Human Molecular Genetics, 2002, Vol. 11, No. 22 2751-2764
© 2002 Oxford University Press
Involvement of survival motor neuron (SMN) protein in cell death
1INSERM U497, 46 rue d'Ulm, Paris 75005, France and 2Signal Transduction Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
Received June 26, 2002; Accepted August 12, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Infantile spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron (SMN)1 gene. We investigated the role of human (h) SMN protein on cell death in PC12 and Rat-1 cells. hSMN prolonged cell survival in PC12 cells deprived of trophic support and in Rat-1 cells induced to die by activation of the proto-oncogene c-Myc, to similar magnitude as Bcl-2 or IAP-2. While hSMN was ineffective in inhibiting apoptosis induced by ultraviolet light (UV) or etoposide treatment in proliferating PC12 or Rat-1 cells, a protective effect was observed in terminally NGF/dBcAMP-differentiated PC12 cells. hSMN inhibited the onset of apoptosis in NGF/dBcAMP-deprived or UV-treated co-differentiated PC12 cells by preventing cytochrome c release and caspase-3 activation, indicating that its effects are through suppression of the mitochondrial apoptotic pathway. Expressing hSMN deleted for exon 7 (
7) or for exons 6 and 7 (6/7), or with the SMA point mutant Y272C, resulted in loss of survival function. Moreover, these mutants also exhibited pro-apoptotic effects in Rat-1 cells. The localization pattern of full-length hSMN in PC12 and Rat-1 cells was similar to that of endogenous SMN: granular labelling in the cytoplasm and discrete fluorescence spots in the nucleus, some of which co-localized with p80 coilin, the characteristic marker of Cajal bodies. However, cytoplasmic and nuclear aggregates were often seen with hSMN7, whereas the hSMN6/7 mutant showed homogenous nuclear labelling that excluded the nucleolus. Thus, our results show that the C-terminal region is critical in suppression of apoptosis by SMN. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinal muscular atrophy (SMA) is the most frequent autosomal recessive neurodegenerative disorder of childhood. It is characterized primarily by loss of motoneurons in spinal cord and brain stem, and by muscular atrophy that results in progressive paralysis of limbs and trunk (1). SMA is classified into three types (I–III) according to the age of onset and the clinical course of the disease, type I being the most severe form (Werding–Hoffman syndrome) (2).
Mutations or deletions in the survival motor neuron gene, SMN1 have been shown to be the cause of SMA (3–5). In humans, two highly homologous copies of the SMN gene, the telomeric SMN1 and centromeric SMN2, are found in a duplicated and an inverted region of chromosome 5q13 (4). Most SMA mutations are situated in the C-terminal region of SMN1, between exons 6 and 7. It has been shown that reduced levels of full-length SMN, particularly in the spinal cord, contribute to loss of motoneurons and increased severity of the disease (6–8). This observation was reproduced in in vivo experimental models of SMA: homozygous SMN-/- mice expressing the human SMN2 transgene (9,10), heterozygous SMN+/- mice (11) and mice with a conditional in vivo deletion of exon 7 (SMNF7/7, Cre+) (12) developed motoneuron degeneration and SMA-like symptoms, the severity depending on the copy number of full-length SMN.
Functionally, experimental evidence indicates that SMN acts as a crucial adaptor or an assembly factor for protein complexes that regulate RNA metabolism (13). Its localization in the cytoplasm and in discrete subnuclear structures, the gems and Cajal (coiled) bodies, appears to be important in its role in the regulation of processes such as snRNP biogenesis, pre-mRNA splicing or transcription (14–17). Recent evidence has shown that SMN translocates to these nuclear bodies by binding to p80 coilin, a signature protein of Cajal bodies, and also to a zinc finger protein, ZPR1 (18–20). In spinal cord neurons, SMN localization at the cytoplasmic face of nuclear pores and its co-localization with cytoskeletal proteins in dendrites and axons suggests that it may also function in nucleocytoplasmic, dendritic or axonal transport (21,22).
SMN is highly conserved across species and ubiquitously expressed in mammalian organisms (23). Its expression levels are high during embryonic development but are reduced postnatally (24,25). Early embryonic lethality that occurs upon homozygous deletion of the SMN gene in mice (26) and a reduction in the Caenorhabditis elegans progeny by disrupting the C. elegans ortholog CeSMN indicates that SMN plays a critical role in development (27). Additionally, in the fission yeast Schizosaccharomyces pombe, drastic reductions in cell growth and in mislocalization of the protein was found with the N- or C-terminal-deleted yeast ortholog ySMN (28,29). Although all these studies support a role for SMN in cell survival, contradictory results have been obtained regarding its involvement in suppressing cell death. The murine homologue of SMN had no effect in motoneurons on cell death induced by glutamate toxicity or trophic factor withdrawal (30). On the other hand, the human SMN was shown to synergize with Bcl-2 to inhibit Fas- or Bax-mediated cell death and, in vivo, to protect CNS neurons against Sindbis virus-induced cell death (31,32). The intracellular mechanisms through which SMN can promote survival have not been elucidated.
In this study, we have analyzed the role of expressing human SMN protein on apoptosis triggered by withdrawal of trophic support in undifferentiated and differentiated PC12 cells or c-Myc induced apoptosis in Rat-l cells. Co-treatment of PC12 cells with nerve growth factor (NGF) and dibutyryl-cAMP (dBcAMP) renders these cells terminally differentiated and dependent on these factors for survival (33). c-Myc activates both proliferation and apoptotic programmes, but it is only in situations of stress such as lack of trophic support, growth inhibition or DNA damage that cells undergo c-Myc-induced apoptosis (34). Importantly, the intracellular apoptotic pathways have been characterized both in PC12 cells lacking trophic support and in Rat-1 cells undergoing apoptosis by c-Myc activation (35,36). We find that expressing hSMN results in inhibition of cell death in both of these cell types. Our results indicate that hSMN delays the onset of apoptosis by acting on mechanisms that mediate cell death through the mitochondrial release of cytochrome c. In addition, we show that the C terminal of hSMN, in particular exons 6 and 7, is important for its cell-death-inhibitory function.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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hSMN inhibits cell death induced by trophic factor deprivation in PC12 cells
Cell death triggered by withdrawal of trophic support in both undifferentiated and differentiated PC12 cells can be suppressed by specific survival factors such as NGF and insulin-life growth factor I (IGF-I) (37) or by overexpression of anti-apoptotic proteins, such as Bcl-2 (S. Vyas, personal observation). In order to determine whether SMN has any effect on cell death induced by trophic withdrawal in PC12 cells, we transiently co-transfected undifferentiated PC12 cells with reporter LacZ and either control (empty vector) or HA-tagged hSMN plasmids. Cell death was quantified by scoring all viable ß-galactosidase-positive PC12 cells in cultures containing serum or deprived of serum for 24 hours. Withdrawal of serum for 24 hours from PC12 cells transfected with control vector resulted in a 45% decrease in the number of ß-galactosidase-positive cells. In contrast, a decrease of 15% was observed in cells that were transfected with HA–hSMN and then deprived of serum for 24 hours (Fig. 1A), showing that hSMN is inhibiting cell death. In parallel, we also transfected cells with IAP-2 and tested its effect on cells deprived of serum for 24 hours. IAP-2, a member of the inhibitor of apoptosis family of proteins, suppresses cell death induced by a wide range of apoptotic stimuli (38). The results of co-transfection with IAP-2 showed a decrease similar to that due to hSMN in the number of ß-galactosidase-positive cells (Fig. 1A).
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Differentiation of PC12 cells with NGF and dBcAMP renders these cells post-mitotic: both morphologically and functionally, they exhibit sympathetic neuron-like characteristics. We have previously shown that these cells become dependent on NGF or dBcAMP for survival: withdrawal of NGF/dBcAMP from culture medium results in significant cell death between 16 and 48 hours (33). To examine whether hSMN is protective against cell death induced by trophic withdrawal in differentiated PC12 cells, two PC12 cell lines stably expressing HA–hSMN (PC12/hSMN 5A and 10A) were generated. The expression of HA-tagged hSMN in these cells was verified by immunofluorescence using anti-HA antibody (data not shown) and SMN overexpression by western blot analysis (Fig. 1B). The effect of NGF/dBcAMP withdrawal was analyzed by quantifying the percentage of apoptotic nuclei in NGF/dBcAMP differentiated control PC12 cells, PC12/hSMN (PC12/hSMN 5A and 10A) and in PC12 cells stably expressing human Bcl-2 (PC12/hBcl-2). The antiapoptotic family members (e.g. Bcl-2 and Bcl-XL) inhibit apoptosis induced by diverse stimuli by preventing the release of cytochrome c from mitochondria and caspase activation (39). In control, differentiated PC12 cells deprived of NGF/dBcAMP for 24 hours, 16%±0.7% of cells had fragmented nuclei, whereas in similarly deprived PC12/hSMN 5A and 10A and in PC12/hBcl-2 cells, 6%±1.0%, 10%±1.39% and 6%±1.0% of cells, respectively, displayed nuclear apoptotic morphology (Fig. 1B). The results therefore indicate that hSMN, like Bcl-2, also protects differentiated PC12 cells against cell death by trophic deprivation.
hSMN inhibits cell death by UV and etoposide treatment in differentiated PC12 cells but not in undifferentiated cells
To explore further the range of antiapoptotic function of SMN, we tested its ability to inhibit ultraviolet light (UV)- and etoposide-mediated cell death in both undifferentiated and differentiated PC12 cells. The effect of these DNA-damaging agents was also analyzed in PC12/hBcl-2 cells and in PC12 cells stably expressing dominant-negative caspase-9 (C9DN). The C9DN contains the C287S point mutation in its active site, and its expression was shown to inhibit procaspase-9 processing as well as cell death induced by etoposide (40). Treatment of undifferentiated PC12 cells with UV or with a range of etoposide concentrations (5, 10 and 20 µM) resulted in cell death that was almost similar in control PC12 and PC12/hSMN cells indicating that hSMN expression has no effect against these DNA-damaging agents (Fig. 1C and D: left). In contrast, there was a significant reduction in cell death in NGF/dBcAMP co-differentiated PC12/hSMN cells treated with these DNA-damaging agents compared with similarly treated co-differentiated PC12 control cells (Fig. 1C and D: right), indicating that SMN is protective against DNA damage in differentiated cells. As predicted from the literature, cell death was inhibited in response to etoposide and UV treatment in undifferentiated and co-differentiated PC12/hBcl-2 and PC12/C9DN cells (Fig. 1C and D). Thus, hSMN is able to inhibit cell death due to DNA damage in differentiated PC12 cells, whereas proliferating, undifferentiated PC12 cells expressing hSMN remain sensitive to these agents.
It is possible that there are differences in apoptotic pathways, activated by DNA damage, between undifferentiated and differentiated PC12 cells. The tumour suppressor protein p53 is activated in response to DNA damage and is also known to induce apoptosis (41,42). Recently, an interaction between p53 and full-length SMN was reported (43). We have previously shown that p53 is activated during apoptosis induced by trophic factor deprivation in both undifferentiated and differentiated PC12 cells (44). Therefore, we examined the expression of p53 during cell death by UV treatment and also the possibility that its activity could be modulated differently in undifferentiated and differentiated PC12/hSMN cells. p53 expression was analyzed by immunofluorescence at 0, 5 and 18 hours after UV treatment in both control and PC12/hSMN cells and, in parallel, cell death was quantified. A significant increase in number of cells displaying p53 immunoreactivity in nuclei was observed 5 hours after UV treatment in both undifferentiated and differentiated PC12 cells (Fig. 1E: right and left, respectively). Interestingly, both the undifferentiated and differentiated PC12/hSMN cells showed a decrease in the percentage of cells exhibiting nuclear p53 immunostaining compared with control PC12 cells. Nevertheless, in spite of a reduction in the number of undifferentiated PC12/hSMN cells showing nuclear localization of p53, there was no difference in cell death between these cells and undifferentiated control PC12 cells.
Cytochrome c release, caspase-3 activation and cleavage of endogenous SMN are inhibited by hSMN expression during cell death in differentiated PC12 cells
Release of cytochrome c from mitochondria is one of the key steps in activation of the caspase-9 cascade (45). Therefore, we analyzed cytosolic cytochrome c levels and caspase-3 cleavage at 4, 8 and 24 hours after deprivation of NGF/dBcAMP or UV treatment in co-differentiated control PC12 and PC12/hSMN cells. In control PC12 cells, there was an increase in cytosolic cytochrome c level already at 4 hours after NGF/dBcAMP deprivation or UV treatment greatly preceding cell death and remaining elevated during cell death (Fig. 2A and B: right). By contrast, in co-differentiated PC12/hSMN cells, both upon NGF/dBcAMP deprivation and UV treatment, the level of cytosolic cytochrome c did not increase until 24 hours (Fig. 2A and B: left). Caspase-3 activation was analyzed using whole-cell lysates with an antibody that only recognizes the cleaved p20 subunit. In control PC12 cells, p20 subunit cleavage product was observed during the period of cell death at 16 and 24 hours after withdrawal of NGF/dBcAMP or UV treatment (Fig. 2A and B: right). In PC12/hSMN cells, the intensity of p20 signal, observed at 24 hours, was significantly less than the control cells (Fig. 2A and B: left). These results suggest that hSMN delays the onset of caspase activation and apoptosis by preventing the release of cytochrome c.
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23 kDa are also observed. The arrow in the right panel indicates endogenous SMN and the arrow in the left panel indicates SMN+hSMN.Proteins that inhibit apoptosis (e.g. Bcl-2, Bcl-XL and Akt) are also caspase substrates that are cleaved during cell death (46,47). Specific cleavage of SMN at a potential caspase cleavage site, Asp-252, was reported during neuronal apoptosis induced by experimental transient focal ischaemia and Sindbis virus infection (32). This cleavage would result in the loss of the C-terminal region of SMN comprising part of exon 6 plus all of exon 7, yielding a truncated 29 kDa protein. In order to determine whether SMN is also a caspase substrate in PC12 cells undergoing cell death, the endogenous SMN protein was analyzed by western blot in differentiated PC12 cells that were deprived of NGF/dBcAMP for 16 and 24 hours. A 29 kDa fragment was observed in trophic-deprived control co-differentiated PC12 cells but not in similarly deprived PC12/hSMN cells (Fig. 2C). Thus, SMN is cleaved into the correct size, implying a role for caspases, and its absence in PC12/hSMN cells suggests that they are not activated.
hSMN inhibits c-Myc-induced apoptosis but does not prevent cell death caused by DNA-damaging agents in Rat-1/MycERTM cells
In PC12 cells, we found that hSMN can inhibit cell death triggered by trophic deprivation in both undifferentiated and differentiated cells, whereas its effect on DNA-damaging agents was limited to NGF/dBcAMP co-differentiated cells. To examine the extent to which the effect of hSMN is cell-type-and/or context-specific, we analyzed its cell death-inhibitory function in Rat-1/MycERTM (TM, tamoxifen) cells. These cells stably express an inducible c-Myc-ER protein (48). In the absence of survival factors such as IGF-1, induction of c-Myc by tamoxifen treatment results in apoptosis (49). Rat-1/MycERTM cells were transiently co-transfected with reporter LacZ plasmid plus control, HA-hSMN or IAP-2 expression plasmids and treated with tamoxifen for 16 hours. Analysis of cell death showed that in control cultures, c-Myc induced 39.8%±2.3% cell death, whereas in parallel cultures transfected with hSMN or IAP-2, there was 19.7%±1.9% and 18.9%±1.19% cell death, respectively (Fig. 3A). Thus, both hSMN as well as IAP-2 inhibit c-Myc-mediated cell death. Ectopic expression of c-Myc in Rat-1/MycERTMcells in low serum conditions results in time-dependent apoptosis of most cells; however, c-Myc can also induce entry into the cell cycle (48). In order to verify that upon ectopic c-Myc expression, hSMN is exerting its effect on cell death rather than on the cell cycle, time-lapse experiments as previously described for these cells (50) were conducted in control Rat-1/MycERTM and in two different Rat-1/MycERTM cell lines (hSMN-3 and 6) stably expressing HA-hSMN. hSMN expression in these cells was verified by immunofluorescence using anti-HA antibody (data not shown) as well as SMN expression by western blot analysis (Fig. 3B1). Time-lapse quantification of cells exhibiting the morphological characteristics of apoptosis revealed a decrease in the number of cells undergoing apoptosis in hSMN-expressing cells compared with control cells (Fig. 3B2), strongly indicating an inhibitory effect of hSMN on cell death induced by c-Myc. Cell death induced by etoposide treatment, in contrast, was not affected by hSMN transfection (Fig. 3C). Similarly, hSMN was also ineffective in preventing cell death by UV irradiation, in contrast to 10% fetal calf serum (FCS) or dominant-negative caspase-9, both of which afforded significant protection (Fig. 3C). These results are in accordance with those for undifferentiated PC12 cells.
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hSMN mutants show proapoptotic activity in Rat-1/MycERTM cells
We examined the effects of the SMA hSMN/Y272C missense point mutant as well as SMN deleted of exon 7 (hSMN
7) and exons 6 plus 7 (hSMN6/7) on c-Myc-induced cell death in Rat-1 cells. Transient transfections of Rat-1 cells with hSMN/Y272C followed by c-Myc induction resulted in greater cell death (52.5%±7.1%) compared with cell death in control cells (Fig. 3A). In addition, time-lapse analysis in two Rat-1/MycERTM cell lines stably expressing hSMN/Y272C (Y272-1 and 2) (cf Fig. 3B1) also showed that this mutation has no inhibitory effect on c-Myc-induced cell death (Fig. 4A). We next analyzed cell death in Rat-1/MycERTMpools of cells that had been transfected with either hSMN7 or hSMN6/7. In these cells, a significant basal increase in cell death was observed even in the absence of an apoptotic stimulus. Greater cell death was consistently observed with hSMN7 (40%±2.17%) in comparison with hSMN6/7 (30%±l.5%) (Fig. 4B). Upon c-Myc activation by tamoxifen treatment, there was no further increase in cell death in hSMN7 cells, but a small increase was seen in Rat-1/MycERTM/hSMN6/7 cells (42%±1.45%). The pro-apoptotic effect was essentially lost by further deleting hSMN, and transfection of exon 5-, 6- and 7-deleted hSMN showed a relatively lower percentage of spontaneous death, which was markedly higher in the presence of tamoxifen (Fig. 4B). The results indicate that not only is the survival effect of hSMN lost by mutating the C terminus but also these mutations confer pro-apoptotic properties upon hSMN.
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hSMN mutants exhibit altered subcellular localization compared with endogenous SMN and transfected full-length hSMN
Deletion of the N terminus (SMN
N27) or exon 7 of SMN results both in perturbation of its function and in modification of subcellular localization (12,51). We have examined the subcellular expression of transfected full-length and mutated hSMN and compared their localization with that of endogenous SMN in PC12 and Rat-1 cells. Immunofluorescence labelling of endogenous SMN revealed a granular labelling in the cytoplasm in Rat-1/MycERTM and PC12 cells (Fig. 5A1 and C1). Moreover, bright fluorescent spots were observed in some but not all nuclei that co-localized with p80 coilin, a specific marker of coiled bodies (Fig. 5A3 and C3). The immunofluorescence results using anti-HA showed that the subcellular localization of transfected HA-hSMN in the cytoplasm and nucleus was same as that of endogenous SMN (Fig. 5B1–3 and D1–3).
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Analysis of Rat-1/MycERTM cells transfected with Myc-tagged hSMN
7 revealed, in many cells, large fluorescence aggregates, mostly in the nucleus (Fig. 6A1). In addition, a significant number of cells exhibited nuclear apoptotic morphology as visualized by Hoechst 33258 staining (data not shown). In PC12 cells, patches of aggregated labeling either in the cytoplasm or in the nucleus were observed (Fig. 6B1 and D1), and, similarly to Rat-1/MycERTM cells, many cells exhibited fragmented nuclei. In contrast to the fluorescent aggregates observed with hSMN7, transfection with Myc-tagged hSMN6/7 showed homogenous labelling, mostly over the nucleus, excluding the nucleolus in both Rat-1 and PC12 cells (Fig. 6C1). The discrete nuclear localization of p80 coilin was not seen in hSMN7 cells (Fig. 6A2 and B2); however, the characteristic p80 coilin speckles were evident in hSMN6/7-expressing Rat-1 and PC12 cells (Fig. 6C2 and D2). Nevertheless, the co-localization with p80 coilin observed in subnuclear structures wit full-length hSMN was lost upon deletion of exon 6 as well as exon 7.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this study, we have characterized the effect of expressing full-length and C-terminal mutated hSMN on cell death in either PC12 or Rat-1 cells. hSMN prolonged survival in both of these cell types: in PC12 cells against trophic factor deprivation and in Rat-1 cells by induction of c-Myc. However, hSMN was ineffective in proliferating PC12 or Rat-1 cells against DNA-damaging agents, whereas terminally differentiated PC12 cells were protected. We also show that the C-terminal region is important for its function in preventing cell death.
The role of SMN in cell survival has been highlighted by studies where the SMN gene has been functionally disrupted in vivo (9,10,26,52). However, the mechanisms by which SMN regulates cell survival and apoptosis have not been characterized. Our findings support the hypothesis that SMN promotes cell survival by suppressing apoptosis.
Activation of caspases is a critical event in the apoptotic programme that is inhibited by IAP-2 and Bcl-2 (38). Procaspase-3, a key executioner caspase, is cleaved to catalytically active enzyme by apical caspase-8 or -9 (53). Trophic factor withdrawal in both undifferentiated and differentiated PC12 cells results in cytochrome c release from mitochondria and procaspase-9 activation (S. Vyas, unpublished). In addition, the release of cytochrome c and the requirement for caspase-9 in c-Myc-induced apoptosis have also been demonstrated (36,54). We found that hSMN suppressed trophic factor-deprived cell death in both undifferentiated and NGF/dBcAMP co-differentiated PC12 cells to a similar extent as the anti-apoptotic proteins, IAP-2 and Bcl-2. c-Myc-induced apoptosis was also inhibited by hSMN, as analyzed either by transiently expressing hSMN or by time-lapse analysis of apoptotic deaths in Rat-1/MycERTM cells stably expressing hSMN. This inhibitory effect of hSMN most likely occurs through suppression of mitochondrial activation of caspases. In control differentiated PC12 cells, either deprived of NGF/dBcAMP or UV-treated, cytochrome c release greatly preceded cell death, the process occurring almost immediately after death stimulus, whereas caspase-3 activation coincided with cell death. We find that in these cells, hSMN delays the onset of apoptosis by inhibiting the release of cytochrome c and cleavage of caspase-3. The action of hSMN on cytochrome c release is likely to be indirect. It can be postulated that hSMN is involved in regulating proteins that determine mitochondrial release of cytochrome c and activation of caspases. Further, indirect evidence that caspase activation is inhibited during trophic withdrawal was provided by the observation that SMN is cleaved into the 29 kDa fragment in control trophic-deprived cells but not in PC12/hSMN cells. The caspase cleavage site, D252A, situated in the C terminus of SMN, is involved in the generation of a 29 kDa SMN fragment (32). The presence of this fragment during cell death in control trophic-deprived cells suggests that SMN is also a target of caspases.
hSMN inhibited cell death induced by UV or etoposide treatment in NGF/dBcAMP co-differentiated PC12 cells but not in proliferating PC12 or Rat-1 cells. It is possible that differentiation process results in modifications of apoptotic pathways that are activated by DNA damage. We analyzed the nuclear expression of the tumour suppressor protein p53, which is known to be activated by DNA damage and also shown to interact with SMN (43,55). We observed a reduction in the percentage of cells exhibiting nuclear localization of p53 in both undifferentiated and differentiated PC12/hSMN cells. Thus, it is possible that in differentiated PC12 cells, hSMN inhibits cell death by decreasing p53 levels. However, the fact that undifferentiated PC12/hSMN cells were not protected by DNA damage suggests that the relationship between SMN, p53 and induction of cell death may also depend on other factors.
Inhibition of cell death by c-Myc was not observed in Rat-1/MycERTM cells expressing hSMN7, hSMN6/7 or point-mutant Y272C. On the contrary, these mutants exhibited pro-apoptotic activity. The results on subcellular localization of hSMN7 revealed both cytoplasmic and nuclear aggregates, while diffuse labelling in the nucleus that excluded the nucleolus was seen with hSMN6/7. Moreover, the discrete co-localization with p80 coilin in the nucleus was not seen, in contrast to full-length hSMN. A defect in coilin assembly and absence of gems in motoneurons, as verified by p80 coilin and SIP1 immunofluorescence, respectively, was reported in mutant SMNF7/7, Cre+ mice (12). Although our results and those of Kerr et al. (32) indicate that experimentally SMN7 induces cell death, this role in SMA disease has not been demonstrated. Evidence to date indicates that full-length SMN protein is a critical determining factor in the development of SMA and that the reduction in full-length SMN is inversely correlated with the severity of the disease (6,8). An important difference identified between the SMN1 and SMN2 genes is a single nucleotide change, C
T in exon 7, which results in skipping of this exon in SMN2 (56,57). Thus, full-length transcripts and protein are produced from SMN1. In contrast, SMN2 transcripts contain only small amounts of full-length SMN, and the majority lack exon 7 (although alternatively spliced products lacking exon 5 or both exons 5 and 7 are also found). Interestingly, milder forms of SMA have increased SMN2 gene dosage (9,10,58), which suggests that SMN7 may not be harmful in vivo. On the other hand, it is possible (as has previously been suggested) that SMN7 protein is degraded more rapidly than full-length SMN (59,60). However, under our experimental conditions, there is endogenous SMN and, most likely, significantly higher amount of transfected SMN7 protein than found either in SMA or in mutant mice carrying one allele of full-length SMN, the other allele being absent or deleted of exon 7. It is possible that, in our case, the transfected mutant proteins interfere with the endogenous function of SMN. It has been shown that exons 2b and 6 are required for SMN self-oligomerization (61). The C-terminal region also contains a YG box domain, which is thought to play a role in the binding of snRNP Sm proteins to SMN, and therefore in the regulation of pre-mRNA splicing (13,16). Thus, transfections of hSMN7 or hSMN6/7 in vitro may result in misfolding of native full-length SMN or alterations in proteins that associate with SMN, which could lead to abrogation of its function in cell survival.
The reasons for motoneuron vulnerability as a consequence of SMN1 gene mutations are not known. In this study, we show that SMN inhibits apoptosis by acting through a mitochondrial apoptotic pathway, and that the C-terminal region is important for its survival function. Further elucidation of the regulation of apoptosis by SMN should lead to understanding the mechanisms of motoneuron loss observed in SMA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture
Rat-1/MycERTM cells, expressing a 4-hydroxytamoxifen (4-OHT)-activatable human c-Myc protein, were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin sulfate and 5 µg/ml puromycin. Undifferentiated PC12 cells were cultivated in RPMI 1640 medium containing 10% FBS and 5% horse serum (HS). They were differentiated in modified L15 medium supplemented with 5% FBS, 10% horse serum and 50 ng/ml NGF. After 3 days in culture, 200 µM dBcAMP was added and the cells were further differentiated for 4–5 days (33).
Transient transfection of Rat-1/MycERTM cells
The method described in Rohn et al. (62) was used. Rat-1/MycERTM cells were seeded in 6-well culture plates (105 cells/well). After 24–48 h of plating, the cells (at least 60% confluent) were transfected with 6 µg lipofectamine (GIBCO-BRL) and 1 µg total DNA (0.2 µg reporter pcDNAlacZ plus 0.8 µg empty plasmid vector or test plasmids) in DMEM medium was removed 5 h later and replaced, after PBS wash, with fresh DMEM medium containing 1% FBS and immediately treated with 100 nM tamoxifen in order to activate c-Myc or 5 µM etoposide (Sigma) or UV irradiation (20 s, 40 J/m2). After 21 h post transfection and treatment, the cells were washed once in PBS, fixed in 1% formaldehyde/0.2%glutaraldehyde in PBS and assayed for ß-galactosidase activity. The transfection efficiency of Rat-1 cells was 
30–35%.
Transient transfection of PC12 cells
Undifferentiated PC12 cells were seeded and transfected with reporter and test expression plasmids using the lipofectamine method as above. The transfection medium was removed 8 h later and replaced with RPMI 1640 medium containing serum for 10 h. Thereafter, cell death was induced as appropriate either by withdrawing serum or by treatment with 5 µM etoposide or UV for 24 h. The cells were assayed for ß-galactosidase activity 42 h post transfection. The transfection efficiency of PC12 cells was 10–15%. We also verified that both of these cell types co-express proteins encoded by reporter and test plasmids. The results of immunofluorescence analysis of GFP as a reporter plasmid and anti-HA staining for HA-hSMN showed that >80% of transfected cells express both the reporter and test plasmids.
Plasmids
hSMN and the mutant SMNY272C were cloned in pcDNA3 vector (In Vitrogen) containing the HA tag at the N-terminal region. SMN deletion mutants lacking exon 7 (SMN7), exons 6 and 7 (SMIN6/7), and exons 5, 6 and 7 (SMN5/6/7) were prepared by PCR, introducing EcoR1/Xbo1 restriction sites and then cloned into pcDNA3 vector containing an N-terminal Myc tag. The hSMN 5' primer was 5'-ATGGCGATGAGCGGCGGCAGT-3' and the 3' primers were (for SMN7) 5'-CATACTGGCTATTATATG-3', (SMN6/7) 5'-TTTCCTTCTGGACCACCA-3' and (SMN5/6/7) 5'-AACATCAAGCCCAAATCTGCTC-3'. The sequences of all these constructs were verified. In addition, we also verified the protein expression by an in vitro assay consisting of 35S-labeled methionine/cysteine and the TNT-coupled transcription/ translation rabbit reticulocyte lysate system (Promega).
Stable cell lines
Rat-1/MycERTM and PC12 cells were transfected with hSMN and hSMN mutants using the lipofectamine method as described for transient transfections. G418-resistant colonies were obtained by treatment of Rat-1/MycERTM with 1 mg/ml G418 for 10 days, whereas PC12 cells were treated with 250 µg/ml G418 for 3 weeks. The expression of hSMN or hSMN mutants was analyzed by western blot for SMN and by immunofluorescence using, where appropriate, either mouse anti-HA antibody (Boehringer) or 9E10 anti-Myc antibody (GIBCO-BRL). Rat-1/MycERTM cells transfected with hSMN7, hSMN6/7 and hSMN5/6/7 mutants were selected in G418 (1.5 mg/ml) medium for 5 days, tested for expression by immunofluorescence and used immediately for analysis.
Immunofluorescence and western blot analysis
For immunofluorescence of SMN, hSMN (HA- or Myc-tagged) and p53, the cells were fixed in 2% paraformaldehyde/PBS for 30 min at 4°C. After three PBS washes and a 15 min incubation at ambient temperature in PBS/50 mM NH4Cl, they were permeabilized successively with 0.3% Triton X-100 in 0.125% gelatin/PBS for 15 min and 0.1% Triton X-100 in 0.125% gelatin/PBS for 30 min. The cells were incubated for 1 h at room temperature with primary antibodies: monoclonal anti-SMN antibody (1 : 400 dilution, clone 8; Transduction Labs), polyclonal anti-coilin rabbit antibody (1 : 200 dilution), monoclonal antibodies to HA or c-Myc tags, (dilution 1:400) or monoclonal anti-p53 antibody (1 : 200 dilution). After PBS washing, the cells were incubated with appropriate secondary antibodies for 1 h and mounted on glass slides using Vectashield. For western blot analysis of SMN and caspase-3, the cells were scraped in Laemmli buffer (62.5 mM Tris–HCl pH 6.8, 1% SDS, 10% glycerol and 5% 2-mercaptoethanol) plus protease inhibitors (Boehringer), boiled and clarified by centrifugation at 10 000gx10 min. The cytoplasmic lysates were prepared to determine cytochrome c levels according to Juin et al. (36). The proteins were fractionated in SDS–PAGE gels and electroblotted, hybridized with primary monoclonal anti-SMIN (1/750), 1/1000 cytochrome c (Pharmingen) and 1 : 2500 anti-caspase-3 (CM-1), followed by secondary HRP-conjugated antibodies. The proteins were detected using enhanced chemiluminescence reagents according to the manufacturer's instructions (Amersham).
Cell death analysis and time-lapse videomicroscopy
Cell death in transient transfection experiments was quantified by counting either all ß-galactosidase-positive viable and dead cells for Rat-1/MycERTM or viable ß-galactosidase PC12 cells (dead cells detach easily). Each condition was done in duplicate and the experiment was repeated at least three times. Death of co-differentiated PC12 cells after trophic withdrawal, etoposide or UV treatment was quantified by staining with Hoechst 33258 (bisbenzimide trihydrochloride), and 300–600 intact and fragmented nuclei were counted for each condition done in quadruplicate for each experiment. Time-lapse videomicroscopic images of Rat-1/MycERTM cells immediately after tamoxifen treatment were acquired on an inverted-phase contrast microscope and collected on sVHS video tape as described previously (63). Apoptotic cells were scored at the time point when they detached and rounded, followed by cell blebbing, cytoplasmic condensation and finally cell fragmentation.
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ACKNOWLEDGEMENTS |
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We thank Dr C. Lorson for hSMN and Y272C hSMN plasmids and useful comments on the manuscript, Dr G. Evan for Rat-1/MycERTM cells, Dr Y. Lazebnik for caspase-9 dominant-negative plasmid, Dr J. Silke for the IAP-2 expression plasmid, Dr A. Srinivasula for CM1 caspase-3 antibody, Professor T. Soussi for p53 antibodies, Dr D. Auby for time-lapse analysis and Dr S. O'Regan for critical reading of the manuscript. We are grateful to the Association Français contre les Myopathies (AFM) and the Institut pour la Recherche sur la Moelle Epinère (IRME) for their financial support.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +33 144323533; Fax: +33 144323654; Email: yyas{at}wotan.ens.fr
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REFERENCES |
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