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The Journal of Neuroscience, July 23, 2003, 23(16):6627-6637
Previous Article | Next Article
Active Transport of the Survival Motor Neuron Protein and the Role of Exon-7 in Cytoplasmic Localization
Honglai L. Zhang,1 Feng Pan,2 Daewha Hong,1,2 Shailesh M. Shenoy,2 Robert H. Singer,2 andGary J. Bassell1 Departments of 1Neuroscience, Rose F. Kennedy Center for Mental Retardation, and 2Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Abstract
Top
Abstract
Introduction
Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by deletion and/or mutation of the survival motor neuron protein Gene (SMN1) that results in the expression of a truncated protein lacking the C terminal exon-7. Whereas SMN has been shown to be an important component of diverse ribonucleoprotein (RNP) complexes, its function in neurons is unknown. We hypothesize that the active transport of SMN may be important for neurite outgrowth and that disruption of exon-7 could impair its normal intracellular trafficking. SMN was localized in granules that were associated with cytoskeletal filament systems and distributed throughout neurites and growth cones. Live cell imaging of enhanced green fluorescent protein (EGFP)-SMN granules revealed rapid, bidirectional and cytoskeletal-dependent movements. Exon-7 was necessary for localization of SMN into the cytoplasm but was not sufficient for granule formation and transport. A cytoplasmic targeting signal within exon-7 was identified that could completely redistribute the nuclear protein D-box binding factor 1 into the cytoplasm. Neurons transfected with SMN lacking exon-7 had significantly shorter neurites, a defect that could be rescued by redirecting the exon-7 deletion mutant into neurites by a targeting sequence from growth-associated protein-43. These findings provide the first demonstration of cytoskeletal-based active transport of SMN in neuronal processes and the function of exon-7 in cytoplasmic localization. Such observations provide motivation to investigate possible transport defects or inefficiency of SMN associated RNPs in motor neuron axons in SMA.
Key words: survival motor neuron protein; spinal muscular atrophy; mRNA transport; mRNA localization; active transport; growth cone; neurite outgrowth
Introduction
Spinal muscular atrophy (SMA) is one of the most common inherited diseases that results in infant death, characterized by a neurodegenerative process affecting primarily-motor neurons of the lower spinal cord (Frugier et al., 2002
). SMA is caused by deletions or mutations of the survival motor neuron protein gene (SMN1) (Lefebvre et al., 1995
SMN is localized to both the nucleus and cytoplasm, and considerable attention has focused on concentration of SMN in nuclear dot-like structures termed gems (Liu and Dreyfuss, 1996
An alternative view of SMN function is that it is an essential component of RNP complexes that are actively transported in neuronal processes. Immunocytochemical studies have localized SMN in dendrites (Bechade et al., 1999
Here we show that SMN was localized in granules that were localized in neurites and growth cones of cultured neurons. SMN granules exhibited rapid, bidirectional movements that were dependent on both microtubules and microfilaments. We further showed that the C terminal exon-7 contains a sequence that is essential for localization of SMN in the cytoplasm. Overexpression of an exon-7 deletion mutant was characterized by abnormal accumulation of SMN in the nucleus and reduced neurite outgrowth. These findings demonstrate the active transport of SMN and the role of exon-7 in cytoplasmic localization, which offers new insight into the biological basis of the neurodegeneration observed in SMA.
Materials and Methods
Cell culture. Two methods were used for primary neuronal culture: embryonic chick forebrain or rat spinal cord. Chick forebrains [embryonic day 8 (E8)] were dissected, trypsinized (0.15% in HBSS) at 37°C for 7 min, and plated on poly-L-lysine and laminin A coated coverslips. Dissociated neurons were cultured for 4 d in N3-conditioned medium with 2% FBS (Zhang et al., 1999For fluorescence in situ hybridization and immunofluorescence analysis, cells were fixed in paraformaldehyde (4% in 1x PBS) for 20 min at room temperature and washed in 1x PBS with 5 mM MgCl2 three times.
Enhanced green fluorescent protein reporter constructs and neuron transfection. Full-length cDNA of the human SMN1 was subcloned into an enhanced green fluorescent protein (EGFP)-C1 vector (BD Biosciences Clontech, Palo Alto, CA) and was designated EGFP-SMN. SMN cDNA with deletions of exon-7, exon-5, or both exon-5 and -7 were generated by PCR via specific primers and then inserted into the EGFP-C1 vectors, respectively, termed EGFP-SMN
Ex7, EGFP-SMN
D-box binding factor 1 (DBF1) was used as a nuclear reporter to investigate the role of SMN exon-7 in localization. DBF1 is a single-stranded DNA binding protein that is normally localized to the nucleus (Smidt et al., 1995
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Table 1. Schematic of the EGFP-reporter constructs for transfection
To identify a targeting motif (tif) within SMN exon-7, three overlapping fragments within the exon-7 were designed: termed "P" for proximal subregion (first 8 amino acids), "M" for middle subregion (middle 9 amino acids), and "D" for distal subregion (last 8 amino acids). These cDNAs were synthesized and inserted into the C terminus of EGFP-DBF1, and designated as EGFP-DBF1/P, EGFP-DBF1/M, and EGFP-DBF1/D (see Fig. 7). A five amino acid sequence, Gln-Asn-Gln-Lys-Glu (QNQKE) from SMN exon-7, was also inserted into the C terminus of EGFP-SMN
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Figure 7. Fusion of SMN exon-7 to the nuclear protein, DBF1, targets it to the cytoplasm. Left panels, EGFP fusion protein signal in nucleus (arrowhead) and cytoplasm (arrows). Right panels, Location of nuclear boundary (arrowheads) using DAPI (red). A, An EGFP-DBF1 transfected neuron presented an exclusive nuclear localization of its EGFP fusion protein (arrowhead). B, DAPI stained nucleus (red). C, In contrast, an EGFP-DBF1/Ex7 transfected neuron showed fluorescence signal in the cytoplasm (arrow) beyond the (D) DAPI stained nucleus (red). E, Proximal segment of exon-7 showed clear nucleus (arrowhead) with two foci and no evidence of EGFP signal accumulation. Cytoplasmic signal (arrows) in the perinuclear region was observed beyond the (F) DAPI stained nuclear border (red). G, A similar pattern was observed when the middle segment of SMN exon-7 was fused to EGFP-DBF1. H, DAPI (red). I, In contrast, the distal segment of the exon-7 was present mostly in the nucleus (arrowhead) with some signal in the perinuclear region (arrow). J, DAPI (red). K, Quantitative analysis of subcellular localization showed the percent of transfected cells with only nuclear signal, signal in both the nucleus and cytoplasm (uniform), and only cytoplasmic signal. L, Schematic of the three subregions of SMN exon-7 (P, proximal; M, middle; D, distal) that were fused to EGFP-DBF1.
Cultured neurons were transfected with the above constructs using DOTAP liposomal reagent (Roche) and cultured for 4 d, as described previously (Zhang et al., 2001
Cytoskeletal disruption experiments. One hour before imaging, transfected neurons were treated with either colchicine (10 µg/ml; Sigma) or cytochalasin-D (5 µg/ml; Sigma). The cells were imaged live in the presence of the drugs. Our previous work has shown that microtubules are depolymerized in these neurons after 1 hr of colchicine treatment. One hour treatment of cytochalasin-D also significantly disrupts actin filaments (Zhang et al., 1999
Similarly, transfected chicken embryonic fibroblasts were subjected to Triton X-100 extraction with or without 30 min treatment with cytochalasin-D or colchicine. Briefly, cytochalasin-D (5 µg/ml) or colchicine (10 µg/ml) was added to the media for 30 min. Cell were rinsed twice with prewarmed PEM buffer (80 mM PIPES, 4 mM EGTA, 1 mM MgCl2, 10% glycerol), then extracted for 30 sec in PEM buffer with 0.5% Triton X-100 and protease inhibitor cocktail (Roche, Hertfordshire, UK). Cells were fixed in 4% paraformaldehyde. Microfilaments were visualized by phalloidin labeled with Texas Red.
In situ hybridization and immunofluorescence analysis. Four amino-modified oligonucleotides (50 bases each), complementary to ribosomal RNA (18 S) of rat, were synthesized on a DNA synthesizer and chemically labeled using biotin succinamide ester (Roche Molecular Biochemicals). In situ hybridization for ribosomal RNA was completed as previously described (Zhang et al., 1999
For detection of endogenous proteins, we used monoclonal antibodies to SMN (BD Biosciences) or tubulin (Sigma). A rabbit polyclonal antibody was used to detect tau (Sigma). All secondary antibodies were affinity-purified donkey antibodies to mouse or rabbit IgG conjugated to a fluorochrome (Jackson ImmunoResearch). Antibody incubations were for 1 hr at room temperature in Tris-buffered saline (TBS) with BSA (1%) and Triton X-100 (0.1%). Coverslips were mounted as described above.
Fluorescence microscopy and digital imaging. Neurons were visualized using a Nikon Eclipse inverted microscope equipped with a 60x Plan-Neofluar objective, phase optics, 100 W mercury arc lamp, and HiQ bandpass filters (Chroma Technology, Brattleboro, VT). Images were captured with a cooled CCD camera (Quantix; Photometrics) using a 35 mm shutter and processed using IP Lab Spectrum (Scanalytics). Fluorescence images of ribosomal RNA (in situ hybridization) and proteins (immunofluorescence) were then acquired with specific filters, including Cy2, Cy3, or Cy5. Exposure time was kept constant and below gray scale saturation. Quantitative analysis of neurite length in each transfection was completed on phase optics from duplicated coverslips. The longest neurites from more than 60 transfected neurons were measured using computer IP Lab software.
For live cell imaging as described previously (Zhang et al., 2001
After colchicine or cytochalasin-D treatment, 10 transfected neurons were imaged for each condition. A region of interest (ROI) in neuronal process was chosen at least 10 µm away from the cell body, between 10 and 50 µm within the processes. One ROI was selected in each imaged neuron, and all granules within the ROI were scored as "directed" if the net displacement was >2 µm in one direction, "nondirected" for oscillatory or bidirectional displacements (<2 µm displacement), and "stationary" if no motility was observed. More than 300 small granules and 100 large granules in each treatment condition were analyzed using a computer SAS program in determination of "frequency" of each granule within the above-defined categories.
To analyze the GFP-SMN signal retained on the cytoskeleton after Triton X-100 extraction, transfected chicken embryonic fibroblasts were viewed on an Olympus BX61 microscope equipped with an Olympus PlanApo 60x, 1.4 NA oil objective. Images were captured by a Roper CoolSnap HQ cooled CCD camera operated by IP lab software. At least 80 cells from two independent experiments were selected individually, and the ROI was analyzed. Average arbitrary fluorescence intensity for each cell was calculated by IP lab software.
Electron microscopy. Pre-embedment immunogold labeling was performed on 4 d cultured in vitro chick forebrain neurons using monoclonal anti-SMN antibody and transmission electron microscopy (TEM). Cells were washed briefly in MEM and then fixed for 15 min (4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium cacodylate). Cells were incubated with primary antibody diluted 1:750 in blocking buffer (TBS, 2% BSA) for 15 hr at 4°C and washed several times in the buffer. Gold-conjugated (1 nm) secondary antibodies (Amersham Biosciences, Arlington Heights, IL) were applied for 3.5 hr incubation and followed by washing several times. Cells were postfixed (2.5% glutaraldehyde) and stained with 1% osmium tetroxide and 1.5% potassium ferricyanide in buffer for 20 min. Ultrasmall gold particles were enhanced (Goldenhance-EM; Nanoprobes) for 10 sec. Cells were stained with 1% aqueous uranyl acetate (pH 4.2) for 20 min, then dehydrated completely. Samples were embedded and sectioned by standard procedures. Sections were stained with 4% uranyl acetate and 0.2% lead citrate (3 min at room temperature). A Jeol 1200 EX TEM was operated at 60 kV to image immunogold labeling.
Western blot. Protein extracts (10 µl) from rat spinal cords and chick forebrains at different embryonic stages were resolved by 12% SDS-PAGE, and fractionated proteins were transferred to Hybond ECL nitrocellulose membrane (Amersham) at 4°C overnight. SMN was detected with a monoclonal antibody (1:500 diluted in TBS buffer). The membrane was washed and incubated with peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch). The signal was developed using ECL detection reagents (Amersham).
Results
Active transport of SMN granules to developing neurites and growth cones
Our previous studies on the localization and active transport of mRNA and mRNA binding proteins have used cultured forebrain neurons from chick embryos as a model system (Zhang et al., 1999
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Figure 1. Western blot analysis of SMN expression from rat spinal cord and chick forebrain at different embryonic stages. A, In rat spinal cord, SMN expression was prominent at embryonic stages and declined developmentally. B, In chick forebrain, SMN was also highly expressed at embryonic stages and declined after birth. A, B, Density ratio of SMN to -actin is shown in the top panel. SMN was detected with a monoclonal antibody and showed a single band at 38 kDa.
To investigate the subcellular localization of SMN, immunofluorescence analysis was performed using the above monoclonal antibody on cultured rat spinal cord and chick forebrain neurons. Spinal cord (E15) and forebrain (E8) neurons were cultured at stages in which SMN is expressed at high levels. SMN was abundant in the cell body and also distributed throughout processes in a highly punctuate or granular pattern in both rat spinal cord neurons (Fig. 2A) and chick forebrain neurons (Fig. 2B). SMN granules were observed in both the minor neurites and the longer axon-like process; the latter was identified with anti-tau reactivity (Fig. 2A). SMN granules within axons were also detected in the growth cone (Fig. 2A, arrowhead). A similar distribution of SMN was observed in chick forebrain neurons (Fig. 2B). These neurons have large, elaborate growth cones that contained many SMN granules (arrowhead).
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Figure 2. Localization and microtubule association of SMN granules in neuronal processes and growth cones. A, Rat spinal cord neurons were cultured for 3 d and fixed for double label immunofluorescence staining with a monoclonal antibody to SMN (red) and a rabbit polyclonal antibody to tau (green), as an axonal marker. SMN formed granules that localized to the axonal process (arrows) and its growth cone (arrowhead). B, Cultured chick forebrain neurons stained with the monoclonal antibody to SMN showed SMN granules (red) within minor neurites (arrows) and growth cones (arrowhead). C, Transfection of EGFP-SMN in chick forebrain neurons similarly revealed granules within processes. Microtubules were detected with a monoclonal antibody and Cy3-labeled anti-mouse antibody (red). Z-series stacks were acquired with a cooled CCD camera, and images were deconvolved (Hazebuster; Vaytek). Higher magnification of two regions (a, b) suggest association of EGFP-SMN granules (yellow, arrows) and microtubules. D, Immunogold detection of SMN (arrows) at the EM levels shows proximity to microtubules (arrowheads) within neuronal processes. E, Rat spinal cord neuron showed colocalization of SMN (green) with ribosomal RNA (red). Ribosomal RNA was detected by biotin-labeled oligonucleotide probes and Cy3-conjugated streptavidin. SMN was stained with a monoclonal antibody and Cy5-conjugated anti-mouse antibody. Higher magnification region (c) indicates SMN colocalization with ribosomal RNA (yellow, arrows).
We performed experiments to determine whether SMN granules were actively transported into processes and associated with cytoskeletal filaments. Fluorescence imaging of neurons transfected with human EGFP-SMN was first done in fixed cells to show that the distribution of EGFP-SMN was similar to that obtained by immunofluorescence. EGFP-SMN was observed in granules within processes (Fig. 2C). Immunofluorescence with anti-tubulin antibody, done on neurons transfected with EGFPSMN, suggested an association or possible coalignment of SMN granules with microtubules, in deconvolved optical sections (Fig. 2C, insets a, b). A similar association was also depicted at the EM level using silver-enhanced immunogold with the anti-SMN antibody (Fig. 2D). Silver-enhanced gold particles were often observed on microtubules. Double-label detection of SMN and biotin-conjugated oligonucleotides to ribosomal RNA (18 S) showed frequent colocalization within neuronal processes and the growth cone (Fig. 2E). These experiments suggest an association between SMN granules and microtubules that could provide the basis for active transport of SMN-containing RNP complexes.
Live cell imaging of cultured forebrain neurons, transfected with EGFP-SMN, revealed granules that exhibited dynamic bidirectional and unidirectional movements in processes (Fig. 3A). The long distance trajectories of several granules in this field are illustrated (long white arrows). Most of the granules showing these types of directed movements tended to be small in size. We also observed larger granules that exhibited predominantly nondirected or oscillatory behavior (short black arrows). A quantitative frame-by-frame analysis was done on both types of granules. Small granules often revealed consistent persistent movements that resulted in long trajectories (>10 µm) in either anterograde (Fig. 3B) or retrograde (Fig. 3C) directions. Both anterograde and retrograde moving granules were often observed to change directions (Fig. 3D). Granules were also observed to continue a trajectory beyond a branch point (Fig. 3A) (see also video 1, available at www.jneurosci.org). Here, a granule took a rapid retrograde trajectory in the primary neurite toward a branch point and then moved anterogradely in the secondary neurite. The average velocity of small granule movements was 1µm/sec, however, instantaneous velocities of >2.5 µm/sec were frequently observed for short durations. In contrast to the persistent movements of small granules, the large granules exhibited oscillatory movements with frequent direction changes resulting in no net displacement (Fig. 3E). Occasionally, these large granules did exhibit bidirectional movements that resulted in small net displacements in one direction (<2 µm). Rates of oscillatory granules were slower than those of persistent granules, with instantaneous velocities typically <0.5 µm/sec.
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Figure 3. EGFP-SMN granules exhibit rapid, bidirectional movements in processes that are dependent on either microtubules or microfilaments. A, Small (S) granules frequently exhibited directed movements, both anterograde and retrograde (long white arrows denote trajectories of several small granules). Granules often moved beyond branch points into secondary neurites (see video 1, available at www.jneurosci.org). Large (L) granules often displayed either oscillatory or bidirectional movements over short distances (short black arrows). B-E, Histograms showing frame by frame analysis of granule velocities. Anterograde movements are positive, and retrograde movements are negative. B, Example of an anterograde trajectory of a small granule. C, Example of small granule with a predominantly retrograde trajectory. D, Example of granule showing bidirectional movements. E, Example of a larger granule that showed predominantly nondirected, oscillatory movements. F, Directed movements of small granules were dependent on microtubules and microfilaments. G, Large granules showing nondirected movements, both oscillatory and bidirectional displacements over short distances, were dependent on actin filaments. *p < 0.01 when compared with untreated control.
We investigated the effects of cytoskeletal perturbing drugs on granule movements in live neurons. Transfected neurons were incubated for 1 hr in the presence of either colchicine (10 µg/ml), to depolymerize microtubules, or cytochalasin-D (5 µg/ml), to disrupt actin filaments. Granules were scored in three categories: directed granules were defined by movements with >2 µm displacement in one direction. Nondirected granules were defined by rapid movements with frequent direction changes and a net displacement of <2 µm. Stationary granules were defined by an absence of dynamic movements between frames. After colchicine treatment, we observed a 47% decrease in the percentage of directed granules, with a corresponding increase in stationary granules (Fig. 3F). There was also a significant decrease (39%) in directed movements after cytochalasin-D treatment (Fig. 3F). These results indicated a role for both microtubules and microfilaments in directed granule movements. However, we noted that the very long trajectories, i.e., >10 µm, were only observed in cytochalasin-D treated cells and not colchicine treated cells, suggesting a role of microtubules in long-distance directed movements. Large granules exhibited predominantly oscillatory movements that were dependent on actin filaments (Fig. 3G). Large granules also exhibited directed movements over short distances, which were dependent on microtubules and actin filaments. These studies show a role of microtubules in directed movements over long distances and a role for actin filaments in short-distance directed movements and oscillatory movements.
Predominant association of SMN with microfilaments in fibroblasts
We investigated whether SMN was physically associated with the cytoskeleton. The approach involves extraction of cells with the non-ionic detergent Triton X-100 (0.5%) in a cytoskeletal preservation buffer before fixation (see Materials and Methods). Under these conditions, soluble proteins and most membrane organelles are extracted, leaving behind cytoskeletal filament systems and bound mRNAs and polyribosomes (Bassell and Singer, 2001Chicken embryonic fibroblasts were transfected with an EGFP-SMN construct and visualized by fluorescence microscopy. GFP-SMN was present in granules that distributed throughout the cytoplasm with moderate enrichment in the fibroblast leading edge in unextracted cells (Fig. 4A,B, arrow), and this distribution was maintained after Triton X-100 extraction (Fig. 4C,D, arrow). Fibroblasts were then treated with either cytochalasin-D or colchicine and then subjected to Triton X-100 extraction and fixation. These experiments showed that the majority of SMN (70%) was released from the cytoskeleton after perturbation of actin filaments (Fig. 4G,I), whereas depolymerization of microtubules released 26% of SMN (Fig. 4E,I), suggesting an association of SMN with both filament systems, but predominantly microfilaments in these cells.
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Figure 4. Association of EGFP-SMN with the actin cytoskeleton in fibroblasts. A, EGFP-SMN was distributed predominantly in the cytoplasm and signal often accumulated at the leading edge of cultured fibroblast (arrow). One or two dots were also observed in the nucleus. B, Corresponding DIC image. C, EGFP-SMN granules were retained after Triton X-100 extraction in buffer (arrow). D, DIC image. E, Retention of EGFP-SMN on the cytoskeleton after colchicine treatment and Triton X-100 extraction (arrows). F, This procedure did not disrupt actin filaments, although microtubules were depolymerized (data not shown). G, In contrast, cytochalasin-D and Triton X-100 extraction released EGFP-SMN, and little signal was apparent. H, Note the disruption of most actin filaments after cytochalasin-D treatment. I, Quantitative analysis of the mean cytoplasmic fluorescence intensity in Triton X-100 extracted cells demonstrated a 70% reduction after cytochalasin-D treatment and a 26% reduction after colchicine treatment.
Role of SMN exon-7 in cytoplasmic localization
The majority of mutations and deletions in SMA are in the C terminal exon-7. A possible role of exon-7 in localization of SMN in neuronal processes has not been elucidated. We hypothesize that exon-7 is necessary for the localization of SMN in neuronal processes. Several truncations of human SMN1 cDNA were constructed and fused to EGFP. These include: an exon-7 deletion (EGFP-SMN
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Figure 5. EGFP-SMN accumulates in the nucleus after deletion of exon-7. SMN constructs, fused to EGFP, were transfected into cultured chick forebrain neurons to investigate the role of specific exons in subcellular distribution. Left panels, Imaged at 0.05 sec exposure times to show nuclear and perinuclear signal. Right panels, Imaged at 0.5 sec exposure time to show fluorescence signal in processes. Nuclei were stained with DAPI (red). A, Transfection of full-length EGFP-SMN revealed numerous granules in the perinuclear region. In the nucleus, one or two foci were often observed. B, In the same cell at longer exposure, granules were apparent in the processes. C, Exon-5 truncation (EGFP-SMN
To study the nuclear localization of SMN constructs with higher resolution, we analyzed optical sections along a Z-series and performed deconvolution that was useful to study spatial colocalization between SMN and coilin, a marker for coiled bodies. Neurons transfected with full-length EGFP-SMN demonstrated one or two foci of SMN that colocalized with coilin (Fig. 6A-C, arrowhead). In the cytoplasm, EGFP-SMN was abundant in the perinuclear region and extended into processes in the form of granules (arrows). Although the exon-7 deletion mutant also showed colocalization with coilin foci in the nucleus (Fig. 6E-G, arrow), there were clearly many other nuclear foci that were positive for SMN but did not contain coilin (Fig. 6E-G, arrows). Another striking pattern of the exon-7 deletion mutant was its complete absence from the cytoplasm and processes. These results showed the importance of exon-7 for cytoplasmic localization and demonstrated an abnormal accumulation of intranuclear foci from exon-7 lacking proteins. We have also performed live cell imaging of EGFP-SMN
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Figure 6. Nuclear aggregates in the SMN exon-7 deletion mutant were not associated with coilin. A, An EGFP-SMN transfected neuron showed a single nuclear foci (arrowhead). In the cytoplasm, EGFP-SMN granules were observed in processes (arrows). B, A coiled body (red) was detected with an anti-coilin antibody in the nucleus. C, EGFP-SMN foci colocalized to a coiled body in an overlay image (yellow, arrowhead). D, DAPI stained nucleus (blue). E, By contrast, an EGFP-SMN
To test whether exon-7 contained a cytoplasmic targeting signal, we fused it to a protein having an exclusive nuclear localization, and we asked whether the protein was redistributed to the cytoplasm. We selected DBF1 as a reporter, a single-stranded DNA binding protein that has been characterized as a transcription factor (Smidt et al., 1995
We designed three partially overlapping subclones of exon-7 (eight to nine amino acids each) to attempt to identify a specific amino acid sequence that was responsible for the cytoplasmic localization of SMN (Fig. 7L). Transfection of EGFP-DBF1 fused to either the proximal (Fig. 7E,F,K) or middle subregion (Fig. 7G,H,K) of exon-7 resulted in strong cytoplasmic localization of EGFP-DBF1, yet the distal subregion had only very weak cytoplasmic localization activity (Fig. 7I-K). A sequence of five amino acids, Gln-Asn-Gln-Lys-Glu (QNQKE), present in the overlapping proximal and middle subregions of exon-7, was shown to redistribute EGFP-DBF1 to the cytoplasm (Fig. 8 A). We then asked if this QNQKE sequence could also redistribute the SMN exon-7 deletion mutant when fused in frame to EGFP-SMN
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Figure 8. Redistribution of nuclear EGFP-SMN
We then investigated if EGFP SMN
The membrane-targeting sequence of GAP-43 was observed to redistribute EGFP-SMN
Reduction of neurite length by overexpression of the exon-7 deletion mutant and rescue by the GAP-43 targeting sequence
Overexpression of SMN
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Figure 9. Decreased neurite length after overexpression of SMN
Discussion
Cytoskeletal-based transport of SMN and other RNPs
Here we report, for the first time, that SMN was localized in granules that distribute throughout processes and growth cones in primary cultured neurons. These granules exhibited rapid, dynamic, and bidirectional movements in processes and growth cones that were characteristic of RNA granules we have previously described (Zhang et al., 2001Our observations of the active transport of SMN and its cytoskeletal association have important implications for understanding the biological basis of SMA. A central objective in SMA research has been to understand the molecular events leading to the neurodegeneration of motor neurons. One idea is that SMN is involved in protection against neuronal cell death and apoptosis where nuclear defects, possibly attributed to altered nuclear-cytoplasmic sorting of SMN, may underlie one aspect of the disease process (Vyas et al., 2002
There is now an emerging literature to document axonal transport defects or impairment in several mouse models of neurodegenerative diseases affecting motor neurons. Defects in tubulin transport or impaired dynein-dynactin function both result in motor neuron degeneration, with denervation of muscle also observed in the latter model (Williamson and Cleveland, 1999
Possible molecular mechanisms for SMN-associated RNPs in neuronal processes
Previous studies have demonstrated direct association between SMN and mRNA binding proteins in neural cells. By yeast two hybrid and coimmunoprecipitation analysis, SMN was shown to bind FUSE binding protein (FBP) (Williams et al., 2000Local protein synthesis in axonal growth cones: an SMN connection?
Our observations on the dynamic localization of SMN granules in developing axons and growth cones in primary neurons and their colocalization with rRNA, coupled with previous observations of SMN in growth cone-like protrusions in postnatal day 19 neuroblastoma cells (Fan and Simard, 2002It is possible that SMN-associated RNP complexes may similarly play a role in the regulation of growth cone behavior in response to local cues and that such mechanisms may be impaired in SMA. It is of interest to note that SMN has been detected at neuromuscular junctions in vivo, where its presence in both nerve and muscle may be essential for the proper development and spatial organization of synapses (Fan and Simard, 2002
In this study, we have established a link between the presence of SMN in the processes, the role of exon-7 and process outgrowth. The SMN
Cytoplasmic localization function of exon-7: implications for SMA
Our results suggest that exon-7 contained a cytoplasmic targeting signal, QNQKE, because it behaved similarly to the cytoplasmic targeting sequence of GAP-43, in that they both could redistribute the SMNMoving away from a nucleocentric viewpoint
The major emphasis on past SMN research has been to understand its role in snRNP and spliceosome assembly (for review, see Terns and Terns, 2001The cytoplasmic function of SMN in the nervous system may be broader in scope than analyzed thus far. We hypothesize that SMN may play a role in the assembly of mRNP complexes that are packaged into granules, the structures or vehicles involved in mRNA transport. We provide new evidence on the axonal transport of SMN, the molecular mechanism of SMN sorting in neurons, and a linkage between the presence of SMN in neurites and their outgrowth. These studies ultimately may provide a plausible model to explain the phenotype of SMA. The primary defect in motor neurons could be attributed to their unusually long axons, which may be dependent more critically on mRNA localization for their outgrowth and/or maintenance. The challenge ahead is clearly to identify specific mRNP complexes that may depend on SMN for their transport or local regulation in developing axons and growth cones.
Footnotes
Received Apr. 2, 2003; revised May. 16, 2003; accepted May. 29, 2003.This work was supported by a Muscular Dystrophy Foundation grant to Honglai Zhang and National Institutes of Health Grant GM55599 to G.B. We thank Phillip Young and Glen Morris for providing SMN monoclonal antibodies, Than Le and Arthur Burghes for SMN1 cDNA, and Marten Smidt for DBF1 cDNA. We thank Michael Plociniak and Andrew Levine for technical assistance and Laura Antar for helpful comments.
Correspondence should be addressed to Dr. Gary Bassell, Department of Neuroscience, Albert Einstein College of Medicine, 1410 Pelham Parkway, Bronx, NY 10461. E-mail: bassell{at}aecom.yu.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/236627-11$15.00/0
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