Human Molecular Genetics, 2001, Vol. 10, No. 2 107-116
© 2001 Oxford University Press
Expression of expanded repeat androgen receptor produces neurologic disease in transgenic mice
1Neurogenetics Branch, National Institutes of Neurological Disease and Stroke, National Institutes of Health, Bethesda, MD, USA, 2Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 208 Bluemle Life Sciences Building, 233 South 10th Street, Philadelphia, PA 19073, USA, 3Graduate Program in Pharmacology and 4Center for Neurodegenerative Disease Research, Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
Received 11 July 2000; Revised and Accepted 7 November 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinal and bulbar muscular atrophy (SBMA) is a motor neuron disease caused by the expansion of a polyglutamine tract within the androgen receptor. This disease is unusual among the polyglutamine diseases in that it involves lower motor and sensory neurons, with relative sparing of other brain structures. We describe the development of transgenic mice, created with a truncated, highly expanded androgen receptor driven by the neurofilament light chain promoter, which develop many of the motor symptoms of SBMA. In addition, transgenic mice created with the prion protein promoter develop widespread neurologic disease, reminiscent of juvenile forms of other polyglutamine diseases. Thus, in these experiments, the distribution of neurologic symptoms depends on the expression level and pattern of the promoter used, rather than on specific characteristics of androgen receptor metabolism or function. The transgenic mice described here develop neuronal intranuclear inclusions (NIIs), a hallmark of SBMA and the other polyglutamine diseases. We have shown these inclusions to be ubiquitinated and to sequester molecular chaperones, components of the 26S proteasome and the transcriptional activator CREB-binding protein. Apart from the presence of NIIs, evidence of neuropathology or neurogenic muscle atrophy was absent, suggesting that the neurologic phenotypes observed in these mice were the result of neuronal dysfunction rather than neuronal degeneration. These mice will provide a useful resource for characterizing specific aspects of motor neuron dysfunction, and for testing therapeutic strategies for this and other polyglutamine diseases.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuron disease (1) characterized by symmetrical muscle weakness, atrophy and fasciculations (for a review of clinical aspects see ref. 2). SBMA patients may also have sensory deficits, which usually remain subclinical. Neurological symptoms start in the 3rd to the 5th decade of life and are slowly progressive, leading in some cases to premature death. In addition, affected males show signs of androgen insensitivity (3). These signs include gynecomastia, which frequently develops during adolescence, as well as reduced fertility and testicular atrophy. Serum testosterone levels are usually normal or increased. Female heterozygous carriers are usually phenotypically normal, although mildly affected females have been reported (4). In addition, subclinical signs such as abnormal electromyography patterns or elevated creatine phosphokinase levels may be observed in carrier females. Consistent with the clinical findings, histological studies of autopsy material have shown marked motor neuron loss and atrophy in the spinal cord and brainstem nuclei. Substantial sensory neuronal degeneration is also seen (5).
SBMA is caused by the expansion of a trinucleotide CAG repeat within the coding region of the androgen receptor (AR) gene (6). The CAG repeat lies in the first exon of the AR gene and its length is highly polymorphic. CAG repeats range in the normal population from 11 to 33 CAGs, with a mean repeat length of 21 (7,8) whereas repeats in SBMA patients range from 40 to 62 CAGs.
The expansion of a CAG repeat encoding a polyglutamine tract has been identified as the pathogenic mutation in a growing number of dominantly inherited neurodegenerative diseases, including Huntington’s disease (HD), spinocerebellar ataxia types 1, 2, 3, 6 and 7 and dentatorubropallidoluysian atrophy (DRPLA) (for a review see ref. 9). In addition to the DNA mutation, these diseases share several other features, implying a common disease mechanism. One of these features is an inverse correlation between CAG repeat length and age at onset. In addition, although most CAG repeat disease genes are expressed ubiquitously throughout the nervous system, and often in other tissues as well, only a limited number of specific neuronal populations are prone to malfunction and degeneration.
A common neuropathological feature of this family of diseases is the presence of neuronal intranuclear inclusions (NIIs) containing the polyglutamine-expanded protein in affected brain regions (10–19). Although inclusions may represent the final physical state of a misfolded protein, their role in disease pathogenesis remains unclear (20,21). Nonetheless, understanding their composition may contribute to an understanding of cellular pathways involved in disease pathogenesis. An additional feature of aggregates in SBMA and in other, but not all, polyglutamine repeat diseases, is the finding that N-terminal epitopes are selectively represented in aggregates (10,11,19), suggesting that proteolytic cleavage may contribute to disease pathogenesis. Indeed, we have found that a truncated, expanded-repeat AR is more toxic to cultured cells than a full-length form with the same repeat length (22,23).
The dominant toxic function of expanded polyglutamines has been clearly demonstrated with transgenic models of other polyglutamine repeat diseases (24–29). The purpose of this study is to produce a transgenic model of SBMA with which to study those aspects of polyglutamine repeat disease that are specific to SBMA. In addition, we sought to test the hypothesis that protein context may contribute to the neuronal selectivity seen in this and other polyglutamine repeat diseases.
Previous attempts to create a transgenic model of SBMA have not resulted in a behavioral or pathological phenotype (30–32). In our earlier studies we used the neuron-specific promoters from the neuron-specific enolase (NSE) and neurofilament light chain (NF-L) genes (30,31) and the ubiquitous inducible promoter Mx (30) to drive the expression of full-length AR constructs containing 24, 45 or 65 repeats in mice. Although expression levels of two to five times those of endogenous AR were obtained with the NSE and NF-L promoters (D.E. Merry et al., unpublished data), transgenic mice carrying the 65 repeat construct developed neither clinical nor subclinical phenotypes up to 2 years of age.
We have now designed a transgene containing only the 5' portion of the AR gene (33), testing the hypothesis that protein truncation increases the efficiency of polyglutamine-induced toxicity, and have expanded the triplet repeat to 112 CAGs in order to advance the manifestations of neuronal degeneration during the short mouse life-span. In addition to the NF-L promoter, we also developed a construct with the prion protein promoter (PrP) (34), which drives much higher and more widespread expression of the transgenic protein. Both types of transgenic mouse have developed pathologic phenotypes that differ in age at onset and spectrum of clinical manifestations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transgenic constructs, founder mice and established lines
The DNA fragments used to create transgenic mice are depicted in Figure 1. We identified four founder animals harboring the NFL-AR112_HA construct and three founders harboring the NFL-AR16_HA construct. All animals were initially fertile and lines were established from two of the expanded repeat lines that were shown to express RNA from the transgene by RT–PCR (NFL112-5, NFL112-16). A line was also established from the highest-expressing normal repeat-containing founder (NFL16-22). One of the expanded repeat lines (NFL112-16) developed infertility after 3 months of age, but long before any neurologic phenotype was observed (see below). Sperm was retrieved from one male animal from this line for in vitro fertilization. The other NFL112 line (NFL112-5) is fertile.
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For PrP-AR112_HA, we identified 10 founders. Four of these animals (numbers 1, 17, 32.2 and 46.2) were infertile. Four founders were fertile for a short time, producing small litters, but did not produce any transgenic offspring (numbers 3, 6, 10.2 and 19). One founder, PrP112-10, was mosaic for the transgene, bred well until 4 months of age and had three transgenic offspring that developed a phenotype too severe to allow breeding. PrP112-10 was euthanized at 7 months of age and his sperm was retrieved for in vitro fertilization. Another founder, PrP112-11, was somewhat fertile, but was probably mosaic and produced only one F1 animal, which was itself infertile. Two founder animals were obtained with PrP-AR16_HA. One of these lines (PrP16-10) expressed detectable levels of transgene protein by western blot and was further expanded.
Phenotype analysis
Mice created with the NFL-AR112_HA construct developed various neurologic symptoms with late onset. Table 1 summarizes the data for those animals/lines for which the transgenic status was confirmed through breeding or expression analysis. Animals from line NFL112-16 exhibited hindlimb clasping when suspended by the tail at 
8 months of age; hindlimb gait abnormalities appeared at 11 months of age, along with a reduction in explorative behavior. The hindlimb gait had a ‘wobbling’ character; in addition, the legs appeared weak and the gait ‘spastic’. The usual smooth sequence of hindlimb movements seen in non-transgenic animals was in these mice interrupted by the temporary freezing of movement at the upstroke of the gait (Fig. 2, top). Muscle weakness was obvious as the mouse stood against the cage wall (Fig. 2, bottom); in just 1–2 s the mouse fell, with buckling at the hip. These motor symptoms slowly increased in severity, until impairment became severe at 12–14 months. The forelimbs became involved late in the disease. From the beginning of the onset of motor symptoms, animals were unable to withstand more than one or two rotations on the rotarod. The abnormal gait in NFL-AR112-16 mice was also characterized by performing a gait analysis, using paint to mark the paws. This analysis revealed an abnormal weight-bearing pattern on the hind feet (Fig. 3, blue). While non-transgenic animals placed only the most distal aspect of the hindpaw on the ground during a footfall, transgenic animals touched the ground with the entire footpad, consistent with hindlimb muscle weakness.
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Transgenic animals from line NFL112-5 showed a milder phenotype than that seen in line NFL112-16. Hindlimb clasping became apparent by 8 months, but animals up to 2 years of age did not show gait abnormalities. However, transgenic animals from NFL112-5 showed impaired performance on the rotarod at 11 months of age (also seen in animals up to 18 months of age) compared with age-matched littermates (Table 2).
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Transgenic animals from lines expressing a normal repeat AR did not develop neurologic phenotypes up to 2 years of age. In addition, they showed no rotarod deficits compared with non-transgenic animals of the same age (data not shown).
A much more severe neurologic phenotype was observed in several of the founders and offspring from PrP-AR112_HA (Table 1). Due to deficits in breeding, most of the behavioral and neuropathological characterization was performed on F1 animals derived from PrP112-10 and on founder PrP112-3. From PrP112-10, three transgenic F1 animals were obtained; all animals developed obvious neurological symptoms at 3 weeks of age. These symptoms included a constant tremor of head and trunk that increased with movement and under stress, irregular short shudders of the entire body, hindlimb clasping and excessive grooming. They also developed a wobbling gait, but without the specific features of spasticity and weakness observed in line NFL112-16. As the disease progressed, several seizures were observed. Rotarod performance of PrP112-10 animals was diminished compared with non-transgenic littermates (data not shown). Death occurred at 6–8 weeks. In addition to the neurologic symptoms, affected mice failed to thrive; although of equal size and weight to their non-transgenic littermates at 3 weeks of age, they were half the weight of their unaffected littermates by 6–8 weeks of age. They also failed to breed. A similar course was observed in three other founders.
Several other PrP112 founder animals and lines developed a subset of these symptoms in a milder form, at a later age or with slower progression. The transgenic founder mice PrP112-3 and PrP112-17 were indistinguishable from non-transgenic founders in behavior and appearance up to 9 months when they developed tremor, hindlimb clasping and gait abnormalities. Their cage activity decreased rapidly and they died within a month. PrP112-11 and its offspring developed very mild and slowly progressive symptoms beginning at 6 months, with death at 12–16 months; fertility was decreased from an early age. No animals expressing a 16 CAG repeat form of the truncated AR developed neurologic phenotypes up to 2 years of age.
Analysis of transgene expression
In the western analysis of NFL112-16 and NFL112-5 lines the expanded-repeat transgenic AR protein was not detected. Transgenic protein was detected in NFL112-16, however, by immunostaining. Analysis of RNA by RT–PCR in brain and spinal cord from NFL112-16 and NFL112-5 revealed moderate and low levels of transgenic RNA, respectively. Western analysis of brain and spinal cord extracts from mice transgenic for the normal repeat NFL-AR16
HA revealed transgenic protein of the expected size in line NFL16-22 at 4 weeks of age (data not shown). The inability to detect transgenic expanded-repeat AR by western analysis likely reflects the relatively small number of expressing neurons within the nervous system, the low level of NF-L-driven expression in each cell and some degree of expanded AR insolubility. In the PrP112 lines, expression of the transgenic expanded-repeat AR protein by western analysis was detected only in animal PrP112-3 and line PrP112-10. In western blots of brain and spinal cord homogenates, the transgene protein was observed both in soluble (monomeric) and insoluble forms (Fig. 4). This pattern was observed with antibodies against both the C-terminal hemagglutinin epitope (Y-11) (Fig. 4) and the N-terminus (ARN-20) (data not shown), whereas an antibody to the expanded polyglutamine stretch (1C2) (35) detected only the monomeric AR (data not shown). As previously observed (33), the monomeric truncated AR112 protein migrated much more slowly on SDS–PAGE than predicted. There was no evidence of proteolysis of the transgenic AR protein in these mice. Transgenic protein of the expected size was detected in one transgenic line expressing the PrP-AR16HA transgene (PrP16-10) (data not shown).
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Neuropathology
Immunohistochemical analysis of AR expression in line NFL112-16 revealed transgenic AR-positive intranuclear inclusions in isolated neurons in several restricted regions of the central nervous system including the brainstem and the cortex and at lower frequency in spinal cord motor neurons (Fig. 5A, Table 1). Inclusions were detected using antibodies both to the N-terminus of the AR (ARN-20) and the C-terminal HA epitope (Y-11).
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Staining of tissue from PrP112-10 revealed NIIs in nearly all neurons (Fig. 5B, Table 1) of the brain and spinal cord. In contrast with previous reports of transgenic mice created with the PrP promoter (27,28,34), expression of the transgene, as revealed by the presence of NIIs, was also observed in cerebellar Purkinje cells (Fig. 5E–G). A similar analysis of the brain of founder mouse PrP112-3 with antibodies to the AR revealed very large inclusions; although inclusions were not present in every neuron, those that were present appeared to nearly fill the entire nucleus (data not shown). We did not observe inclusions in testis or muscle (quadriceps or gastrocnemius) of another severely affected PrP112 founder (PrP112-46.2), although NIIs were observed in brain sections from this mouse. Compared with PrP112-10 and PrP112-3, the density of NIIs in brain and spinal cord sections from line NFL112-16 was much lower. This probably reflects the lower transgene expression levels in these mice. NIIs were not detected in brain or spinal cord from transgenic mice of the normal repeat lines PrP16-10 (Fig. 5C) and NFL16-22 (data not shown). Indeed, although transgenic AR protein from these mice was detected by western analysis, the transgenic protein was barely detectable by immunostaining.
In order to understand the pathogenesis of disease in this mouse model, we characterized inclusions with regard to their composition. Characterization of inclusions was performed on mice derived from PrP112-10, on PrP112-3 and on mice derived from NFL112-16. Using antibodies to a number of different candidate proteins, we found that inclusions were ubiquitinated (Fig. 5F) and contained several molecular chaperones, including HDJ-2 and Hsc70 (Fig. 5D and E). Staining with an antibody to the inducible Hsp70 was only weakly positive for inclusions in a very small subset of neurons (data not shown). In addition, brain inclusions were positive for an antibody to the 20S proteasome ‘core’ (Fig. 5G) and more weakly positive with an antibody to the 19S regulator ATPase subunit 6b (Tbp7) (data not shown). An antibody to the subunit PA28
of the 11S proteasome regulator did not detect inclusions.
The transcriptional activator CREB-binding protein (CBP) is known to interact with the full-length AR (36,37). We found CBP within a proportion of inclusions in the brain of mouse PrP112-3 (Fig. 5H). Not only did these inclusions stain positively with anti-CBP antibody, the nuclei of such cells showed lower diffuse staining than their non-inclusion-containing neighbors, suggesting a true sequestration of this protein. The finding of CBP within AR inclusions is likely to be quite specific for this particular transcriptional co-activator. Other transcriptional activators and co-activators, including SRC-1, c-fos, c-jun and p53, were not found in inclusions (data not shown). Staining of spinal cord tissue from a PrP112-10 F1 transgenic mouse with thioflavine-S (Fig. 5I) revealed a low frequency of stained inclusions, suggesting that at least a subset of inclusions contained protein in a ß-pleated sheet conformation reminiscent of amyloid.
Analysis of paraffin sections of brain and spinal cord by hematoxylin and eosin staining failed to reveal any overt signs of degeneration in either NF-L or PrP transgenic lines (data not shown). In addition, glial fibrillary acidic protein (GFAP) staining did not reveal evidence of gliosis. In order to assess motor neuron loss in line NFL112-16, we analyzed ventral roots from lumbar L4 and L5 sections for axon number. Loss of axons was not observed, nor was there evidence of pathology to proximal motor roots. Analysis of quadriceps, gastrocnemius, masseter, levator ani and bulbocavernosus muscles of transgenic mice by hematoxylin and eosin staining revealed no evidence of fiber atrophy. Additional analyses with ATPase and NADH stains showed no evidence of fiber type grouping (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have used a truncated, highly expanded repeat (112Q) AR to create neurologic disease in transgenic mice. Expression of the transgene using the prion protein promoter resulted in widespread neurologic disease. Mice developed a variety of symptoms, ranging from mild tremor with foot clasping to loss of motor function, severe tremor, handling-induced seizures, weight loss and death. In contrast, the restricted expression that results from the NF-L promoter produced a motor disease exhibiting many of the aspects of SBMA. In addition, these mice developed gait abnormalities suggestive of hindlimb spasticity, a sign of upper motor neuron disease not typically seen in SBMA patients. Neither of these phenotypes was observed in mice transgenic for the full-length, moderately expanded (45Q, 65Q) AR driven by either the NF-L or the NSE promoters, the inducible Mx promoter or the endogenous AR promoter (30–32). These findings show that protein truncation, highly expanded repeats and/or high levels of expression are required for the modeling of this motor neuronopathy.
In the transgenic mice that we describe, the effects of polyglutamine expression on specific brain regions depends on the expression pattern of the respective promoter. The multiform and severe disease observed in PrP transgenic mice is consistent with the widespread and robust expression obtained with this promoter. In contrast, the NF-L promoter drives protein expression only in selected neuronal populations, with highest expression in the spinal cord anterior horn, brain stem, cerebral cortex and sensory neurons of the dorsal root ganglia. Consistent with this predominantly motor expression pattern, NFL-AR112 transgenic mice develop a phenotype confined to motor systems. The finding of upper motor neuron manifestations, in addition to lower motor neuron disease, is in keeping with the expression of the transgene in cortical neurons.
When creating the transgenic construct with a substantially truncated version of the AR, the possibility existed that some of the specific aspects of AR metabolism that contribute to disease pathogenesis would be lost, which might result in neuronal dysfunction and disease quite different from SBMA. However, when expressed in motor neurons with the NF-L promoter, the truncated, expanded AR produces neurologic disease that resembles SBMA in many respects. In addition, although the PrP transgenic mice do not show an exclusively motor phenotype, they share features with animal models of other polyglutamine diseases. Their phenotype resembles in particular that of the transgenic HD mice created with the same promoter driving expression of an N-terminal huntingtin fragment with 82 glutamines (27). Both types of transgenic mouse develop hindlimb clasping, tremors, similar gait abnormality and impaired rotarod performance. Both have early onset of symptoms and a shortened lifespan; the somewhat longer survival of the HD mice might be due to the shorter repeat length of the transgene or the different protein context. The expression patterns of these two variant forms of PrP promoter mice differ slightly, however: unlike the PrP-AR112 mice (Fig. 5E–G), the PrP-HD mice did not express the transgene in cerebellar Purkinje cells. The neurological symptoms in our PrP transgenic mice also resemble those of HD transgenic mice in which a highly expanded N-terminal huntingtin fragment is expressed from the HD promoter (25). This finding is likely due to the widespread expression patterns of both the huntingtin and prion protein promoters, resulting in disease in overlapping neuronal populations.
The use of a truncated form of the expanded AR in these studies has a precedent in the neuropathological studies of SBMA autopsy material (19,38). These studies revealed NIIs that were detected with antibodies only to N-terminal (ARN-20) but not to more C-terminal epitopes, suggesting that these were either missing or masked (19,38). Similar findings in studies of autopsy material of HD and DRPLA (10,28,39) support the idea of a toxic fragment hypothesis, whereby a truncated polyglutamine-containing peptide is produced that creates or exacerbates the polyglutamine toxicity in affected neurons. The recent development of mouse models of DRPLA and HD, in which both neuropathology and temporal course of disease correlate with the nuclear accumulation and aggregation of truncated fragments (28,39), supports this hypothesis. Whether this process contributes to the pathogenic mechanism in SBMA has yet to be determined. However, the inclusions that we have observed in our PrP and NF-L transgenic mice are similar to those in SBMA patients (19) and other mouse models of polyglutamine diseases (26,27,40,41) in that they are ubiquitinated and sequester several heat shock proteins and proteasome components. Thus, at least this aspect of disease pathogenesis, which represents the histological evidence of the misfolding and targeting of a polyglutamine-containing AR fragment for degradation, is likely to be the same as that occurring in SBMA. It is possible that the metabolism of the full-length AR protein contributes to both the disease process and the motor neuron specificity of SBMA; such a role of the full-length protein metabolism may account for the differences with those mouse models in which neuronal degeneration is observed (24,42). This issue will be further investigated in our ongoing experiments using highly expanded, full-length AR driven by the PrP promoter.
Another feature of disease phenotype in both mouse models created here is reduced fertility. Because of the small numbers of mice obtained from each line, we have to date been unable to determine the reason for this infertility. In male NF-L transgenic mice with motor phenotype, perineal muscle weakness appeared to contribute to this infertility, although no muscle atrophy of the levator ani or the bulbocavernosus was observed using several histological methods. As the spinal nucleus of the bulbocavernosus is one of several motor nuclei innervating the perineal muscles, a toxic effect of the transgene on this motor population would be expected to affect motor function resulting in penile dysfunction. Our further studies will help in determining whether motor neurons in this nucleus are included in the disease process and to which extent their malfunction contributes to the observed infertility. The reduced fertility in SBMA patients, which is usually attributed to androgen insensitivity, may also be due in part to motor dysfunction.
The characterization of NIIs in our AR transgenic mouse models reveals similarities with those found in spinal cord tissue from SBMA patients (19) as well as in tissue from patients with other polyglutamine diseases (10,11,13,16,18,19,41,43). The finding of CBP in NIIs indicates that other proteins are also drawn into aggregates, either as a function of their normal interactions with the AR, or as a function of becoming misfolded and targeted to the 26S proteasome for degradation. Such sequestration may alter the normal functioning of pathways dependent on these proteins (44) and may contribute to disease pathogenesis.
The finding of molecular chaperones, along with components of the 26S proteasome, in NIIs of these transgenic mice, suggests that the expanded AR is misfolded and targeted for degradation by the 26S proteasome. However, the presence of NIIs also indicates that this system is inefficient in ridding neurons of the mutant protein, which accumulates in the nucleus. The observed effects of molecular chaperones on aggregation and cellular toxicity (45, our unpublished data) suggest that this family of proteins represents a potential therapeutic target for SBMA treatment. We will test this idea through crosses of transgenic mice overexpressing the HDJ-2 or Hsp70 molecular chaperones with additional transgenic lines that we are currently creating. In addition, recent evidence from a conditional mouse model for HD (29) suggests that decreasing the cellular load of expanded polyglutamine protein, either through decreasing its production or through increasing its degradation, should prove to be a valid therapeutic strategy for treating any polyglutamine disease. The latter of these therapeutic approaches may be used on the mice reported here and on mice transgenic for the full-length AR protein that we are currently creating.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transgene constructs and genotype analysis
Transgene constructs were prepared in the following way: the EcoRI–PvuII AR
HA cDNA fragments from 16 or 112 CAG repeat constructs from Merry et al. (33) were isolated, filled in with Klenow (New England Biolabs) to create blunt ends and ligated to either a ClaI-digested and Klenow-filled-in human pGCHNF-L DNA (46,47) or an XhoI-digested mouse prion protein DNA (34). We previously engineered the NF-L promoter construct to contain a 1.5 kb fragment from the mouse ß-globin gene [isolated from the NSE-AR24 construct described by Bingham et al. (30)], containing splice donor and acceptor signals and a polyadenylation signal. This was done by isolating a 1.5 kb XbaI ß-globin fragment from NSE-AR66, produced as by Bingham et al. (30), creating blunt ends with Klenow (New England Biolabs) and ligating to pGCHNFL DNA that had been digested with SalI and filled in with Klenow to create blunt ends. The prion protein promoter construct had previously been engineered by removing the coding sequences (34). Excised fragments of transgene DNA were gel-purified, then further column-purified (Elutip-D; Schleicher and Schuell). DNAs were injected into fertilized oocytes, derived from a C57Bl6 x SJL mating, by the Transgenic and Chimeric Mouse Facility at the University of Pennsylvania. Mice were maintained by breeding to C57Bl6/SJL F1 animals (Jackson Laboratories).
DNA from mice was prepared from tail biopsies according to a standard protocol (48). Transgenic mice within the NF-L lines were identified by PCR [forward primer from AR sequence 3' to the hemagglutinin tag (hARfin), coupled with a reverse primer from ß-globin sequences 3' to the AR cDNA (3'BG)]. Transgenic animals within the PrP lines were identified either by PCR [forward primer from the PrP promoter region (PP5), coupled with a reverse primer from AR sequence 5' to the CAG repeat (AR3), or forward primer from AR sequence 3' to the hemagglutinin tag (hARfin), coupled with a reverse primer from PrP sequences 3' to the transgene (PP3)] or by Southern blot analysis using the ARHA cDNA as a probe.
Western blot analysis
Brain and spinal cord were removed and flash-frozen in liquid nitrogen. Frozen tissue was pulverized in a mortar and pestle on dry ice and homogenized in 10 vol of sample buffer (20 mM DTT, 4% SDS, 160 mM Tris–HCl pH 6.9, 20% glycerol, 0.004% bromophenol blue). Lysates were then sonicated three times for 10 s using a Branson cup sonifier. Aliquots (50 µl) of protein lysates were electrophoresed by SDS–PAGE and transferred to nitrocellulose (Immobilon-P) using a semi-dry transfer apparatus. Western hybridization was performed using antibodies to the AR (ARN-20, Santa Cruz Biotechnology; 1C2, Chemicon International) (35), and to the HA epitope (Y11, Santa Cruz Biotechnology). Detection was performed with ECL (Amersham).
RT–PCR analysis
Total RNA was isolated from mouse brain and spinal cord using Trizol (Life Technologies). One microgram of RNA was treated with DNAse I (Life Technologies) and then incubated with AMV reverse transcriptase (Stratagene) under conditions recommended by the manufacturer. PCR was carried out with an AR forward primer at the 3' end of the cDNA (ARfin) and reverse primers from PrP (PP3) or NF-L (3'TG) sequences 3' to the AR. Samples were electrophoresed on a 1% agarose gel, and bands visualized with ethidium bromide. Control primers detecting glyceraldehyde 3-phosphate dehydrogenase RNA were also used. A reaction lacking reverse transcriptase was performed for each sample–primer pair combination.
Behavioral testing
Animals were tested on a Rotamex Rotarod (Columbus Instruments) on three consecutive days during the light phase of the 12 h light–dark cycle. We performed three trials each day, with breaks of at least 1 h between tests. In each trial, four mice were placed in separate chambers on the resting rod before rotation was initiated. After 5 s of constant slow rotation the speed increased gradually over the course of 5 min from 4 to 40 r.p.m. The timer was stopped either automatically if the mouse fell from the rod as detected by an infrared light sensor, or manually in cases when the mouse gripped the rod and started rotating with it.
Footprint analysis was performed as described (49). Front and hind paws were dipped in red and blue non-toxic water-soluble tempera paint, respectively. When placed at one end of a dark tunnel, the mouse passed through to the other end leaving colored footprints on a replaceable white paper strip.
Immunohistochemistry and histology
For immunohistochemical analysis, brain and spinal cord tissue from transgenic mice and non-transgenic littermates was snap-frozen in OCT without prior fixation. Frozen sections (7 µm) were fixed in 4% paraformaldehyde for 10 min, washed in phosphate-buffered saline (PBS), blocked in 1.5% goat serum in PBS for 20 min, then incubated with the appropriate antibody and diluted in 1.5% goat serum in PBS overnight at 4°C. Slides were washed in PBS, then incubated with either FITC- or rhodamine-conjugated secondary antibody (Jackson ImmunoLaboratories), or for immunohistochemistry, with a biotinylated secondary antibody. For immunohistochemistry experiments, secondary antibody hybridization and peroxidase development were performed using a Vectastain ABC kit (diaminobenzidine) (Vector Laboratories). Antibodies used included anti-AR (ARN-20) and anti-HA (Y-11; Santa Cruz Biotechnology), anti-Hsp70 (Hsp72), anti-Hsc70 (Hsp73) (StressGen Biotech), anti-HDJ-2/DNAJ (clone KA2A5.6; NeoMarkers), anti-ubiquitin (Dako), anti-20S proteasome ‘core’, anti-19S regulator ATPase subunit 6b (Tbp7) and anti-subunit PA28 of the 11S proteasome regulator (Affiniti Research Products). Anti-CBP antibody (C-1) was obtained from Santa Cruz Biotechnology.
For thioflavine-S staining, paraformaldehyde-fixed, paraffin-embedded tissue sections were deparaffinized and hydrated through graded ethanols, washed in 0.01 M PBS pH 7.3 for 5 min, immersed in 0.05% potassium permanganate/PBS for 20 min, rinsed well in PBS and destained in 0.2% potassium metabisulfite/0.2% oxalic acid/PBS until clear. Tissue sections were then rinsed well in PBS and immersed in freshly prepared 0.0125% thioflavine-S/40% ethanol/60% PBS for 3 min in the dark. Differentiation was performed with 50% ethanol/50% PBS followed by extensive rinsing in PBS and water and mounting with Vectashield with DAPI mounting medium (Vector Laboratories).
Histological analysis was performed on hematoxylin and eosin-stained paraffin sections of brains and spinal cords from transgenic mice and their non-transgenic littermates. Quadriceps, gastrocnemius, masseter, levator ani and bulbocavernosus muscles were dissected and embedded in OCT embedding medium in isopentane. Muscle was sectioned at 7 µm intervals and used for hematoxylin and eosin, ATPase and NADH staining as described (50).
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ACKNOWLEDGEMENTS |
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The authors would like to thank Dr David Borchelt and Dr Jean-Pierre Julien for the prion protein promoter and neurofilament light chain promoter constructs, respectively, Addis Taye and Larry Bish for technical assistance and Dr Donald Schotland and Anne Sorling for assistance with studies of muscle pathology. We also thank Dr Kenneth Fischbeck for helpful discussions and for critical reading of the manuscript and Dr Jean Richa (University of Pennsylvania Transgenic and Chimeric Mouse Facility) for transgene injections and expert advice. This work was supported by the National Institutes of Health R01-NS32214 (D.E.M.) and by the Muscular Dystrophy Association (D.E.M.). J.D. is supported by NIH Training Grant AG00255.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 215 503 4907; Fax: +1 215 923 9162; Email: diane.merry@mail.tju.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Kennedy, W.R., Alter, M. and Sung, J.H. (1968) Progressive proximal spinal and bulbar muscular atrophy of late onset: a sex-linked recessive trait. Neurology, 18, 671–680.
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