Human Molecular Genetics, 2000, Vol. 9, No. 5 849-858
© 2000 Oxford University Press
Nuclear targeting defect of SMN lacking the C-terminus in a mouse model of spinal muscular atrophy
Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM, CNRS, ULP, BP163, 67404 Illkirch cedex, CU de Strasbourg, France
Received 4 January 2000; Revised and Accepted 25 January 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Deletion of the murine survival of motor neuron gene (SMN) exon 7, the most frequent mutation found in spinal muscular atrophy (SMA) patients, directed to neurons but not to skeletal muscle, enabled generation of a mouse model of SMA providing evidence that motor neurons are the primary target of the gene defect. Moreover, the mutated SMN protein (SMN
C15) is dramatically reduced in the motor neuron nuclei and causes a lack of gems associated with large aggregates of coilin, a coiled-body-specific protein. These results identify the lack of the nuclear targeting of SMN as the biochemical defect in SMA. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by degeneration of motor neurons of the spinal cord associated with symmetrical limb and trunk paralysis with muscle atrophy. SMA represents a frequent recessive autosomal disorder and is one of the most common genetic causes of death in childhood (1,2). A positional cloning strategy allowed the identification of the survival of motor neuron gene (SMN) as the SMA-determining gene (3,4). SMN exists in duplicate, with only five nucleotides distinguishing it from its highly homologous copy gene, SMNc (4–6). In SMA, 95% of patients carry homozygous deletion or conversion events of the SMN exon 7 and the remaining 5% carry intragenic mutations within the SMN gene (reviewed in ref. 7). SMN encodes a protein of 294 amino acids which locates both to the cytoplasm and to a nuclear structure called a gem (for gemini of coiled bodies) (8). SMN forms part of a large protein complex that contains the SMN-interacting protein 1 (SIP1) and Sm proteins of the spliceosomal uridine-rich small ribonucleoproteins suggesting that SMN plays a key role in spliceosomal biogenesis by bringing together the components of the spliceosomal complex (8–11).
In contrast to the human, the mouse SMN gene is not duplicated, which may explain the fact that the conventional knockout of the SMN gene results in embryonic lethality (12–14). To gain insight into the pathogenesis of SMA and to understand the function of SMN inferred from the SMA phenotype, we carried out a conditional deletion of the murine SMN exon 7, the most frequent mutation found in SMA patients, using the Cre/loxP recombination system of bacteriophage P1 (15,16).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Homozygous deletion of SMN exon 7 in all cell types results in early embryonic lethality
We used a targeting vector that includes two loxP sites flanking the SMN exon 7 and a NeoR gene (Fig. 1). Through homologous recombination, mice heterozygous or homo- zygous for the loxP-flanked SMN exon 7 allele (SMNF7) appear similar in size and morphology to wild-type mice. At the RNA level, no alternative splicing of SMN exon 7 was detected by RT–PCR amplification analysis of RNA extracted from various tissues of the SMNF7/F7 mice, indicating that neither the NeoR gene nor the loxP sites have a deleterious effect on SMN splicing (Fig. 2). Mice heterozygous for the SMNF7 allele were initially crossed with a strain carrying the Cre recombinase transgene driven by the cytomegalovirus (CMV) promoter (17). Cre recombinase expression in germline led to the generation of mice carrying a heterozygous deletion of SMN exon 7 (SMN
7/+). SMN7/+ mice gained weight normally after birth and have remained indistinguishable from wild-type littermates to date (12 months of age). Histological examination of skeletal muscle did not reveal any morphological changes (data not shown). The Cre-mediated deletion of the SMN exon 7 resulted in SMN transcripts lacking exon 7 as determined by sequencing of RT–PCR amplification products of RNA extracted from various tissues including the spinal cord and skeletal muscle of SMN7/+ mice (Fig. 2). To determine whether the homozygous deletion of SMN exon 7 in all cell types would lead to embryonic lethality or to a milder phenotype, SMN7/+ mice were intercrossed. These crosses generated no live SMN7/7 mice out of 50 newborns analysed (Table 1). To determine the timing of the SMN7/7 lethality, embryos were analysed during gestation. At 9 days post-coitum, a total of 53 embryos, including 13 with almost complete resorbtion was analysed (Fig. 2). Normal embryos showed the expected ratio for genotypes (+/+) or (SMN7/+), whereas all resorbed embryos were homozygous for the SMN7 mutation (Fig. 2 and Table 1) indicating that homozygous deletion of SMN exon 7 in all cell types results in embryonic lethality during early development.
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Homozygous deletion of SMN exon 7 directed to neurons leads to the generation of mice affected with an SMA phenotype
To induce neuron-restricted exon 7 deletion, we generated transgenic mice harbouring the Cre recombinase gene driven by the promoter of the rat neuron-specific enolase gene (NSE) (18). The transgenic NSE-Cre lines (NSE-23 and -39) were crossed to the strain harbouring the SMNF7 allele and Cre-mediated deletion of SMN exon 7 was quantified through semi-quantitative PCR amplification analyses of genomic DNA extracted from various tissues of double transgenic mice (NSE-Cre, SMNF7/+). Of the two lines, only one, NSE39-Cre, generated a high level of Cre-mediated exon excision in neuronal tissues and this line was studied further (Fig. 3). In situ hybridization using a Cre recombinase riboprobe was performed and revealed an expression pattern that was restricted to neurons in the adult spinal cord whereas in skeletal muscle, no Cre recombinase expression was detected (Fig. 3). These findings led us to use the NSE39-Cre transgenic line for the conditional targeting of SMN in neurons but not in the skeletal muscle.
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NSE39-Cre mice were crossed with mice carrying an SMN
7 allele. F1 offspring carrying both the NSE39-Cre transgene and SMN7 allele were selected and crossed with SMNF7/F7 mice. Twenty-eight of 105 (26.6%) mice carried both the NSE39-Cre recombinase transgene and the SMNF7/7 genotype as expected (termed SMNF7/7, Cre+). Mice carrying this genotype can be easily distinguished from their control littermates by severe motor defect associated with tremors, both of which are evident at 2 weeks of age (Fig. 4). Mutant mice exhibit an extremely reduced life expectancy, dying at a mean age of 25 days (range 17–36 days, n = 10). Morphological analysis on transverse sections of several skeletal muscles including gastrocnemius, intercostal and sternomastoid revealed the presence of groups of atrophic muscle fibers and angular fibers intermixed with normal-sized fibers in SMNF7/7, Cre+ mice using haematoxylin and eosin staining (Fig. 5). These findings are consistent with a denervation of skeletal muscles of neurogenic origin which was further confirmed by the presence of a marked extrajunctional labeling of acetylcholine receptors in SMNF7/7, Cre+ animals (Fig. 5). A morphological analysis on transverse semithin sections of spinal cord was performed on 2-week-old control and SMNF7/7, Cre+ mice using toluidine blue staining. In mutant mice, the striking feature was the pronounced morphological changes of nuclei of motor neurons (Fig. 5). A marked increase in toluidine blue staining was observed within the nuclei at the nucleo-cytoplasmic boundary suggesting the presence of indentations of the nuclear membrane (Fig. 5). In addition, some motor neurons display a granular aspect of the cytoplasm (Fig. 5). To determine whether these changes were associated with a loss of motor neurons, a quantification of motor neuron number was performed at the lumbar level of the spinal cord but no significant loss of motor neurons of the anterior horns was detected in 2-week-old mutant mice (data not shown). In 4-week-old SMNF7/7, Cre+ mice, toluidine blue or DAPI staining of spinal cord sections revealed the presence of vacuolation of the cytoplasm associated with a marked deformation or fragmentation of motor neuron nuclei (Fig. 6).
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Prominent and frequent indentations of the nuclear membrane are associated with the lack of gems associated with large aggregates of coilin in motor neuron nuclei
To test whether the neuromuscular changes are associated with an abnormal expression of SMN transcript, RT–PCR amplification analysis of RNA extracted from the spinal cord and skeletal muscle of mutant or wild-type mice was performed using primers flanking SMN exon 7 (Fig. 2). The transcript analysis of SMN revealed a marked reduction of the full-length transcript associated with an increased amount of the SMN
7 transcript in the spinal cord but not in skeletal muscle of SMNF7/7, Cre+ mice (Fig. 2). Immunoblotting of proteins prepared from the spinal cord of SMNF7/7, Cre+ and control mice was performed using the monoclonal antibody directed against the N-terminal part of the SMN protein (2B1) (8). In SMNF7/7, Cre+ mice, the SMN protein is present with an apparent size and amount similar to that observed in control mice (Fig. 7). These results strongly suggest that the truncated SMN transcript is translated into a protein lacking the last C-terminal 15 amino acids (SMNC15) although the resolution of western blot gel is not sufficient to distinguish the wild-type protein from a protein lacking 15 amino acids. To determine whether the subcellular localization of the truncated protein is similar to that of the wild-type, the monoclonal antibody 2B1 was further used in an immunofluorescence microscopy analysis of spinal cord sections derived from mutant and control mice. In the spinal cord of SMNF7/7, Cre+ mice, the 2B1 monoclonal antibody stained the cytoplasm but failed to recognize the localization of SMN in gems of motor neurons (Fig. 7). SIP1, a protein interacting with the N-terminal part of SMN and colocalized with SMN in gems, was immunostained using a monoclonal antibody specific to SIP1 (2E17) (9). In the spinal cord of control mice, SIP1 antibody revealed uniform staining of the nucleus in addition to gems in motor neurons (Fig. 7). In the spinal cord of SMNF7/7, Cre+ mice, SIP1 antibody failed to detect gems in motor neurons (Fig. 7). These findings provide strong evidence of an absence of gems in the motor neurons of mutant mice. We therefore tested whether the lack of gems has any effect on the structure or organization of coiled bodies, a nuclear structure most often found to be associated with gems. A polyclonal anti-p80 coilin, a coiled-body-specific protein (19) was used in an immunofluorescence microscopy analysis and detected several small foci corresponding to coiled bodies in motor neuron nuclei of control mouse (Fig. 7). In the spinal cord of SMNF7/7, Cre+ mice, anti-p80 coilin antibody stained large nuclear aggregates providing evidence for a defect in the coilin assembly into coiled bodies (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Through a conditional gene-targeting approach, we were able to circumvent embryonic lethality resulting from homozygous deletion of SMN exon 7 in all cell types. The mice carrying a deletion of SMN exon 7 directed to neurons display abnormal motor behavior characterized by severe defects in both spontaneous and induced motor activities which are evident at 2 weeks of age. These defects appear to be progressive as mutant mice develop more pronounced motor disability with age and die usually at a mean age of 25 days. Morphological analysis of several skeletal muscles, including those involved in motor activity or respiratory function, revealed the presence of a muscle denervation process of neurogenic origin, a constant feature found in human SMA (20). The morphological analysis of the spinal cords of SMNF7/
7, Cre+ mice showed the presence of indentations in the motor neuron nuclear membranes which have also been reported to occur with a less pronounced extent in neurodegenerative diseases caused by abnormal polyglutamine repeat expansion (21); however, unlike these diseases no neuronal inclusion was observed in SMN mutant mice using toluidine blue staining. In 4-week-old mutant mice, the presence of nuclear fragmentation suggests the presence of an apoptotic process although the abnormal changes of the cytoplasm do not allow exclusion of the involvement of another degenerative process. Further investigations including electron microscopic examination should enable to clarify this point. It is clear that the presence of motor neuron nuclear changes is associated with pronounced neuronal dysfunction as demonstrated by the denervation of skeletal muscle fibers, the targeted cells of motor neurons, despite the absence of motor neuron loss. The morphological changes occurring in response to the lack of SMN have not been reported in human SMA suggesting that they likely represent the early events of neuronal degeneration which lead later to motor neuron loss, a late manifestation such as that found in autopsy material of SMA patients (20). Interestingly, Monani et al. (22) found that motor neuron loss is a late manifestation of the disease phenotype of mutant mice that they generated, which is in agreement with our results (22). Previous reports have suggested that a defect of muscular origin might be responsible for motor neuron degeneration in SMA (23,24). Our results demonstrate that an SMN gene defect directed to neurons but not to skeletal muscle leads to an SMA phenotype in vivo indicating that motor neurons are the primary target of the SMN gene defect in SMA. However, it cannot be ruled out that an SMN gene defect in both spinal cord and muscle could modulate the SMA phenotype. Conditional SMN gene targeting restricted to skeletal muscle or to both spinal cord and skeletal muscle should contribute to further exploring this issue.
The pronounced effect of SMNC15 on the organization of both gems and coiled bodies was unexpected. The dramatic reduction of SMNC15 within the motor neuron nuclei of mutant mice indicates that the C-terminus of SMN (amino acids 274–288) represents an important domain required for the SMN targeting into gems. Interestingly, the absence of SIP1 assembly into gems strongly suggests that SMN is involved in the normal pathway of gem formation in vivo. Previous studies indicated that gems were distinct from coiled bodies, though they were most often found to be closely associated (8). The lack of gems in mutant mice led us to examine coiled body organization. The presence of large nuclear aggregates of coilin indicates that the lack of SMN and/or gems has a pronounced effect on coiled body formation. Therefore, the generation of a mouse model of SMA, carrying the most frequent mutation found in human SMA, allowed us to determine that SMNC15 causes a defect in nuclear targeting of SMN which cannot proceed further the normal pathway of gem/coiled body formation, thus identifying the biochemical defect in SMA. In human, coiled bodies were found to be present in SMA fetuses suggesting that the full-length SMN protein which remains present in SMA patients, but not in mutant mice, is likely able to maintain coiled body structure (25). In human SMA, the highly homologous SMNc gene remains expressed although the alternative splicing of exon 7 specific to SMNc transcripts leads to the production of a truncated protein lacking the C-terminus as the murine mutated protein (4,6). Molecular agents able to promote nuclear targeting of the mutant SMN into gems should represent an attractive therapy.
The generation of animal models of SMA required sophisticated genetic strategies to avoid embryonic lethality. In a first strategy, two groups have created mouse lines carrying a transgene which contains the human SMNc gene (22,26). Mice carrying a low copy number of SMNc transgene and harbouring a homozygous null SMN allele (SMN–/–) (14,27) develop a severe phenotype consistent with human type I SMA. Crosses of SMN–/– mice with those carrying a high copy number of SMNc transgene lead to the absence of abnormal phenotype, which demonstrates that an increased copy number of SMNc reduces the severity of the phenotype, data consistent with those observed in human SMA. These animal models will be very useful in testing molecules able to upregulate the SMNc gene (22,26).
The second strategy described in the present study provided important and complementary information: (i) motor neurons are the target cells of the SMN gene defect in SMA; (ii) the most frequent gene mutation found in human SMA causes a defect in nuclear targeting of SMN into gems; (iii) the observation of a severe denervation process without marked loss of motor neurons strongly suggests that motor neuron loss is a late manifestation of the disease; and (iv) the mutant mice display an intermediate phenotype resembling human type II or III SMA. They should represent therefore an appropriate model for the design of therapeutic strategies to protect from or prevent neuronal death in SMA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gene targeting, generation of loxP-flanked SMN exon 7 mice and mating
The targeting construct derived from 129Sv total genomic DNA was electroporated into embryonic stem (ES) cells, and of 406 G418-resistant ES cells two clones containing the specifically targeted allele (SMNF7) through homologous recombination were injected into blastocysts which were transferred into pseudopregnant foster mothers. One clone gave a high percentage of chimeras. Chimeras were bred to C57BL/6J females and mice harbouring the targeted allele were identified following genotype analysis of agouti offspring using two sets of primers: PHR5-XG3 (5'-TTC TCT TGA TTC CCA CTT TGT GGT TC-3'; 5'-CTG GCC CAA ATC ACA ACA TAA-3'); or ex7sou1-GS8 (5'-AGA AGG AAA GTG CTC ACA TAC AAA TT-3'; 5'-TGT CTA TAA TCC TCA TGC TAT GGA G-3'). Heterozygous SMNF7 mice were intercrossed, and a strain homozygous for the SMNF7 allele was established.
Mice heterozygous for the SMNF7 allele were initially crossed with a strain carrying the Cre recombinase transgene driven by the CMV promoter (17). Offspring carrying both the SMNF7 allele and CMV-Cre transgene were selected and crossed with C57BL/6J mice. A heterozygous deletion of the SMN exon 7 (SMN7) was detected in offspring carrying both CMV-Cre and the mutated allele. Backcross of CMV-Cre, SMN7/+ with C57BL/6J mice allowed to select mice carrying a heterozygous deletion of SMN exon 7 in all cell types but lacking the CMV-Cre transgene (SMN/+).
Plasmid construction, generation of a Cre recombinase transgenic line and quantification of Cre-mediated deletion
A 2100 bp EcoRI–HindIII restriction fragment of the pNSE-CAT plasmid, containing the rat NSE gene promoter (18), was cloned into the pGS5 plasmid which contains the Cre recombinase gene separated from the promoter by the rabbit ß-globin intron and followed by the SV40 polyadenylation signal. Subsequently, the purified NotI fragment was microinjected into the pronuclei of fertilized eggs from (C57BL/6J x SJL) F1 mice and implanted in pseudopregnant foster mothers. The parental transmission of the transgene was confirmed both by PCR amplification using primers Cre1 (5'-CCG GTC GAT GCA ACG AGT GAT-3') and Cre2 (5'-ACC AGA GTC ATC CTT AGC GCC-3') and Southern blotting. The PCR product (790 bp) was used as a probe for Southern blot analyses of DNA extracted from tail biopsy.
To test the efficiency of DNA excision by the Cre recombinase, transgenic mice were crossed with mice carrying the SMNF7 allele and double transgenic mice were selected. Genomic DNA was extracted from a variety of adult tissues. The wild-type and SMNF7 alleles were simultaneously amplified by PCR using one set of primers (ex7sou1-GS8). The efficiency of excision was estimated by comparing the intensity of the band amplified from the SMNF7 allele with that of the band amplified from the wild-type allele which differs only in the absence of the loxP site. After 15 cycles, PCR products were separated by agarose gel electrophoresis, transferred to Nylon-N+ membrane and hybridized using the wild-type PCR product as a probe. Intensity of bands was quantified using the Bio-Rad (Hercules, CA) GS700 densito- meter and Molecular Analyst software.
For in situ hybridization experiments, a BamHI–XhoI restriction fragment of the Cre recombinase gene derived from the pGS5 plasmid was subcloned into pBluescript plasmid KS(–). Sense and antisense RNA synthesis was performed using T3 or T7 RNA polymerase and labeled with [
-35S]CTP as previously described (28).
Morphological and immunofluorescence analyses
For toluidine blue staining of the spinal cord, the mice were anaesthetized with nembutal and perfused with 0.9% NaCl and 2.5% glutaraldehyde, 0.5% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Tissue samples were dissected and immersed in the same fixative for 2 h, rinsed in PB and postfixed in 2% osmium tetroxide. After three washes with phosphate-buffered saline, each sample was dehydrated in a graded series of ethanol and embedded in Epon. Semithin transverse sections of spinal cord (1 µm) were stained with toluidine blue and examined under a Zeiss Axiophot microscope. Quantification of motor neuron number was performed at the lumbar level of the spinal cord using double labelling of nuclei and cytoplasm by DAPI and anti-choline acetyl transferase antibody (anti-ChAT, 1:800 dilution; Chemicon, Temecula, CA), respectively. Motor neurons were counted when large DAPI-stained nuclei-associated ChAT labelling of the cytoplasm were observed. Immunostaining of SMN, SIP1 and coilin was performed on tissues fixed with 4% paraformaldehyde and incubated with either monoclonal antibody anti-SMN (2B1, 1:500), anti-SIP1 (2E17, 1:100) or rabbit polyclonal anti-p80 coilin (204,10, 1:500) and revealed by Cy3-conjugated anti-mouse or rabbit IgG (Jackson Laboratories, West Grove, PA) then counterstained with DAPI. Confocal laser scanning microscopy analysis was performed using a Sarastro 2000 laser confocal system (Molecular Dynamics, Sunnyvale, CA) mounted on a Nikon Optiphot-2 upright microscope. Series of optical sections were taken at 0.2–0.3 µm steps.
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ACKNOWLEDGEMENTS |
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We are grateful to P. Kastner for providing the PHR68 plasmid, D. Metzger for the PGS5 plasmid and CMV-Cre transgenic strain, J. Sutcliffe for the pNSE-CAT plasmid, G. Dreyfuss for the anti-SMN and anti-SIP1 antibodies and J. Sleeman for the anti-p80 coilin antibody. We thank J.L. Mandel, A. Sobel and F. Rieger for invaluable help and Julia C. Young for useful comments. We thank the animal facility staff for animal care, the ES cell culture, microinjection and the sequencing facility services. We are grateful to J. Molgo and L. Faille for help in confocal laser microscopy analysis. All animal procedures were performed in accordance with institutional guidelines. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, Rhône-Poulenc Rorer, the Association Française contre les Myopathies (AFM) and Families of SMA (USA). T.F., F.D.T., C.D. and P.M. are recipients of fellowships of Andrew’s Buddies (USA), INSERM, GENOPOLE and Families of SMA (USA), respectively.
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FOOTNOTES |
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+ These authors contributed equally to this work
§ Present address: Laboratoire de Neurogénétique Moléculaire, INSERM E9913, GENOPOLE, 2 rue Gaston Crémieux CP 5724, 91057 Evry cedex, France
¶ To whom correspondence should be addressed. Tel: +33 1 60 87 45 52; Fax: +33 1 60 87 45 50; Email: j.melki@genopole.inserm.fr
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REFERENCES |
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 S Rudnik-Schoneborn, R Heller, C Berg, C Betzler, T Grimm, T Eggermann, K Eggermann, R Wirth, B Wirth, and K Zerres Congenital heart disease is a feature of severe infantile spinal muscular atrophy J. Med. Genet., October 1, 2008; 45(10): 635 - 638. [Abstract] [Full Text] [PDF]
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V. L. McGovern, T. O. Gavrilina, C. E. Beattie, and A. H.M. Burghes Embryonic motor axon development in the severe SMA mouse Hum. Mol. Genet., September 15, 2008; 17(18): 2900 - 2909. [Abstract] [Full Text] [PDF]
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S. Kariya, G.-H. Park, Y. Maeno-Hikichi, O. Leykekhman, C. Lutz, M. S. Arkovitz, L. T. Landmesser, and U. R. Monani Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy Hum. Mol. Genet., August 15, 2008; 17(16): 2552 - 2569. [Abstract] [Full Text] [PDF]
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T. O. Gavrilina, V. L. McGovern, E. Workman, T. O. Crawford, R. G. Gogliotti, C. J. DiDonato, U. R. Monani, G. E. Morris, and A. H.M. Burghes Neuronal SMN expression corrects spinal muscular atrophy in severe SMA mice while muscle-specific SMN expression has no phenotypic effect Hum. Mol. Genet., April 15, 2008; 17(8): 1063 - 1075. [Abstract] [Full Text] [PDF]
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 J. Vitte, C. Fassier, F. D. Tiziano, C. Dalard, S. Soave, N. Roblot, C. Brahe, P. Saugier-Veber, J. P. Bonnefont, and J. Melki Refined Characterization of the Expression and Stability of the SMN Gene Products Am. J. Pathol., October 1, 2007; 171(4): 1269 - 1280. [Abstract] [Full Text] [PDF]
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 A. Schmid and C. J. DiDonato Animal Models of Spinal Muscular Atrophy J Child Neurol, August 1, 2007; 22(8): 1004 - 1012. [Abstract] [PDF]
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C. R. Heier, R. G. Gogliotti, and C. J. DiDonato SMN Transcript Stability: Could Modulation of Messenger RNA Degradation Provide a Novel Therapy for Spinal Muscular Atrophy? J Child Neurol, August 1, 2007; 22(8): 1013 - 1018. [Abstract] [PDF]
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 L. R. Simard, M-C Belanger, S. Morissette, M. Wride, T. W. Prior, and K. J. Swoboda Preclinical validation of a multiplex real-time assay to quantify SMN mRNA in patients with SMA Neurology, February 6, 2007; 68(6): 451 - 456. [Abstract] [Full Text] [PDF]
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 L. A. Major, J. Hegedus, D. J. Weber, T. Gordon, and K. E. Jones Method for Counting Motor Units in Mice and Validation Using a Mathematical Model J Neurophysiol, February 1, 2007; 97(2): 1846 - 1856. [Abstract] [Full Text] [PDF]
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A. Tarrade, C. Fassier, S. Courageot, D. Charvin, J. Vitte, L. Peris, A. Thorel, E. Mouisel, N. Fonknechten, N. Roblot, et al. A mutation of spastin is responsible for swellings and impairment of transport in a region of axon characterized by changes in microtubule composition Hum. Mol. Genet., December 15, 2006; 15(24): 3544 - 3558. [Abstract] [Full Text] [PDF]
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 N. Salah-Mohellibi, G. Millet, I. Andre-Schmutz, B. Desforges, R. Olaso, N. Roblot, S. Courageot, G. Bensimon, M. Cavazzana-Calvo, and J. Melki Bone Marrow Transplantation Attenuates the Myopathic Phenotype of a Muscular Mouse Model of Spinal Muscular Atrophy Stem Cells, December 1, 2006; 24(12): 2723 - 2732. [Abstract] [Full Text] [PDF]
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 C. Girard, H. Neel, E. Bertrand, and R. Bordonne Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation Nucleic Acids Res., May 31, 2006; 34(10): 2925 - 2932. [Abstract] [Full Text] [PDF]
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 B. Renvoise, K. Khoobarry, M.-C. Gendron, C. Cibert, L. Viollet, and S. Lefebvre Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells J. Cell Sci., February 15, 2006; 119(4): 680 - 692. [Abstract] [Full Text] [PDF]
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S. Jablonka, K. Karle, B. Sandner, C. Andreassi, K. von Au, and M. Sendtner Distinct and overlapping alterations in motor and sensory neurons in a mouse model of spinal muscular atrophy Hum. Mol. Genet., February 1, 2006; 15(3): 511 - 518. [Abstract] [Full Text] [PDF]
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 R. Olaso, V. Joshi, J. Fernandez, N. Roblot, S. Courageot, J. P. Bonnefont, and J. Melki Activation of RNA metabolism-related genes in mouse but not human tissues deficient in SMN Physiol Genomics, January 12, 2006; 24(2): 97 - 104. [Abstract] [Full Text] [PDF]
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F. Gabanella, C. Carissimi, A. Usiello, and L. Pellizzoni The activity of the spinal muscular atrophy protein is regulated during development and cellular differentiation Hum. Mol. Genet., December 1, 2005; 14(23): 3629 - 3642. [Abstract] [Full Text] [PDF]
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J. Jarecki, X. Chen, A. Bernardino, D. D. Coovert, M. Whitney, A. Burghes, J. Stack, and B. A. Pollok Diverse small-molecule modulators of SMN expression found by high-throughput compound screening: early leads towards a therapeutic for spinal muscular atrophy Hum. Mol. Genet., July 15, 2005; 14(14): 2003 - 2018. [Abstract] [Full Text] [PDF]
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T. T. Le, L. T. Pham, M. E.R. Butchbach, H. L. Zhang, U. R. Monani, D. D. Coovert, T. O. Gavrilina, L. Xing, G. J. Bassell, and A. H.M. Burghes SMN{Delta}7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN Hum. Mol. Genet., March 15, 2005; 14(6): 845 - 857. [Abstract] [Full Text] [PDF]
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 M M K Muqit, J Moss, C Sewry, and R J M Lane Phenotypic variability in siblings with type III spinal muscular atrophy J. Neurol. Neurosurg. Psychiatry, December 1, 2004; 75(12): 1762 - 1764. [Abstract] [Full Text] [PDF]
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J. M. Vitte, B. Davoult, N. Roblot, M. Mayer, V. Joshi, S. Courageot, F. Tronche, J. Vadrot, M. H. Moreau, F. Kemeny, et al. Deletion of Murine Smn Exon 7 Directed to Liver Leads to Severe Defect of Liver Development Associated with Iron Overload Am. J. Pathol., November 1, 2004; 165(5): 1731 - 1741. [Abstract] [Full Text] [PDF]
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 W. Rossoll, S. Jablonka, C. Andreassi, A.-K. Kroning, K. Karle, U. R. Monani, and M. Sendtner Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of {beta}-actin mRNA in growth cones of motoneurons J. Cell Biol., November 24, 2003; 163(4): 801 - 812. [Abstract] [Full Text] [PDF]
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 H. L. Zhang, F. Pan, D. Hong, S. M. Shenoy, R. H. Singer, and G. J. Bassell Active Transport of the Survival Motor Neuron Protein and the Role of Exon-7 in Cytoplasmic Localization J. Neurosci., July 23, 2003; 23(16): 6627 - 6637. [Abstract] [Full Text] [PDF]
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Y. B. Chan, I. Miguel-Aliaga, C. Franks, N. Thomas, B. Trulzsch, D. B. Sattelle, K. E. Davies, and M. van den Heuvel Neuromuscular defects in a Drosophila survival motor neuron gene mutant Hum. Mol. Genet., June 15, 2003; 12(12): 1367 - 1376. [Abstract] [Full Text] [PDF]
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J.-C. Lesbordes, C. Cifuentes-Diaz, A. Miroglio, V. Joshi, T. Bordet, A. Kahn, and J. Melki Therapeutic benefits of cardiotrophin-1 gene transfer in a mouse model of spinal muscular atrophy Hum. Mol. Genet., June 1, 2003; 12(11): 1233 - 1239. [Abstract] [Full Text] [PDF]
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J. E. Sleeman, L. Trinkle-Mulcahy, A. R. Prescott, S. C. Ogg, and A. I. Lamond Cajal body proteins SMN and Coilin show differential dynamic behaviour in vivo J. Cell Sci., May 15, 2003; 116(10): 2039 - 2050. [Abstract] [Full Text] [PDF]
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S. Nicole, B. Desforges, G. Millet, J. Lesbordes, C. Cifuentes-Diaz, D. Vertes, M. L. Cao, F. De Backer, L. Languille, N. Roblot, et al. Intact satellite cells lead to remarkable protection against Smn gene defect in differentiated skeletal muscle J. Cell Biol., May 12, 2003; 161(3): 571 - 582. [Abstract] [Full Text] [PDF]
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 P. E. Polak, F. Simone, J. J. Kaberlein, R. T. Luo, and M. J. Thirman ELL and EAF1 are Cajal Body Components That Are Disrupted in MLL-ELL Leukemia Mol. Biol. Cell, April 1, 2003; 14(4): 1517 - 1528. [Abstract] [Full Text] [PDF]
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D. Charvin, C. Cifuentes-Diaz, N. Fonknechten, V. Joshi, J. Hazan, J. Melki, and S. Betuing Mutations of SPG4 are responsible for a loss of function of spastin, an abundant neuronal protein localized in the nucleus Hum. Mol. Genet., January 1, 2003; 12(1): 71 - 78. [Abstract] [Full Text] [PDF]
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 A. Buj-Bello, V. Laugel, N. Messaddeq, H. Zahreddine, J. Laporte, J.-F. Pellissier, and J.-L. Mandel The lipid phosphatase myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice PNAS, November 12, 2002; 99(23): 15060 - 15065. [Abstract] [Full Text] [PDF]
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S. Vyas, C. Bechade, B. Riveau, J. Downward, and A. Triller Involvement of survival motor neuron (SMN) protein in cell death Hum. Mol. Genet., October 15, 2002; 11(22): 2751 - 2764. [Abstract] [Full Text] [PDF]
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 S. Massenet, L. Pellizzoni, S. Paushkin, I. W. Mattaj, and G. Dreyfuss The SMN Complex Is Associated with snRNPs throughout Their Cytoplasmic Assembly Pathway Mol. Cell. Biol., September 15, 2002; 22(18): 6533 - 6541. [Abstract] [Full Text] [PDF]
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L. Fan and L. R. Simard Survival motor neuron (SMN) protein: role in neurite outgrowth and neuromuscular maturation during neuronal differentiation and development Hum. Mol. Genet., July 1, 2002; 11(14): 1605 - 1614. [Abstract] [Full Text] [PDF]
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 C. Soler-Botija, I. Ferrer, I. Gich, M. Baiget, and F. Tizzano Neuronal death is enhanced and begins during foetal development in type I spinal muscular atrophy spinal cord Brain, July 1, 2002; 125(7): 1624 - 1634. [Abstract] [Full Text] [PDF]
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C. Cifuentes-Diaz, S. Nicole, M. E. Velasco, C. Borra-Cebrian, C. Panozzo, T. Frugier, G. Millet, N. Roblot, V. Joshi, and J. Melki Neurofilament accumulation at the motor endplate and lack of axonal sprouting in a spinal muscular atrophy mouse model Hum. Mol. Genet., June 1, 2002; 11(12): 1439 - 1447. [Abstract] [Full Text] [PDF]
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W. Rossoll, A.-K. Kroning, U.-M. Ohndorf, C. Steegborn, S. Jablonka, and M. Sendtner Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons? Hum. Mol. Genet., January 1, 2002; 11(1): 93 - 105. [Abstract] [Full Text] [PDF]
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C. J. DiDonato, C. L. Lorson, Y. De Repentigny, L. Simard, C. Chartrand, E. J. Androphy, and R. Kothary Regulation of murine survival motor neuron (Smn) protein levels by modifying Smn exon 7 splicing Hum. Mol. Genet., November 1, 2001; 10(23): 2727 - 2736. [Abstract] [Full Text] [PDF]
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 M. D. Hebert, P. W. Szymczyk, K. B. Shpargel, and A. G. Matera Coilin forms the bridge between Cajal bodies and SMN, the Spinal Muscular Atrophy protein Genes & Dev., October 15, 2001; 15(20): 2720 - 2729. [Abstract] [Full Text] [PDF]
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C. Cifuentes-Diaz, T. Frugier, F. D. Tiziano, E. Lacene, N. Roblot, V. Joshi, M. H. Moreau, and J. Melki Deletion of Murine SMN Exon 7 Directed to Skeletal Muscle Leads to Severe Muscular Dystrophy J. Cell Biol., March 5, 2001; 152(5): 1107 - 1114. [Abstract] [Full Text] [PDF]
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S. Jablonka, M. Bandilla, S. Wiese, D. Buhler, B. Wirth, M. Sendtner, and U. Fischer Co-regulation of survival of motor neuron (SMN) protein and its interactor SIP1 during development and in spinal muscular atrophy Hum. Mol. Genet., March 1, 2001; 10(5): 497 - 505. [Abstract] [Full Text] [PDF]
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L. Pellizzoni, B. Charroux, J. Rappsilber, M. Mann, and G. Dreyfuss A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II J. Cell Biol., January 8, 2001; 152(1): 75 - 86. [Abstract] [Full Text] [PDF]
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D. A. Kerr, J. P. Nery, R. J. Traystman, B. N. Chau, and J. M. Hardwick Survival motor neuron protein modulates neuron-specific apoptosis PNAS, November 8, 2000; (2000) 230364197. [Abstract] [Full Text]
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P. J. Young, N. t. Man, C. L. Lorson, T. T. Le, E. J. Androphy, A. H.M. Burghes, and G. E. Morris The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in self-association and SIP1 binding Hum. Mol. Genet., November 1, 2000; 9(19): 2869 - 2877. [Abstract] [Full Text] [PDF]
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U. R. Monani, D. D. Coovert, and A. H.M. Burghes Animal models of spinal muscular atrophy Hum. Mol. Genet., October 1, 2000; 9(16): 2451 - 2457. [Abstract] [Full Text] [PDF]
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 J. Wang and G. Dreyfuss A Cell System with Targeted Disruption of the SMN Gene. FUNCTIONAL CONSERVATION OF THE SMN PROTEIN AND DEPENDENCE OF Gemin2 ON SMN J. Biol. Chem., March 23, 2001; 276(13): 9599 - 9605. [Abstract] [Full Text] [PDF]
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J. Wang and G. Dreyfuss Characterization of Functional Domains of the SMN Protein in Vivo J. Biol. Chem., November 21, 2001; 276(48): 45387 - 45393. [Abstract] [Full Text] [PDF]
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D. A. Kerr, J. P. Nery, R. J. Traystman, B. N. Chau, and J. M. Hardwick Survival motor neuron protein modulates neuron-specific apoptosis PNAS, November 21, 2000; 97(24): 13312 - 13317. [Abstract] [Full Text] [PDF]
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