenesis (10), perhaps as recently suggested, via the loss of the protein’s ability to regulate BDNF, a potent neuroprotectant (11).
In the present study, we directly test the consequence of introducing the HD mutation on a background of severely reduced huntingtin levels by assessing mice bearing hypomorphic neo-Hdh CAG knock-in alleles that survive to adulthood. We report the striking finding of a rapidly progressive, lethal neurological disorder, without detectable striatal involvement, in compound heterozygous Hdh knock-in mice engineered to express low levels of mutant huntingtin with 111 glutamines (Q111) and huntingtin with 20 glutamines (Q20). In the absence of a Q111 segment, mice expressing a low level of Q20 huntingtin do not show progressive disease, but have brain deficits that reflect abnormal development (3). Thus, consistent with a neuroprotective role for huntingtin in HD, the novel neurologic disease caused by low levels of mutant huntingtin on this sensitized background shows that knowledge of the impact of potential agents on huntingtin levels will be critical for the development of effective therapies for HD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In previous studies we generated a series of mouse Hdh CAG expansion knock-in alleles that mimic the human normal (HdhQ20) and HD disease alleles (HdhQ50–111), by altering the normal mouse HdhQ7 gene, containing a 7-glutamine stretch, to have a human HD exon 1 region encoding 20 (normal) or 50–111 (disease) glutamines (3,6). In each case an intermediate targeted-allele was made that contains neomycin (neo) in the promoter region, resulting in greatly reduced levels of the engineered Hdh allele (3). Furthermore, it was found that homozygous neo-Hdh containing knock-in mice with 50 or more glutamine repeats were not viable (3), whereas some HdhneoQ20 homozygotes survived. Therefore, to assess the consequences of mutant huntingtin on a low-huntingtin background, we used the less severe neo-20 allele to attempt to generate adult mutant HdhneoQ20/HdhneoQ111 mice.
HdhneoQ20/HdhneoQ111 mice have developmental defects but can survive to adulthood
While breeding HdhneoQ20/HdhQ7 mice to HdhneoQ111/HdhQ7, we observed that numerous small pups were born (58/360), and that they usually did not survive through weaning unless larger littermates were removed soon after birth. By thinning the litters, many of the smaller pups survived weaning and all were found to have the genotype HdhneoQ20/HdhneoQ111 (n = 50). HdhneoQ20/HdhneoQ111 mice weighed significantly less than control littermates [HdhQ7/HdhQ7 (wild-type), HdhneoQ20/HdhQ7 and HdhneoQ111/HdhQ7] at 1 week postnatal [HdhneoQ20/neoQ111, 3.1 ± 0.3 g (n = 6) versus controls, 4.7 ± 0.1 g (n = 6)] and throughout adult life (Fig. 1). The HdhneoQ20/HdhneoQ111 mice, although small were well formed and appeared to move normally until adulthood (2 months).
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We next produced HdhneoQ20/HdhneoQ20 and HdhneoQ20/Hdhnull (carrying an Hdh allele that has exons 4 and 5 deleted, previously called Hdhex4/5) compound heterozygous mutants that express only very low levels of huntingtin with a human normal polyglutamine length, for comparison with the HdhneoQ20/HdhneoQ111 mice. Both intercrosses gave rise to small pups (HdhneoQ20/HdhQ7 x HdhneoQ20/HdhQ7, 17/138; HdhneoQ20/HdhQ7 x Hdhnull/HdhQ7, 28/265), which often died within a few days of birth. After thinning the litters by removing larger pups, a number of smaller pups survived through weaning and all were either HdhneoQ20/HdhneoQ20 (n = 14) or HdhneoQ20/Hdhnull (n = 16) mutants. Both HdhneoQ20/HdhneoQ20 and HdhneoQ20/Hdhnull mice were smaller than control littermates throughout life (Fig. 1). We also investigated the viability of HdhneoQ111/HdhneoQ111 mice using an HdhneoQ111/HdhQ7 intercross but no small pups were seen in four litters (0/39), as expected since HdhneoQ50/HdhneoQ50 mice were previously shown to die by birth (3). Thus, mice expressing low levels of Q20 huntingtin from one or two alleles can survive to adulthood, but are smaller than normal, whereas mice expressing low levels of only Q111 huntingtin, even from two alleles, do not survive.
HdhneoQ20/HdhneoQ111 mice express low levels of Q20 and Q111 huntingtin
Immunoblot analysis was performed to confirm that a reduced level of huntingtin expression was present from both CAG knock-in alleles in the HdhneoQ20/HdhneoQ111 mice (Fig. 2). The monoclonal huntingtin antibody mAb-2166 detected the full-length (350 kDa) Q7 and Q20 versions of huntingtin in brain extracts from HdhneoQ20/HdhneoQ111, HdhneoQ20/HdhneoQ20, HdhneoQ20/Hdhnull mice and control mice lacking neo and expressing normal levels of Q7 or Q20 huntingtin (HdhQ7/HdhQ7, HdhQ7/Hdhnull, HdhQ7/HdhQ111 and HdhQ20/HdhQ111). The level of Q20 huntingtin was similar in HdhneoQ20/HdhneoQ111 and HdhneoQ20/Hdhnull mice, and was 50% of the level detected in HdhneoQ20/HdhneoQ20 mice, and 10-fold lower than the level detected in extracts from the neo-excised control (HdhQ20/HdhQ111), which appropriately expressed these proteins. Q111 huntingtin was not detected in HdhneoQ20/HdhneoQ111 brain extracts using mAb-2166, although it was detected in brain extracts from control mice expressing normal levels of Q111 huntingtin. To confirm that Q111 huntingtin was present in HdhneoQ20/HdhneoQ111 mice, the sensitive mAb-1F8 antibody, specific for long soluble-glutamine segments (12), was used and did detect Q111 huntingtin in HdhneoQ20/HdhneoQ111 brain extracts, albeit at a level greatly reduced compared to that in the neo-excised controls.
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HdhneoQ20/HdhneoQ111 mice develop a lethal progressive movement disorder
Variable movement abnormalities were observed in all three lines of compound heterozygous mutants from 2 months of age, which were usually mild (Table 1). In addition, a number of severely affected HdhneoQ20/Hdhnull mice died within 1 month of weaning (n = 5) and were therefore not followed longitudinally. The abnormalities observed in all three mutant lines at young ages were similar, with stiff tail and hind-limb clasping during tail suspension (Fig. 3A) being most common. The HdhneoQ20/HdhneoQ20 and HdhneoQ20/Hdhnull mice had movement abnormalities that were variable from week to week, and did not noticeably worsen over time.
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Significantly, the HdhneoQ20/HdhneoQ111 mice demonstrated a progressive increase in movement problems with age, ultimately causing lethality (Table 1). The initial mild movement abnormalities (age
2 months), namely stiff tail and clasping of the hind limbs when suspended in the air by the tail (Fig. 3A), were similar to those seen in mice expressing only reduced levels of Q20 huntingtin at all ages. In most HdhneoQ20/HdhneoQ111 mice, the phenotype progressed over 9–10 months (Table 1) and other movement abnormalities became apparent, including limb stiffness or paralysis, reduced left–right alternation of the hind limbs (‘hopping’) during walking (Fig. 3B), resting tremors, difficulties walking after handling and seizure-like episodes. At the most advanced stage, most mice became hypokinetic and died prematurely or had to be killed when flagged by veterinary staff as being severely hypokinetic or paralyzed. Additionally, we found that HdhneoQ20/HdhneoQ111 mice were sensitive to anesthesia-induced death during MRI studies (10/25 HdhneoQ20/HdhneoQ111 mice versus 3/20 controls), tolerating only 50–80% of the dose delivered to littermate controls. None of these abnormal behaviors was observed in the control HdhQ7/HdhQ7 (n = 12), HdhneoQ20/HdhQ7 (n = 6) or HdhneoQ111/HdhQ7 mice (n = 8) that expressed normal levels of huntingtin from at least one wild-type HdhQ7 allele. The progressive disease, and in particular the paralysis, was also not observed in any of the HdhneoQ20/Hdhnull mice (0/9) and in only one of the HdhneoQ20/HdhneoQ20 mice (1/7), which suddenly, and not progressively, developed movement problems at age 10 months, including seizure-like episodes following tail suspension, and an unusual manner of moving in hyperactive bursts, followed by periods of inactivity during which resting tremors were apparent. To confirm the progressive decline revealed by our general scoring system, gait parameters in the HdhneoQ20/HdhneoQ111 mice were measured from footprint patterns of individual mice (13) at an early [PRE, (0;0) to (1;1); see Materials and Methods] and a more advanced [POST, (2;2)] stage of disease (n = 5)]. The footprint tracings displayed obviously abnormal patterns at the more advanced stage, including dragging hind limbs and a hopping gait that lacked normal alternating left–right steps (Fig. 3C). No significant differences were found between PRE and POST measurements of stride length and hind- or fore-base widths (Fig. 3D). However, we did observe marked changes in the uniformity of step alternation, resulting in an increase in the distance between front and hind footprint (OL: PRE = 0.6 ± 0.3 cm versus POST = 1.4 ± 0.2 cm; n = 5, P = 0.0006), and a decrease in the hind footprint left–right alternation (LR: PRE = 2.8 ± 0.3 cm versus POST = 0.7 ± 0.5 cm; n = 5, P = 0.001) (Fig. 3D).
Some HdhneoQ20/HdhneoQ111 and HdhneoQ20/Hdhnull mice have enlarged ventricles
To determine whether the three lines of Hdh mutant mice had obvious morphological brain defects, histological analysis was performed on mice surviving to adulthood. Microscopic examination of cresyl-violet stained coronal sections revealed that the brains from a number of HdhneoQ20/Hdhnull (n = 3/7) and HdhneoQ20/HdhneoQ111 (n = 2/6) mice had enlarged lateral ventricles compared to normal control littermates, consistent with our previous finding of overtly abnormal brain development resulting from drastically reduced huntingtin (3). Notably, none of the HdhneoQ20/HdhneoQ20 mice, that express a higher level of Q20 huntingtin, had this dilated ventricle phenotype (n = 0/5). Evaluation of cresyl-violet stained coronal sections did not uncover other overt morphologic defects.
Since the HdhneoQ20/HdhneoQ111 mice developed a progressive neurological disorder, we used magnetic resonance micro-imaging (µMRI) to monitor ventricle size of individual animals during disease progression to determine whether any change in ventricle size occurred with time. Similar to the histology, µMRI analysis of brain morphology, in mice aged between 2 and 19 months, showed that 50% of the HdhneoQ20/HdhneoQ111 mice had enlarged lateral and third ventricles, extending from the anterior striatum to the level of the hippocampus (Fig. 4A and B). Furthermore, the HdhneoQ20/HdhneoQ111 mice showed a range of ventricle sizes (Fig. 4C). Significantly, despite a progressive worsening of the neurological phenotype over time, no discernible change in the size of the ventricles was observed in individual mice followed for several months (n = 7; Fig. 4D), and mice with normal sized ventricles nevertheless had the progressive disease. In addition, in a group of asymptomatic 1-month-old HdhneoQ20/HdhneoQ111 animals, enlarged ventricles were observed in approximately the same ratio (3/7; data not shown). We conclude that the enlarged ventricles, seen in some HdhneoQ20/Hdhnull and HdhneoQ20/HdhneoQ111 mice, are the result of developmental defects similar to those seen in other neo containing Hdh CAG knock-in mice, expressing reduced levels of Q50 huntingtin (3).
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The HdhneoQ20/HdhneoQ111 neurological phenotype is independent of detectable striatal pathology
To determine whether the progressive neurologic disease in HdhneoQ20/HdhneoQ111 mice was associated with pathology in medium spiny striatal neurons, as is seen in mice expressing normal levels of mutant huntingtin in Hdh CAG knock-in mice with neo excised (5–7; Wheeler et al., submitted for publication), we evaluated striatal sections for disease correlates with a huntingtin antibody EM48, that detects a form of nuclear huntingtin, and with a glial fibrillary acid protein (GFAP) antibody for any reactive gliosis that usually accompanies neuronal atrophy (6). Striatal sections from 12-month-old HdhneoQ20/HdhneoQ111 mice [n = 4; movement score (2;2) or higher] revealed only rare neurons with EM48-reactive diffuse nuclear huntingtin and/or intranuclear inclusions, whereas no nuclear huntingtin was detected in control, 12-month-old littermates (n = 4), or in asymptomatic 1-month-old HdhneoQ20/HdhneoQ111 mice (n = 3) (data not shown). In addition, all mice in which nuclear huntingtin was detected also showed abundant cytoplasmic huntingtin in many striatal neurons, using the HF1 huntingtin antibody (data not shown). Furthermore, no evidence of cell loss, apoptotic cells or GFAP-immunoreactive astrocytes was detected in any of these mice (data not shown). Therefore, the pathogenic process that underlies the rapid neurologic dysfunction that is caused by mutant huntingtin on a background of low overall huntingtin appears to be distinct from the dominant striatal-specific disease process in precise genetic mouse models of HD.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice engineered to express reduced levels of huntingtin with a 20 polyglutamine segment tract, a normal human length, reveal that low huntingtin expression is associated with abnormal development and movement disorders. HdhneoQ20/Hdhnull and HdhneoQ20/HdhneoQ111 mice have a reduced body size and
50% of the animals have a variable enlargement of the cerebral ventricle volume. Strikingly, the HdhneoQ20/HdhneoQ111 mice expressing a similar low level of Q20 huntingtin as HdhneoQ20/Hdhnull mice, but which in addition express a very low level of mutant Q111 huntingtin, develop a rapid progressive movement disorder between 3 and 12 months of age that results ultimately in death. Longitudinal µMRI analysis of brain and ventricle volume proved to be a sensitive and reproducible method for assessing differences in brain morphology in individual mice, and was important in establishing the lack of correlation between the early dilated ventricle phenotype and the progressive neurological disease, and to demonstrate no increase in ventricle size with age. While the mild movement abnormalities that start at an early age may be a consequence of a developmental defect, the fact that the severe progressive movement disorder was only observed in HdhneoQ20/HdhneoQ111 mice demonstrates a critical role for mutant huntingtin in the disease. However, the rapid hind-quarter paralysis and premature death does not seem to involve the striatum. One possibility is that low-huntingtin renders neurons in the hindbrain or spinal cord particularly sensitive to the effects of the toxic polyglutamine tract. Thus, the disease process caused by mutant huntingtin on the low-huntingtin background appears to be distinct from the dominantly inherited late onset striatal-specific neuronal atrophy and subtle gait deficits found in Hdh CAG knock-in mice that are models of human HD (5–7; Wheeler et al., submitted for publication).
On the other hand, the movement disorder in HdhneoQ20/HdhneoQ111 mice is similar to the rapid decline reported in transgenic mice engineered to greatly overexpress either an N-terminal truncated fragment or a full-length copy of mutant human huntingtin with a highly expanded glutamine stretch (13–16). This raises the question of whether neuronal and/or motor dysfunction are the same in these two cases and can be achieved either by overexpressing an N-terminal fragment on a background of normal huntingtin, or by expressing full-length mutant huntingtin on a background of overall reduced levels of huntingtin.
While the slow striatal-specific neurodegeneration in HD patients and Hdh CAG knock-in mice is likely to be initiated by a novel activity of the mutant protein, our results indicate that severely reduced huntingtin levels provides a background that sensitizes neurons to the toxic effects of mutant huntingtin, yielding a rapid disease. Consistent with huntingtin’s role as a neuroprotective protein, shown by conditional Hdh inactivation leading to atrophy of striatal neurons (10), loss of huntingtin as a consequence of the disease process may hasten neuronal cell death. Recent evidence suggests that huntingtin’s neuroprotective function may involve up-regulation of BDNF, a potent neuronal survival factor (11).
In summary, we have shown that reduced huntingtin sensitizes the CNS to the toxic effects of even a very low level of mutant huntingtin, provoking a novel severe neurologic disorder. This striking finding raises the possibility that therapeutic strategies for HD, which inadvertently or purposefully alter huntingtin levels may render additional neurons sensitive to the harmful effects of the mutant protein, provoking potentially severe neurologic as well as developmental side effects. This finding may well have implications for all neurodegenerative diseases caused by the expression of expanded polyglutamine proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice
The generation by homologous recombination in embryonic stem (ES) cells of heterozygous neo-containing CAG knock-in mice bearing the HdhneoQ20 and HdhneoQ111 alleles, encoding human glutamine tracts in mouse huntingtin with 20 and 111 residues, respectively, with a neomycin (neo) cassette in the upstream Hdh promoter has been described previously (3). The Hdhnull knock-out allele (previously called Hdhex4/5), which completely eliminates huntingtin expression, was reported in Duyao et al. (8). Three lines of compound heterozygous mice on a Swiss Webster background were generated by interbreeding knock-out and CAG knock-in mice with neo: HdhneoQ20/HdhneoQ20 from HdhneoQ20/HdhQ7 intercrosses; HdhneoQ20/Hdhnull from HdhneoQ20/HdhQ7 x HdhQ7/Hdhnull; and HdhneoQ20/HdhneoQ111 from HdhneoQ20/HdhQ7 x HdhneoQ111/HdhQ7 matings. The presence of the appropriate targeted Hdh alleles in each offspring was determined by Southern blot analysis and a PCR assay for CAG repeat size of tail DNA as described previously (3,8).
Magnetic resonance micro-imaging (µMRI)
All in vivo imaging experiments were performed on a 7 Tesla µMRI system. Mice were anesthetized with intraperitoneal injections of a ketamine (120 mg/kg body weight)/xylazine (20 mg/kg) mixture. Anesthetized mice were placed in a custom holder, securing the head during µMRI. The body temperature was maintained above 30°C during µMRI using a water-circulating warming pad.
Images were acquired using a T2-weighted spin-echo sequence (echo time, TE = 30 ms; repetition time, TR = 2 s) providing high spatial resolution images (78 x 78 x 700 µm) in a reasonable scanning time (35 min). For each mouse, 15 sequential coronal slices were acquired covering the entire head. Quantitative measurements of brain and ventricular volume were made using NIH Image (Version 1.62, National Institutes of Health, Bethesda MD). The area of the mouse brain was measured in each µMR image over the entire length of the cerebral cortex, from the caudal edge of the olfactory bulb to the rostral edge of the cerebellum. Areas of the lateral and third ventricles were also measured in each slice: a threshold grayscale value was selected and the contiguous ventricular area above this threshold was measured in each image. The brain and ventricular volumes were then calculated, adding the areas in each slice. The ventricular size of each mouse was normalized and expressed as a percentage of the brain volume, and is referred to as the relative ventricle volume.
Movement tests
General scoring system. Hind-limb clasping during tail suspension has been used previously to assess disease in transgenic mice overexpressing mutant huntingtin (13–15) and in Hdh conditional knockouts (10). In this study, hind-limb clasping behavior was scored on an scale from 0 to 3: 0 = normal; 1 = clasps hind limbs within 30 s of being suspended in the air; 2 = clasps hind limbs within 5 s but recovers quickly when released after 30 s; 3 = clasps hind limbs within 5 s and has difficulty recovering when released after 30 s.
General observation of movement was scored on a similar scale from 0 to 3: 0 = normal; 1 = one movement abnormality; 2 = two movement abnormalities; 3 = three or more movement abnormalities. The movement abnormalities observed were: tail stiffness or paralysis, hind-limb stiffness or paralysis, reduced left–right alteration of the hind limbs (‘hopping’) during walking, resting tremors, difficulties walking after handling and seizure-like episodes. In addition, one of the HdhneoQ20/Hdhnull mice suddenly displayed an unusual walking style, consisting of alternating hyperactive bursts and prolonged periods of inactivity accompanied by resting seizures.
Mice were assessed bi-weekly and assigned a dual number score: (hind-limb clasping; general movement), where (0;0) is normal and (3;3) is most advanced. Each mouse was observed for 5 min every other week. First, the mouse was placed on a weigh scale and its body weight recorded. Next, the tail suspension test was run holding the mouse by the tail for 30 s and then releasing it gently on an open surface. The general movement score was recorded after subsequent observation of the mouse for 1–3 min on the open (1 x 1 m) surface, and then for an additional 1–2 min after placing the mouse back in its cage. No special equipment, other than a weigh scale and stop watch, was used to perform these tests. Some of the more advanced HdhneoQ20/HdhneoQ111 mice, especially after seizure-like episodes following the tail suspension test, were reluctant to walk, preferring to sit still for long periods of time (more than several minutes), and had to be gently prodded in order to observe their walking. It was not possible to perform these tests blinded to the genotypes because of the large difference in size between HdhneoQ20/HdhneoQ111, HdhneoQ20/HdhneoQ20 and HdhneoQ20/Hdhnull mice and their control littermates (Figs 1 and 3A).
Gait test. In addition to the scoring system described above, a quantitative test was implemented to assess changes in gait of individual HdhneoQ20/HdhneoQ111 mice as disease progressed (13). In this gait test, the fore and hind feet were coated with two colors of non-toxic paint, and the mice were allowed to walk along a 50 cm-long, 10 cm-wide runway. After three training runs a fresh sheet of white paper was placed on the floor of the runway for each final test run. The resulting footprint tracings were analyzed, measuring four parameters described previously (13): (i) stride length; (ii) hind-base width; (iii) fore-base width; (iv) overlap between fore and hind footprints. In addition, the forward distance between left and right hind footprints was measured to assess the degree of alternation between left and right steps. Gait parameters were determined by drawing parallel lines through the center of each footprint and measuring the distance (in centimeters) between appropriate lines (Fig. 3C). Each measurement represented the average over three steps. Differences between gait parameters measured at two time points were tested statistically using a Student’s t-test.
Immunoblot analysis
Soluble protein extracts were made from brain tissue from mice killed at 1 month of age by hypotonic lysis (50 mM Tris–Cl pH 7.5, 10% glycerol, 5 mM magnesium acetate, 0.2 mM EDTA with 0.5 mM DTT, 170 µg/ml PMSF, 10 µg/ml leupeptin and 2 µg/ml aprotinin) and were directly electrophoresed in 6% SDS–polyacrylamide gels. Proteins were transferred from the gel by electroblot to a Protran membrane. Membranes were incubated overnight at 4°C with either mAb-2166 (huntingtin amino acids 181–812) (Chemicon) at 1:2500, mAb-1F8 [recognizing soluble long polyglutamine tracts (12) at 1:10 000, or, as a protein-load control mAb-anti-spectrin (Sigma) at 1:1000]. ECL antibody detection was used (Amersham Pharmacia). Densitometric analysis (Bio-Rad GelDoc 1000) was performed on the huntingtin bands detected with mAb-2166 and normalized against measurements of the corresponding spectrin controls for each band.
Histology and evaluation of striatum for HD-like pathology
Mice were perfused intracardially with 4% paraformaldehyde, after which the brains were dissected, post-fixed for 6 h in 4% paraformaldehyde at 4°C, cryopreserved in sucrose and sectioned (40 µm thick coronal sections) on a cryostat. Gross brain morphology was evaluated microscopically by inspection of cresyl-violet stained sections. Immunostaining protocols for GFAP, EM48 and HF1, on 40 µm thick frozen sections, were as described previously (6). Apoptosis was analyzed, on 40 µm thick frozen sections, using an indirect TUNEL labeling kit (Boehringer-Mannheim).
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ACKNOWLEDGEMENTS |
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We thank Dr Jean-Paul Vonsattel for his advice on the evaluation of the neuropathology in these mice, Dr X.-J.Li for EM48 antibody and Lori Lebel and Olivier Jin for technical assistance. We also thank Dr Mark Zervas for critical review of the manuscript. This research was supported by NIH grants NS38461 and GM57467 (D.H.T.) and NS32765 and the Huntington’s Disease Society of America (M.E.M.). A.L.J. is an investigator of the Howard Hughes Medical Institute. V.C.W. was supported by a fellowship from the Hereditary Disease Foundation.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA. Tel: +1 212 263 7262; Fax: +1 212 263 8214; Email: turnbull@saturn.med.nyu.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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