The survival motor neuron protein in spinal muscular atrophy
The survival motor neuron protein in spinal muscular atrophyDaniel D. Coovert1, Thanh T. Le2, Patricia E. McAndrew3, John Strasswimmer6,7, Thomas O. Crawford8, Jerry R. Mendell4, Susan E. Coulson5, Elliot J. Androphy6,7, Thomas W. Prior3,4 and Arthur H. M. Burghes1,2,4,*
1Department of Molecular Genetics, College of Biological Sciences, 2Department of Medical Biochemistry, 3Department of Pathology and 4Department of Neurology, College of Medicine and 5Molecular Cellular and Developmental Biology Program, College of Biological Sciences, Ohio State University, Columbus, OH 43210, USA, 6Department of Dermatology, New England Medical Center, Boston, MA 021113, USA, 7Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA and 8Department of Neurology, Johns Hopkins Medical School, Baltimore, MD 21205, USA
Received May 15, 1997;Revised and Accepted May 28, 1997
The 38 kDa survival motor neuron (SMN) protein is encoded by two ubiquitously expressed genes: telomeric SMN (SMNT) and centromeric SMN (SMNC). Mutations in SMNT, but not SMNC, cause proximal spinal muscular atrophy (SMA), an autosomal recessive disorder that results in loss of motor neurons. SMN is found in the cytoplasm and nucleus. The nuclear form is located in structures termed gems. Using a panel of anti-SMN antibodies, we demonstrate that the SMN protein is expressed from both the SMNT and SMNC genes. Western blot analysis of fibroblasts from SMA patients with various clinical severities of SMA showed a moderate reduction in the amount of SMN protein, particularly in type I (most severe) patients. Immunocytochemical analysis of SMA patient fibroblasts indicates a significant reduction in the number of gems in type I SMA patients and a correlation of the number of gems with clinical severity. This correlation to phenotype using primary fibroblasts may serve as a useful diagnostic tool in an easily accessible tissue. SMN is expressed at high levels in brain, kidney and liver, moderate levels in skeletal and cardiac muscle, and low levels in fibroblasts and lymphocytes. In SMA patients, the SMN level was moderately reduced in muscle and lymphoblasts. In contrast, SMN was expressed at high levels in spinal cord from normals and non-SMA disease controls, but was reduced 100-fold in spinal cord from type I patients. The marked reduction of SMN in type I SMA spinal cords is consistent with the features of this motor neuron disease. We suggest that disruption of SMNT in type I patients results in loss of SMN from motor neurons, resulting in the degeneration of these neurons.
Proximal spinal muscular atrophy (SMA) is an autosomal recessive disorder that results in destruction of the [alpha] motor neurons in the anterior horn of the spinal cord. SMA has an estimated incidence of 1 in 10 000 live births with a carrier frequency of 1 in 40 (1 ). The childhood SMAs can be classified into three groups based on age of onset and clinical course (2 ). Type I SMA is the most severe form with onset before the age of six months and death occurring within the first two years of life. Type II SMA is intermediate in severity with onset of symptoms before 18 months of age and patients never gaining the ability to walk. Individuals with type III SMA, the mildest form that typically presents after the age of 18 months, are able to stand and walk.
All three forms of proximal SMA have been mapped by linkage analysis to chromosome 5q12 (3 -11 ) in a region that contains multiple copies of both genes and markers (12 -18 ). Analysis of these multicopy markers showed that specific alleles associate with SMA (18 -21 ), the loss of a copy of the marker in ~50% of severe SMA cases (20 -21 ), and deletions of all copies of the marker on some SMA chromosomes (15 ,21 ). Three cDNAs that detect deletions in SMA patients were simultaneously reported (16 -18 ). Two of these cDNAs, neuronal apoptosis inhibitory protein (NAIP) and XS2G3, detect the same deletion in 50% of type I SMA cases, and appear to mark the extent of deletion (16 ,17 ,21 -22 ). The third cDNA, survival motor neuron (SMN), is encoded by two nearly identical genes, telomeric SMN (SMNT) and centromeric SMN (SMNC). These genes can be distinguished from one another by base pair changes in exons 7 and 8 (18 ,24 ). The SMNT gene is not detectable in 95% of SMA cases due either to gene deletion or conversion of the sequences in the SMNT gene to those of the SMNC gene (18 ,22 -35 ). Conversion events predominate in mild SMA chromosomes whereas deletion of SMNT is associated with severe SMA chromosomes (33 -35 ). Cases of SMA that have detectable SMNT have been identified, and were shown to possess several small mutations in SMNT. These mutations include deletions that disrupt splicing of exon 7 (18 ), deletion of 4 or 5 bp in exon 3 (32 ,36 ), an 11 bp duplication in exon 6 (37 ), and five different missense mutations in exons 6 and 7 (18 ,35 ,38 -40 ). Dosage analysis of the SMNT gene indicates that the majority of these small mutation cases are compound heterozygotes and that current methods have detected ~75% of non-deletion/conversion mutations (35 ,40 ). These data clearly indicate that SMNT is the SMA gene and that the region encoded by exons 6 and 7 is important for normal function of the SMNT gene product in motor neurons.
Recent studies have demonstrated that antibodies against SMN detect a 38 kDa protein that is localized in the cytoplasm and the nucleus (41 ). Nuclear SMN is found in a structure named gems for `Gemini of the coiled bodies' (41 ). These novel structures, which are detected by anti-SMN antibodies, are ~0.1-1 [mu]m in size, and appear to be intimately associated with the nuclear coiled bodies. Coiled bodies are subnuclear structures believed to have a role in mRNA metabolism. The gems appear to interact directly with the coiled bodies and undergo similar changes in response to environmental and metabolic conditions of the cell (41 ). This suggests that gems function in concert with the coiled bodies, and may also share a role in RNA processing (41 ). At present, there are no other markers for the gem structures and their function is unknown.
The effect of the loss of the SMNT gene on the levels of SMN protein in SMA patients has not been reported. In this paper, we use a panel of antibodies to analyze the SMN protein in SMA patient tissue. We show that both the SMNT and SMNC genes produce SMN protein. Secondly, the number of gems as detected by SMN antibodies correlates with the phenotype of the patient. Thirdly, type II patients have significantly more gems as detected with SMN antibodies than type I patients even though the copy number of the SMNC and SMNT genes are equivalent in both patients. This indicates that not all SMNC genes are functionally equivalent. Finally, we show that the amount of SMN protein is reduced in tissues of SMA patients. Type I SMA patients show a 100-fold reduction of SMN in spinal cord. Therefore, the disruption of the SMNT gene in type I SMA patients causes a reduction of the SMN protein in the motor neurons and results in motor neuron degeneration.
The SMN gene is ubiquitously expressed. We, therefore, examined the SMN protein in lymphoblasts, fibroblasts, spinal cord and muscle of SMA and non-SMA individuals. We developed a chicken polyclonal antibody to SMN (SMN-C3) by injection of a His-tagged SMN bacterial fusion protein that lacked exon 5 into chickens. We also used a rabbit antibody raised against peptide sequences (amino acids 270-289 of SMN) in the C-terminal domain (SMN-CT) (Strasswimmer et al., manuscript in preparation), and a previously reported monoclonal antibody 2B1 (41 ) for analysis of SMN. Figure 1 shows a western blot of HeLa cells demonstrating that all three antibodies detect the same 38 kDa protein. A fourth antibody, SMN-NT, raised against peptide sequences (amino acids 10-29 of SMN) from the N-terminal domain (Strasswimmer et al., manuscript in preparation), is also shown in Figure 1 . We found that the SMN-C3 and SMN-CT were the most effective at detecting SMN protein on western blots. The monoclonal antibody 2B1 and affinity purified SMN-NT were effective for immunocytochemistry studies.
We first examined the levels of the SMN protein in primary fibroblasts derived from normal individuals, SMA carriers and SMA patients of various phenotypic severity. Fibroblasts were chosen as they were available and facilitated the determination of gem number and protein level in the same cells. The level of SMN protein is significantly lower in primary fibroblasts than in HeLa cells. Western blot analysis of SMN in these primary fibroblasts lines demonstrated a lower level of SMN in SMA patients, particularly in type I patients (Fig. 2 , Table 1 ). The total reduction of SMN in the type I child (3813) compared to his mother (3814) is ~90%. This would suggest a significant reduction of SMN levels in fibroblasts derived from SMA patients compared to carrier individuals. Because the number of copies of the SMN genes could influence the levels of SMN observed, we have indicated the gene copy number in the controls and patients for both SMNT and SMNC (Table 1 ). Furthermore, the SMNC gene produces SMN protein. This is demonstrated by the detection of the SMN protein in SMA patients lacking the SMNT gene.
The SMNT gene has been shown by mutation analysis to be the SMA determining gene (18 ,32 -40 ). In humans, the SMN cDNA is encoded by two genes, SMNT and SMNC (18 ). These genes can be distinguished at the DNA and RNA level by nucleotide changes in exons 7 and 8 that do not alter the encoded amino acids (18 ). Alternative splicing has been shown to occur in the SMN genes in humans. The majority of transcripts (~90%) from the SMNT gene are full length. The remainder (~10%) are missing exon 5 (18 ,37 ,42 ). The SMNC gene produces all of the SMN isoforms, including transcripts without exon 5, transcripts without exon 7, transcripts lacking both exons 5 and 7, as well as full length SMN. The full length transcript accounts for only 20-30% of the total transcripts from the SMNC gene (18 ,37 ,42 ). Therefore, one effect of the loss of the SMNT gene is the reduction in the total amount of full length transcript. However, the amount of full length transcript could vary in a particular patient due to the variable copy number of SMNC (35 ). The importance of the full length SMN transcript is also implicated by studies of the SMN homologue in mouse. The murine genome contains one copy of the SMN gene per chromosome, which does not undergo alternative splicing, and therefore only produces the full length SMN isoform (43 -44 ). Gennarelli et al. (42 ) determined the levels of SMN isoforms in muscle tissue from patients of varying phenotypic severity, but did not observe any differences between the phenotypic groups. However, the isoform levels were measured relative to each other and did not take into account the variant copy number or expression levels of SMNC genes, because no mRNA external to SMN was used to calibrate the level of various SMN isoforms.
In this paper, we demonstrate that both the SMNC and SMNT genes produce protein and the status of each contributes to disease severity. Nearly all SMA patients used in this study had no detectable SMNT genes regardless of their clinical SMA phenotype. The only exception to this is the type III patient shown in Figure 4 D. As seen in Table 1 , patients with types I and II SMA can have an identical copy number of SMNC. However, in all cases type II SMA patients clearly had many more detectable gems than type I patients. Gems, which lie adjacent to the coiled bodies in the nucleus, are the punctate structures detected by SMN antibodies. Gems are believed to have a role in RNA processing (41 ). As seen in Table 1 , type II patients produce many more gems than type I SMA patients who have the identical SMNC copy number. This suggests that not all SMNC genes are equivalent. That is, some SMNC genes can produce sufficient SMN for gem formation, albeit at a reduced number.
The exact difference between these SMNC alleles is not known. The most likely explanation is that in type II SMA patients, one of the SMNC loci is generated by gene (sequence) conversion of an SMNT gene to an SMNC gene. Expression of the appropriate form of SMN protein from this converted SMNT gene is reduced, but still sufficient for the formation of gems. In type I SMA patients, however, the loss of SMNT is most often due to deletion. The SMNC gene remaining in the type I patients is not capable of expressing the appropriate form, or amounts of SMN for the formation of gems (33 ,34 ). Indeed, previous studies using markers that flank the SMN region indicate that deletions of SMNT are specific to type I chromosomes and conversions of SMNT genes to SMNC genes predominate in typeII/III chromosomes (20 -22 ,33 ,34 ). Analysis of SMNC copy number distribution in normals and SMA carriers indicates a gain of one SMNC gene in type II/III SMA chromosomes. The gain of SMNC genes in mild SMA cases is consistent with gene conversion predominating in mild chromosomes (34 ,35 ). Furthermore, Campbell et al. (33 ) provide physical evidence that conversion and not deletion has occurred in mild SMA chromosomes. Chimeric genes have also been identified in SMA patients where exon 8 of the SMNT gene is joined to exon 7 of the SMNC gene (25 ,28 ,29 ). This can occur by one of two general mechanisms: first, deletion of the region between the SMN genes and fusion of the 5' end of SMNC with the 3' end of SMNT or second, conversion of the sequences in the SMNT genes to those in the SMNC gene. The former would be a severe allele because there is a deletion of the majority of the SMNT gene (27 -29 ).
It has been suggested that the copy number of SMNC could modify the phenotypic severity (18 ,30 ). However, the correlation of SMNC copy number to severity is not absolute. We have shown, by immunocytochemical and western blot analysis, that type II patients with exactly the same SMNC copy number as a type I patient produce considerably more gems and SMN. This strongly suggests that there are differences between SMNC genes in type I and II patients. One explanation is that there are critical sequences 5' of exon 7 which are not present or are mutated in SMNC genes in type I patients. For instance, conversion events that occur in type II SMA would not exchange the entire gene but just the 3' end, retaining the critical sequences in the 5' end of the SMN gene. Sequence analysis of the SMNC and SMNT genes is critical to resolve whether any difference between these two genes exists in the 5' region. Therefore, differences between the SMNC genes present in a severe chromosome and a mild chromosome remain to be resolved. Based on the presence of a modifying SMNC gene and a non-modifying SMNC gene, we have suggested a modification of the previous models of SMA (20 ,21 ,29 ,33 ). In this model we suggest that no copies of modifying SMNC genes occur in type I SMA, one copy occurs in type II SMA and two in type III SMA (34 ).
SMA is a disorder that affects motor neurons. The level of expression in other adult tissues is variable, with high levels of expression present in the brain, liver and kidney, moderate levels in cardiac and skeletal muscle, and the lowest levels in fibroblasts and lymphocytes. In type I patients, a mutation in SMNT has a modest effect on the level of expression in fibroblasts and skeletal muscle, and yet the number of detectable gems in fibroblasts is an indicator of the severity of the disease. That is, a low number of gems correlates with the severe form of SMA and higher numbers correlate with milder forms of SMA. Gems are nuclear structures of ~0.1-1 [mu]m in size that lie adjacent to the coiled bodies (41 ). Gems have been suggested to have a role in RNA metabolism (41 ). The loss of SMN from the gems in SMA has a catastrophic effect on the motor neuron cell, but exactly what metabolic pathways are disrupted is unknown.
The tissue that shows the most significant alteration in the expression of SMN is the spinal cord. We have shown here that the levels of SMN are normally high in spinal cord but are reduced 100-fold in samples from type I SMA patients. The loss of SMN in spinal cord critically affects the motor neurons, and may explain the specificity of this disease for the [alpha] motor neurons. This effect could be due to the exquisite sensitivity of motor neurons to the loss of SMN or that the SMNT gene contains an element lacking in SMNC that allows expression in motor neurons throughout life, including the time when the neuron makes contact with the muscle. Detailed analysis of the elements controlling SMN expression will be required to resolve this question.
Patients were diagnosed with spinal muscular atrophy using the criteria outlined by the International SMA Consortium. Lymphocytes were obtained from blood samples by fractionation in Lymphocyte Separation Medium (Organon Teknika). Muscle biopsies were originally obtained for diagnostic purposes. Tissue including spinal cord material was obtained at post-mortem from a total of four type I SMA patients, two ALS patients, one patient with Multiple Sclerosis (MS) and two patients who were originally referred as SMA, however analysis of SMNT gene copy number showed two copies of the SMNT gene. The SMNT gene is not detectable in 95% of 5q SMA cases. Therefore, according to Hardy-Weinberg equilibrium, virtually all the remaining 5q SMA individuals should have a heterozygous deletion (35 ). Both of these cases have two copies of the SMNT; we have not detected any other alterations in the SMNT gene from these patients. Hence, these two patients most likely have an unclassified motor neuron disease resembling SMA. For the remaining patients, the diagnosis of SMA was confirmed by demonstrating the absence of SMNT using the method described by McAndrew et al. (35 ). Primary fibroblasts from type I (four cell lines), type II (eight lines), type III (one line), and one normal (96-2842) were obtained and grown in culture using standard protocols.
Fibroblasts were plated at a density of 105 on gelatinized coverslips in a 150 mm plate. The subconfluent cultures were obtained by growing the cells for 24 h after plating. The confluent cultures were prepared as above, except cells were not fixed until the cells had reached confluency by visual inspection. The cells were fixed for 7 min in ice cold 50% methanol-50% acetone. Fixation by paraformaldehyde/sucrose methods showed similar results (Strasswimmer et al., unpublished observations). The coverslips were then air dried for 30 min and stored at -70oC until used. The cells on the coverslips were rehydrated with phosphate buffered saline (PBS) for 5 min, and then blocked with 1* BLOCK [1% fetal bovine serum, 1% horse serum, 0.1% bovine serum albumin (BSA) in PBS] for 30 min. The 2B1 monoclonal antibody (41 ) was diluted 1:500 in the 1* BLOCK solution and applied to the coverslips for 1 h in a humidity chamber. The coverslips were extensively washed with PBS (6 * 5 min) and the secondary antibody (goat anti-mouse TRITC conjugate, Sigma Chemical) diluted 1:300 in 1* BLOCK was applied. After 1 h, the coverslips were again extensively washed with PBS. The coverslips were mounted on Superfrost slides (Fisher Scientific) using DAPI II (Vysis) diluted 1:3 in antifade solution (Vector Research). The cells were then examined on a Zeiss microscope equipped 63* objective and a dual band pass filter set suitable for DAPI/TRITC fluorescence.
Tissue samples and cell pellets were dissolved in blending buffer (10% sodium dodecyl sulfate (SDS), 62.5 mM Tris pH 6.8, 5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonylfluoride (PMSF) (added fresh). The protein concentration of the samples were determined using BCA kit (Pierce, Rockford IL) according to the manufacturer's instructions. An equal volume of sample buffer (62.5 mM Tris pH 6.8, 10% glycerol, 10% 2-mercaptoethanol, 0.4 mg Bromophenol Blue) was added to the remaining supernatant. The samples were boiled for 2 min and stored at -20oC until use. Samples were electrophoresed on a 12% polyacrylamide gel, transferred to Immobilon-P (Millipore) in a Hoeffer TE50 transblot apparatus at 7 V/cm constant voltage for 1.5 h in 192 mM glycine, 25 mM Tris, 0.1% SDS, 20% methanol. The Immobilon was blocked for 1 h at room temperature in 5% instant milk, 1% BSA in 25 mM Tris pH 7.5, 500 mM NaCl (TBS). The primary antibody was diluted 1:1000 SMN-NT, SMN-CT (Strasswimmer et al.), anti-[beta]-tubulin (Amersham), or 1:10 000 SMN-C3 in 1% milk, 0.2% BSA in TBS containing 0.2% Tween-20 (TTBS), and placed on the blot for 1 h. The blot was washed four times 10 min each with TTBS. The secondary antibody was diluted into 1% milk, 0.2% BSA in TTBS as above. The sheep anti-mouse and donkey anti-rabbit HRP conjugated antibodies (Amersham) were used at 1:1000 dilution. A donkey anti-chicken F(ab')2 antibody (Jackson Immunoresearch) was diluted 1:20 000. For the spinal cord immunoblots, the diluted secondary antibodies were preadsorbed against a human spinal cord acetone powder preparation (45 ). The blots were incubated with the secondary antibodies for 1 h at room temperature and washed as above. After a final 15 min wash with TBS, the blot was visualized using ECL chemiluminescent reagent (Amersham) or Pierce Ultra stable reagent according to the manufacturer's instructions. The blots were exposed to Kodak X-OMAT LS film for the detection of the chemiluminescent emissions. Blots were stripped and reprobed according to the manufacturer's protocols.
The levels of SMN were determined by scanning western blots on an AGFA Arcus II flat bed scanner set for the highest quality. The images were imported into BioMax 1D image analysis software (Kodak) which was calibrated with a T-14 optical density scale. The level of SMN relative to [beta]-tubulin was determined by dividing the scanned density of SMN bands by the scanned density of [beta]-tubulin for each lane. These ratios were then normalized to that of the control sample.
An SMN expression construct for the isolation of fusion protein was assembled by cloning a cDNA lacking exon 5 into the pRSETC vector (Invitrogen). The removal of the proline rich sequence encoded by exon 5 of SMN was performed in order to enhance the expression of the bacterial fusion protein. The fusion protein was purified under denaturing conditions on the TALON resin (Clontech) according to the manufacturer's instructions. Injections of the affinity purified fusion protein into chickens was carried out by Cocalico Biologicals (Lancaster, PA) using standard protocols (45 ). Titers of the crude serum were determined according to standard methods (45 ). Antibodies were purified by incubating the antibodies with nitrocellulose squares saturated with purified fusion protein. After 1 h, the antibody was eluted in 500 [mu]l of 20 mM glycine pH 2.4, 1 mM ethylene glycol-bis([beta]-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The supernatant was neutralized with 125 [mu]l of 1 M Tris pH 8. The titer was determined using standard methods (45 ).
We are truly grateful to all the SMA families for their kind cooperation and to all the clinicians for their help in providing patient material. We thank Dr G.Dreyfuss at the Howard Hughes Medical Institute for the 2B1 monoclonal antibody and Dr K.Rammohan for his invaluable assistance. We especially thank all the members of the Burghes Laboratory and Jeanne Coovert for help with the manuscript. This work was supported by Families of SMA (Chicago, IL) and the Muscular Dystrophy Association of America. We would also like to acknowledge the Kennedy/Hopkins NICHD Mental Retardation core grant HD24061 for establishing some of the fibroblast and lymphoblast cell lines.
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*To whom correspondence should be addressed at: Department of Neurology, Ohio State University, 452 Means Hall, 1654 Upham Drive, Columbus, OH 43210, USA. Tel: +1 614 293 8434; Fax: +1 614 293 4688; Email: burghes.1{at}osu.edu
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