Laminin α2 deficiency and muscular dystrophy; genotype-phenotype correlation in mutant mice

Neuromuscular Disorders 13 (2003) 207–215 www.elsevier.com/locate/nmd

Laminin a2 deficiency and muscular dystrophy; genotype-phenotype correlation in mutant mice L.T. Guo a,1, X.U. Zhang a,1, W. Kuang a,1, H. Xu a,1, L.A. Liu a,1, J.-T. Vilquin b, Y. MiyagoeSuzuki c, S. Takeda c, M.A. Ruegg d, U.M. Wewer e, E. Engvall a,1,* a

The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA b Institut de Myologie, Hospitalier Pitie-Salpetriere, Paris, France c Department of Molecular Therapy, National Institute for Neuroscience, Kodaira, Japan d Department of Pharmacology/Neurobiology, Biozentrum, University of Basel, Basel, Switzerland e Institute of Molecular Pathology, University of Copenhagen, Denmark Received 13 June 2002; received in revised form 7 October 2002; accepted 1 November 2002

Abstract Deficiency of laminin a2 is the cause of one of the most severe muscular dystrophies in humans and other species. It is not yet clear how particular mutations in the laminin a2 chain gene affect protein expression, and how abnormal levels or structure of the protein affect disease. Animal models may be valuable for such genotype-phenotype analysis and for determining mechanism of disease as well as function of laminin. Here, we have analyzed protein expression in three lines of mice with mutations in the laminin a2 chain gene and in two lines of transgenic mice overexpressing the human laminin a2 chain gene in skeletal muscle. The dy 3K/dy 3K experimental mutant mice are completely deficient in laminin a2; the dy/dy spontaneous mutant mice have small amounts of apparently normal laminin; and the dy W/dy W mice express even smaller amounts of a truncated laminin a2, lacking domain VI. Interestingly, all mutants lack laminin a2 in peripheral nerve. We have demonstrated previously, that overexpression of the human laminin a2 in skeletal muscle in dy 2J/dy 2J and dy W/dy W mice under the control of a striated muscle-specific creatine kinase promoter substantially prevented the muscular dystrophy in these mice. However, dy W/dy W mice, expressing the human laminin a2 under the control of the striated muscle-specific portion of the desmin promoter, still developed muscular dystrophy. This failure to rescue is apparently because of insufficient production of laminin a2. This study provides additional evidence that the amount of laminin a2 is most critical for the prevention of muscular dystrophy. These data may thus be of significance for attempts to treat congenital muscular dystrophy in human patients. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Laminin a2 chain gene; Muscular dystrophy; Mice

1. Introduction Muscular dystrophy is a group of disorders characterized by degeneration, regeneration, and fibrosis of striated muscle [1–6]. Over 30 genes have been identified causing muscular dystrophy when mutated. Many of these genes encode molecules necessary for the structural integrity of muscle cells and/or for muscle cell adhesion. Among the most severe forms of muscular dystrophy are the ones in which the major laminin in the muscle basement membrane, laminin containing the a2 chain, is affected [7,8]. Many distinct mutations in laminin a2 chain gene (LAMA2) have been reported [9–18]. Some of these mutations resulted in complete absence of laminin a2, while others * Corresponding author. Tel.: 11-858-646-3100; fax: 11-858-646-3199. E-mail address: [email protected] (E. Engvall). 1 http://www.burnham.org.

resulted in partial deficiency. The putative transcript or protein product has not always been characterized in cases where mutations in the LAMA2 gene have been identified. Therefore, it is not clear how particular mutations in LAMA2 affect protein expression, and how abnormal levels or structure of the protein affect disease [19]. An allelic series of mutations in experimental animals may facilitate genotypephenotype correlation and shed new light on disease processes [20]. Laminin a2 deficiency causes muscular dystrophy in many other species than humans, including dogs [20,21], cats [22], and mice. A number of spontaneous and experimental mutations in laminin a2 exist in the mouse, including dy/dy, dy 2J/dy 2J, dy 3K/dy 3K, dy W/dy W, and dy Pas/dy Pas. The dy/dy and dy 2J/dy 2J mice were characterized a long time ago without knowledge of the molecular defect [23,24], and the mice were thought to be models for dystrophin-deficient

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Duchenne Muscular Dystrophy. It was much later that the dy/dy mice were shown to be deficient in laminin a2 and not in dystrophin [25,26]. The dy/dy mice express a laminin a2 polypeptide of apparent normal size but in very small amounts, and the mice suffer from a very severe form of muscular dystrophy. The mutation in the dy/dy mice has not yet been identified. Next, it was shown that the dy 2J/dy 2J mice had a mutation in the Lama2 gene that resulted in abnormal splicing and production of a laminin a2 polypeptide that lacked the N-terminal domain VI [27,28]. The truncated laminin a2 was present in substantial amounts in the skeletal muscle of the dy 2J/dy 2J mice, and the muscular dystrophy of the mice was less severe than that of dy/dy mice. The dy Pas/dy Pas mice completely lack laminin a2 due to an insertion of a retrotransposon [29]. Two experimental Lama2 mutant mice have been generated by homologous recombination in embryonic stem cells, the dy 3K/dy 3K [30] and the dy W/dy W [31]. Both were intended to generate null alleles. Transgenic mice have been generated to express laminin a2 specifically in skeletal muscle of laminin a2-deficient mice with the help of the muscle-specific creatine kinase (MCK) promoter [31]. High level expression of the transgene in dy W/dy W mice prevented the development of muscular dystrophy in the mice and increased their wellbeing and longevity but, as expected, did not correct the neuropathy [31]. However, the transgenic dy W/dy W mice did develop a mild form of myopathy characterized by elevated serum creatine kinase (CK) levels and the presence of centrally located muclei in muscle fibers [31]. It was unclear if the myopathy resulted from the neuropathy in these mice, from too much or too little laminin a2 being produced in the muscle, or from the abnormal timing of expression. A promoter active earlier in myogenesis than the MCK promoter may be advantageous for expression of laminin a2, and the desmin promoter may be such a promoter [32]. We show here, that the dy 3K/dy 3K mouse is indeed a null mutant, as originally reported [30], but the dy W/dy W is not; a very small amount of truncated laminin a2 is produced in the dy W/dy W muscle. The desmin promoter used to generate a new line of transgenic mice provided striated musclespecific expression of laminin a2, but the amount of laminin a2 was not sufficient to correct the dystrophy in dy W/dy W mice. The present and previous data indicate, that nearphysiological amounts of laminin a2 are needed in muscle to prevent or correct muscular dystrophy, and that high levels of laminin a2 may be difficult to obtain with current techniques. Nevertheless, low levels of laminin a2 may be better for muscle function than complete absence.

2. Materials and methods 2.1. Dystrophic and transgenic mice dy 3K/dy 3K [30] and dy W/dy W [31] mice were generated as

reported. Samples of muscle from dy/dy mice (Jackson Laboratories) were provided by Dr Frederica Montanaro. The generation of transgenic mice expressing human laminin a2 chain in skeletal muscle (MCK-LAMA2) and breeding to the dy W/dy W mice to generate dy W/dy W; MCK-LAMA2 mice have been described [31]. We here generated mice expressing a human laminin a2 with the help of a striated muscle-specific desmin promoter [32]. The plasmid pHuDes2.2, which contains the portion of the human desmin promoter that drives the expression in striated muscle but not in smooth muscle, was a gift from Dr Zhen Li [32]. A 0.7 Kb KpnI/HindIII DNA fragment and a 1 Kb KpnI/BglII DNA fragment were released from pHuDes2.2 and inserted into the HindIII/BamHI sites of a plasmid, which contains the human laminin a2 complementary DNA (cDNA) sequence from ATG to a NotI digestion site at nt 119 [33] to generate the plasmid pDesLS. pDesLS was digested by SalI/NotI, and a 1.9 Kb DNA fragment containing the desmin promoter plus the 5 0 end of human laminin a2 cDNA was ligated with the 3 0 end of the human laminin a2 cDNA from the SalI/NotI digested plasmid pCCLMCK-ha2 [31]. This generated a plasmid pDesLC, which contains the desmin promoter and the full length human laminin a2 cDNA. A 13.4 Kb Des-LAMA2 cDNA fragment was released with SalI/SwaI, and the purified fragment was microinjected into fertilized oocytes from mice of strain FVB/N. Viable embryos were implanted into pseudopregnant recipients and allowed to develop to term. Five founders were identified and used for breeding. The transgenic offspring with the highest expression of laminin a2 in skeletal muscle were identified by immunofluorescence with human specific antibodies and used to develop a line of transgenic mice: 1/1; Des-LAMA2. These mice were subsequently mated with heterozygous 1/dy W mice to obtain, after two generations of breeding, dy W/dy W; DesLAMA2 transgenic mice that were homozygous for the dy W mutation and heterozygous for Des-LAMA2.

2.2. Recombinant laminin a 2 domain VI and antibody production Recombinant mouse laminin a2 domain VI with a histidine tag at the C-terminus was produced in HEK 293EBNA cells. cDNA coding for residues 1–324 [34] was generated by reverse transcription-polymerase chain reaction (RTPCR) using RNA isolated from mouse heart. The cDNA was inserted into the pCEP4 vector (Invitrogen), and DNA was transfected into 293 cells with the help of lipofectamine. After selection and cloning, a clone of cells expressing high levels of domain VI was chosen for large scale culture. Domain VI was purified from serum-free culture medium by affinity chromatography on Ni-Sepharose. An antiserum was produced by injection of 0.5 mg of purified domain VI, emulsified in Freund’s adjuvant, subcutaneously into rabbits at monthly intervals. The antiserum

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Fig. 1. Indirect immunofluorescence on leg muscle from different mutant mice. Affinity purified, polyclonal rabbit antibodies to domains VI and G and monoclonal mouse antibodies to domain IVa–IIIa were used to detect the corresponding domains in wild type (wt) and in mutant dy/dy, dy W/dy W, and dy 3K/dy 3K mice. Muscle from dy/dy mice have reduced levels of apparently full length laminin a2; muscle from dy W/dy W mice have further reduced levels of laminin a2 that lacks domain VI; dy 3K/dy 3K muscle has no detectable laminin a2.

was absorbed with bovine serum albumin and affinity purified on purified domain VI coupled to Sepharose. 2.3. Other antibodies Antibodies against laminin a2 G domain repeats 4–5 (‘anti-80 kD’) and against a peptide sequence in G repeat 2 (‘anti-300 K’) have been described and used previously [31]. Antiserum against recombinant G-domain was a kind gift of Dr Peter Yurchenco [35]. Monoclonal rat antibody 4H8-2 to an epitope within domain IVa–IIIa in human and mouse laminin a2 [10] was purchased from Alexis Biochemicals. Mouse monoclonal antibodies 5H2 and 2G9 against human laminin a2 G-domain repeat 4–5 have been described [36]. 2.4. Immunofluorescence Samples of muscle were collected and immediately frozen in isopentane chilled in liquid N2. Ten mm sections were cut on a cryostat, dried at room temperature, fixed in ice cold acetone for 10 min, and washed in phosphate buffered saline, PBS. Antibodies were diluted in 10 % normal goat serum, applied to the sections at 1:200 (4H8)

or 1:100 (anti-G repeat 4–5 and anti-domain VI), and incubated over night in the cold. After washing in PBS, goat anti-rat IgG or anti-rabbit IgG (Jackson Immuno Research Laboratories, Inc) diluted 1:200 in 10% normal goat serum in PBS was applied and incubated for 1 h at room temperature. Slides were washed in PBS and sections were mounted and examined in an inverted fluorescent microscope. The monoclonal antibodies 5H2 and 2G9 were used as undiluted hybridoma culture medium together with a kit (‘M.O.M.’) from Vector. 2.5. Immunoblotting Samples of mouse skeletal muscle were ground to a powder in liquid N2, mixed with sample buffer (0.125 M Tris–HCI, 4% sodium dodecyl sulfate (SDS), 10% glycerol, 1% b-mercaptoethanol, and 0.1 M phenylmethylsulphonylfluoride), and boiled for 5 min. For some experiments, muscle samples were extracted with EDTA to solubilize laminin, and glycoproteins including laminin were enriched by binding to Concanavalin A Sepharose after addition of CaCl2. Twenty mg of SDS-extracted protein or EDTAextracted and ConA-enriched protein were loaded on 4–

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kinase activity with the CK520 kit from Sigma. Values between groups were compared by using non-parametric Mann–Whitney test. 3. Results 3.1. Laminin a 2 expression in mutant mice

Fig. 2. (a) Immunoblotting of muscle extract from wild type, heterozygous 1 /dy W, and homozygous dy W/dy W mice. Extract from muscle was prepared as described in Section 2 and fractionated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and stained with polyclonal antibodies. (b) dy W/dy W mice express Lama2 with a G-domain but not with a domain VI. RT-PCR with RNA from muscle from homozygous mutant dy W/dy W mice and wild type littermates with primers from domain VI or the G domain.

12% acrylamide gels and electrophoresed. Proteins were then transferred electrophoretically to nitrocellulose membranes (BioRad). The membranes were treated with 10% non-fat milk powder in Tris buffer and then incubated with antibodies at 1:500 (anti-80 and anti-300 K) or 1:100 (2G9 hybridoma culture medium). After washing, the membranes were incubated with 1:2000 goat anti-rabbit or anti-mouse IgG labeled with peroxidase. Bound peroxidase was detected by enhanced chemiluminescene with a kit from Amersham Pharmacia Biotech. 2.6. RT-PCR Total RNA was isolated from leg muscle of 6 weeks old mice with TRIzol reagent (GibcoBRL). First strand cDNA were synthesized using oligo-dT and SUPERSCRIPT II, RNase H Reverse Transcriptase (GibcoBRL). Specific primers were designed according to published mouse cDNA sequence (GenBank, MMU12147). Twenty-two pairs of primers corresponding to various domains of laminin a2 chain were used. At least one intron was included for each expected PCR product, in order to eliminate effects of possible genomic DNA contamination. Each PCR reaction was programmed according the melting temperatures of primers. Generally, DNA was denatured initially at 948 for 5 min, followed by 35 cycles of 948 for 30 s, 55–628 for 60 s, and 728 for 60 s. RT-PCR products were analyzed on 1% agarose-gels. 2.7. Measurement of serum levels of creatine kinase and statistical analysis A total of 20 ml of serum was used to measure creatine

We used affinity-purified rabbit polyclonal antibodies to domain VI and G and rat monoclonal antibodies to domain IVa–IIIa to re-evaluate and compare the expression of laminin a2 in muscle from wild type and mutant mice (Fig. 1). Muscle from wild type mice was positively stained with all antibodies as expected. Muscle from dy/dy mice was also positive with all antibodies, but the intensity of the staining was much lower than that of muscle from wild type mice. This is in agreement with dy/dy mice producing small amounts of apparently normal laminin a2 in muscle [25,26]. When muscle from dy W/dy W mice was tested, no staining was obtained with antibodies to domain VI as expected. However, with antibodies to domains IVa–IIIa and G, some staining of low intensity was seen. This pattern of staining suggested, that the dy W/dy W mice may not be complete knockouts but may express a small amount of a truncated laminin a2. The dy 3K/dy 3K muscle was negative with all antisera, indicating that these mice are completely null for laminin a2, as reported [30]. 3.2. The dy W/dy W mouse expresses small amounts of truncated laminin a 2 To confirm and characterize further the expression of laminin a2 in dy W/dy W mice shown by the staining patterns, we performed immunoblotting and RT-PCR. In accordance to previous results [31], we could not detect the 300 or 80 kD segments of laminin a2 in dy W/dy W muscle by immunoblotting (Fig. 2a). Furthermore, we could not detect any lower molecular weight products of the 300 kD segment (not shown), as would be expected from a possible truncation of the laminin a2 chain as suggested by the immunofluorescence results. Apparently, the amount of laminin a2 in dy W/dy W muscle is very low, and/or the protein is degraded. However, based on the staining with the various antibodies, some protein should be expressed in muscle, and a transcript coding for domain G but not domain VI should be expressed. Therefore, we performed RT-PCR to detect the corresponding sequences in messenger RNA (mRNA). Fig. 2b shows the results with primers from the domains VI and G. No PCR product could be detected representing domain VI in dy W/dy W muscle, but PCR fragments of different intensities representing the G domain were detected in all samples of varying amounts. The results suggest that dy W/dy W mice express mutant laminin a2, which lacks domain VI but contains the G domain. To attempt to define the nature of the transcript(s) further we performed PCR with overlapping primers, representing

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Fig. 3. Lack of detectable laminin a2 in peripheral nerve of dy W/dy W mice. Muscle tissue with peripheral nerve was stained with monoclonal antibody 4H8. dy W/dy W and dy W/dy W; Des-LAMA2 muscle show staining in skeletal muscle but not in peripheral nerve.

sequences in domains IV, III, and II/I. However, we could not detect any such PCR fragments in samples from homozygous mutant mice; we only detected the fragments in samples from wild type and heterozygous mice (not shown). It has been noted before that laminin a2 can not be detected in peripheral nerve in dy/dy and dy 2J/dy 2J mice, although it is detectable in skeletal muscle in these mice [25,37]. The dy W/dy W mice, although they did contain detectable laminin a2 G domain in skeletal muscle, did not contain any G domain (Fig. 3) or domain IVa–IIIa (not shown) in peripheral nerve. In this context it is of interest to note that several attempts to express a laminin a2 transgene in peripheral nerve have failed. In these experiments, three different promoters were used, the P0 [38] and glial fibrillary acidic protein (GFAP) [39] promoters and a partial collagen VI promoter [40]. However, in no case was any detectable laminin a2 produced (data not shown). 3.3. Generation of a new laminin a 2 transgenic mouse using a desmin promoter To address questions about the possible importance of timing and level of Lama2 expression for muscle function, we generated a new line of transgenic mice that expressed the human laminin a2 chain in skeletal muscle with the help of a striated muscle-specific partial desmin promoter [32]. Human laminin a2 was detected extracellulary in skeletal muscle of the mice (Fig. 4a), indicating assembly of the human laminin a2 chain with mouse laminin b and g chains. The staining observed was patchy with some fibers being more strongly stained than others. In dy W/dy W; DesLAMA2 mice, muscle fibers were often larger than those in

wild type mice or dy W/dy W mice indicating hypertrophy. Hematoxylin and eosin staining (Fig. 4b) also showed areas of hypertrophy, in addition to areas of degeneration and regeneration similar to what was seen in dy W/dy W mice (Fig. 4b). To evaluate the general condition of the transgenic dy W/ W dy ; Des-LAMA2 mice, we used weight gain as a measure of general wellbeing. The dy W/dy W dystrophic mice stop gaining weight at 2–3 weeks of age [31,41], when muscle degeneration becomes acute. The dy W/dy W; Des-LAMA2 trangenic mice showed a slight tendency to gain more weight than the dy W/dy W mice without the transgene but were still far behind wild type litter mates in postnatal growth (not shown). As the transgenic mice were generated on FVB/N background, and the dy W/dy W mice were of a mixed 129/SVJ and C57BL/ 6J background, the weights and weight gains of even the wild type litter mates were highly variable. To address more specifically the state of muscle, serum levels of CK were used instead to measure muscle integrity in the mice of different genotypes. The homozygous mutant dy W/dy W mice with the Des-LAMA2 transgene had somewhat lower levels of CK than those without (Fig. 4c), suggesting that the expression of human laminin a2 chain increased muscle stability but only to a small degree. However, the differences were not statistically significant (P ¼ 0:1). That the Des-LAMA2 gene did not have the same beneficial effect on dy W/dy W muscle as the MCK-LAMA2 gene [31] suggested that the level of expression of laminin a2 may be too low. Therefore, we compared the amount of laminin a2 in muscle from mice of the two transgenic lines by using monoclonal human-specific antibodies in immunoblotting. Fig. 4d shows that muscle from DesLAMA2 mice contained about 1/3 of the amount of human

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Fig. 4. (a) Indirect Immunofluorescence on muscle from transgenic mice with monoclonal antibodies to human laminin. Mice with the Des-LAMA2 transgene express human laminin a2 with a somewhat uneven distribution around muscle fibers. (b) Histology of muscle from transgenic mice. Hematoxylin and eosin staining of section of fresh frozen muscle from Des-LAMA2 transgenic mice on a wild type or dy W/dy W background. dy W/dy W mice show characteristic fiber size variation, degeneration, some regeneration, and fibrosis. dy W/dy W mice with the transgene show also fibers size variation with more large hypertrophic fibers and show less fibrosis. (c) Serum creatine kinase in wild type and dy W/dy W mice with or without the Des-LAMA2 transgene at 6 weeks. Bars show mean 1/2 standard deviation of the levels in the indicated number of mice (n). Expression of human laminin a2 driven by the desmin promoter does not prevent muscle damage as evidenced by increased levels of CK. (d) Immunoblotting of transgenic mouse muscle with human-specific monoclonal antibodies. Human laminin 80 kD fragment of laminin a2 can be detected in both MCK-LAMA2 (MCK; 2nd lane) and Des-LAMA2 (DES; 3rd lane) lines of transgenic mice, but not in wild type mice (WT; 4th lane), and the amount of the fragment is higher in the mice with the MCK promoter. The endogenous mouse IgG heavy chain band at 50 kDa, detected by the anti-mouse IgG secondary antibody conjugate, served as internal loading control.

laminin in the MCK-LAMA2 mice. Judging from the high CK values in dy W/dy W; Des-LAMA2 mice, this amount is not enough to prevent the pathology of muscle.

4. Discussion The dystrophic mice, dy/dy, dy 2J/dy 2J, dy 3K/dy 3K, dy W

/dy W, and dy Pas/dy Pas, constitute a allelic series ranging from complete absence of laminin a2 (dy 3K/dy 3K and dy Pas/dy Pas) to reduced levels (dy/dy), to truncated protein (dy 2J/dy 2J), to reduced levels of truncated protein (dy W/dy W) (Table 1). To this series can now be added the transgenic dy W/dy W; DesLAMA2, as they produce a normal laminin a2 chain at subnormal levels, too low to prevent the development of muscular dystrophy in mice.

Table 1 Mice with spontaneous or experimental mutations in laminin a2 Mouse strain

Type of mutation

Protein product

Phenotype

Reference

dy dy 2J dy 3K dy W dy Pas dy W; Des-LAMA2 dy W; MCK-LAMA2

Unknown Point mutation Insertion LacZ, Neo insertion Retrovirus insertion Transgene addition Transgene addition

Normal low level Near-normal lacks domain VI No protein Low level lacks domain VI No protein Normal human low level Normal human; moderate level

Severe Less severe Severe Severe Severe Severe Mild to none

Michelson et al. [23] Xu et al. [27] Miyagoe et al. [30] Kuang et al. [31] Besse et al. [29] This communication Kuang et al. [31]

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4.1. Low level and abnormal laminin a 2 expressed in dy W/ dy W mice The dy W/dy W mice were generated with the intent of creating a null-mutant mouse [31]. The LAMA2 gene was interrupted with the LacZ and Neo genes, so that the LacZ gene could be used as reporter gene to follow expression of laminin a2 during muscle development and regeneration in heterozygous and homozygous mice [42]. Although the expression of the reporter gene seemed to mimic the expression of LAMA2, it was noted that the sensitivity of detection of b-galactosidase was not very high [42]. It was unclear if the low expression of LacZ reflected genuinely low expression of LAMA2 or was caused by other factors, such as the LacZ being silenced or eliminated. Later, we noticed that antibodies raised against purified G-repeats 4– 5 sometimes weakly stained dy W/dy W mouse muscle in immunofluorescence. In contrast, affinity purified antibodies to recombinant domain VI did not stain the dy W/dy W muscle. As the G-repeat antiserum was raised against a fragment of laminin a2 purified from human placenta [36], and this fragment may have contained impurities of unknown nature, it was impossible to ensure the absolute specificity of the antiserum. In contrast, production of domain VI as a recombinant protein in 293 cells allowed to control the purity of the immunogen and the specificity of the antiserum much more rigorously. The lack of staining of dy W/dy W muscle with these antibodies indicated that dy W/dy W mice were completely deficient in laminin a2. With the availability of the monoclonal antibody 4H8, which reacts very strongly with an epitope in the domain IVa–IIIa region of both human and mouse laminin a2 [10], it became apparent that dy W/dy W mice may not be ‘knockouts’ but contain a laminin a2 with at least the domains IVa–IIIa and G. Alternatively, the result could be explained by the expression of a protein that crossreacts with 4H8. Other laminins related to laminin a2 are expressed in dy/dy mice [43–45], but these laminins do not crossreact with the 4H8 antibody. The staining results with various antibodies are consistent with the dy W/dy W mouse expressing low amounts of a truncated laminin a2. These data were corroborated by the RT-PCR results, showing expression of mRNA encoding domain G but not domain VI. The lack of detection of a specific truncated protein in immunoblotting, and of specific PCR products for other domains than the G domain, may suggest that multiple abnormal splice products rather than a single splice product may be produced. Multiple alternative splice products of the dystrophin gene have been demonstrated in cells originating from dystrophin null mice [46]. Although the exact genetic aberration in dy W/dy W mice is not known, based on the low expression of abnormal protein, the dy W/dy W mouse adds another mouse strain to the dy series and another model for laminin a2deficient muscular dystrophy and for laminin a2 function.

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4.2. Comparison of dy W/dy W and dy 2J/dy 2J mice The laminin a2 protein expressed in the dy W/dy W mice resembles the laminin a2 in the dy 2J/dy 2J mice in that it lacks domain VI, but it is present at lower levels in muscle. The relatively well expressed but truncated laminin a2 in dy 2J/dy 2J mice shows the importance of domain VI for basement membrane formation, because basement membranes are defective in the dy 2J/dy 2J mice [27]. Nevertheless, the dy 2J/dy 2J mice have a relatively mild form of muscular dystrophy. They have a longer life span [24] and at least the females can be bred and produce and rear offspring [27]. The dy W/dy W mice have much lower levels of their truncated laminin a2 than the dy 2J/dy 2J mice, and the disease in these mice appears to be as severe as that of the dy 3K/dy 3K mice, which completely lack laminin a2 [30,31]. Although a direct comparison of dy 3K/dy 3K and dy W/dy W has not been carried out, the severe phenotype in dy W/dy W mice may be related more to the low amounts of laminin a2 protein than to the lack of domain VI in the protein. This raises the question as to how much laminin is needed to prevent muscle degeneration. 4.3. Insufficient laminin a 2 production driven by a Desmin promoter The dy W/dy W; Des-LAMA2 transgenic mice were generated with the intention to express the normal LAMA2 transgene earlier in myogenesis than could be accomplished with the MCK promoter. We have not confirmed the intended early expression, because it became apparent that we failed to obtain sufficient levels of expression of LAMA2 in the desmin promoter mice in order to avert muscle degeneration. The phenotype of the mice shows that levels of laminin a2, that are high enough to be detected by both immunofluorescence and immunoblotting but are subnormal, are not sufficient in preventing muscular dystrophy and early death. In addition, the expression of the transgene was variable between muscle fibers. The reason for this apparent mosaic expression is not known but has been noted for other transgenes [47]. We did see a slight improvement in overall wellbeing and in muscle stability, as measured by serum CK levels, in the dy W/dy W mice with the transgene compared to those without. We conclude that this improvement is due to the low but definite expression of laminin a2. In this respect, the dy W/dy W; Des-LAMA2 mice resemble the dy/ dy mice, but with a little more laminin. We propose that the dy W/dy W; Des-LAMA2 mice be regarded as yet another member of the allelic dy series of mice (Table 1). The present and previous experiments have been aimed at replacing a mutant mouse laminin a2 chain in mice with a normal human laminin a2 chain. This strategy worked relatively well with the MCK promoter [31] and less well with the desmin promoter (this study). The human laminin a2 chain may not assemble as efficiently with the mouse laminin b and g chains as the mouse laminin a2. It is also

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possible that the endogenous mutant laminin a2 in the dy W/ dy W mouse acts in a dominant negative fashion on basement membrane assembly. To fully understand the effects of levels of expression of a therapeutic laminin a2 gene on muscle, it would be important to perform experiments with the mouse gene in the dy 3K/dy 3K null mutant mice. 4.4. Tissue specific expression of laminin a 2 Normally, laminin a2 is expressed in peripheral nerve in addition to striated muscle. However, the expression of laminin a2 in nerve appears to be controlled differently from the expression in muscle, because the peripheral nerves are completely negative for laminin a2 in the dy W/ dy W mice as well as in both the dy/dy and dy 2J/dy 2J lines of mice [25,27,37]. Interestingly, all our attempts to express the laminin a2 cDNA in peripheral nerves with three different proven promoters failed. The mechanisms for expression or assembly of laminin a2 in muscle and nerve appear different, as may be the case also in humans [16]. Perhaps an element necessary for Schwann cell expression of laminin a2 is lacking in the mutant dy, dy 2J, and dy W genes as well as from the human laminin a2 cDNA. In order to correct laminin a2 deficiency in the future, it may be necessary to determine precisely what factors normally determine the levels and sites of expression of laminin a2 not only in muscle but also in other tissues where laminin a2 is normally expressed. Acknowledgements We thank Dr Greg Lemke for the P0 promoter, Dr Lennart Mucke for the GFAP promoter, Dr Giorgio Bressan for the collagen VI promoter, Dr Peter Yurchenco for antibodies to laminin a2 G domain, Dr Frederica Montanaro for dy/dy muscle tissue, and Dr Ling Wang for generating transgenic mice. This work was supported by grants from the Japan Ministry of Education, Culture, Sports, Science and Technology (10557065), and from the Human Frontier Science Project to ST, from the Swiss Foundation for Research on Muscle Diseases to MAR, from the European Union (Quality of Life and Management of Living Resources; contract no. QLG1-CT-1999-00870) to UMW, and from the National Institutes of Health to EE. References [1] Voit T. Congenital muscular dystrophies: 1997 update. Brain Dev 1998;20:65–74. [2] Dubowitz V. Congenital muscular dystrophy: an expanding clinical syndrome. Ann Neurol 2000;47:143–144. [3] Cohn RD, Campbell KP. Molecular basis of muscular dystrophies. Muscle Nerve 2000;23:1456–1471. [4] Tubridy N, Fontaine B, Eymard B. Congenital myopathies and congenital muscular dystrophies. Curr Opin Neurol 2001;14:575–582. [5] O’Brien KF, Kunkel LM. Dystrophin and muscular dystrophy: past, present, and future. Mol Genet Metab 2001;74:75–88.

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