SM-20 Is a Novel Mitochondrial Protein That Causes Caspase-dependent Cell Death in Nerve Growth Factor-dependent Neurons*

Elizabeth A. LipscombDagger §, Patrick D. Sarmiere§, and Robert S. Freeman||

From the Dagger  Department of Environmental Medicine and  Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication, September 13, 2000, and in revised form, October 27, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sympathetic neurons undergo protein synthesis-dependent apoptosis when deprived of nerve growth factor (NGF). Expression of SM-20 is up-regulated in NGF-deprived sympathetic neurons, and ectopic SM-20 is sufficient to promote neuronal death in the presence of NGF. We now report that SM-20 is a mitochondrial protein that promotes cell death through a caspase-dependent mechanism. SM-20 immunofluorescence was present in the cytoplasm in a punctate pattern that colocalized with cytochrome oxidase I and with mitochondria-selective dyes. Analysis of SM-20/dihydrofolate reductase fusion proteins revealed that the first 25 amino acids of SM-20 contain a functional mitochondrial targeting sequence. An amino-terminal truncated form of SM-20 was not restricted to mitochondria but instead localized throughout the cytosol and nucleus. Nevertheless, the truncated SM-20 retained the ability to induce neuronal death, similar to the wild type protein. SM-20-induced death was accompanied by caspase-3 activation and was blocked by a general caspase inhibitor. Additionally, overexpression of SM-20, under conditions where cell death is blocked by a general caspase inhibitor, did not result in widespread release of cytochrome c from mitochondria. These results indicate that SM-20 is a novel mitochondrial protein that may be an important mediator of neurotrophin-withdrawal-mediated cell death.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sympathetic neurons undergo apoptosis when deprived of their survival factor, nerve growth factor (NGF).1 Inhibitors of RNA or protein synthesis block this death, suggesting that gene expression is important for apoptosis in these cells (1, 2). Consistent with this idea, a small number of genes have been shown to increase in expression in neurons after NGF withdrawal (3-5). Among these, c-jun and cyclin D1 have been implicated as upstream regulators of neuronal death (4-9). Recently, we found that expression of the SM-20 gene is also up-regulated in NGF-deprived sympathetic neurons (10). SM-20 expression is also induced in sympathetic neurons treated with the anti-tumor agent cytosine arabinoside or with inhibitors of phosphatidylinositol 3-kinase, agents that cause apoptosis in the presence of NGF (11, 12). Although a requirement for SM-20 in cell death remains to be established, overexpression of SM-20 is sufficient to induce cell death in neurons maintained in the continual presence of NGF (10). These findings suggest that SM-20 may function as a regulator of neuronal death.

Regulation of SM-20 expression also occurs in muscle cells and in response to activation of the p53 protein in fibroblasts. SM-20 mRNA levels increase in vascular smooth muscle cells stimulated with growth factors and are up-regulated when muscle cells are induced to differentiate (13, 14). In vivo, SM-20 mRNA levels are elevated in muscle cells in response to injury to the blood vessel wall (15). SM-20 expression also increases after activation of a temperature-sensitive p53 protein in rat embryo fibroblasts (16). Activation of p53 in these cells results in both growth arrest and apoptosis. Interestingly, stable expression of SM-20 in tumor cells lacking functional p53 resulted in greatly reduced numbers of colonies in colony formation assays, suggesting that SM-20 might act downstream of p53 in either growth-arrest or apoptosis (16).

Although the biochemical function of the SM-20 protein is unknown, it contains a 218-amino acid region that is closely related to sequences in the Caenorhabditis elegans Egl-9 protein (17). Mutations in the egl-9 gene had been originally shown to disrupt normal egg laying in nematodes (18). More recently, egl-9 was implicated as a mediator of toxin-induced neuromuscular paralysis in C. elegans infected with the pathogenic bacterium Pseudomonas aeruginosa (17).

To help elucidate the function of SM-20, its subcellular localization was investigated. We report that the amino terminus of SM-20 contains a mitochondrial targeting sequence that localizes SM-20 to mitochondria. Because mitochondria have a prominent role in the regulation of cell death (19), we tested the significance of the mitochondrial targeting of SM-20 on its ability to promote neuronal death. Our results indicate that targeting of SM-20 to mitochondria is not required for SM-20-induced death. In addition, we find that caspase-3 activation occurs during SM-20-induced cell death. In contrast to Bax, expression of SM-20 in neurons maintained in the presence of a general caspase inhibitor did not lead to detectable cytochrome c release from mitochondria. These results indicate that SM-20 is a novel mitochondrial protein that may be an important mediator of neurotrophin-withdrawal mediated cell death.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- CV-1 (ATCC; Manassas, VA) and NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone; Indianapolis, IN), 100 units/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Inc.). Primary cultures of sympathetic neurons were prepared from the superior cervical ganglia of embryonic day 21 rats as described previously (12). Cultures were maintained in vitro for 5-6 days in media consisting of 90% minimal essential medium, 10% fetal bovine serum (Harlan Bioproducts; Madison, WI), 2 mM L-glutamine, 20 µM uridine, 20 µM fluorodeoxyuridine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 ng/ml NGF (Harlan Bioproducts).

Expression Vectors and Transient Transfections-- The expression vectors containing the SM-20 open reading frame in Rc/cytomegalovirus (CMV) (Invitrogen; Carlsbad, CA) and the SM-20/green fluorescent protein (GFP) cDNA in pcDNA3 (Invitrogen) were described previously (10). For expressing T7 epitope-tagged forms of SM-20, dihydrofolate reductase (DHFR), SM-20(60-355), and SM-20/DHFR fusions, complementary oligonucleotides encoding the T7 epitope tag (Novagen; Madison, WI) flanked by restriction sites for EcoRI-NdeI (5') and EcoRV (3') were annealed and ligated into Bluescript KS+ (Stratagene; La Jolla, CA) between the EcoRI and EcoRV sites. The various cDNAs were inserted into the T7-modified Bluescript vector upstream of the T7 epitope using EcoRI and NdeI. The SM-20 sequences used to make SM-20(60-355), SM-20 (1-25)/DHFR, and SM-20(1-50)/DHFR encoded an amino-terminal methionine and were generated by polymerase chain reaction using Pfu polymerase (Stratagene). The EcoRI to EcoRV fragments containing each cDNA fused at its 3' end to the T7 epitope were then subcloned into pcDNA3. For SM-20(60-355)/GFP, a DNA fragment consisting of an amino-terminal methionine followed by amino acids 60-355 of SM-20 was amplified using Pfu polymerase fused to the 5' end of GFP and inserted into pcDNA3 between the EcoRI and EcoRV sites. All amplified DNAs were sequenced to confirm that they were correct.

CV-1 cells (2 × 105 cells) were plated onto glass coverslips in 35-mm plastic tissue culture dishes 24 h before transfection with LipofectAMINE (Life Technologies, Inc.) as described by the manufacturer. NIH3T3 cells (1 × 107 cells) were plated onto 100-mm tissue culture dishes and transfected with LipofectAMINE. Cells were used for experiments on the second day after transfection.

Mitochondrial Labeling-- CV-1 cells were incubated for 5 h in media containing 1 µM Mitotracker Green FM (Molecular Probes; Eugene, OR), a mitochondria-selective dye that is retained in fixed cells. To establish the specificity of this dye for labeling mitochondria, cells were plated on grid coverslips and then stained with Mitotracker Green FM as described above. After staining, the cells were visualized using a fluorescein isothiocyanate (FITC) filter set (lambda ex = 450-490 nm, lambda em = 520-560 nm), and images were captured. The same cells were then stained with the nonfixable mitochondria-specific dye Rhodamine 123 (20), and images were obtained using a tetramethylrhodamine isothiocyanate (TRITC) filter set (lambda ex = 546 ± 10 nm, lambda em > 590 nm). In every cell, the fluorescence of Mitotracker Green FM and Rhodamine 123 coincided completely. For labeling of mitochondria in sympathetic neurons, Mitotracker Orange (25 nM, Molecular Probes) was added to the culture medium for 15 min, after which images of live cells were captured using a Leica confocal microscope and TCS-NT software.

Immunofluorescence-- CV-1 cells were fixed and permeabilized with 4% paraformaldehyde containing 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 min and then incubated in block buffer consisting of 3% goat serum and 0.2% bovine serum albumin in PBS. To detect SM-20, DHFR, or SM-20/DHFR proteins, cells were incubated with either a 1:200 dilution of rabbit polyclonal anti-SM-20 antibody (10) or 0.2 µg/ml anti-T7 monoclonal antibody (Novagen) in block buffer. Coverslips were then rinsed with PBS and incubated with TRITC-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Jackson ImmunoResearch; West Grove, PA) at 15 µg/ml in block buffer. The cells were counterstained with Hoechst 33258 (Molecular Probes) and then mounted onto slides for viewing by epi-fluorescence with a Nikon Diaphot 300 microscope. Digital images were captured using a Dage-MTI CCD camera and Scion Image software.

For dual immunofluorescence, CV-1 cells were first incubated with IgG-purified anti-SM-20 primary antibody and TRITC-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch) as described above. After extensive washing of the coverslips, antibodies to cytochrome oxidase I (A-6403; Molecular Probes) or cytochrome oxidase VI (A-6401; Molecular Probes) were added at 1 µg/ml or 4 µg/ml in block buffer, respectively, followed by FITC-conjugated goat anti-mouse IgG at 15 µg/ml in block buffer. Secondary antibodies used in the double-labeling experiments were cross-absorbed against mouse or rabbit IgG.

For visualization of active caspase-3 and cytochrome c, sympathetic neurons were fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C and then incubated in block buffer consisting of 5% goat serum and 0.3% Triton X-100. Primary antibodies to active caspase-3 (Pharmingen; San Diego, CA) and cytochrome c (Pharmingen) were added at 0.5 µg/ml in PBS containing 1% goat serum and 0.3% Triton X-100, and the neurons were labeled overnight at 4 °C. The appropriate FITC-conjugated secondary antibodies were diluted to 15 µg/ml in PBS containing 1% goat serum and 0.3% Triton X-100, and the cultures were incubated for 2 h. Cultures were then rinsed in PBS and counterstained with Hoechst 33342 (Molecular Probes).

Subcellular Fractionation, Immunoprecipitation, and Immunoblotting-- CV-1 cells stably expressing SM-20 were preincubated for 30 min in media lacking cysteine and methionine and then incubated for 4 h in the same media containing 0.5 mCi/ml Tran35S-label (ICN; Costa Mesa, CA) and 10% dialyzed calf serum. For preparing whole cell lysates, cells were rinsed twice with cold PBS and then lysed in radioimmune precipitation (RIPA) buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet-P40, 1% deoxycholate, 0.1% SDS, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin) for 15 min at 4 °C. Cell lysates were clarified by centrifugation at 10,000 × g for 10 min, preabsorbed with protein A-Sepharose (Amersham Pharmacia Biotech), and then incubated with anti-SM-20 antibody or, in some cases, antibody that had been preincubated with excess blocking peptide. Immune complexes were precipitated with protein A-Sepharose, washed four times with RIPA buffer buffer, separated by 12.5% SDS-polyacrylamide gel electrophoresis, and analyzed using a PhosphorImager and ImageQuant software (Molecular Dynamics; Sunnyvale, CA).

Mitochondrial-enriched fractions were prepared essentially as described by others (21, 22). For these experiments, NIH3T3 cells were used rather than CV-1 cells because higher levels of SM-20 expression were obtained (note that both CV-1 and NIH3T3 cells showed the same pattern of SM-20 immunofluorescence). Cells were transiently transfected with SM-20/Rc-CMV or an empty control vector and metabolically labeled as described above. After labeling, each dish was rinsed twice with PBS before adding ice-cold homogenization buffer (17 mM MOPS, 2.5 mM EDTA, 250 mM sucrose, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin). The cells were then scraped from the dishes, homogenized in a Dounce homogenizer (Kontes; Vineland, NJ) for 20-25 strokes, and then centrifuged at 500 × g for 10 min at 4 °C to pellet nuclei. The supernatants were then centrifuged at 10,000 × g for 15 min. The resultant pellets from this centrifugation were resuspended in homogenization buffer and centrifuged again at 10,000 × g for 15 min, yielding a mitochondria-enriched heavy membrane pellet that also contains lysosomes, golgi, and rough endoplasmic reticulum. The mitochondria-enriched fraction was resuspended in RIPA buffer and subjected to immunoprecipitation with anti-SM-20 antibody as described above. A soluble cytosolic fraction was prepared from the supernatants obtained after the 10,000 × g spin by further centrifuging the supernatants at 150,000 × g for 60 min. To verify that the heavy membrane fraction was enriched for mitochondria, 25 µg of protein from the heavy membrane and cytosolic fractions were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat dry milk in Tris-buffered saline before labeling overnight with primary antibody against cytochrome c (Pharmingen) at 5 µg/ml in 1× Tris-buffered saline containing 1% nonfat dry milk and 0.05% Tween 20. The blots were then incubated with biotinylated goat anti-mouse IgG (Jackson ImmunoResearch) at 0.3 µg/ml and detected using a streptavidin-conjugated alkaline phosphatase assay kit (Bio-Rad).

Intracellular Microinjections and Quantitation of Cell Death-- Sympathetic neurons were microinjected with expression plasmids and scored for viability as described previously (12). Neurons were injected directly into the nucleus with each plasmid at 50 µg/ml in a buffer containing nonfixable rhodamine-dextran (survival experiments) or lysine-fixable rhodamine-dextran (immunofluorescence experiments) to permit visualization of injected cells. For some experiments, the neurons were treated immediately after microinjection by including the general caspase inhibitor BOC-Asp-CH2F (BAF) (Enzyme Systems Products; Dublin, CA) at 50 µM in the culture medium. The number of successfully injected (rhodamine-positive) neurons was determined 12-15 h after injection. Three days after injection, cells were stained with the fluorescent DNA binding dye Hoechst 33342 (Molecular Probes) and evaluated for survival by counting the number of rhodamine-positive cells that were phase-bright with smooth and intact neurites, a discernible nucleus and diffusely stained chromatin. In contrast, dying cells are characterized by condensed or undetectable chromatin, fragmented neurites, and atrophic cell bodies. Percent survival is reported as the number of healthy cells divided by the number of rhodamine-positive cells counted 12-15 h after injection.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Localization of SM-20 to Mitochondria-- To determine where the SM-20 protein is localized in cells, we first performed immunofluorescence on transfected cells using a polyclonal anti-peptide antibody directed against the carboxyl terminus of SM-20. This antibody specifically recognizes recombinant SM-20 protein, and it immunoprecipitates SM-20 from cells transfected with an SM-20 expression plasmid but not from cells transfected with an empty vector (10). CV-1 cells were chosen for these experiments because of their flattened morphology and large cytoplasmic volume and because transient expression of SM-20 does not lead to death in these cells (use of NIH3T3 cells yielded identical results, data not shown). SM-20 immunofluorescence occurred in a punctate pattern throughout the cytoplasm of expressing cells (Fig. 1, panels B and E). In contrast, neighboring untransfected cells or cells transfected with an empty expression vector did not show detectable immunofluorescence.



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Fig. 1.   Localization of SM-20 to mitochondria. CV-1 cells were transiently transfected with SM-20-Rc/CMV plasmid DNA. Dual immunofluorescence was performed for SM-20 and cytochrome oxidase I (panels A-C) 48 h after transfection. Primary antibodies for cytochrome oxidase I (panel A) and SM-20 (panel B) were detected using FITC- and TRITC-conjugated secondary antibodies, respectively. Other cells were labeled with Mitotracker Green FM and then subjected to immunofluorescence with anti-SM-20 antibody (panels D-F). Yellow in the overlay images (panels C and F) appears where the red fluorescence of SM-20 colocalizes with the green fluorescence of either cytochrome oxidase I (panel A) or Mitotracker Green FM (panel D). Bar = 10 µm.

The distribution of SM-20 immunofluorescence appeared similar to the distribution of mitochondria described previously in CV-1 cells (23). Therefore, experiments were performed to determine whether SM-20 expression colocalizes with known mitochondrial markers. In dual-labeling experiments, SM-20 immunofluorescence completely coincided with the labeling generated by a monoclonal antibody that recognizes cytochrome oxidase subunit I, an inner mitochondrial membrane protein (Fig. 1, panels A-C). SM-20 immunofluorescence also colocalized with a second mitochondrial protein, cytochrome oxidase subunit VI (data not shown).

The SM-20 localization pattern was also compared with the staining pattern of the mitochondria-selective dye Mitotracker Green FM. SM-20 immunofluorescence colocalized perfectly with mitochondria stained by Mitotracker Green FM (Fig. 1, panels D-F). The colocalization of SM-20 with two distinct mitochondrial proteins and a mitochondria-selective dye strongly suggests that SM-20 is localized to mitochondria in these cells.

Identification of a 33-kDa Processed Form of SM-20 in Mitochondria-- The SM-20 open reading frame predicts a 355-amino acid protein with a calculated molecular mass of 39.8 kDa (13). When whole cell lysates were prepared from CV-1 cells stably expressing SM-20 and immunoprecipitated with anti-SM-20 antibody, proteins of ~33 and 40 kDa were specifically recovered (Fig. 2A). Pre-incubating the antibody with the cognate peptide blocked immunoprecipitation of both proteins. Moreover, proteins of the same sizes were detected in cells expressing a T7 epitope-tagged form of SM-20 (tagged at its carboxyl terminus) when an antibody specific for the T7 epitope was used for the immunoprecipitation (data not shown). These results indicate that both the 33 and 40 kDa proteins are products of the SM-20 cDNA. Because antibodies specific for the carboxyl terminus of SM-20 recognize the 33-kDa protein, it most likely corresponds to a processed form of SM-20 lacking amino-terminal sequences.



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Fig. 2.   A 33-kDa processed form of SM-20 is enriched in mitochondria. A, whole cell extracts prepared from metabolically labeled CV-1 cells (lane 1) or from CV-1 cells stably expressing SM-20 (lanes 2 and 3) were immunoprecipitated with a SM-20 anti-peptide antibody. Immunoprecipitations were done in the presence (+) or absence (-) of excess blocking peptide corresponding to the last 14 amino acids of SM-20. The positions of molecular mass markers are indicated. The arrows indicate the presence of ~40- and 33-kDa proteins in SM-20-expressing cells that were specifically immunoprecipitated in the absence of the blocking peptide (lane 2). B, cytosolic (Cyto) and mitochondria (Mito)-enriched fractions were prepared from NIH3T3 cells transiently transfected with a SM-20 expression vector (lanes 2 and 4) or with pcDNA3 (lanes 1 and 3) and metabolically labeled for 4 h with Tran35S-label. Fractions were immunoprecipitated with anti-SM-20 antibody, and then the immunoprecipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis. Note that the 33-kDa protein (lower arrow) is present only in the mitochondria-enriched fraction. The cytosolic and mitochondrial-enriched fractions were immunoblotted with anti-cytochrome (cyt.) c antibody to confirm that a fraction enriched in mitochondrial proteins was obtained.

Proteins that are synthesized in the cytosol and then targeted to mitochondria often contain mitochondrial-targeting sequences at their amino termini. Mitochondrial targeting sequences are typically 15-35 residues long, rich in hydroxylated and positively charged amino acids, and devoid of acidic residues (24). Inspection of the SM-20 protein sequence revealed that the first 59 amino acids contain 13 hydroxylated residues, 13 basic residues, and no acidic residues. Because mitochondrial targeting sequences are often proteolytically removed during import into mitochondria (25), we suspected that the 33-kDa protein represented a processed form of SM-20 present in mitochondria. NIH3T3 cells transiently transfected with SM-20 cDNA or a control plasmid were used to prepare cytosolic and mitochondria-enriched protein fractions. Immunoprecipitation of these fractions with anti-SM-20 antibody revealed that the 33-kDa SM-20 protein was the predominant form present in the mitochondria-enriched fraction (Fig. 2B). In contrast, the soluble cytosolic fraction contained almost exclusively the full-length SM-20 protein. Because the antibody used to detect SM-20 recognizes its carboxyl terminus, these results provide evidence that an ~7-kDa polypeptide is removed from the amino terminus of SM-20 when it is targeted to mitochondria.

SM-20 Contains an Amino-terminal Mitochondrial Targeting Sequence-- To determine whether the amino terminus of SM-20 is required for its mitochondrial localization, we constructed a truncated form of SM-20 (SM-20(60-355)) expected to encode a protein similar in size to the 33-kDa form of SM-20 detected by immunoprecipitation. As predicted, SM-20(60-355) was not targeted to mitochondria in CV-1 cells but, instead, was diffusely dispersed throughout the cytosol and nucleus, similar to DHFR (Fig. 3). Thus the loss of 59 amino-terminal residues apparently disrupted a mitochondrial targeting sequence within SM-20.



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Fig. 3.   An amino-terminal deletion of SM-20 prevents its mitochondrial localization. Expression vectors encoding DHFR tagged with a carboxyl-terminal T7 epitope (A), SM-20(60-355)/T7 (SM-20 minus its first 59 amino acids) (B), or full-length SM-20 (C) were transfected into CV-1 cells. Indirect immunofluorescence was performed 48 h later using anti-T7 antibody or anti-SM-20 antibody. Both DHFR and SM-20(60-355)/T7 show diffuse cytoplasmic and nuclear fluorescence in marked contrast to the punctate distribution of the full-length SM-20. Bar = 10 µm.

To ascertain whether sequences within the amino-terminal end of SM-20 are capable of targeting a normally cytosolic protein to mitochondria, the first 50 amino acids of SM-20 were fused to the amino terminus of DHFR. When expressed in CV-1 cells, the fusion protein (SM-20(1-50)/DHFR) colocalized with Mitotracker Green FM, indicating that SM-20(1-50)/DHFR was efficiently targeted to mitochondria (Fig. 4, panels A-C). A second fusion protein consisting of the first 25 amino acids of SM-20 fused to DHFR (SM-20(1-25)/DHFR) was also targeted to mitochondria (Fig. 4, panels D-F). These data indicate that the amino-terminal 25 residues of SM-20 can function as a mitochondrial targeting sequence.



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Fig. 4.   SM-20 contains an amino-terminal mitochondrial targeting sequence. CV-1 cells transfected with expression vectors for SM-20(1-50)/DHFR (panels A-C) and SM-20(1-25)/DHFR fusion proteins (panels D-F) were incubated with Mitotracker Green FM (panels A and D). Indirect immunofluorescence using anti-T7 antibody and TRITC-conjugated secondary antibody was performed to detect the T7-tagged fusion proteins (panels B and E). Yellow in the overlay images (panels C and F) appears where the red fluorescence of the fusion proteins colocalizes with the green fluorescence of Mitotracker Green FM. Bar = 10 µm.

Mitochondrial Targeting of SM-20 Is Not Required for Its Ability to Induce Cell Death in Sympathetic Neurons-- SM-20 mRNA levels and SM-20 protein synthesis increase in sympathetic neurons undergoing apoptosis (10). Despite this, SM-20 protein levels are still relatively low in these cells, and this has impeded our attempts at confirming the localization of the endogenous SM-20 in neurons. To determine whether SM-20 can be targeted to mitochondria in neurons, we microinjected sympathetic neurons with a cDNA encoding a SM-20/GFP fusion protein. The distribution of SM-20/GFP in sympathetic neurons visualized with confocal microscopy coincided with the labeling pattern of Mitotracker Orange, a mitochondria-selective dye (Fig. 5, panels A-C). The mitochondrial labeling was specific to the full-length SM-20 protein; SM-20(60-355)/GFP was diffusely localized throughout the cytosol and nucleus and did not colocalize with Mitotracker Orange (Fig. 5, panels D-F).



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Fig. 5.   SM-20, but not SM-20(60-355), is localized to mitochondria in sympathetic neurons. Sympathetic neurons were microinjected with expression vectors encoding SM-20/GFP (panels A-C) or SM-20(60-355)/GFP fusion proteins diluted to 50 µg/ml in injection buffer without rhodamine-dextran (panels D-F). Cells were labeled with the mitochondria-selective dye Mitotracker Orange 24 h after microinjection (panels A and D). Images of live cells were acquired by confocal microscopy. The green fluorescence in cells injected with SM-20/GFP fusion constructs is shown in panels B and E. These images are superimposed with the red fluorescence of Mitotracker Orange in panels C and F. The letter n represents the position of the nucleus in the injected neurons. Bar = 10 µm.

Expression of SM-20 in sympathetic neurons maintained in the presence of NGF induces cell death in approximately half of the microinjected neurons (10). To determine whether the mitochondrial localization of SM-20 is necessary for its ability to promote neuronal death, we microinjected sympathetic neurons with expression plasmids for SM-20(60-355)/GFP or, as a control, GFP. Three days after microinjection, most GFP-injected neurons appeared healthy with phase-bright cell bodies and readily discernible nuclei and nucleoli (Fig. 6A). In contrast, most of the SM-20(60-355)/GFP-injected neurons displayed morphological features of apoptosis, including atrophic cell bodies and condensed or degraded chromatin. Similar results were obtained when the SM-20(60-355) cDNA fused to the T7 epitope tag was expressed in the neurons. Quantifying these results showed that greater than 60% of the SM-20(60-355)-injected neurons contained condensed or degraded chromatin compared with only 25% of the control cells injected with beta -galactosidase (LacZ) (Fig. 6B). The extent of cell death induced by SM-20(60-355) in these experiments is comparable with the level of cell death produced by microinjection of full-length SM-20 (10). Since SM-20(60-355) is not targeted to mitochondria in sympathetic neurons, the mitochondrial localization of SM-20 does not appear to be critical for its death promoting activity.



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Fig. 6.   SM-20(60-355) induces cell death in NGF-maintained sympathetic neurons. A, sympathetic neurons were microinjected with GFP (panels a-c) or SM-20(60-355)/GFP (panels d-i) expression plasmids and, after 72 h, stained with the DNA binding dye Hoechst 33342. Injected cells were identified by the green fluorescence of GFP (panel b) or SM-20(60-355)/GFP fusion protein (panels e and h). The Hoechst-stained nuclei of the same cells are shown in panels c, f, and i, and phase-contrast images are shown in panels a, d, and g. The white arrows indicate the injected cells and their corresponding phase-contrast and Hoechst-stained nuclei. Bar = 15 µm. B, NGF-maintained neurons were microinjected with expression vectors encoding LacZ or SM-20(60-355). After 72 h, the injected cells were stained with Hoechst 33342 and scored for viability as described under "Experimental Procedures." Data are the mean ± S.E. from six independent experiments with 200-300 neurons scored per injected DNA in each experiment. The mean survival of SM-20-injected cells was significantly less than that of LacZ-injected cells (*, two-tailed t test, p = 0.004).

SM-20-promoted Death in Sympathetic Neurons Is Caspase-dependent and Leads to Activation of Caspase-3-- Sympathetic neurons deprived of NGF undergo caspase-dependent apoptosis that can be blocked by the general caspase inhibitor BAF (26). To investigate whether SM-20-induced death is also caspase-dependent, we microinjected sympathetic neurons with expression vectors containing SM-20 or LacZ and then incubated the cells in NGF-containing media with or without BAF (50 µM). After 3 days, the SM-20-injected neurons maintained in the absence of BAF displayed morphological features of apoptosis, including condensed, crescent-shaped or degraded chromatin (Fig. 7A, panels a-c). In contrast, SM-20-injected neurons maintained in the presence of BAF were healthy with intact cell bodies and diffuse chromatin (Fig. 7A, panels d-f). Greater than 70% of SM-20-injected neurons maintained in the presence of BAF survived after 72 h compared with 38% in the absence of BAF (Fig. 7B). LacZ-injected cells, maintained under the same conditions, showed no significant difference in percent survival. Thus SM-20-induced neuronal death is caspase-dependent and can be blocked by a general caspase inhibitor.



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Fig. 7.   The cell death induced by SM-20 in sympathetic neurons is blocked by a general caspase inhibitor. A, sympathetic neurons were microinjected with a SM-20 expression plasmid and treated with (panels d-f) or without (panels a-c) 50 µM BAF for 72 h. Injected cells were identified by inclusion of a non-fixable rhodamine-dextran dye in the injection buffer (panels b and e). The white arrows indicate the injected cells and their corresponding phase-contrast (panels a and d) and Hoechst-stained nuclei (panels c and f). Bar = 15 µm. B, NGF-maintained neurons were microinjected with expression vectors encoding LacZ or SM-20 and incubated in the presence or absence of 50 µM BAF. After 72 h, the injected cells were stained with Hoechst 33342 and scored for viability as described under "Experimental Procedures." Data are the mean ± S.E. from four independent experiments. The mean survival of SM-20-injected cells in the absence of BAF was significantly less than that of SM-20-injected cells in the presence of BAF (two-tailed t test, p = 0.0013).

To test whether SM-20-induced death leads to activation of effector caspases such as caspase-3, sympathetic neurons expressing SM-20, LacZ, or Bax were analyzed by immunofluorescence using an antibody that recognizes activated caspase-3. As shown in Fig. 8 (panels a-d), LacZ-injected neurons appeared healthy and lacked active caspase-3 immunofluorescence. In contrast, both SM-20- and Bax-injected neurons showed significant staining for active caspase-3 (Fig. 8, panels e-l). The percentage of injected cells expressing activated caspase-3 at 72 h was significantly greater for both SM-20 (18.5 ± 1.5%) and Bax (47.2 ± 9.8%) when compared with LacZ-injected cells (6.3 ± 1.7%). For SM-20-injected neurons (see Fig. 7B) and neurons injected with Bax or deprived of NGF (data not shown), the percentage of cells with activated caspase-3 was less than the fraction that underwent cell death, as assayed by visualizing Hoechst-stained nuclei. Thus, activated caspase-3 may only accumulate to detectable levels in cells with advanced chromatin condensation and degradation. Taken together, these data suggest that SM-20-induced cell death is caspase-dependent and that it involves activation of caspase-3.



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Fig. 8.   Expression of SM-20 in sympathetic neurons leads to caspase-3 activation. NGF-maintained sympathetic neurons were microinjected with expression plasmids encoding LacZ (panels a-d), SM-20 (panels e-h), or Bax (panels I-l). Injected cells were identified by inclusion of a fixable rhodamine-dextran dye in the injection buffer (panels b, f, and j). After 3 days, the cells were fixed, and immunofluorescence was performed using anti-activated caspase-3 antibody (panels c, g, and k). The white arrows indicate the injected cells and their corresponding phase-contrast (panels a, e, and i) and Hoechst-stained nuclei (panels d, h, and l). Bar = 15 µm.

SM-20 Expression Does Not Cause Cytochrome c Release from Mitochondria-- The release of cytochrome c from mitochondria in sympathetic neurons after NGF withdrawal is thought to contribute to caspase activation (27). Once in the cytosol, cytochrome c can form a complex with Apaf-1 and caspase-9, resulting in the activation of caspase-9 and, eventually, downstream caspases such as caspase-3 (28). To determine whether ectopic expression of SM-20 can cause release of cytochrome c from mitochondria, we performed cytochrome c immunofluorescence on NGF-maintained neurons microinjected with expression plasmids for SM-20, LacZ, or Bax. For these experiments, the injected neurons were incubated in the presence of BAF (50 µM), which prevents the final stages of cell death caused by NGF withdrawal without blocking cytochrome c release (27). Greater than 85% of SM-20- or LacZ-injected neurons showed a punctate mitochondrial pattern after labeling with an anti-cytochrome c antibody, indicating that cytochrome c was not released from mitochondria in these cells (Fig. 9, A and B). In contrast, ~75% of Bax-injected neurons displayed a diffuse cytoplasmic staining pattern that closely resembled the cytochrome c immunofluorescence observed after NGF deprivation (27), consistent with a role for Bax as an upstream regulator of cytochrome c release (29). These data indicate that SM-20 expression, by itself, is not sufficient to cause cytochrome c release.



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Fig. 9.   Absence of detectable cytochrome c release from mitochondria in SM-20-injected sympathetic neurons. A, sympathetic neurons were microinjected with LacZ (panels a-c), SM-20 (panels d-f), or Bax (panels g-i) expression vectors and maintained in BAF-containing media. After 24-48 h, the cells were fixed, and immunofluorescence was performed using anti-cytochrome c antibody (panels c, f, and i). Injected cells were identified by inclusion of a fixable rhodamine-dextran dye in the injection buffer (panels b, e, and h). The white arrows indicate the injected cells and their corresponding phase-contrast images (panels a, d, and g). Bar = 15 µm. B, NGF and BAF-maintained neurons were microinjected with expression vectors encoding LacZ, SM-20, or Bax. After 48 h, the percentage of injected cells (rhodamine-positive) showing a loss of punctate mitochondrial cytochrome c labeling was determined. Data are mean ± S.E. from 3-7 independent experiments. The mean number of SM-20-injected cells showing release of cytochrome c was not significantly different than that for LacZ (two-tailed t test, p > 0.2).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In nonneuronal and neuronal cells, SM-20 immunofluorescence colocalized with mitochondria-selective dyes as well as the immunofluorescence for two distinct mitochondrial proteins. Deletion of 59 amino acids from the amino terminus of SM-20 resulted in the truncated protein being distributed uniformly throughout the cell. Consistent with its mitochondrial localization, the amino-terminal 25 amino acids of SM-20 were found to be sufficient to target a normally cytosolic protein to mitochondria. In addition to the expected 40-kDa SM-20 protein, both CV-1 cells and NIH3T3 cells expressed a 33-kDa form of SM-20. In cell fractionation experiments, the smaller SM-20 protein was present predominantly in the mitochondria-enriched fraction. This result, together with the presence of an amino-terminal mitochondrial targeting sequence in SM-20, suggests that the mitochondrial targeting sequence is proteolytically removed when SM-20 is imported into mitochondria. Together, these results identify SM-20 as a novel mitochondrial protein.

SM-20 immunocytochemistry was previously detected in a punctate pattern in the cytoplasm of vascular smooth muscle cells (15). The SM-20 immunostaining was shown to be distinct from that of alpha -actin, but efforts to further localize SM-20 were not undertaken. The appearance of SM-20 immunostaining in smooth muscle cells was very similar to the distribution of SM-20 in neurons and CV-1 cells that we observed. The identification of a mitochondrial targeting sequence in SM-20 suggests that the punctate distribution of SM-20 detected previously in smooth muscle cells reflects targeting of the endogenous SM-20 protein to mitochondria.

Although SM-20 is predominantly localized to mitochondria, a truncated form of SM-20 lacking its mitochondrial targeting sequence unexpectedly retained the ability to induce death in sympathetic neurons, suggesting that endogenous SM-20 may not need to be localized to mitochondria to induce cell death. One explanation for this apparent discrepancy is that SM-20 might be released from mitochondria into the cytosol after NGF withdrawal, analogous to the release of cytochrome c (27, 30). Expression of truncated SM-20(60-355) that is present in the cytoplasm could mimic this event. An alternative explanation is that endogenous SM-20 does not get released from mitochondria during cell death and that it is the mitochondrial form of SM-20 that contributes to apoptosis. Expression of SM-20(60-355) may result in a small amount of SM-20(60-355) being imported into mitochondria or binding to undefined proteins present on the surface of mitochondria, which in this case may be sufficient to promote death.

To further examine the mechanism of SM-20-induced cell death, we investigated the role of SM-20 in caspase activation and in cytochrome c release from mitochondria. As with NGF-withdrawal-induced death, SM-20-induced death was efficiently blocked by the general caspase inhibitor BAF. In addition, we detected activated caspase-3 in a significant fraction of SM-20-injected neurons, suggesting that SM-20 may directly or indirectly participate in the activation of this caspase in dying neurons. The release of cytochrome c from mitochondria occurs after NGF withdrawal and has been suggested to contribute to cell death in this paradigm (27, 30). In contrast to Bax, ectopic expression of SM-20 did not cause widespread cytochrome c release. Since SM-20 does not act by directly causing cytochrome c release, our results suggest that SM-20 functions during cell death in a pathway that is either downstream or independent of cytochrome c release.

Although SM-20 does not resemble known mammalian proteins, it contains a 218-amino acid region that is 43% identical (61% similar) to a portion of the protein encoded by the C. elegans egl-9 gene (17). Deletions and mutations that disrupt the egl-9 gene confer resistance to a P. aeruginosa-derived toxin that causes a lethal neuromuscular paralysis in C. elegans. The genetics in this model indicate that the toxin results in aberrant activation of Egl-9 rather than in its inactivation. Consistent with a role for Egl-9 as a regulator of neuronal signaling or muscle contraction, the egl-9 promoter is most active in muscle cells and in neurons (17). Expression of the rat SM-20 gene is also greatest in muscle-containing tissues and in brain, suggesting that the function of SM-20/Egl-9 in these tissues may be conserved (13). In humans, several SM-20-related genes or pseudogenes appear to be present on distinct chromosomes (31).2 Future studies will examine the ability of these SM-20-related proteins, including Egl-9, to induce cell death as well as their subcellular localization.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Daniel Donoghue for providing the DHFR plasmid, Dr. Eugene Johnson for the Bax plasmid, Dr. Patricia Hinkle for providing NIH3T3 cells, and Dr. Jane Chisholm for expert assistance with confocal microscopy. We also thank Daphne Hasbani and Leah Larocque for excellent technical assistance.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant NS34400.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by NIEHS, National Institutes of Health institutional training Grant ES07026.

|| Supported in part by the Paul Stark Endowment at the University of Rochester. To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-273-4893; Fax: 716-273-2652; E-mail: Robert_Freeman@urmc.rochester.edu.

Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M008407200

2 D. M. Hasbani and R. S. Freeman, unpublished observation.


    ABBREVIATIONS

The abbreviations used are: NGF, nerve growth factor; BAF, BOC-Asp-CH2F; DHFR, dihydrofolate reductase; GFP, green fluorescent protein; LacZ, beta -galactosidase; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; MOPS, 4-morpholinepropanesulfonic acid; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate; RIPA, radioimmune precipitation.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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