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Human Molecular Genetics, 2004, Vol. 13, No. 2 181-190
DOI: 10.1093/hmg/ddh017

Myopathy phenotype in transgenic mice expressing mutated PABPN1 as a model of oculopharyngeal muscular dystrophy

Hirotake Hino1,2, Kimi Araki1, Eiichiro Uyama2, Motohiro Takeya3, Masatake Araki4, Kumiko Yoshinobu4, Koichiro Miike1, Yasuhiro Kawazoe1, Yasushi Maeda2, Makoto Uchino2 and Ken-ichi Yamamura1,*

1Department of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan, 2Department of Neurology Advanced Biomedical Sciences, Faculty of Medical and Pharmaceutical Sciences, Graduate School of Medical Science, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan, 3Department of Cell Pathology, Kumamoto University School of Medicine, Kumamoto, 860-0811, Japan and 4Department of Bioinformatics, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan

Received August 26, 2003; Revised October 30, 2003; Accepted November 9, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized clinically by progressive ptosis, dysphagia and limb weakness, and by unique intranuclear inclusions in the skeletal muscle fibers. The disease is caused by the expansion of a 10-alanine stretch to 12–17 alanine residues in the poly(A)-binding protein, nuclear 1 (PABPN1; PABP2). While PABPN1 is a major component of the inclusions in OPMD, the exact cause of the disease is unknown. To elucidate the molecular mechanism and to construct a useful model for therapeutic trials, we have generated transgenic mice expressing the hPABPN1. Transgenic mice lines expressing a normal hPABPN1 with 10-alanine stretch did not reveal myopathic changes, whereas lines expressing high levels of expanded hPABPN1 with a 13-alanine stretch showed an apparent myopathy phenotype, especially in old age. Pathological studies in the latter mice disclosed intranuclear inclusions consisting of aggregated mutant hPABPN1 product. Furthermore, some TUNEL positive nuclei were shown around degenerating fibers and a cluster of it in the lesion in necrotic muscle fibers. Interestingly, the degree of myopathic changes was more prominent in the eyelid and pharyngeal muscles. Further, muscle weakness in the limbs was apparent as shown by the fatigability test. Nuclear inclusions seemed to develop gradually with aging, at least after 1 week of age, in model mouse muscles. We established the first transgenic mouse model of OPMD by expressing mutated PABPN1, and our model mice appear to have more dramatic alternations in myofiber viability.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive ptosis, dysphagia, and varying degrees of limb muscle weakness (1–5). Its pathological hallmark is the presence of nuclear inclusions comprising clusters of 8.5 nm tubular filaments restricted to the skeletal muscle fibers (6). Other characteristics include predominant involvement of the levator palpebra superioris and pharyngeal muscle, which show severe dystrophic changes (7), and the presence of scattered rimmed vacuoles in affected muscles in more than 90% of patients (8). In contrast to other muscular dystrophies, necrosis of muscle fibers is rare (3–8).

OPMD is caused by the abnormal expansion of a (GCG)6 trinucleotide repeat at the 5' end of the coding region of the poly(A)-binding protein, nuclear 1 gene (PABPN1 : PABP2). The (GCG)6 codes for the first six alanines in a homopolymeric stretch of 10 alanines. In most patients, the (GCG)6 repeat is expanded to (GCG)8–13 (9) and insertional or duplicative mutations such as (GCG)6+GCA(GCG)2, +GCA(GCG)3, +(GCA)2(GCG)2 or +(GCA)3(GCG)2 rarely occur in other patients (10–12). Thus, disease is associated with expansions of 12–17 uninterrupted alanines located at the N-terminus of this protein. PABPN1 is an abundant nuclear protein that is known to bind the pre-mRNA polyadenylation site in the 3' poly(A) tail, and plays a role in mRNA polyadenylation and control of the length of poly(A) tails. The (GCG)9 expansion found in three cluster populations including French Canadian (13), Bukhara Jews (14) and Hispanic New Mexicans (15) is the most frequent mutation among at least 33 countries in the world.

The pathogenic process caused by mutated PABPN1 with polyalanine expansion remains undetermined. As indicated in at least nine neurodegenerative disorders caused by polyglutamine repeat expansions, including Huntington's disease (HD), spinocerebellar ataxia 1 (SCA1) and dentatorubral pallidoluysian atrophy (DRPLA), polyalanine expansion might induce a misfolding of protein with an increased propensity for aggregation by conferring a toxic gain of function on the disease protein (16,17). Immunohistochemical analyses revealed that the filamentous inclusions in the OPMD muscle nuclei contain PABPN1, ubiqutin and subunits of proteasome (18–20). COS-7 cells expressing mutant PABPN1 with 17 alanine residues showed similarities with polyglutamine disorders, in that the mutant proteins facilitated aggregate formation and enhanced cell death (21,22). Furthermore, inactivation of oligomerization of mutant PABPN1 by deleting the C-terminal oligomerization domain (23) or overexpression of various types of chaperones (22) reduces cell death. This evidence indicates that intranuclear inclusions and residues outside the polyalanine stretch may play important roles in pathogenesis of the disease. However, the pathogenic mechanism of cell death in OPMD muscles is still unclear. For the study of polyglutamine disorders, there is now a wide ranges of mouse models that can provide important insights into processes associated with disease pathogenesis (24–26). By contrast, no animal models for OPMD have been reported.

In general, there are two methods for establishing a genetically manipulated mouse model: one is the introduction of exogenous transgene by microinjection into pronuclei of fertilized eggs; the other is the creation of a knock-in or knock-out using embryonic stem cells. Since OPMD is considered to be caused by a gain of function type of mutation, we chose the transgenic strategy to overexpress mutant PABPN1 protein with (GCG)9 expansion. The (GCG)9 expansion has been chosen in this study because it is standard for OPMD (9,13–15). To obtain high-level expression of the introduced PABPN1 gene, we used a chicken ß-actin (CAG) promoter, which expresses transgene ubiquitously but predominantly in muscle (27,28).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Establishment of transgenic mice expressing different levels of hPABPN1 in muscle
We prepared two constructs; one carries normal hPABPN1 cDNA with a (GCG)6 repeat, and the other carries mutant hPABPN1 cDNA with a (GCG)9 expansion (Fig. 1A). The cDNAs were driven by CAG promoter, which is known to be a strong and ubiquitous promoter. Transgenic mice were generated by pronuclear microinjection of these constructs, and nine and eight transgenic mice out of 43 and 49 founder mice were obtained with the normal and mutant PABPN1 gene, respectively. Although all transgenic founder mice were confirmed by PCR and Southern blot analysis, only four (N19, N26, N33, N35) and three (M38, M41, M43) founder mice with the normal and mutant PABPN1 construct, respectively, showed germline transmission of the transgene to the F1 generation (Fig. 1B and C). In order to examine whether the transgene was expressed in each of the F1 mice, we performed northern blot analysis using mRNAs from skeletal muscle, in which the CAG promoter works strongly. As shown in Figure 1D, mRNA was expressed in two normal (N33 and N35) and three mutant lines (M38, M41 and M43), while it was not expressed in two normal lines (N19 and N26). To examine the tissue specificity of the transgene expression, we performed northern blot analysis using mRNAs from the brain, lung, heart, liver, spleen, kidney, small intestine, colon and muscle. The results demonstrated ubiquitous expression in all transgenic lines (data not shown). To compare the protein levels of PABPN1 in the transgenic lines, we performed western blot analysis using an anti-hPABPN1 antibody. As shown in Figure 1E, high levels of expression were observed in lines N33, M41 and M43, and low levels were observed in lines N35 and M38 similar to the wild type, in which the endogenous mouse PABPN1 protein was detected due to cross-reactivity.



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  		Figure 1. Construction of the transgene and establishment of the transgenic mouse lines expressing normal and mutant hPABPN1. (A) The hPABPN1 transgenic expression cassette. The full-length normal or mutant hPABPN1 cDNA was cloned into the expression vector pCAGGS, which carries the cytomegalovirus enhancer and chicken ß-actin promoter (CAG) and the polyadynylate DNA fragments used for Southern and northern blotting, respectively. Four (N19, N26, N33 and N35) and three (M38, M41 and M43) transgenic mouse lines expressing normal and mutant hPABPN1 cDNA, respectively, were established. S, SalI; E, EcoRI; H, HindIII. (B, C) Detection of transgene. PCR (B) and Southern blot analysis (C) of mouse tail DNA from each of the transgenic lines. N, negative control; P, positive control. (D) Northern blot analysis of mRNA from skeletal muscle of each of the transgenic lines. N, negative control. (E) Western blot analysis. The expression of PABPN1 in wild-type mice, transgenic mice, and COS-7 cell extracts transfected with the normal and expanded PABPN1 constructs. Equal amounts of protein lysates from mouse muscle and cell extracts from COS-7 cells were immunodetected using anti-PABPN1 antibody. WT, wild-type mouse; P1, COS-7 cells transfected with normal PABPN1 constructs, P2, COS-7 cells transfected with the expanded PABPN1 constructs.

 
Transgenic mouse lines expressing a normal hPABPN1 (N33 and N35) and the line expressing low-levels of mutant hPABPN1 (M38) appeared to develop normally, and about 50% of the offspring were hemizygotes for the transgene. However, the percentage of hemizygotes in the lines expressing high-levels of mutant hPABPN1 (M41 and M43) at 4 weeks of age was about 25%. In contrast, the frequency of hemizygotes at embryonic day (E) 19.5 was 50%, indicating no embryonic lethality in M41 and M43 lines. Therefore, we investigated the survival rate and the increase of body weight of M43 mice to compare with that of normal littermates from birth to the age of 4 weeks. As shown in Figure 2A, half of the M43 mice died before 3 weeks of age, while most of the normal littermates survived. Although the body weight of M43 mice was the same as that of normal littermates at birth, the rate of weight gain gradually diminished, resulting in significantly lower body weights relative to those of normal littermates at the age of 3 weeks (Fig. 2B). Macroscopic and histological experiments were carried out for M43 transgenic mice at 4 weeks of age. However, we could not find any abnormalities in the examined internal organs, including the brain, lung, heart, liver, spleen, kidney, small intestine and colon, except for the skeletal muscles.



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  		Figure 2. Phenotypes of M43 transgenic mice. (A) Survival curves of M43 F2 transgenic mice and normal littermates. M43 mice have significantly reduced life spans compared with their normal littermates (NL). (B) Growth retardation in M43 survival mice. M43 F2 transgenic mice from birth to the age of 4 weeks show reduction of the rate of body weight gain. (C) Load resistance time (LRT) of 3-month-old M43 and normal littermates. Difference in LRT between M43 F2 transgenic mice (6.3±2.1) and their normal littermates (16.4±5.8 min). The data were analyzed statistically using Student's t-test. Values presented are mean±SD; *P<0.05; **P<0.01.

 
To evaluate muscle weakness in M43 transgenic mice, we performed a physical examination. Mice were positioned hanging head-down on a wire net and the time until they fell off (load resistance time, LRT) was measured. Average LRT in normal littermates was 16.4±5.8 min, whereas average LRT in M43 mice was 6.3±2.1 min, which is significantly shorter than that in normal littermates (Fig. 2C). Thus, M43 mice showed apparent muscle fatigability on the LRT test, indicating significant weakness of the limbs muscles.

Muscle pathology in transgenic mice expressing mutant PABPN1
Histological sections of soleus muscles from transgenic and normal mice at the age of 6 months were stained with hematoxylin and eosin (H&E). We observed no histological abnormalities in the transgenic mice of the N33 (Fig. 3B) and N35 lines (Fig. 3C) and M38 lines expressing low levels of mutant hPABPN1 (Fig. 3D) as in wild-type mice (Fig. 3A); their muscle fibers were homogeneous in shape and size, and almost all nuclei were situated at the periphery of muscle fibers, as in normal human muscle fibers. The frequency of internal nuclei was less than 1% in the soleus muscles. By contrast, the muscles of M41 (Fig. 3E) and M43 mice (Fig. 3F), which expressed high levels of mutant hPABPN1, showed increased variability in the size of muscle fibers, a high frequency of internal nuclei, proliferation of the endomysial connective tissue, and occasional cytoplasmic vacuoles mimicking rimmed vacuoles (Fig. 4A). All of these findings were indicative of muscle degeneration and regeneration. Wide range areas of apparent necrosis were also observed (Fig. 8C), but the frequency was very low. M41 and M43 mice appeared to have more dramatic alternations in myofiber viability than OPMD patients. The frequency of internal nuclei in the soleus muscle was 5–15% at the age of 6 months, increased with age, and reached more than 40% at the age of 18 months (Fig. 4B). M43 mice showed neither ptosis nor dysphagia externally, while pharynx (Fig. 4D) and eyelid muscles (Fig. 4F) showed apparent variability in shape and size compared with pharynx (Fig. 4C) and eyelid muscles (Fig. 4E) of N33 mice. In addition, we measured serum creatine kinase (CK) levels of 6-month-old M43 mice and their normal littermates. The serum CK levels of M43 mice (n=8) and of normal littermates (n=15) were 60.9±13.0 and 52.8±24.8 U/l, respectively. This result is consistent with the observation that the level of CK is generally normal in patients with OPMD (4). Thus, M41 and M43 transgenic mice that expressed high levels of the mutant PABPN1 protein seemed to be the most useful models for OPMD. We chose N33 and M43 lines in the following studies.



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  		Figure 3. Histology of the soleus muscle in transgenic and wild-type mice. H&E staining of frozen sections of soleus muscle in 6-month-old wild-type (A), N33 (B), N35 (C), M38 (D), M41 (E) and M43 (F) mice. N33 (B), N35 (C) and M38 (D) did not show any significant abnormalities compared with wild-type mice (A). However, M41 (E), and M43 mice (F) mice showed apparent myopathic changes: increased variability in the size of muscle fibers, high frequency of internal nuclei, and proliferation of endomysial connective tissue. The bar represents 50 µm.

 


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  		Figure 4. Comparison of muscle histology of N33 and M43 transgenic mice. H&E staining of frozen sections of the soleus muscle in 18-month-old M43 mice (A and B). The soleus muscle showing a typical rimmed vacuole (A) and remarkably increased central nuclei (B). The bar in A represents 10 µm, in B represents 50 µm. H&E staining of frozen sections of pharynx muscle (C, D) and eyelid muscle (E, F) in 1-year-old N33 (C, E), and M43 mice (D, F). Both the pharynx muscle (D) and eyelid muscle (F) show dystrophic changes: marked variability in fiber size with clusters of small atrophic fibers, prominent endomysial connective tissue, and proliferating adipose tissue. The bar in C–F represents 100 µm.

 


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  		Figure 8. TUNEL staining. Sections of the soleus muscle of 1-year-old N33 mice (n=5) and M43 mice (n=10) were analyzed by TUNEL and hematoxylin staining. In the muscle from N33, scattered TUNEL-positive nuclei (arrow) were detected rarely in the interstitium (A). In the muscle from M43, some TUNEL-positive nuclei (arrow) were detected around degeneration fibers but not inside myofibers (B), and cluster of TUNEL-positive nuclei were detected in more severe degeneration fibers (C). The bar represents 50 µm.

 
Immunohistochemical analysis in wild-type and transgenic N33 and M43 mice
PABPN1 is usually identified within the nuclei of all muscle cells throughout the nucleoplasm, excluding the nucleolus, by staining with anti-PABPN1 antibody (18,29,30). In OPMD patients, an aggregated mutant PABPN1 product is detected in several muscle fiber nuclei, appearing as bright inclusion bodies. In order to clarify the localization and expression level of human PABPN1 protein in the transgenic mice, we stained the sections of the soleus muscle from wild-type, N33 and M43 mice with polyclonal anti-human PABPN1 antibody.

In wild-type mice, weak and diffuse intranuclear immunoreaction was observed, probably due to a cross-reaction to mouse endogenous PABPN1 (Fig. 5A and D). The staining pattern was quite similar to that of normal human muscle tissue. Compared with wild-type mice, the muscle nuclei of N33 and M43 mice showed extremely strong immunoflorescence (Fig. 5B and C). The muscle nuclei in N33 mice showed bright patchy stains in nuclear speckles (Fig. 5E), similar to that previously reported in COS-7 cell lines (22). On the other hand, the muscle nuclei in M43 mice showed a pattern of intranuclear inclusions (Fig. 5F), closely resembling that of OPMD patients (18–20). These results indicated that only overexpressed mutant PABPN1 protein, and not normal PABPN1 protein, aggregated in the muscle nuclei.



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  		Figure 5. Detection of PABPN1 protein by immunofluorescence analysis in the soleus muscle. Sections of the soleus muscle of 6-month-old wild-type (A), N33 (B, D) and M43 mice (C, E) were stained with anti-human PABPN1 antibodies. In the all sections, the immunoreactivity was localized in the nuclei. Wild-type (A) showed weak signal due to cross-reaction with the endogenous mouse PABPN1 protein. The muscles of two transgenic mice were strongly stained but showed different patterns. N33 (D) was stained in a bright patchy pattern in nuclear speckles, and staining in M43 (F) showed a pattern of intranuclear inclusions.

 
Electron microscopy shows intranuclear inclusions in the muscle of M43 mice
To obtain conclusive evidence of the existence of intranuclear inclusions—the distinctive hallmark of OPMD—we performed toluidine blue staining for epon-embedded semi-thin section of the soleus muscle of wild-type (Fig. 6A and D), N33 (Fig. 6B and E), and M43 (Fig. 6C and F) mice at the age of 6 months old. There were no abnormalities in the muscle nuclei of N33 mice (Fig. 6B and E) as well as wild-type mice (Fig. 6A and D). By contrast, only the M43 mouse muscle nuclei had a number of unique bright nuclei (Fig. 6C), which were occupied by an intranuclear clear zone, corresponding to large collections of filamentous inclusions (Fig. 6F). To clarify the time course of the formation of nuclear inclusions, we examined the soleus muscles from 1- and 3-week-old wild-type (Fig. 6G and I) and M43 mice (Fig. 6H and J). At 1 week of age, there was no nuclear inclusion in either wild-type (Fig. 6G) or M43 mice (Fig. 6I). At 3 weeks of age, intranuclear inclusions were found in the muscle fibers of M43 (Fig. 6J), but not in wild-type mice (Fig. 6H). These results indicate that intranuclear inclusions develop as early as 3 weeks of age.



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  		Figure 6. Detection of intranuclear inclusions in muscle nuclei of transgenic mice by staining with toluidine blue. Semi-thin sections of the soleus muscle of 6-month-old wild-type (A, D), N33 (B, E), and M43 mice (C, F) stained with toluidine blue. Muscle fiber nuclei (arrow) of M43 mice (C) showed large inclusion bodies with a pale appearance (F) as compared with N33 mice (B). Developmental change of soleus muscle (G–J). Sections from 1-week-old wild-type (G) and M43 mice (H) showed no difference and were normal. The muscle nuclei of 3-week-old M43 mice (J) were pale compared with wild-type mice (I) demonstrating the appearance of inclusion bodies. The eyelid muscle of 6-month-old wild-type (K) and M43 mice (L) stained with toluidine blue. The eyelid muscle nuclei of M43 (L) had inclusion bodies. The bar represents 10 µm.

 
We also examined the eyelid muscle of 6-month-old wild-type (Fig. 6K) and M43 (Fig. 6L) mice. Almost all the nuclei in M43 mice revealed pale zones, indicative of typical intranuclear inclusions (Fig. 6L). The inclusions were never observed in the fibroblasts, smooth muscle cells, endothelial cells or cells in other internal organs.

To confirm whether this area consisted of inclusion bodies with aggregated PABPN1, we examined the muscle nuclei of M43 mice by electron microscopy. As shown in Figure 7A and C, intranuclear inclusions consisting of clusters of ~8.5 nm unbranched tubular filaments were observed in the nuclei of the muscle fibers of M43 mice. In comparison with typical intranuclear inclusions of human OPMD muscles (Fig. 7B and D), the filament length in the M43 line seemed to be shorter than that in human OPMD (up to 0.25 µm). The inclusion filaments were less condensed and the formation of palisades or tangles was less evident under higher magnification. However, the appearance of a clear zone in the nuclei under lower magnification (Fig. 7A) was quite similar to that of OPMD muscles. Taken together, M43 transgenic mouse could be a good model for human OPMD.



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  		Figure 7. Electron microscopic analysis of the inclusion body. Soleus muscle of 6-month-old M43 (A, C) showing intranuclar inclusions, and biopsied deltoid muscle from OPMD patient with (GCG)11/(GCG)6 genotype obtained under informed consent (B, D). (A, B) x5000 magnification; (C, D) x75 000 magnification. The appearance with clear zones in the nuclei under lower magnification was quite similar between transgenic mouse muscle and OPMD muscle. Under higher magnification, the filaments of inclusions in the latter were less condensed, and the formation of palisades or tangles was less evident.

 
Detection of nuclear DNA fragmentation of soleusmuscles from M43 mice
Immunohistocehmical detection of apoptotic cells in the soleus muscles from N33 and M43 mice at the age of 6 months was carried out using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay. Both in N33 (Fig. 8A) and M43 mice muscles, scattered TUNEL-positive nuclei were detected rarely in the interstitium around non-degenerating fibers. In M43 mice, TUNEL-positive nuclei were slightly increased around degeneration fibers (Fig. 8B), and a cluster of TUNEL-positive nuclei was detected in muscle fibers showing more severe degeneration (Fig. 8C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have successfully produced transgenic mice expressing mutant hPABPN1 caused by polyalanine expansion, which showed myopathologic features similar to those of OPMD patients. The fact that the muscle nuclei of M43 mice expressing high levels of mutant hPABPN1 had intranuclear inclusions closely resembling those of OPMD patients is the most important evidence for establishing this mouse model of OPMD. The affected nuclei were restricted to the skeletal muscles, including the eyelid muscles and pharynx muscles, although mutant hPABPN1 gene was expressed in many tissues. Interestingly, the degree of muscle degeneration paralleled aging in the transgenic mice. Thus, we clearly established the first animal model of OPMD. The muscle specimen from 18-month-old mice showed apparently more severe myopathic changes. The OPMD model mice may have the more dramatic effect on myofiber viability than OPMD patients. Accordingly, our transgenic mouse model of OPMD can be a very useful resource for testing therapeutic strategies in the future.

We also generated two control transgenic mouse lines expressing normal hPABPN1. Although the degree of protein expression in N33 muscles was the same as that of M41 and M43 mice that carry mutant PABPN1 cDNA, the N33 mice did not show any myopathic changes or intranuclear inclusions in similarity with N35 mice expressing low levels of normal hPABPN1 protein. Thus, normal hPABPN1 may not aggregate to form inclusion bodies, even if the protein is produced at high levels. A similar observation has been reported in an experiment using COS-7 cells overexpressing normal hPABPN1 gene (21). Interestingly, M38 mice expressing mutant PABPN1 at low levels showed a completely normal phenotype. Therefore, we hypothesize that a certain level of expression of the expanded polyalanine stretch may be necessary to form intranuclear aggregation of PABPN1 in the muscle of transgenic OPMD model mice.

In human heterozygous patients with the (GCG)9/(GCG)6 genotype, the symptoms usually appear in the sixth decade with progressive swallowing difficulties and eyelid drooping (9,13). Proximal dominant muscle weakness is followed by a long disease course with gradual progression (5,31). In our transgenic mouse model, it is difficult to determine the age of onset of initial symptoms, because of the difficulty of examining swallowing and eyelid drooping. Therefore, we used a muscle fatigability test for the evaluation. The results indicated that limb muscle weakness occurred in M43 transgenic mice. On histological examination, the time course of nuclear inclusion formation in skeletal muscle was observed as early as 3 weeks of age. Thus, the disease onset in transgenic mice seems to be earlier than that in human OPMD. The early formation of inclusion bodies in transgenic mice may be due to the overexpression of mutant hPABPN1 by the strong CAG promoter. The M43 transgenic mice expressed much higher levels of mutant hPABPN1 not only in comparison with endogenous normal PABPN1, but also with mutant hPABPN1 in OPMD patients. As shown in Figure 6B and E, the mice expressing high levels of mutant hPABPN1 formed intranuclear inclusion bodies among a number of muscle fibers, resulting in dramatic muscle fiber changes. Actually, in homozygous (GCG)9 patients with OPMD, the symptoms usually start between the age of 21 and 36 (32).

It is notable that about 50% of M43 mice died by the age of 3 weeks with growth retardation. The cause of these deaths was uncertain. M43 transgenic mice did not show any histopathological abnormalities in the internal organs, except for skeletal muscle. Since the intranuclear inclusions were detected in mice older than 3 weeks, it is possible that dysphagia might have occurred immediately after birth, resulting in malnutrition followed by death.

Current data from OPMD cell models indicates the possibility that overexpression of various types of chaperones reduces aggregate formation and cell death (22). In addition, complementary data suggest that the inactivation of oligomerization of mutant PABPN1 prevents intranuclear aggregation and reduces cell death. This evidence corroborates the hypothesis that intranuclear aggregation of the mutant PABPN1 protein is likely to be a critical event in initiating OPMD pathogenesis. Several investigations have suggested that the mutant proteins are much more prone to aggregate formation than the wild-type counterparts and induce more cell death (22,23). The OPMD model mice revealed that TUNEL-positive nuclei were detected during degeneration process. On the other hand, other experiments using human muscle samples from OPMD patients indicate that cell death may be caused by a non-apoptotic mechanism (31). This raises the possibilities of a species-specific effect in mice and/or forced expression effect by a strong promoter in mutant hPABPN1 mice. We speculate that the cell death phenomenon may be emphasized in our model mice because they showed a more rapid and progressive degeneration on myofiber than human OPMD patients. Degeneration has generally been accepted to occur by necrotic pathways, but during the phase of acute muscle degeneration in mdx mouse, apoptosis precedes necrotic cell death during the phase of acute muscle degeneration (33). In human Duchenne muscular dystrophy, apoptotic nuclei were detected in mainly in the interstitum, and apoptotic cells in the interstitium were identified as inflammatory cells and activated satellite cells (34). Satellite cells were related to repair or regeneration of skeletal muscle, and Pax7 localized in satellite cells and their daughter myogenic precursor cells (35). In our preliminary experiments, we stained the adjacent sections of Figure 8C with Pax7, but TUNEL-positive nuclei were negative (data not shown). Taken together, further evidence is needed to clarify the mechanism of OPMD cell death, and our model mouse is a useful tool for this purpose and for constructing therapeutic trials.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of transgene
The normal and mutant hPABPN1 cDNA were described previously (36). The PABPN1 cDNA was subcloned into the EcoRI site of the transgenic expression vector pCAGGS (37). In the pCAGGS vector, the CAG promoter sequence is located upstream of the EcoRI site and the rabbit ß-globin poly(A) sequence is located downstream. The resulting plasmid was digested with SalI and HindIII to isolate the transgene cassette consisting of the CAG promoter, the hPABPN1 cDNA, and a rabbit ß-globin poly(A) sequence. To generate transgenic mice, the DNA fragments were separated by agarose gel electrophoresis and passed through a Sephaglas Band Prep Kit (Amersham Biosciences, NJ, USA). The recovered DNA was resuspended in 1 mM Tris–HCl (pH 7.5, containing 0.1 mM EDTA) at 5 ng/µl and used for microinjection.

Production of transgenic mice
BDF1 mice of either sex were purchased from CLEA Japan (Tokyo, Japan). For microinjection, fertilized eggs were collected and pronuclear injection was performed according to the standard procedure (38). Transgenic founder mice were then back-crossed with C57BL/6 mice at the Center for Animal Resources and Development.

Genotyping by PCR and Southern blot analysis
DNA was purified from mouse tails by lysis for 5 h at 55°C in a buffer containing proteinase K, followed by phenol–chloroform extraction and isopropanol precipitation. To detect the transgene, 0.1 µg of genomic DNA was incubated under the following PCR conditions: 94°C for 1 min; 94°C for 1 min, 58°C for 2 min, 72°C for 2 min for 28 cycles; then 72°C for 10 min. The two primer sets were; 5'-ACCTTCTGATAG-GCCG-3' and 5'-ACCAGCAGAAGAGCTGGAAG-3'. For Southern blot analysis, 6 µg of genomic DNA were digested with the appropriate enzymes, electrophoresed on a 1% agarose gel and blotted onto a nylon membrane (Roche Diagnostics, Tokyo, Japan). Hybridization was performed using a DIG labeled probe of full-length hPABPN1 cDNA using DIG DNA labeling and detection kit (Roche Diagnostics).

Northern blot analysis
A 0.1 µg sample of mRNA was extracted from transgenic and control mouse tissues, and separated on 1% agarose gels containing 18% formaldehyde and then blotted onto a nylon membrane (Roche Diagnostics). After prehybridization with SDS buffer (50% formamide, 0.1% N-laurylsarcosine, 2% SDS, 4xSSC, 2% blocking reagent) for 2 h at 68°C, hybridization was performed overnight with RNA probe and a detection kit (Roche Diagnostics). Subclonoes of HincII/EcoRI fragment at 3' end of hPABPN1 cDNA were subcloned into pBluescriptsII and linearized to transcribe digoxigenin-labeled antisense RNA probe.

Western blot analysis
Quadriceps femoris muscle was homogenized in NP-40 Lysis Buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) and 1% Protease Inhibitor Cocktail (Nacarai Tesque Inc., Kyoto, Japan) on ice. After the clarification by centrifugation at 15 000 rpm for 5 min, the 16 µg of supernatants were separated on 12% SDS–polyacrylamide gels and then electrophoretically transferred to PVDF membranes (Millipore Corporation, MA, USA). The membranes were blocked with 5% milk in PBS and immunoreacted with a rabbit polyclonal antibody against PABPN1 overnight at 4°C. After washing with PBS containing 0.1% Tween-20, the blots were incubated with rabbit IgG antibody. Immunoreactive bands were visualized with ECL plus Western Blot Detection Kit (Amersham Biosciences, NJ, USA).

LRT measurement
To determine the fatigability, the mice were positioned with their head hanging down on a wire net (10 cm in diameter) 30 cm above the floor, and the time until they fell off was measured. The LRT measurement was made only once unless it was less than 60 s.

Histopathological analysis
H&E staining and immunocytochemistry were performed on 8 µm transverse cryostat sections of frozen mouse soleus muscle following quick immersion in isopentane chilled in liquid nitrogen and storage at –80°C. PABPN1 was visualized using a polyclonal serum previously described by Krause et al. (30). For immunocytochemistry, sections were mounted on slides and fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min and washed three times for 10 min each in PBS containing 0.1 M glycine. The sections were then rinsed in PBS containing 0.1% Tween 20 (PBST) and incubated with a rabbit polyclonal PABPN1 antibody in a humid chamber overnight at 4°C. After washing in PBST (three times for 15 min each), the samples were incubated with FITC-conjugated anti-rabbit IgG antibody (Molecular Probes, Inc., OR, USA) for 30 min, washed in PBST (twice for 10 min each), rinsed in PBS (5 min) and mounted in Vectashield (Vector, Peterborough, UK). TUNEL analysis was performed using an in situ apoptosis detection kit (Wako Pure Chemical Industries, Osaka, Japan) according to the instructions of the manufacturer.

Electron microscopy
Muscle biopsies were fixed with 10% paraformaldehyde in 0.2 M sodium cacodylate and 2.5% glutaraldehyde and post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate for 2 h at room temperature. After dehydration in a graded series of ethanol and propylene oxide, the samples were embedded in epoxy resin. For light microscopy, 1 µm sections of the samples were stained with 1% toluidine blue. Ultra-thin sections stained with uranyl acetate and lead citrate were observed with an H-7500 electron microscope (Hitachi, Tokyo, Japan).


   ACKNOWLEDGEMENTS
 
The authors thank Drs Y.-J. Kim and K. Arahata (National Center of Neurology and Psychiatry, Tokyo, Japan) for providing human PABPN1 cDNA, Drs U. Kuhn and E. Wahle (Universitat Halle, Germany) for providing anti-PABPN1 antibody, and S. Okamoto, M. Nakata and I. Kawasaki for technical assistance with the histopathological examination. This work was supported, in part, by a Grants-in-aid on Priority Areas to K.Y., and by Grants-in-aid (C) to E.U. from the Ministry of Education, Science, Culture and Sports of Japan Scientific Research and the Research Grant (11-1) for Nervous and Mental Disorders to M.U. from the Ministry of Health, Labour and Welfare, Japan.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +81 963736083; Fax: +81 963736599; Email: yamamura{at}gpo.kumamoto-u.ac.jp


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TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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