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From the Molecular Neurogenetics Laboratory,* Institut National de la Santé et de la Recherche Médicale (INSERM), E-223 Université d’Evry, Evry; the Service d’Anatomie Pathologie, Centre Hospitalier Sud-Francilien, Evry; the Hôpital Saint Vincent de Paul, Paris; CNRS FRE-2401, the Institut de Biologie, Collège de France, Paris; and the Laboratoire de Biologie Clinique,¶ Centre Hospitalier Sud-Francilien, Evry, France
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Abstract |
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Materials and Methods |
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Mice carrying Smn exon 7 flanked by two loxP sites (SmnF7) were generated through homologous recombination in embryonic stem cells.7
Transgenic line expressing the Cre recombinase transgene driven by both the mouse albumin regulatory elements and the 
-fetoprotein enhancers was used for hepatocyte-specific expression of the Cre recombinase (Alfp-Cre).11
(Alfp-Cre, SmnF7/F7) mutant mice were generated by crossing homozygous (SmnF7/F7) mice to those carrying the Cre recombinase transgene (Alfp-Cre) and the (SmnF7/+) genotype. Mice were maintained on C57BL/6J genetic background. (SmnF7/+) or (SmnF7/F7) mice from litter were used as controls. Animals were genotyped by PCR amplification of DNA extracted from tail biopsies.7,12
Ex7sou1 and GS8 primers were used to amplify the wild-type (Smn+, 430 bp), the SmnF7 allele (630 bp) or both. The transmission of the Alfp-Cre transgene was confirmed by PCR amplification of DNA using primers Cre1 and Cre2.12
All animal procedures were performed in accordance with institutional guidelines (agreement numbers A91–228-2 and 3429). Mice were maintained in a pathogen-free animal house.
Analysis of Cre Recombinase Transgene Activity
To characterize the pattern of Cre recombinase activity, Rosa26 reporter mice were used (R26R).13 ß-galactosidase expression was assayed on tissues of adult or whole embryos carrying both R26R locus and Alfp-Cre transgene. Double transgenic mice were identified using PCR amplification analysis of DNA for Cre and R26R transgenes as previously described.13 Noon of the day of the plug was considered as 0.5 days p.c. Embryos were dissected in 0.1 mol/L phosphate buffer (PB) then fixed in 0.2% glutaraldehyde, 5 mmol/L EGTA, 2 mmol/L MgCl2 in 1X PB for 10 minutes at 4°C. Fixed embryos were washed three times in 2 mmol/L MgCl2, 1% Nonidet P-40 (NP-40) in 1X PB and incubated for 3 to 4 hours at 37°C in 1 mg/ml X-Gal, 5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide in the washing solution as described above. After staining, embryos were rinsed twice in the washing solution. Tissues from adult mice were frozen in isopentane cooled on liquid nitrogen. Frozen sections of 10 µm were fixed in 2% formaldehyde and 0.2% glutaraldehyde in 1X PB for 10 minutes at 4°C. Fixed tissues were washed twice in 1X PB, and incubated overnight at 37°C in the staining solution described above without NP-40. After staining, tissues were washed twice in 1X PB and counterstained with safranine.
To test the efficiency of Smn exon 7 excision by the Cre recombinase, Alfp-Cre transgenic mice were crossed to 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).7
The efficiency of excision was estimated by comparing the intensity of the band amplified from the SmnF7 allele with that from the wild-type allele which differs only by the absence of the loxP site. Detection of the Smn
7 allele was performed by PCR amplification using primers pHR5 and GS8. 7
Morphological and Immunohistochemical Analyses
Liver, spinal cord, or skeletal muscle samples of adult mice or whole embryos were fixed in fresh 10% formalin for at least 24 hours at 4°C. At least three mutant or control mice or embryos were analyzed. Samples were processed for routine light histochemistry. Fixation was followed by dehydration using a series of graded concentrations of alcohol and toluene and embedding in paraffin. Five-µm sections were de-waxed, washed and stained with hematoxylin eosin safran (HES), periodic acid-Schiff (PAS), Perls staining, and Masson method. Sections were observed under light microscope. The slides were examined by three different observers in blind manner.
For immunolabeling experiments, liver samples were frozen in Tissue-Tek (Sakura) on dry ice. For -fetoprotein (AFP) immunostaining and TdT-mediated x-dUTP nick end labeling (TUNEL) analyses, 5-µm frozen sections were prepared from liver of control and mutant embryos. For AFP immunostaining, sections were fixed in cold acetone for 10 minutes and incubated in blocking solution containing 3% donkey serum in PBS for 30 minutes, then with the anti-AFP antibody (1:40, Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at +4°C. After washing in PBS, sections were incubated with Cy3-conjugated anti-goat antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature. For CD68 labeling, sections were fixed with formalin, blocked, then incubated with anti-CD68 (1:200, clone PG-M1, DAKO, Denmark). The TUNEL assay was performed using the In Situ Cell Death Detection kit (Fluorescein, Roche Applied Science, Penzberg, Germany). For Ki67 immunostaining, 7-µm frozen sections were prepared and immediately fixed in Zamboni’s fixative (2% paraformaldehyde and 15% picric acid in PB) for 10 minutes.14
Sections were washed in Tris-buffered saline (TBS), pH 7.6 and incubated in blocking solution containing 3% donkey serum in TBS for 10 minutes, and then with the anti-Ki67 antiserum (1:200, Novocastra Laboratories Ltd.) for 1 hour at room temperature. After washing in TBS, sections were incubated with FITC-conjugated anti-rabbit antibodies (1:1000, Jackson ImmunoResearch Laboratories) for 45 minutes at room temperature. Sections were then mounted with Vectashield (Vector Laboratories) and observed under Zeiss Axiophot fluorescence microscope.
Transcript Analysis
Total RNA was extracted from freshly isolated tissues using the Trizol procedure (Gibco-BRL). First-strand cDNA synthesis was performed by using either random or oligo(dT) primers. PCR amplification analysis of single-strand cDNA was performed using primers flanking Smn exon 7 (ex5sou2, 5'- TGC TGG ATG CCC CCG TTC CCT TCA- 3' and ex8sou, 5'- GGC ACG CTC TGC TGC TGA CTT AG - 3') and revealed Smn transcripts containing (350 bp) or lacking exon 7 (290 bp). Transcript analysis of genes encoding transferrin receptor 1 (TFR1), transferrin receptor 2 (TFR2), HFE, hepcidin 1 and 2 (HAMP1 and 2) was performed using the following primers. TFR1: TFR1-Ex2F, 5'-GTAGATGGAGATAACAGTCATGTG-3' and TFR1-Ex5R, 5'- AGTTACCATTT GACTGCACTATG-3'; TFR2: TFR2-Ex1F, 5'-CACAAGCATGGAGCAACGTTGG-3' and TFR2-Ex6R, 5'-CTTTTAGGTCCTGTAGGTCCTC-3'; HFE: HFE-Ex2, 5'-GGACCATCAT GGGCAACTATAAC-3' and HFE-Ex6, 5'-TGTTAAGACATAGCCACCCATGG-3', HAMP1: HEPC1-F, 5'-CCTATCTCCATCAACAGATGAGAC-3' and HEPC1-R, 5'-GATGTGGCTCT AGGCTATGTTTTG-3'; HAMP2: HEPC2-F, 5'-CCTATCTCCAGCAACAGATGAGAC-3' and HEPC2-R, 5'-TTCTTCACAACAGATACCACAGGA-3'. As internal control for RT-PCR amplification analysis, aldolase A cDNA was co-amplified using primers aldo 1 (5' - TAA GAA GGA TGG AGC CGA CTT TG -3') and aldo 600 (5'- GCG AGG CTG TTG GCC AGG GCG CG-3'). RT-PCR products were separated by agarose gel electrophoresis and labeled with ethidium bromide.
Serum Level of Liver Proteins
Serum enzymatic activities of transaminases and 
-glutamyl transferase (-GT) from control (SmnF7/F7) or (Alfp-Cre, SmnF7/+) mice were evaluated according to standard procedures. Blood was collected by retro-orbital puncture of anesthetized mice and the serum was stored at –80°C before measurement. Serum activities of transaminases, -GT and dosage of -fetoprotein and albumin were determined from blood samples of SMA patients according to standard procedures and after informed consent of families or patients.
Immunoblotting Experiments
Frozen liver of (SmnF7/+), (SmnF7/F7), (Alfp-Cre, SmnF7/+) or (Alfp-Cre, SmnF7/F7) mice were rapidly frozen and crushed in liquid nitrogen using a mortar and pestle. Pulverized tissue samples were transferred into 5 volumes of a buffer containing 25 mmol/L sodium phosphate (pH 7.2), 5 mmol/L ethylene diamine tetraacetic acid (EDTA) and 1% sodium dodecyl sulfate (SDS) supplemented with protease inhibitor cocktail (Sigma, St. Louis, MO), and boiled for 5 minutes. Protein sample (25 to 50 µg) was mixed with an equal volume of 2X Laemmli buffer, electrophoresed, and then transferred. For SMN and actin immunodetection, anti-SMN and anti-actin monoclonal antibodies were used (1:5000, Transduction Laboratories, Lexington, CA; 1:10000, Chemicon International Inc., Temecula, CA, respectively). After washes in PBS, 0.05% Tween 20, membranes were incubated with anti-mouse IgG conjugated to horseradish peroxydase (1:5000) and the immune complexes were revealed using chemiluminescent detection reagents (Pierce, Rockford, IL). For AFP and albumin immunodetections, goat anti-AFP and anti-albumin antibodies were used (1:500, Santa Cruz Biotechnology; 1:15000, Bethyl Lab. Inc., Montgomery, TX) and revealed by anti-goat IgG conjugated to horseradish peroxidase (1:500).
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Results |
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To characterize the Cre recombinase expression pattern of Alfp-Cre transgene, (Alfp-Cre) transgenic mice were crossed to either Rosa26 reporter mice (R26R) or mice harboring SmnF7 allele. In adult transgenic mice carrying both Alfp-Cre and R26R transgenes, liver stained uniformly blue with standard ß-galactosidase incubation times, indicating a specific and strong Cre recombinase activity in liver (Figure 1A)
. No ß-galactosidase activity was detected in other tissues including lung, kidney, brain, heart, or spleen (Figure 1A)
. These results were further supported by PCR amplification analysis of genomic DNA extracted from various tissues of adult mice carrying both Alfp-Cre transgene and SmnF7/+ genotype. Cre-mediated deletion of Smn exon 7 was observed in liver but not in other tissues, indicating that deletion of Smn exon 7 was restricted to liver (Figure 1C)
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and data not shown). In other tissues, no ß-galactosidase activity was observed at that stage or in older embryos (Figure 1B
and data not shown). These findings led us to use the Alfp-Cre transgenic line for the targeting of Smn in liver but not in the other tissues. Homozygous Deletion of Smn Exon 7 Directed to Liver Leads to Late Embryonic Lethality
To generate mutant mice carrying homozygous deletion of Smn exon 7 directed to liver (Alfp-Cre, SmnF7/F7) in a 1:4 ratio, mice homozygous for the SmnF7 allele (SmnF7/F7) were crossed to mice carrying both the Alfp-Cre transgene and (SmnF7/+) genotype. These crosses generated no living mutant mice carrying (Alfp-Cre, SmnF7/F7) genotype of 201 newborns analyzed (Table 1
and Figure 2A
). To determine the timing of the lethality, embryos were analyzed during gestation. Until 16 days p.c., embryos showed the expected ratio for the various genotypes, then progressive lack of embryos carrying the (Alfp-Cre, SmnF7/F7) genotype was observed (Figure 2A)
. These data indicate that homozygous deletion of Smn exon 7 directed to liver leads to late embryonic lethality from 18 days p.c. to birth.
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. Using primers flanking Smn exon 7, RT-PCR analysis of Smn in liver of mutant embryos revealed a marked reduction of the full-length transcript (Figure 3A)
. Surprisingly, Smn7 transcripts were detected in liver of heterozygous but not homozygous mutant mice suggesting that cells carrying homozygous Cre-mediated deletion of Smn exon 7 do not survive (Figure 3A)
. Immunoblotting of proteins extracted from liver of 18 days p.c. mutant (Alfp-Cre, SmnF7/F7) or control embryos (SmnF7/+ or SmnF7/F7) was performed using a monoclonal antibody directed against the N-terminus of the SMN protein. Dramatic reduction of SMN was observed in liver of mutant embryos, which correlated with the marked decrease of full-length Smn transcripts (Figure 3B)
. The residual amount of the SMN protein likely resulted from non-deleted liver or hematopoeitic cells.
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Macroscopic examination of (Alfp-Cre, SmnF7/F7) mutant embryos revealed a dramatic reduction of liver mass (Figure 2C)
. From 16 to 21 days p.c., weight of liver was markedly diminished in mutant embryos (Alfp-Cre, SmnF7/F7; 10 mg ± 1.3 SE, 86% reduction at 18 days p.c., P < 0.0001) when compared with controls of the same age (SmnF7/+ or SmnF7/F7; 73 mg ± 2.1 SE, Figure 2B
). Hematoxylin and eosin staining of liver sections of 18 days p.c. mutant and control embryos was performed. At 18 days p.c., the morphology of liver from mutant mice revealed markedly reduced number of hepatocytes and bile ducts, liver tissue being mainly composed of hematopoeitic cells (Figure 4)
. No liver fibrosis was observed as determined by Masson method (data not shown). The morphology of the remaining hepatocytes was similar to that of control embryos and no nuclear fragmentation was observed. TUNEL staining did not reveal any increased number of apoptotic cells in mutant when compared to control embryos (Figure 4
and data not shown). Marked reduction in number of hepatocytes and bile ducts strongly suggested that homozygous mutation of Smn resulted in dramatic impairment of liver development.
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-fetoprotein was evaluated. Dramatic reduction of albumin and -fetoprotein expression was observed in (Alfp-Cre, SmnF7/F7) mutant embryos when compared to that of (SmnF7/+) control embryos and actin expression (Figure 3)
. Consistently, immunolabeling experiments performed on frozen liver sections revealed very few hepatocytes labeled by -fetoprotein antibody in mutant embryos as compared to control embryos. These results indicate further a marked reduction of functional hepatocyte number (Figure 4)
. Defect of liver functions was also suspected by the absence of blood clot after umbilical cord section of dissected mutant but not of control embryos. These data suggest a defect in hemostasis, which could be responsible for neonatal lethality during delivery.
To ascertain whether liver regeneration occurred in mutant embryos, immunohistochemical experiments were performed on frozen liver sections using antibody directed against Ki67, an antigen specific to cell cycle division. The density of Ki67-positive cells was similar in both control and mutant embryos suggesting that liver regeneration did not occur in mutant embryos (Figure 4)
. Altogether, these data strongly suggest that Cre-mediated deletion of Smn exon 7 occurred in hepatoblasts leading to marked defect of liver development associated with lack of liver regeneration. The presence of rare hepatocytes likely results from cells that do not express the Cre recombinase in agreement with the low amount of SMN in mutant embryos.
Mutant Embryos Display Massive Iron Overload Restricted to Liver
Surprisingly, Perls staining showed massive iron overload in liver of 16 days p.c. mutant embryos when compared to control embryos of the same age (Figure 5)
. Examination of other tissues including brain, intestine, kidney, pancreas, bone, or heart from mutant embryos did not reveal any accumulation of iron indicating that it was restricted to liver (data available on request). To determine whether iron accumulated in hepatocytes or reticulo-endothelial cells, serial sections of liver were labeled with Perls and anti-CD68, a marker specific to reticulo-endothelial cells. Iron overload was observed in both types of cells. The same labeling was applied to lung and revealed high proportion of reticulo-endothelial cells without any iron accumulation. These results indicate that iron overload occurred in hepatocytes and reticulo-endothelial cells of liver (data available on request).
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. No abnormal spliced products were detected in mutant liver when compared to control. Similar results were obtained using either random or oligo(dT) primers for cDNA synthesis.
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Lack of Dominant Negative Effect of Heterozygous Deletion of Smn Exon 7 in Vivo
To determine whether heterozygous deletion of Smn exon 7 might have any deleterious effect on liver, (Alfp-Cre, SmnF7/+) mice were analyzed. Macroscopic examination of liver from (Alfp-Cre, SmnF7/+) embryos did not reveal any abnormalities (Figure 2B)
and ratio of liver to total body weight of heterozygous or control adult mice did not differ significantly (mean of 3.7% in SmnF7/F7 and 3.9% in Alfp-Cre, SmnF7/+, Student’s t-test, P = 0.34). In addition, histological analysis of liver from (Alfp-Cre, SmnF7/+) adult mice did not show any change when compared to control mice (SmnF7/F7, data not shown). No iron accumulation was detected in liver of adult heterozygous mice (data not shown). RT-PCR analysis of RNA extracted from liver of heterozygous mice (Alfp-Cre, SmnF7/+) revealed the presence of truncated transcripts lacking exon 7 and immunoblotting experiments showed as expected half dose of SMN in liver of (Alfp-Cre, SmnF7/+) adult mice when compared to control mice and actin expression (Figure 3)
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Serum activities of enzymes expressed in liver including transaminases and -glutamyl transferase (-GT) did not reveal any change in either young (1- to 2-month-old, n = 11) or older heterozygous mice (7- to 13-month-old, n = 7) when compared with controls of the same age (SmnF7/F7, n = 9, Table 2
). Finally, immunoblotting experiments revealed no increase of -fetoprotein protein amount in (Alfp-Cre, SmnF7/+) mice with respect to that of (SmnF7/F7) control of the same age and actin expression (Figure 3C)
. These data indicate that heterozygous deletion of Smn exon 7 is sufficient for normal liver function and did not provide any evidence for liver necrosis nor for regeneration as determined by liver enzyme or -fetoprotein analyses, respectively.
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-GT, transaminases and dosage of -fetoprotein and albumin were evaluated in serum of four patients affected with SMA type II, the intermediate form of SMA. In these patients, onset of symptoms occurred within the first year of life.20
They were able to sit unaided but not to walk. Patients carried homozygous deletion of SMN1 exon 7 (data not shown). Serum analysis was performed at a mean time of 13.3 years after the onset of symptoms (11 to 17 years). No change was observed indicating that the residual amount of SMN in liver of SMA patients is sufficient for normal liver function during natural SMA disease course (Table 2)
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Discussion |
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-fetoprotein associated with the absence of Smn7 transcripts strongly suggest that hepatocytes carrying homozygous Cre-mediated deletion of Smn exon 7 do not survive. Negative TUNEL staining excludes apoptosis as the pathogenic mechanism involved in cell loss. Surprisingly, no liver regeneration was observed from the remaining hepatocytes or hepatoblasts as determined by both the marked reduction of -fetoprotein and the absence of increased number of dividing cells in liver of mutant embryos. It can be hypothesized that hepatocyte progenitors entering into cell cycle division lead to the generation of new hepatocytes in which Cre recombinase activity will induce deletion of Smn and cell loss.
These results demonstrate that Smn transcripts lacking exon 7 (Smn7), which represent the predominant transcript isoform encoded by the SMN2 gene in human,3
are not able to compensate for the lack of full-length Smn transcripts and lead to loss of function. Loss of function rather than dominant negative effect of Smn7 was further supported by the analysis of heterozygous mutant mice. Heterozygous deletion of Smn exon 7 in liver, which results in the expression of both full-length and truncated transcripts, did not result in any changes in either morphology or function of liver. In these mice, the presence of half dose of SMN protein strongly suggests an instability of Smn7 protein as previously suspected.8
These results indicate that half dose of SMN is sufficient for normal liver function and exclude the hypothesis of a dominant negative effect of Smn7 on wild-type allele in vivo. These data are consistent with previous in vivo data in either mice8
or human SMA carriers.
The most striking feature found in mutant embryos was massive iron deposition in the liver. Examination of other organs did not provide any evidence for iron accumulation in other tissues. Moreover, examination of tissues in which homozygous deletion of Smn exon 7 had been previously targeted, namely skeletal muscle and neurons, did not reveal iron overload.7-9 Therefore, genes encoding proteins predominantly expressed in liver and playing an essential role in iron homeostasis might be regarded as targets of SMN defect, a protein involved in RNA processing in cell systems (5 for review). Marked reduction or lack of transcripts encoding TFR2, TFR1, HFE, and HAMP1 was observed in mutant liver embryos, which could be ascribed to marked reduction of functional hepatocytes in mutant embryos. Several spontaneous or induced mutations have been reported in murine genes encoding proteins involved in iron metabolism. Interestingly, iron accumulation in liver was observed from 1-month-old mutant mice but not in embryos lacking hepcidin or HFE21,22 excluding both genes as candidates to account for liver iron deposition in Smn mutant embryos. In mice carrying homozygous mutation of Tfr2 or ß2-microglobulin genes, liver iron accumulation was observed from 1 to 8 months of age but no data are available in liver of mutant embryos.23,24 However, these genes have been regarded as candidates in human neonatal hemochromatosis (OMIM 231100), a rare syndrome in which congenital cirrhosis or fulminant hepatitis in early infancy is associated with marked iron deposition in the liver and extrahepatic tissues. Autosomal recessive inheritance has been suggested in familial forms. No pathogenic mutations in ß2-microglobulin, HFE, and haem oxygenases 1 and 2 genes have been identified in multiplex families.25 In the same way, mutations of the TFR2 gene have been identified in juvenile but not in neonatal form of hemochromatosis.26 Our data and previous studies in various mouse models of hemochromatosis or in human inherited neonatal hemochromatosis did not provide any rational candidate gene to account for iron accumulation in liver embryos deficient for SMN. Evidence from human patients with neonatal hemochromatosis strongly suggests that iron accumulation might be secondary to disrupted hepatocellular development resulting from various non-inherited insults.27 In Smn mutant mice, severe impairment of liver development could be responsible for defect of various liver functions including iron regulation leading to liver iron overload. Alternatively, liver being the major site of iron storage, iron accumulation could result from marked loss of hepatocytes no longer able to ensure this function.
Importantly, this study indicates that the lack of full-length SMN protein in various mouse cell types including neurons, skeletal muscle fibers, or hepatocytes leads to severe cell dysfunction. However, in human SMA, the disease phenotype is restricted to degeneration of the neuromuscular system and no liver dysfunction had been reported so far. How to explain the phenotypic difference in response to SMN defect in liver between human and mouse? Murine Smn gene is present as a single copy while in humans, SMN1 is duplicated in a highly homologous copy SMN2 (4 for review). The residual amount of SMN produced by full-length transcripts of the SMN2 gene, which remains present in patients, is likely sufficient to ensure normal functions of various organs including liver but not motor neurons. Motor neuron is one of the largest cells in the body by both volume and surface area. Such large cellular unit requires molecular machinery able to produce and regulate molecules, including proteins and RNA, from the cell body to the neuromuscular junction through motor axons. Mild impairment in production or stability of molecules involved in these processes could have detrimental effect on axonal growth. This hypothesis is consistent with features found in mouse or Drosophila carrying mutation of Smn9,28 or in in vitro experiments.29,30 Identifying defective transcripts encoding proteins involved in or regulating structural specificities of motor neurons should contribute to elucidate SMA pathogenesis.
However, other neurons including sensory neurons are also very large in size. Recently, involvement of these neurons has been described in severe cases of SMA.31 Skeletal muscle has also been recently shown to be involved when lack of murine SMN has been directed to that tissue.8,10 Although motor neurons are the predominant cell type affected in the human disease, data from mouse and human suggest that other tissues might be involved during prolonged SMA disease course or in very severe form of SMA. Our results reinforce the need of therapeutic approaches targeted to all tissues in human SMA. In addition, liver function of patients should be carefully investigated and followed up before and during therapeutic trials.
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Acknowledgements |
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Footnotes |
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Supported by INSERM, the Association Française contre les Myopathies (AFM), Families of SMA (USA), and the Fondation Bettencourt Schueller.
Accepted for publication July 27, 2004.
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