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1998 Oxford University Press Human Molecular Genetics, 1998, Vol. 7, No. 12 1927–1933 The distribution of SMN protein complex in human fetal tissues and its alteration in spinal muscular atrophy P. Burlet, C. Huber, S. Bertrandy, M. A. Ludosky1, I. Zwaenepoel, O. Clermont, J. Roume2, A. L. Delezoide, J. Cartaud1, A. Munnich and S. Lefebvre* Unité de Recherches sur les Handicaps Génétiques de l’Enfant, INSERM Unité 393, IFREM, Institut Necker, Hôpital des Enfants Malades, 149 rue de Sèvres, 75743 Paris cedex 15, France, 1Département de Biologie Supramoléculaire et Cellulaire, Institut Jacques Monod, CNRS, Université Paris 7, Paris, France and 2Unité de Foetopathologie, Hôpital Saint-Antoine, Paris, France Received June 23, 1998; Revised and Accepted August 7, 1998 Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of motor neurons of the spinal cord and muscular atrophy. SMA is caused by alterations to the survival of motor neuron (SMN) gene, the function of which has hitherto been unclear. Here, we present immunoblot analyses showing that normal SMN protein expression undergoes a marked decay in the postnatal period compared with fetal development. Morphological and immunohistochemical analyses of the SMN protein in human fetal tissues showed a general distribution in the cytoplasm, except in muscle cells, where SMN protein was immunolocalized to large cytoplasmic dot-like structures and was tightly associated with membrane-free heavy sedimenting complexes. These cytoplasmic structures were similar in size to gem. The SMN protein was markedly deficient in tissues derived from type I SMA fetuses, including skeletal muscles and, as previously shown, spinal cord. While our data do not help decide whether SMA results from impaired SMN expression in spinal cord, skeletal muscle or both, they suggest a requirement for SMN protein during embryo–fetal development. INTRODUCTION The survival of motor neuron (SMN) gene has been identified as the disease-causing gene in spinal muscular atrophy (SMA), a frequent autosomal recessive neuromuscular disorder (1/6000 live births) characterized by degeneration of motor neurons of the spinal cord and progressive proximal muscular atrophy (1–5). The childhood SMAs have been divided into three forms, based on age of onset and clinical severity: type I (acute form, Werdnig–Hoffmann disease), type II (intermediate) and type III SMA (Kugelberg–Welander disease) (6). In >90% of cases, the disease results from absence of the SMNt gene and SMA patients retaining the SMNt gene carry intragenic mutations (1,2,7–10). Further analyses revealed that the absence of SMNt was associated with a reduction in gene dosage in type I SMA but not in type III SMA, indicating that the SMNt gene was replaced by another copy of the SMNc gene (1,11,12). However, severe forms have also been associated with an increased number of SMNc copies, suggesting that other factors may modulate clinical severity (13). Thus, loss of the SMNt gene in SMA patients could be due either to gene deletion or gene conversion. Yet, rare cases with no detectable SMNt gene (<1%) have been reported in asymptomatic relatives of haploidentical SMA type II and III patients (14,15). The SMNt gene belongs to a duplicated element (500 kb) located in a region of chromosome 5q13 prone to large scale rearrangements (11) and a highly homologous copy gene (SMNc) maps proximal to the disease gene (1). A tight correlation between clinical severity and SMNc protein level was demonstrated in tissues and cell lines derived from SMA patients (16,17) and strong SMN protein expression was shown in normal fetal lower motor neurons (16). The SMN gene encodes a protein of hitherto unclear function, detected in the cytoplasm and in nuclear bodies called gem and found to interact with RNA-binding proteins (18). More recently, SMN protein has been shown to form a complex with spliceosomal small nuclear ribonucleoproteins (snRNP) (19) and to be involved in biogenesis of the spliceosomal snRNPs (20). Here, we have studied SMN protein expression in various human tissues during normal fetal and postnatal development and showed a reduction in SMN protein level in the postnatal period. Interestingly, SMN protein in fetal muscle cells was concentrated in large particles distributed within the cytoplasm and similar in size to gem. A great reduction in SMN protein level was observed in all tissues of type I SMA fetuses, including skeletal muscles. RESULTS Western blot analyses Immunoblotting experiments on normal human fetal tissues using the monoclonal anti-SMN antibody 4B3 revealed that the SMN protein had the same apparent molecular size (38 kDa) in *To whom correspondence should be addressed. Tel: +33 1 44 49 51 63; Fax: +33 1 47 34 85 14; Email: [email protected] 1928 Human Molecular Genetics, 1998, Vol. 7, No. 12 Figure 1. SMN protein expression in control and SMA tissues. (A) The monoclonal anti-SMN antibody 4B3 revealed a 38 kDa protein in the various human fetal tissues tested. (B) Immunoblot studies of total protein preparations from fetal (F) (25 µg) and postnatal control tissue samples (P) (75 µg). SMN protein expression was reduced in postnatal compared with fetal tissues. (C) Western blot analyses of muscle tissues from control and type I SMA fetuses. (D) Immunoblot analyses of muscle tissues from control, type II and type III SMA fetuses. Total protein preparations from SMA fetuses showed a marked reduction in SMN protein level as compared with three control fetuses (C1, C2 and C3). Incubations with monoclonal anti-actin and anti-β-tubulin antibodies were performed as internal controls for both loading conditions and tissue expression. lymphoblastoid cell lines (16), skeletal muscle, heart, kidney, thymus, brain, pancreas and lung (Fig. 1A). The relative amount of SMN protein was higher in kidney and brain and similar in skeletal muscle, heart and thymus of 16-week-old fetuses. The amount of SMN protein in skeletal muscle, heart, kidney and brain was relatively higher during fetal life than in the postnatal period, as compared with actin and β-tubulin protein levels (Fig. 1B). Interestingly, the drop in protein level during the postnatal period was less pronounced in kidney compared with other tissues. The SMN protein level was markedly deficient in fetal muscle tissues from all three forms of SMA, being more reduced in severe than milder forms, as compared with age-matched controls under our experimental conditions (Fig. 1C and D). Immunolocalization of SMN protein in human fetal tissues Immunofluorescence detection using monoclonal anti-SMN antibody 4B3 in human fetal tissues (thymus, kidney, lung and brain) revealed that SMN was present as large gems in the nucleus and was uniformly distributed throughout the cytoplasm, with no preferential compartmentation (Fig. 2). No detectable SMN immunostaining was noted in tissues of type I SMA fetuses (Figs 2 and 3F). Morphological and immunohistochemical analyses of normal fetal limb showed large dot-like SMN immunolabelling in the cytoplasm of myotubes and myofibres, and SMN was occasionally concentrated in small gems in the nucleus (Fig. 3A–E). The size of these large cytoplasmic structures was estimated to be similar to that of gems in the nucleus, i.e. 0.1–1.0 µm (18). A strong SMN immunostaining was also detected around blood vessels in endothelial cells (Fig. 3B). Neither nuclear nor cytoplasmic SMN immunostaining could be detected in muscle cells from a SMA type I fetus (Fig. 3F). The availability of the Sol 8 mouse muscle cell line (21) allowed in vitro studies of SMN protein immunolocalization in the myoblast stage and during its terminal differentiation into multinucleated myotubes (Fig. 3G and H). Strong SMN immunostaining was detected throughout the cytoplasm during the myoblast proliferative state (Fig. 3G). In contrast, a dramatic reduction in SMN immunodetection was observed in a cell culture differentiated into multinucleated myotubes (Fig. 3H). Subcellular localization of SMN protein in the particulate cellular fraction The subcellular localization of the SMN protein in skeletal muscle from human fetal limb was further investigated using the velocity sedimentation procedure (Fig. 4A; see Materials and Methods). Immunoscreening of the different subcellular fractions revealed a specific SMN enrichment in the pellet of the 142 000 g centrifugation (P142 fraction, Fig. 4B). In order to further characterize SMN protein interactions, the P142 fraction was 1929 Human Genetics, 1998, 7, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 12 1929 Figure 2. Immunofluorescence staining of SMN in control and SMA fetal tissues. Haematoxylin staining of serial tissue sections showed the morphological aspect of various tissues (A). Monoclonal anti-SMN antibody 4B3 stained both the cytoplasm and the nuclear gems (arrows) in control thymus, kidney, lung and brain (B). No detectable immunostaining of the SMN protein was observed in tissues from type I SMA fetuses (C). Bar 10 µm. either treated with the non-ionic detergent Nonidet P-40 (Fig. 4C) or analysed by discontinuous sucrose gradient centrifugation (Fig. 4D). The SMN protein was recovered in the pellet (P), indicating that neither treatment could dissociate SMN from the heavy sedimenting particulate fraction. Our results further support the view that SMN is strongly associated with a multimeric protein complex with no interaction with membrane components. These data complement the observations made 1930 Human Molecular Genetics, 1998, Vol. 7, No. 12 Figure 3. Immunohistological and immunofluorescence analyses of SMN protein expression in muscle cells. Combined haematoxylin staining and immunohistochemical experiments using the anti-SMN monoclonal antibody 4B3 were performed on human skeletal muscle sections from control fetuses at 16 (A) and 24 weeks gestation (B and C). Immunofluorescence analyses of human skeletal muscle sections with the same antibody also revealed a dot-like immunolocalization in the cytoplasm and, occasionally, small gems (arrows) in the nucleus of fetal muscle cells at 8 (D) and 16 weeks gestation (E, DAPI staining of the nucleus). No detectable SMN immunostaining was shown in a SMA type I fetus at 16 weeks gestation (F). Mouse myogenic cell line Sol 8 in the myoblast proliferative state (G), and differentiated into multinucleated myotubes (H), was immunostained with anti-SMN monoclonal antibody. The immunodetection of SMN protein was performed using anti-mouse IgG–FITC (A–D) or anti-mouse IgG–Cy3 (G–H). Bar 10 µm. using fractionation of HeLa cell cultures (19). This study hopefully represents a step towards characterization of tissuespecific partners of SMN and unravelling of its function(s). DISCUSSION SMN protein function has hitherto been unclear and the pathogenesis of SMA is still unknown. Yet the disease is caused by alterations to the telomeric copy of the SMN gene (1–10) and its severity is tightly correlated with the amount of protein encoded by its highly homologous gene copy, SMNc (16,17). Recent studies identified an interaction of SMN with RNAbinding proteins (18,19) and its involvement in the biogenesis of spliceosomal snRNPs (20). The present study has shown that the SMN protein level undergoes a marked drop in the postnatal period in the human tissues tested, such as skeletal muscle, heart and brain. Also, analysis of the Sol 8 mouse muscle cell line showed a marked decay in SMN immunodetection upon in vitro cell differentiation. These observations suggest that SMN expression undergoes hitherto unknown developmental and/or hormonal regulation (22). We have also shown that in fetal muscle cells SMN protein 1931 Human Genetics, 1998, 7, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 12 1931 Figure 4. Subcellular fractionation and immunoblot studies of control fetal skeletal muscle. (A) Flow chart for subcellular fractionation by centrifugation. (B) Western blot analyses with anti-SMN, anti-emerin and anti-SERCA1 antibodies were carried out on skeletal muscle protein homogenate (total) and centrifugation fractions (P14, P30, P142 and S142) were prepared as described in (A). The SMN protein was highly enriched in the P142 fraction. (C) Immunoblot analyses with anti-SMN and anti-SERCA1 antibodies of the pellet (P) and the supernatant (S) prepared from P142 in an extraction buffer containing NP-40 (0.5%). SERCA1 is released in the supernatant, while SMN remains associated with the pellet. (D) Immunoblot analyses using anti-SMN and anti-αSARC antibodies were performed on the interface of the two sucrose layers (I) and on the pellet (P). αSARC was detected at the interface while SMN protein was still found in the pellet. is concentrated in large cytoplasmic dot-like structures similar in size to gem (0.1–1.0 µm; 18). To our knowledge, cytoplasmic structures as large as gem have never been described before. This immunolocalization of SMN in skeletal muscle contrasts with results obtained in other normal tissues such as thymus, kidney, lung and brain or liver and spinal cord (16) or even in cell cultures (17,18), where a general staining throughout the cytoplasm and large gems in the nucleus were detected. This distribution might account for a shorter nuclear half-life of SMN protein. SMN protein was shown to associate directly with SIP1 (SMN-interacting protein 1) and to form a complex with spliceosomal snRNP proteins in HeLa cells (19). Further experiments will be necessary to determine whether accumulation of SMN protein in large dot-like structures in the cytoplasm of myotubes and myofibres from fetal limb could be due to post-transcriptional modification of SMN and/or to tissue-specific partners of SMN. Immunohistochemical analyses of fetal liver, fetal spinal motor neurons (16) and of cultured fibroblasts (17) revealed a lack of and significant reduction in the number of gems in SMA types I and III, respectively. Here we have extended the range of tissues tested and shown that SMN protein is greatly reduced in all tissues derived from type I SMA fetuses. It has recently been reported that SMN protein levels are markedly reduced in spinal cord of types I and III SMA fetuses (16) and of type I SMA patients (17). Immunoblot analyses showed that in postnatal skeletal muscle derived from type I SMA patients, the amount of SMN protein was also markedly reduced, but not in a type III patient (17). Interestingly, the observation of a significant reduction in SMN in muscle from type III SMA fetuses suggests that expression and/or the stability of SMNc protein in muscle tissue may differ between the pre- and postnatal periods. The lack of or the marked reduction in SMN in skeletal muscle of types I or II and III SMA fetuses might be related to the pathological defects of muscle fibres in SMA patients (23,24). These experiments lead to the observation that in SMA the SMN protein is markedly reduced prior to onset of the disease, while SMN protein is strongly expressed in normal tissues. Our present data do not help in deciding whether SMA results from impaired SMN expression in spinal cord, skeletal muscle or both, but they raise the hypothesis that SMA is an embryo–fetal rather than a postnatal disease. Strong SMN expression in human fetal life along with its conservation among species (9) and its absolute requirement in early mouse embryos (25) give strong support to the view that SMN plays a major role in cell survival. Ubiquitous SMN expression and its role in RNA metabolism (26) contrast with the very specific clinical expression of the disease. One can hypothesize that SMN protein deficiency alters a subset of tissue-specific RNAs or another function(s) in a developmental manner. Characterization of SMN protein complexes should contribute to a better understanding of SMN function(s) and the pathogenesis of SMA. MATERIALS AND METHODS Patients A total of six unrelated control fetuses were selected for immunohistochemical and immunoblotting analyses. Three fetuses predicted to be affected with type I, three with type II and two with type III SMA at 13–18 weeks gestation were included in this study. The probands were diagnosed as SMA according to the clinical criteria of the International SMA Consortium (6). All SMA fetuses were homozygous for the absence of SMN exon 7 and retained the SMNc gene (1). Embryos were obtained from legally terminated pregnancies according to the French ethical committee recommendations. Production of monoclonal antibodies against SMN protein Anti-SMN antibodies 4B3 and 0B5 were produced by immunization of Balb/c mice with a bacterial fusion protein starting at amino acid position 85 of the SMN protein expressed in vector pET30 (Novagen). Hybridoma and ascite fluid preparations were performed according to standard procedures (Coval Ab, France). Hybridomas were selected based on the immunoreactivity of the secreted antibodies in western blotting and immunohistochemical analyses of control and SMA samples. Morphological and immunofluorescence analyses Immunostaining of cryosections (4 µm) from non-muscle fetal tissues (thymus, kidney, lung and brain) fixed in 3% formaldehyde-buffered solution was carried out as previously described (16). Fetal muscle sections were prepared from paraffinembedded specimens for immunofluorescence experiments 1932 Human Molecular Genetics, 1998, Vol. 7, No. 12 using the NEN TSA System and immunohistochemical studies using the DAKO CSA system. Sections were stained with haematoxylin solution for morphological analyses or stained with 1 µg/ml 4,6-diamidino-2-phenylindole (DAPI) (Sigma) to visualize the nucleus. The human tissue sections were incubated with monoclonal anti-SMN antibody 4B3 ascite fluid at a 1:1000–4000 dilution (0.25–1 µg/ml) and detected using 1:100 anti-mouse IgG–fluorescein isothiocyanate (FITC) (Biosys). The Sol 8 mouse muscle cell line was cultured and in vitro differentiated according to previously published procedures (21). The cells were fixed with 3% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), permeabilized with 0.5% Triton and pre-incubated in blocking solution (5% sheep serum and 1% BSA). Immunodetection was performed with 1:1000 monoclonal anti-SMN antibody (Transduction Laboratories) using 1:400 sheep anti-mouse IgG–indocarbocyanide (Cy3) (Sigma). The immunofluorescence was observed using a Leica DM microscope equipped with a ×10/0.30 or ×100/1.30 objective and dual bandpass filter for DAPI/FITC or DAPI/rhodamine fluorescence. organelles and cell debris. The supernatant was centrifuged in a cold SW41Ti rotor (Beckman Instruments) at 30 000 g for 30 min. The pellet P30 contained heavy microsomes from the endoplasmic and sarcoplasmic reticulum. The P30 supernatant was further centrifuged at 142 000 g for 35 min. The resulting pellet (P142) was resuspended in sucrose buffer and layered on a discontinuous gradient of 0.303 M sucrose on top of 1.0 M sucrose in buffer A. The membrane fraction at the interface of the two sucrose layers and the particulate cellular fraction (pellet, P) were collected after centrifugation at 150 000 g in a SW41Ti rotor for 16 h at 4C. Immunodetection with anti-emerin monoclonal antibody indicated its specific nuclear enrichment. The weak signal in other fractions reflected the strong homogenization conditions. The anti-SERCA1 monoclonal antibody revealed labelling of the P30 (heavy microsomes) and P142 (light microsomes and particulate cellular) fractions. The membranous component of the sarcoplasmic reticulum (heavy microsomes) was probably detected in the P142 fraction, possibly because of its abundance and its poor fractionation under our homogenization conditions. Immunoblot analyses The various human fetal tissues and postnatal skeletal muscles were homogenized in ice-cold extracting buffer containing 20 mM pyrophosphate, 20 mM phosphate monohydrate, 1 mM MgCl2, 0.303 M sucrose, 0.5 mM EDTA (pH 7.4) in the presence of protease inhibitors [2 mM phenylmethylsulfonyl fluoride (PMSF), 3 µg/ml pepstatin, 3 µg/ml anti-papain, 15 µg/ml benzaminidine and 40 µg/ml leupeptin]. Following a Bradford protein assay (Bio-Rad Laboratories) using BSA as standard, the protein homogenates were diluted in Laemmli sample buffer, separated by SDS–PAGE (27) and electrotransferred onto Immobilon-P membranes (28). Whole tissue protein preparations of normal adult heart, kidney and brain were obtained from Clontech as electrophoresis-ready solutions. Triplicate immunoblots were incubated with monoclonal antibodies directed against α-sarcoglycan (α-SARC) (1:100 dilution), emerin (1:250 dilution), sarcoplasmic reticulum Ca2+-dependent ATPase 1 (SERCA1) (1:250 dilution), actin (1:4000 dilution) and β-tubulin (1:4000 dilution) as recommended by the suppliers (Novocastra Laboratories and Amersham). The monoclonal anti-SMN antibody 4B3 was used at a 1:1000 dilution. The immunoblots were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000–10 000; Amersham) or with the biotin–streptavidin horseradish peroxidase system (1:10 000 dilution; Vector Laboratories) and detected using chemiluminescent reagents (ECL; Amersham). The autoradiographs were scanned using a computerized densitometer as previously described (16). Subcellular fractionation by centrifugation Flash frozen fetal muscle samples (500–750 mg) were homogenized with a tight fitting glass pestle in 10 vol ice-cold 0.303 M sucrose in 20 mM pyrophosphate, 20 mM phosphate monohydrate, 1 mM MgCl2, 0.5 mM EDTA, pH 7.4, buffer (buffer A) in the presence of protease inhibitors (2 mM PMSF, 3 µg/ml each pepstatin and anti-papain, 15 µg/ml benzaminidine and 40 µg/ml leupeptin). Fractionation was performed by velocity sedimentation as previously described (29). Briefly, the homogenate was centrifuged at 14 000 g for 15 min in a refrigerated bench centrifuge. The corresponding pellet (P14) contained nuclei, ACKNOWLEDGEMENTS We thank the patients, families and doctors who have contributed to this project and without whom this study would not have been possible. We thank J.Melki and C.Fallet for tissues derived from SMA fetuses, D.Montarrat and C.Pinset for providing the Sol 8 mouse muscle cell line, M.Fardeau and F.Tomé for helpful discussions, M.C.Gubler and E.Viegas-Pequignot for equipment facilities and M.Recouvreur, V.Raclin and Y.Deris for expert technical assistance. We thank the members of the International SMA Consortium for stimulating discussions. S.B. is the recipient of a MRT Predoctoral Fellowship. I.Z. was a pre-doctoral summer student. This work was supported by Association Française contre les Myopathies (AFM), Action Concertée des Sciences du Vivant (ACC-SV2) and Programme Hospitalier de Recherche Clinique. Work in the J.Cartaud laboratory was supported by a grant from AFM and Sciences de la Vie, CNRS. 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