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Published as doi: 10.1096/fj.06-7136com.
 (The FASEB Journal. 2007;21:1492-1502.)
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The spinal muscular atrophy gene product regulates neurite outgrowth: importance of the C terminus

Jeroen van Bergeijk*, Katharina Rydel-Könecke*, Claudia Grothe*,{dagger} and Peter Claus*,{dagger},1

* Department of Neuroanatomy, Hannover Medical School, and

{dagger} Center for Systems Neuroscience (ZSN) Hannover, Hannover, Germany

1Correspondence: Department of Neuroanatomy, Hannover Medical School, OE 4140, Carl-Neuberg-Str.1, 30625 Hannover, Germany. E-mail: claus.peter{at}mh-hannover.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spinal muscular atrophy is a neurodegenerative disease accompanied by a loss of motoneurons. Either mutations or deletions in the survival of motoneuron (SMN) gene are responsible for this defect. SMN is an assembly protein for RNA-protein complexes in the nucleus and is also found in axons of neurons. However, it is unclear which dysfunctions of SMN are important for disease progression. In this study we analyzed the contributions of different SMN regions for localization and neuronal differentiation associated with outgrowth of neurites. Suppression of endogenous SMN protein levels significantly decreased the growth of neurites. Down-regulation of the interacting protein gemin2 had the opposite effect. Surprisingly, selective overexpression of the SMN C-terminal domain promoted neurite outgrowth similar to full-length protein and could rescue the SMN knock-down effects. The knock-down led to a significant change in the G-/F-actin ratio, indicating a role for SMN in actin dynamics. Therefore, our data suggest a functional role for SMN in microfilament metabolism in axons of motoneurons.—van Bergeijk, J., Rydel-Könecke, K., Grothe, C., Claus, P. The spinal muscular atrophy gene product regulates neurite outgrowth: importance of the C terminus.


Key Words: survival of motoneuron • neurodegeneration • actin • PC12 cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SPINAL MUSCULAR ATROPHY (SMA) is a neurodegenerative disease caused by progressive degeneration of motoneurons in the spinal cord. SMA is caused by mutations and deletions in the survival of motoneuron gene SMN1 (1 2 3) . The severity of the disease is related to the amount of functional SMN protein coded by the second gene SMN2, which mainly expresses an exon 7 truncated form of SMN (4 , 5) , and the to number of SMN2 gene copies (6 7 8) . The C-terminally truncated form of SMN without exon 7 can cause severe muscular dystrophy when expressed in muscle in the absence of functional protein (9) . However, in the presence of full-length SMN, overexpression of the truncated protein can even be beneficial for the SMA phenotype probably due to oligomerization between both forms and, as a consequence, stabilization of the heterotypic complexes (10) .

SMN is an assembly factor for small nucleolar RNP particles (snoRNPs) and small nuclear RNPs (snRNPs) involved in splicing. Additional data show a putative role of SMN in transcription (11 12 13 14) . Certain mutations of the tudor domain, the central interaction domain of SMN (amino acid residues 90–160), inhibit binding of snRNP core factors (Sm proteins) (15) . Deletion of the first 27 N-terminal amino acid residues of SMN also leads to inhibition of snRNP assembly and splicing (16 , 17) . Regarding the intracellular distribution, SMN localizes to the cytoplasm and to the nucleus in structures called gems (gemini of Cajal bodies) as well as partly to Cajal bodies (18 19 20) . In the cytoplasm, SMN is observed as part of a complex with spliceosomal Sm proteins and a group of proteins designated gemins2–8 (21 22 23) . Further, SMN interacts with an intracellular isoform of fibroblast growth factor 2 (FGF-2) and is associated with at least a subset of snRNPs (24 , 25) . In motoneurons, endogenous FGF-2 is expressed and exhibits neurotrophic activity after exogenous application (26 , 27) , indicating the importance of the SMN/FGF-2 complex for the survival and regeneration of these cells.

Although SMN is expressed ubiquitously, exclusively motoneurons degenerate related to SMN1 defects. A motoneuron represents a unique cell type, since it exhibits the longest axons in the body. SMN localizes to axons as well as to growth cones and branch points of developing neurons (28) . During development, motoneurons and glial cells have to precisely regulate axon outgrowth directed to the muscular target tissue, while they have to fulfill regulated transport of molecules along the axons. In terms of neuropathology, motoneurons show signs of a retrograde axonal degeneration, with degeneration of the cell bodies as a secondary consequence (29 , 30) . SMN can be defined as a general assembly protein for RNA-protein complexes critically involved in survival and maintenance of motoneurons with their very long axons (e.g., in the axonal transport of RNA molecules as shown for ß-actin mRNA) (12 , 31 32 33) . Defects in axonal functions seem to play an important role in the pathophysiology of spinal muscular atrophy (29 , 32 , 34 , 35) . It is unclear whether nuclear and/or axonal dysfunctions caused by SMN deficiency contribute to SMA pathology (36) . To elucidate the molecular pathology of SMA, it is not only required to investigate the nuclear functions of SMN with respect to splicing, but also axonal functions of SMN (e.g., by analyzing axonal interaction partners of SMN).

PC12 cells are phaeochromocytoma cells of sympatho-adrenal origin and represent a useful model for the analysis of neuronal differentiation (32 , 37) . Treatment of undifferentiated PC12 cells with nerve growth factor (NGF) drives differentiation into a sympathetic neuronal phenotype and outgrowth of neurites. In the present study we show that SMN expression is not coregulated with expression of Sm and other interacting proteins during neuronal differentiation of PC12 cells, indicating different functions in this developmental context. Using this cell line, we analyzed the capacity of various regions of SMN to promote neurite outgrowth. Overexpression of full-length SMN resulted in significantly longer neurites compared with a C-terminal truncated form. Furthermore, functional knock-down of endogenous SMN decreased neurite lengths accompanied by a change of the ratio between free G-actin and filamentous F-actin. These data suggest direct involvement of SMN in neurite growth by regulation of microfilament assembly. In contrast to suppression of SMN protein levels, knock-down of the interacting protein gemin2 had the opposite effect on neurite outgrowth. Surprisingly, the C terminus of SMN, encoded by exons 6 and 7, was able to promote neurite outgrowth independently from other domains and to rescue the effects of SMN knock-down.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids
Full-length human SMN was cloned by polymerase chain reaction (PCR) from clone I.M.A.G.E. Consortium CloneID IMAGp998P0910174Q2 from RZPD (Berlin, Germany) (38) in pCMV-SPORT6 comprising the complete human cDNA for SMN as template. Deletion mutants were cloned by PCR into pGEM-T (Promega, Madison, WI, USA) and verified by sequencing. The primers contained EcoRI/SalI restriction sites for subsequent cloning into pEGFP-N2 (Clontech, Palo Alto, CA, USA). For expression of gemin2 a complete cDNA in pCMV-SPORT6 was used (clone ID IRAVp968A05103D6 from RZPD). Constructs were transfected into PC12 cells by liposomal transfection with Metafectene (Biontex, Martinsreid/Planegg, Germany) according to the manufacturer’s recommendations.

Cell culture and analysis of protein expression
PC12 cells were cultivated in PC12-medium containing Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% (v/v) fetal calf serum (FCS), 10% (v/v) horse serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1 mM sodium pyruvate, and 6 mM L-glutamine on collagen I. After transfection with SMN-enhanced GFP (EGFP) constructs, cells were cultivated in PC12-medium without FCS but with 1% (v/v) horse serum and 100 ng/ml NGF to induce neuronal differentiation (39) . For analysis of SMN expression and associated proteins, PC12 cells were cultivated for the indicated periods with or without NGF. After lysis of cells, the protein concentrations were measured by a BCA assay (Pierce, Rockford, IL, USA) and equal amounts of protein were analyzed by SDS-PAGE/Western blot as described before (24) .

PC12 neurite outgrowth assay
After 3 days of differentiation with NGF, the lengths of the longest neurites of PC12 cells were measured morphometrically using the AnalySIS (Soft Imaging Systems, Münster, Germany) software package (39) . For visualization of neurites, cells were cotransfected with a vector encoding the red fluorescent protein DsRed2 (Clontech). For analyses of cells after transfection, only fluorescent cells were measured.

siRNA experiments
An shRNA vector construct in pSupp (Imgenex, San Diego, CA, USA) expressing a double-stranded short RNA fragment coupled by a short hairpin loop under the control of a U6 promoter was generated. Two complementary oligonucleotides were synthesized comprising each of 20 nt of the SMN coding sequence (capital letters), followed by a hairpin sequence (small letters in italics) and the same coding sequence as a reversed complement (sense strand oligonucleotide PC127-F: 5'-tcg agG AGA AAC CTG TGT CGT GGT Tga gta ctg AAC CAC GAC ACA GGT TTC TCt ttt t-3'; antisense strand PC128-R: 5'-cta gaa aaa GAG AAA CCT GTG TCG TGG TTc agt act cAA CCA CGA CAC AGG TTT CTC c-3'). The coding sequence represents nucleotides 353–371 downstream from the start codon of the rat SMN sequence (GenBank accession no. NM_022509). The hairpin sequence also contains a restriction recognition site of ScaI to screen for plasmid clones with inserts. For cloning into the XhoI/XbaI sites of the pSupp vector after annealing of the oligonucleotides, sites for the respective restriction enzymes were included at the ends (sequences in small letters above at 5'and 3' ends). The resulting plasmid pSupp.SMN was sequenced to verify correctness of cloning. PC12 cells were transfected by electroporation (each 3x106 cells, 975 µF, 350 V; EasyjecT, Equibio), enriched in a 750 µg/ml geneticin containing medium, and the effects on the amounts of SMN protein were evaluated by Western blot as described before (24) . An empty pSupp vector without an insert was used as a control (pSupp.control). Another shRNA construct for down-regulation of SMN (pSupp124/125) was not functional. The siRNA to down-regulate rat gemin2 (GenBank accession no. NM_053389.1) was designed against a region between nucleotides 351–375 (sequence of sense strand: 5'-GGCAGCAGCAACAAGUGAUACAGUU-3'). Single strands were synthesized, annealed to form a duplex, and transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). A scrambled sequence was used as a control (sequence of sense strand: 5'-GGCCGACACAAGUGACAUAAGAGUU-3'). PC12 cells were cotransfected with pDsRed2 (Clontech) vector expressing a red fluorescent protein in the whole cell to clearly identify transfected cells and to label neurites for measurement of lengths as describe above.

Immunocytochemistry and fluorescence microscopy
Cells were fixed and processed as described before (40) and mounted in Prolong Gold (Molecular Probes, Carlsbad, CA, USA). Confocal microscopy was performed at 19°C on a Leica TCS SP2, equipped with Leica acquisition software, using oil immersion objectives HCX PL APO CS40 (40x/numerical aperture 1.25) and HCX PL APO BL (63x/numerical aperture 1.4). Images were assembled with Adobe Photoshop.

G-/F-actin assay
To determine G-/F-actin ratios (41) , cells were lysed in prewarmed (37°C) LAS buffer (50 mM PIPES pH 6.9, 50 mM NaCl, 5 mM MgCl, 5 mM EGTA, 1 mM ATP, 5% (v/v) glycerol, 0.1% (v/v) IGEPAL CA-630, 0.1% (v/v) Triton X-100, 0.1% (v/v) Tween 20, 0.1% (v/v) ß-mercaptoethanol, 1 x 10–3 % (v/v) antifoam C, 2% (v/v) Complete EDTA-free protease inhibitor (Roche, Nutley, NJ, USA), homogenized, and incubated for 10 min at 37°C. Controls were also treated with F-actin-enhancing solution (1 µM phalloidin; Sigma, St. Louis, MO, USA) and F-actin-depolymerization solution (10 µM cytochalasin D, Sigma), respectively. Lysates were then centrifuged at 380 g for 5 min. The supernatants were removed and centrifuged for 100,000 g at 37°C for 1 h. After ultracentrifugation, supernatants containing the G-actin fraction were transferred to reaction cups and placed on ice. The pellet containing the F-actin was resuspended in one initial sample volume in 10 µM cytochalasin D with 2% (v/v) Complete protease inhibitors (Roche). Protein concentrations of the pellet fractions were measured with BCA assay (Pierce) and equal amounts of each sample were mixed with a 2 x sample buffer for SDS-PAGE and subsequent Western blot. The blots were probed with anti-actin antibody (BD Biosciences, Bedford, MA, USA) at a 1:2500 dilution.

Antibodies
The monoclonal anti-ß-tubulin antibody developed by M. Klymkowsky was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, IA, USA. Monoclonal anti-SMN was obtained from BD Biosciences. Antibodies against the following proteins were used: mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (mAb) (Chemicon, Temecula, CA, USA), monoclonal antigemin2 (Sigma), mouse anti-Sm Ab-1 (clone Y12) (Abcam, Cambridge, MA, USA), mouse anti-protein-arginine methyl transferase 5 (PRMT5) (Abcam), mouse anti-coilin (BD Biosciences), anti-neurofilament pan antibody (Biotrend, Cologne, Germany, USA), mouse anti-actin Ab-5 (BD Biosciences), mouse anti-green fluorescent protein (GFP) (Roche), and mouse anti-ßIII-tubulin (Upstate, Lake Placid, NY, USA). For immunofluorescence, a secondary goat anti-mouse antibody conjugated to Alexa Fluor 555 (Molecular Probes) was applied.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of SMN and associated proteins during neuronal differentiation
We analyzed the effects of neuronal differentiation by NGF in PC12 cells on protein expression of SMN and some of its interaction partners. Results of at least three independent experiments were analyzed by Western blots and subsequently evaluated densitometrically (Fig. 1 A). The housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as a loading control, did not display changes of expression levels (Fig. 1A, B ). The 68 kDa neurofilament protein (NF-68) became up-regulated upon stimulation of cells with nerve growth factor for 24 h (Fig. 1A, B ). An increase in NF-68 mRNA levels in PC12 cells has been reported as a consequence of neuronal differentiation (42) . Similarly, SMN was significantly up-regulated in PC12 cells during neuronal differentiation within 3 days of continuous NGF induction (Fig. 1A, B ). Gemin2 and SmB are two binding partners of SMN in the SMN complex (43) . Gemin2 did not display changes in expression levels (Fig. 1A, B ). SMN interacts with and assembles Sm proteins on small nuclear RNAs (snRNA) into a ring-like structure (44) . We examined the level of one of the Sm proteins (SmB) and found that it was not altered during differentiation of PC12 cells (Fig. 1A, B ). Sm proteins are methylated at arginine-rich motifs in the so-called methylosome by the protein arginine methyl transferase PRMT5 (45) . Therefore, we investigated changes in PRMT5 expression on neuronal stimulation. Like other components of the SMN complex, no significant differences could be detected. Although Sm proteins are in a common complex with SMN, neither SmB nor PRMT5 were coregulated with SMN. In the nucleus, SMN interacts with coilin, the main constituent of Cajal bodies (46) . During the course of neuronal differentiation, coilin levels decreased (Fig. 1A, B ), indicating a putative concomitant change in Cajal body number and structure (47) . The results demonstrate that SMN is not coregulated with other components of the SMN complex and methylosome during differentiation of PC12 cells. Instead, SMN becomes strongly up-regulated, suggesting an independent role for this protein.


Figure 1
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  		Figure 1. Expression of SMN and associated proteins during neuronal differentiation of PC12 cells. A) PC12 cells were treated with nerve growth factor (NGF) for 24 h and 72 h, respectively, or untreated (0 h) and harvested after the times indicated. Cells were lysed and equal amounts of total cell lysates were analyzed by SDS-PAGE/Western blots with antibodies against the indicated proteins. An antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control. B) Protein levels were quantified by densitometry, normalized against GAPDH, and analyzed statistically by a Mann-Whitney test (*P<0.05). Data points represent means with SEs of the mean (SEM). The experiments were repeated at least three times.

Functional knock-down of endogenous SMN reduces neurite outgrowth
Patients with SMA show reduced levels of functional SMN protein. To mimic this effect and to establish a cell culture model for this neurodegenerative disease, we designed a vector expressing a small hairpin RNA (shRNA) that is subsequently cleaved by the RISC complex to function as a small interfering RNA (siRNA) (48) . Expression of the shRNA in the vector pSupp.SMN is under the control of the U6 promoter upon transfection of the construct into PC12 cells. Since the primary transfection rate was 28 ± 0.6%, higher numbers of transfected cells could be achieved after selection with geneticin (42.5±0.7%; data not shown) since the plasmid used also carried a selectable marker for mammalian cells. A decrease in SMN protein levels was tested by Western blot (Fig. 2 A). Densitometrical analysis of the SMN signals showed a significant decrease of 53 ± 15% relative to ßIII-tubulin and compared to cells transfected with control vector (pSupp.control) on day 3 after transfection and neuronal differentiation (Fig. 2B ). The data demonstrated that SMN protein levels could be suppressed in PC12 cells by the shRNA construct to levels that mimic the reduced SMN protein levels in SMA patients. However, since neuronal differentiation resulted in up-regulation of SMN protein levels (Fig. 1A, B ), the siRNA machinery had to counteract this increase of expression. To study the effects of suppression on neurite outgrowth, neurite lengths were measured by morphometrical analysis of maximum neurite lengths (39) . To ensure that only transfected cells were included in the statistical analysis and to allow measurement of neurite lengths, PC12 cells were cotransfected with a plasmid coding for the red fluorescent protein DsRed2. Suppression of SMN levels resulted in highly significant shorter neurites compared with the control (Fig. 2C, D ). These data demonstrate that endogenous SMN plays a critical role in regulating neurite growth.


Figure 2
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  		Figure 2. Knock-down of endogenous SMN and neurite outgrowth. A) PC12 cells were transfected with a shRNA construct against SMN (pSupp.SMN) and with a control construct (pSupp.control), respectively. After treatment with NGF for the indicated times, cells were lysed and equal amounts of proteins were analyzed by SDS-PAGE/Western blot with antibodies against SMN and ßIII-tubulin (as a control for loading and as an unregulated protein). Note the increase in SMN levels upon neuronal differentiation in cells transfected with the control shRNA, as also shown in Fig. 1 . B) Analysis of three independent knock-down experiments. Bands were densitrometrically analyzed and SMN/tubulin ratios were calculated for normalization. SMN/tubulin ratios of control and knock-down cells (0 h) were set to zero. Asterisk denotes a significant difference (*P<0.05; Mann-Whitney test; n=3 independent experiments). C) Representative images of neuronal differentiated PC12 cells transfected with the control construct (pSupp.control) and with the shRNA construct against SMN (pSupp.SMN) showing different neurite lengths. Cells were cotransfected with a vector coding for the red fluorescent protein DsRed2 for visualization of neurites. Bar, 25 µm. D) Neurite lengths of PC12 cells after transfection with the control shRNA construct (control) and shRNA against SMN (knock-down). Asterisks denote significant differences (**P<0.01; Mann-Whitney test; n=3 independent experiments). For quantification, at least 140 neurites were measured in three independent experiments. Data points represent means with SEs of the mean (SEM).

SMN knock-down changes the G-/F-actin ratio of neuronal differentiated cells
Changes in neurite outgrowth and growth cone dynamics are intimately linked to regulation of the actin cytoskeleton (49) . First, we asked whether down-regulation of SMN causes changes of actin expression. Three days after transfection and neuronal differentiation, cells were harvested and protein levels were determined by Western blot. SMN was successfully down-regulated (Fig. 3 A). No changes in GAPDH or actin expression could be observed (Fig. 3A ). Actin levels relative to GAPDH levels did not change significantly (Fig. 3B ).


Figure 3
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  		Figure 3. Knock-down of SMN influences the actin cytoskeleton. A) PC12 cells were transfected with the shRNA control construct or the shRNA plasmid against SMN, respectively, and differentiated for 72 h in the presence of NGF. Lysates were subsequently analyzed by Western blot with antibodies against GAPDH, SMN, and actin, respectively. B) Western blots of five independent experiments were densitrometrically analyzed and actin protein levels were calculated relative to GAPDH expression. No significant difference between control and knock-down cells could be detected. Knock-down of SMN did not influence total actin levels. C) G-/F-actin levels were determined by Western blot after ultracentrifugation of control and knock-down cell lysates. Pelleted F-actin was treated with the depolymerization agent cytochalasin D for subsequent analysis of actin amounts. The fractions were subsequently analyzed by SDS-PAGE/Western blot with an anti-actin antibody. Lower panel: Western blots of cell lysates treated as additional controls with cytochalasin D and the F-actin-stabilizing substance phalloidin, respectively, before centrifugation. S, supernatant fraction containing G-actin; P, pellet fraction containing F-actin. D) Densitometry of blots revealed a significant change of G-/F-actin ratios in SMN knock-down cells. Data points represent means with SEs of the mean (SEM). n = 3 independent experiments; *P < 0.05 (Mann-Whitney test).

Second, our observation that SMN levels influence neurite outgrowth and data about SMN association with actin (33) prompted us to determine whether SMN affects actin metabolism. PC12 cells were transfected with SMN shRNA and control vector, respectively, and differentiated for 3 days with NGF. The amounts of free globular G-actin and filamentous F-actin were measured by an in vivo assay using differential precipitation of F-actin (pellet fraction) and G-actin (supernatant) after ultracentrifugation of cell lysates. The pelleted F-actin was treated with the depolymerization agent cytochalasin D for subsequent analysis of actin amounts by Western blot after ultracentrifugation of samples. The fractions were subsequently analyzed by SDS-PAGE and Western blot with an anti-actin antibody (Fig. 3C ). As additional controls for the detection system, we treated control samples with cytochalasin D and the F-actin-stabilizing substance phalloidin, respectively, before centrifugation (Fig. 3C ). The Western blots of this control showed an increased signal in the pellet fraction for phalloidin-treated lysates, as F-actin was stabilized. As expected, control treatment showed a marked decrease of signal in the pellet fraction (Fig. 3C ). Ratios of G-/F-actin were calculated after densitometry of the bands. The results showed that SMN knock-down leads to a significant change in the G-/F-actin ratio compared with cells transfected with the control vector (Fig. 3C, D ). Although we could not detect a general change in actin expression (Fig. 3A, B ), the portion of F-actin increased as a result of SMN protein loss. These results demonstrate that SMN exhibits a direct impact on actin metabolism in neuronal cells.

The SMN interaction partner gemin2 shows antagonistic effects on the regulation of neurite outgrowth
Gemin2 is a core component of the SMN complex and binds directly to the N terminus of SMN (50 , 51) . We asked whether gemin2 is involved in the regulation of neurite outgrowth. A gemin2 siRNA was designed and used in knock-down experiments in PC12 cells. A scrambled siRNA was used as a control. Endogenous gemin2 proteins could be down-regulated to 25 ± 8% of control levels in differentiated cells after 3 days in culture (Fig. 4 A, B). However, this down-regulation had only a small effect on neurite outgrowth as revealed by the neurite outgrowth assay. In contrast to down-regulation of SMN, gemin2 knock-down resulted in slightly larger neurites (Fig. 4C ). In contrast and antagonistic to the effects of SMN, gemin2 overexpression showed significantly shorter neurites.


Figure 4
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  		Figure 4. Knock-down and overexpression of the SMN interaction partner gemin2. A) Western blots of PC12 cells transfected with siRNA against gemin2 or with control siRNA. B) Densitometry of n = 4 independent experiments revealed successful knock-down of gemin2. *P < 0.05 (Mann-Whitney test). C) Neurites of 150 cells of n = 3 independent experiments were measured for subsequent analysis of neurite length in control and gemin2 knock-down cells, respectively. **P < 0.01 (Mann-Whitney test). D) In contrast to the knock-down, overexpression of gemin2 demonstrated increased neurite lengths. Neurites of 160 cells were analyzed in n = 3 independent experiments. ***P < 0.001 (Mann-Whitney test). Data points represent means with SEs of the mean (SEM).

SMN deletion mutants localize differentially to cellular compartments in PC12 cells
To investigate the functional domain of the SMN protein responsible for neurite outgrowth, we generated a series of SMN deletion mutants and cloned these fragments in-frame with EGFP to generate mutant SMN-EGFP fusions (Fig. 5 ). These constructs were transfected into PC12 cells prior to neuronal differentiation with NGF. After 3 days, cells were fixed and counterstained with anti-ß-tubulin to visualize neurites by confocal microscopy (Fig. 6 ). Transfection with full-length SMN protein, comprising all amino acid residues of human SMN (SMN1–294-EGFP), displayed nuclear gems in the nuclei as well as diffuse cytoplasmic signals and, to some extent, cytoplasmic aggregates, which could reflect sites of snRNP biogenesis (Fig. 6A, E ). Similarly, SMN28–294-EGFP displayed nuclear gems (data not shown). In cells transfected with an SMN mutant without the C terminus (SMN1–239-EGFP) nuclear foci were still present but appeared more aggregated. A diffuse nuclear and cytoplasmic staining could be seen with this mutant (Fig. 6E ). Addition of the amino acid sequence QNQKE, encoded by exon 7 and originally described as a cytoplasmic targeting signal (33) , behind residue 239 of this mutant (SMN1–239-QNQKE-EGFP) showed nuclear foci as well as diffuse signals in the nucleus (data not shown). With regard to neurites and growth cones, we could not detect a differential localization compared with SMN1–239. A C-terminal construct (SMN235–294-EGFP) did not form nuclear foci as could be observed for SMN1–294-EGFP and SMN1–239-EGFP fusion proteins (Fig. 6E ). Other mutants tested (SMN-EGFP deletion constructs comprising amino acid residues 1–90, 1–123, and 119–239) displayed the same localization patterns as the EGFP control (data not shown). As a control, EGFP consistently showed nuclear enrichment to some degree in PC12 cells, but otherwise a diffuse staining (Fig. 6A, E ). With regard to localization to neurites and growth cones, full-length SMN1–294-EGFP could be observed in a punctuate pattern (Fig. 6D ), which is consistent with a localization in granules (33) . The localization of signals of SMN235–294-EGFP and SMN1–239-EGFP resembled EGFP staining. However, the C-terminal fragment SMN235–294-EGFP displayed intense, diffuse signals in neurites and growth cones, whereas the C-terminal truncated SMN1–239-EGFP showed less fluorescence in the growth cones (Fig. 6D ).


Figure 5
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  		Figure 5. Schematic representation of the SMN deletion constructs used in this study. Full-length human SMN (SMN1–294) as well as various SMN deletion mutants were fused to enhanced green fluorescent protein (EGFP). Numbers denote amino acid residues of human SMN. Numbers in boxes represent the numbers of exons.


Figure 6
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  		Figure 6. Localization of SMN-EGFP deletion mutants in PC12 cells. Full-length and various SMN deletion mutants were cloned in-frame with EGFP and transfected into undifferentiated PC12 cells. After 3 days of differentiation with NGF, cells were fixed, processed for immunocytochemistry with anti-ß-tubulin or SMN antibody (secondary antibody anti-mouse IgG conjugated to Alexa 555), and analyzed with a Leica TCS SP2 confocal microscope. Numbers denote the region of SMN present in the individual constructs with SMN1–294 referring to full-length SMN (comprising amino acid residues 1 to 294). Representative examples are shown for each construct. Whole cells are shown to demonstrate localization in the nuclei as well as neurites (A–C). SMN positive nuclear foci are clearly visible in cells expressing pEGFP-SMN1–294 and pEGFP-SMN1–239 (E). Full-length SMN is also visible in granules in growth cones, but none of the mutants formed these structures in neurites or growth cones (D). With regard to nuclear localization, quantitative analyses of SMN revealed in 93% and 89% of pEGFP-SMN1–294 and pEGFP-SMN1–239, respectively, transfected cells a distribution pattern in nuclear foci (n=44 for pEGFP-SMN1–294 and n=46 for pEGFP-SMN1–239). In the other cells a diffuse nuclear staining was detectable. The majority of pEGFP-SMN235–294 transfected cells (69%, n=85 cells analyzed) showed no nuclear foci, whereas the reminder contained some diffuse aggregates (not shown). Immunocytochemistry shows the localization of endogenous SMN in differentiated PC12 cells (F). In the nucleus, SMN-positive nuclear foci are visible. Growth cones demonstrate enrichment and a granular localization pattern of SMN. A–C, F) left, 25 µm; D–F) right, 5 µm.

The C terminus of SMN promotes neurite outgrowth
In an attempt to evaluate contributions of individual SMN protein domains to the promotion of neurite outgrowth, PC12 cells were cotransfected with SMN deletion constructs (Fig. 5) together with a plasmid coding for the red fluorescent protein DsRed2. Expression of this fluorescent protein allowed identification of transfected cells (rate of cotransfection 94±6%, data not shown) as well as labeling of neurites to measure their length. After transfection cells were differentiated in the presence of NGF and maximum neurite lengths were measured after 3 days. Overexpression of human full-length SMN (SMN1–294) led to a significant increase in neurite length compared with control transfections with the vector only (Fig. 7 ). A C-terminal deleted mutant protein (SMN1–239 encoded by exons 1 to 5) failed to promote neurite outgrowth defining a C-terminal functional domain important for regulation of process growth (Fig. 7) . Moreover, the C-terminal fragment comprising the amino acid residues 235–294 of human SMN (mutant SMN235–294; encoded by exons 6 to 7) was able to promote neurite outgrowth independently from other SMN domains, arguing that this effect could be independent of the function of SMN as a protein involved in snRNP splicing complex assembly. The tudor domain, originally described in the Drosophila tudor protein (52) , mediates Sm protein binding but is not present in the C terminus of SMN (15) . Most of the tudor domain was comprised by mutant SMN119–239 and did not stimulate neurite outgrowth when overexpressed (Fig. 7) . An N-terminal truncated mutant SMN29–294 also significantly enhanced process outgrowth in PC12 cells (Fig. 7) . N-terminal-deleted SMN is defective in some aspects of pre-mRNA splicing (16) , but displays a comparable neurite growth-promoting effect like the full-length wild-type protein. The presence of N-terminal sequences in the mutants SMN1–90 and SMN1–123 were not sufficient to promote neurite outgrowth compared with the effects of full-length SMN (Fig. 7) .


Figure 7
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  		Figure 7. Neurite outgrowth assay in PC12 cells transfected with SMN mutants revealed neurite outgrowth-promoting activity of the SMN C terminus. After transfection and neuronal differentiation of PC12 cells for 3 days, maximum lengths of neurites were determined morphometrically. The relative increase was calculated for each mutant compared with the lengths measured upon transfection with EGFP as a control. Asterisks denote significant differences (**P<0.01; Student’s t test; n=30). Data points represent means with SEs of the mean (SEM).

Neurite outgrowth is rescued with the C terminus after knock-down of SMN
Next we asked whether a knock-down of SMN levels resulting in suppression of neurite outgrowth can be rescued by overexpression of the SMN C terminus. Undifferentiated PC12 cells were cotransfected with SMN shRNA vector (pSupp.SMN) or control vector (pSupp.control). In addition, cells were cotransfected with pEGFP control vector or vectors expressing the C terminus (pSMN235–294EGFP) and a C-terminal truncated SMN (pSMN1–239EGFP), respectively, as used in the overexpression experiment. Western blots of lysates revealed successful down-regulation of SMN (Fig. 8 A) as well as overexpression of the truncated SMN proteins comprising amino acid residues 235–294 and 1–239. In these controls, the C-terminal SMN fragment 235–294 could only be detected by an antibody directed against the GFP tag, since the epitope for the anti-SMN antibody was not present. Neurite lengths were measured after 3 days in culture with NGF (Fig. 8B ). Whereas the SMN deletion construct SMN1–239 without the C terminus did not show prolonged neurites compared with the control, the expression of the C terminus rescued significantly neurite outgrowth. These results clearly demonstrate the particular role of the C-terminal domain in regulating neurite outgrowth; the other part of the SMN protein was not sufficient to promote this effect.


Figure 8
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  		Figure 8. Neurite outgrowth is rescued by overexpression of the C terminus after knock-down of SMN. A) PC12 cells were transfected with the shRNA construct for knock-down of SMN (pSupp.SMN) or with the control construct (pSupp.control) and cotransfected with pDsRed2 as well as pEGFP, the SMN C-terminal construct pSMN235–294EGFP, or the construct comprising the first 239 amino acid residues of SMN (pSMN1–239EGFP) and pDsRed2. Cells were differentiated for 3 days in the presence of NGF, harvested, and lysates were analyzed by SDS-PAGE/Western blot. Blots were probed with antibodies against GAPDH, SMN, and GFP. The overexpressed C terminus SMN235–294 was visible only with the anti-GFP antibody, since the epitope for the monoclonal SMN antibody was not present in this construct. Endogenous SMN was successfully down-regulated whereas expression of the deletion constructs was up-regulated. B) Rescue of neurite outgrowth after down-regulation of SMN and expression of the C terminus of SMN. Relative changes of neurite lengths were determined as before. At least 200 cells of n = 3 independent experiments were evaluated for each condition. *P < 0.05; **P < 0.01; ***P < 0.001 (Mann-Whitney test). Data points represent means with SEs of the mean (SEM).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PC12 cells are a useful system to study neuronal differentiation and growth of neuronal processes. In this study we demonstrate for the first time a specific role of the SMN C-terminal domain (encoded by exons 6 and 7) for neurite outgrowth. Transfection of PC12 cells with the full-length SMN construct resulted in increased neurite formation in the presence of NGF. Furthermore, we show that a functional knock-down of endogenous SMN by a shRNA construct elucidated the opposite effect. A mutant lacking the C-terminal sequence (comprising amino acid residues 1–239, encoded by exons 1–5) failed to increase neurite outgrowth. Intriguingly, transfection of PC12 cells only with the SMN C terminus increased neurite lengths to the same extent as transfection with full-length SMN. These data assign a functional role for neurite outgrowth to a domain comprising amino acid residues 235–294 of SMN. Moreover, neurite outgrowth can be rescued in an SMN knock-down situation by overexpression of the C terminus. Consistent with our results, a neurite growth-promoting role of the full-length SMN protein has been demonstrated (32) as well as a reduction in neurite length by overexpression of an exon 7 deletion mutant in chick forebrain neurons (33) . Here we provide a more complex molecular analysis of the SMN domain structure with regard to neurite formation. The results could argue for distinct functions of SMN complexes used in snRNP biogenesis for splicing and SMN in axons or dendrites since, as components of snRNPs, Sm proteins bind to a region within the tudor domain (15) that is not present in the C-terminal construct. The particular role of the C terminus for neurite outgrowth is also supported by the finding that a construct similar to ours without exon 6 and 7 failed to sustain cell viability, whereas a mutant without the tudor domain was able to restore viability (50) . Oligomerization of SMN molecules may not represent the mechanism responsible for increased neurite length observed by overexpression of the C terminus, since the other mutants (e.g., SMN1–123) did not show prolonged neurites when overexpressed. These mutants could bind with the N-terminal oligomerization domain encoded by exon 2b (51) . The C-terminal mutant we used in our study is still able to bind to full-length SMN protein (data not shown) since it comprised the second dimerization domain of residues 249–278 in exon 6 (4) . However, dimerization with endogenous SMN cannot explain the unique ability of the C terminus, because in the knock-down situation endogenous SMN is suppressed and therefore an association with the overexpressed C terminus is reduced.

What are the unique features of SMN’s C terminus to promote neurite outgrowth? SMN localizes to axons, dendrites, and growth cones of neuronal cells (28 , 33 , 53) . SMN complexes seem to lack Sm proteins, but contain gemin2 as well as gemin 6 and 7 in neurites of PC12 cells (53) . However, in one study gemin2 did not colocalize with SMN in granules, although colocalization in neurites could also be demonstrated (28) . Here we show that neuronal differentiation of PC12 cells is accompanied by SMN up-regulation whereas SmB and PRMT5 protein levels, for example, are not changed. This may reflect a higher requirement of SMN during neuritogenesis, which is probably needed independently of its role in snRNP assembly. However, in whole spinal cord tissue lysates SMN and gemin2 were coregulated, reflecting a different developmental context as well as several cell types represented in this tissue (28) . With regard to localization, differentiation of a PC12-derived cell line UR61 was accompanied by recruitment of SMN to Cajal bodies (47) . Axonal complexes of SMN have been described with profilin IIa, the main neuronal profilin isoform (53 , 54) and heterogeneous nuclear ribonucleoprotein (hnRNP-R) (31 , 32) . Both of these directly interacting proteins are involved in ß-actin metabolism in neurites. Profilin IIa binds to the proline-rich motifs of SMN located mainly in the sequence encoded by exon 5 (54) and is able to promote actin filament polymerization (55) , but can also sequester free actin monomers (56) . Intriguingly, in an in vitro actin polymerization assay, SMN had a modulatory role by reducing the inhibitory effect of profilin IIa on spontaneous actin polymerization (53) . SMN has also been shown to be associated with the actin cytoskeleton in fibroblasts (33) . Our C-terminal construct comprised a part of the Pro5 motif of the tripartite profilin IIa binding region. Residual binding of this protein to the proline-rich motif in the deletion construct could account for the effects observed with respect to neurite outgrowth. Regulation of actin polymerization is crucial for neurite formation (33 , 57 , 58) . We analyzed changes in globular (G) and filamentous (F) actin levels and found an increase of F-actin compared with controls upon down-regulation of SMN (Fig. 3) . This phenocopies the effect of profilin IIa overexpression, where a significant shift toward polymerized actin was observed (59) . Combining our results, the observed neurite phenotype in SMN knock-down cells is accompanied by a change in F-actin, suggesting that downstream actin-regulating proteins are involved in neuritogenesis. Decreasing the levels of SMN probably increases the amounts of free profilin IIa, which in turn induces a more rapid polymerization of monomeric G-actin into F-actin. Moreover, it has been shown in a straightforward study that SMN and F-actin colocalize in growth cones (35) . The cellular consequence of an increase or stabilization of filamentous actin is a suppression of neurite extension and sprouting (59) . In vitro assays also demonstrated that SMN together with profilin IIa had a positive effect on actin polymerization (53) .

The protein hnRNP-R binds directly to ß-actin mRNA through its RNA recognition motif and regulates the amount of ß-actin mRNA in axons of motoneurons by its interaction with SMN (32) . SMN mutants lacking regions encoded by exon 5/exon 7 failed to bind hnRNP proteins (31) . Consistently, the axonal role of SMN has also been observed in an SMA mouse model. These animals display a lack of axonal sprouting as well as neurofilament accumulation in terminal axons (30) . The axonal SMN complex could also assist in cotranslational folding of actin during local protein synthesis in axonal growth cones. Intra-axonal protein synthesis has been described in motoneurons and seems to be required for structural integrity of the growth cone (60) . SMN has been linked to the dynamics of growth cones and also localized to these structures (33 , 35) .

In the emerging scenario of axonal functions, SMN plays a key role in actin metabolism in axons and growth cones. First, SMN may influence actin polymerization and stabilization by binding to profilin IIa. Second, SMN is important for the translocation of ß-actin mRNA to neurites via hnRNP-Q/-R complexes. Third, SMN could indirectly facilitate actin assembly by interactions with so far unidentified proteins involved in actin metabolism.

Is the SMN/gemin complex involved in regulation of neurite outgrowth? Our findings indicate that this SMN complex (61) may not directly regulate this cellular function. First, SMN and gemin2 are regulated differently during neuronal differentiation of PC12 cells (Fig. 1) . SMN is up-regulated whereas gemin2 levels do not change, indicating a differential requirement for these factors during neuronal differentiation. Second, both factors show opposing effects on neurite outgrowth after up- and down-regulation, respectively (Figs. 2 , 4) . Gemin2 knock-down displayed prolonged neurites whereas knock-down of SMN resulted in shorter neurites. The gemin2 effects could be explained by an SMN-independent and yet undefined function of this molecule in regulating neurite outgrowth or in a change of the stochiometry of the SMN complex. Depletion of gemin2 could lead to an increase of free SMN molecules available for SMN complex independent functions. This would be similar to the effects observed when SMN becomes overexpressed.

SMN is involved not only in snRNP assembly and splicing, but also in axonal processes during neuronal differentiation. Our results clearly demonstrate for the first time a function of the SMN C-terminal region in neurite outgrowth. This function appears to be independent of the role of SMN as an assembler of nuclear RNP complexes. To clarify, whether mutated SMN in patients with SMA affects nuclear as well as axonal functions as two parts of a common functional scenario, further efforts are needed to analyze the role of SMN in motoneurons.


   ACKNOWLEDGMENTS
 
We thank Hella Brinkmann and Maike Wesemann for expert technical assistance and Dr. Lars Klimaschewski, Dr. Kirsten Haastert, and Alexander-Francisco Bruns for discussions and comments on the manuscript. We are also grateful to Drs. I. Just and R. Gerhard from the Department of Toxicology for use of an ultracentrifuge. This work has been supported by grants from the Fritz Thyssen Stiftung, Germany, and Röchling-Stiftung, Germany, to P.C.

Received for publication September 4, 2006. Accepted for publication December 25, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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