Article |
Address correspondence to Umrao R. Monani, Department of Molecular and Cellular Biochemistry, 363 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. Tel.: (614) 688-4759. Fax: (614) 292-4118. E-mail: monani.2{at}osu.edu; or Arthur H.M. Burghes, Department of Molecular and Cellular Biochemistry, 363 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. Tel.: (614) 688-4759. Fax: (614) 292-4118. E-mail: burghes.1{at}osu.edu
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Abstract |
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Key Words: SMA; SMN; mouse model; motor neurons; transgene
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Introduction |
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The 38-kD SMN protein is ubiquitously expressed (Coovert et al., 1997; Lefebvre et al., 1997), often localizing in dot-like structures termed gems. Gems overlap with, or are in close proximity to, coiled bodies (Liu and Dreyfuss, 1996; Young et al., 2000). SMN is thought to be important in snRNP biogenesis and mRNA splicing (Pellizzoni et al., 1998), but is most likely a multifunctional molecule (Fischer et al., 1997; Liu et al., 1997; Buhler et al., 1999; Strasswimmer et al., 1999; Charroux et al., 1999; Campbell et al., 2000; Gangwani et al., 2001; Jones et al., 2001; Pellizzoni et al., 2001a,b; Terns and Terns, 2001; Young et al., 2001). However, it is not clear which of SMN's functions is critical specifically to motor neurons.
In keeping with its housekeeping functions, an Smn knockout in mice is embryonic lethal (Schrank et al., 1997). To create a viable animal model of SMA, the SMN2 gene was introduced into Smn-/- mice (Hsieh-Li et al., 2000; Monani et al., 2000). One to twocopy SMN2;Smn-/- mice exhibit a type I SMA phenotype. 816 copies of SMN2 completely ameliorate the disease phenotype. Inducing SMN2 to produce higher levels of SMN protein therefore seems an attractive option in treating SMA. A number of groups have reported molecules capable of inducing SMN2 to produce higher levels of SMN (Baron-Delage et al., 2000; Andreassi et al., 2001; Chang et al., 2001; Zhang et al., 2001). It is essential that these be tested in appropriate animal models before human clinical trials are undertaken. Although the existing animal models have been extremely important in furthering the understanding of the pathophysiology of the SMA disease process, it is clear that a more suitable animal model is needed to address the next generation of in vivo experiments. In this study, we show that an SMN A2G missense mutation rescues the severe SMA phenotype in low copy SMN2;Smn-/- mice but is unable to rescue Smn-/- embryonic lethality in the absence of the SMN2 gene. Increased SMN protein in SMN A2G;SMN2;Smn-/- mice affects the timing of motor neuron loss and results in animals with a mild SMA phenotype. Mild SMA mice exhibit muscle atrophy, motor neuron degeneration, and abnormal electromyographs (EMGs). These characteristics, in particular the electrophysiology, serve as easily assayed outcome measures in therapeutic strategies in SMA. Importantly, we also show that SMN A2G is unable to efficiently self-associate, a property required for the formation of "active" SMN complexes. We suggest that mutant oligomers are unstable and fail to bind components that are essential for one or more of SMN's functions. This results in a rapid turnover of the protein, underlining the importance of some, albeit low levels of, full-length SMN in the SMN complex and provides insight into the mechanism involving functional complex formation.
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Results |
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Decreased self-association of mutant A2G SMN molecules
SMN self-associates using domains encoded by exons 2b and 6 (Lorson et al., 1998; Young et al., 2001). We investigated the effect of the missense A2G mutation on self-association. A GSTSMN A2G fusion protein was immobilized on glutathione-linked agarose beads and then incubated with 35S-labeled SMN A2G protein. Bound fractions were washed extensively and resolved by SDS-PAGE. As controls, similar self-association studies were performed with full-length SMN and 7 SMN. Our results show that SMN A2G molecules self-associate with an efficiency between that of full-length SMN and
7 SMN (Fig. 2 A) as do other mild SMA mutations (Lorson et al., 1998). This is consistent with the mild SMA phenotype we see in patients carrying this mutation and our SMN A2G;SMN2;Smn-/- mice. To determine the effect of the A2G mutation on the ability of the mutant protein to bind native full-length (FL) SMN and Sm proteins, GSTSMN and GSTSmN fusion proteins immobilized on agarose beads were incubated with labeled SMN A2G. Bound fractions were washed as described above. As a comparison, the ability of
7 SMN and FL-SMN each to bind to SmN and native SMN was assessed. As expected, SMN A2G binds FL-SMN with a higher affinity than does
7 SMN but with a lower affinity than does native SMN (Fig. 2 B). Interestingly, the ability of SMN A2G to bind the Sm protein SmN was greatly decreased even though the mutation does not lie in the Sm binding domain (Fig. 2 C).
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Electrophysiological studies and evidence of axonal sprouting in type III SMA mice
To further characterize the muscle weakness in our type III SMA mice, EMG recordings were made on 46-mo-old SMN2;Smn-/- animals heterozygous for the mutant SMN A2G transgene. Recordings were made on resting muscle as it is difficult to obtain controlled, voluntary muscle contraction in mice. Normal muscle is electrically silent at rest and no spontaneous activity is detected after cessation of movement of the recording needle. In both of the type III (1SMN A2G;SMN2;Smn-/-) animals we tested, we found abnormal spontaneous activity of single muscle fibers (fibrillation potentials) and of motor units (fasciculation potentials) accompanied occasionally by biphasic positive sharp waves (Fig. 5, A and B). These observations made on multiple pelvic (cranial tibial, vastus lateralis, and gastrocnemius; see Fig. S2, available online at http://www.jcb.org/cgi/content/full/jcb.200208079/DC1) and thoracic (suprascapular) muscles are a clear indication of denervation and provide a simple diagnosis of this process, as previously demonstrated in human patients (Dubowitz, 1995). We did not see abnormal electrical activity in age-matched type III SMA animals homozygous for the A2G transgene, indicating that increased levels of SMN from the A2G transgene correct this abnormality. This is consistent with data from some very mildly affected type III SMA patients (Hausmanowa-Petrusewicz and Karwanska, 1986). To assess the nature of the muscle weakness in mild SMA mice, we analyzed evoked compound action potentials and motor nerve conduction velocities (MNCVs) of the tibial nerve (Fig. 5 C). Although nerve conduction velocity tests on type III SMA (30.1 ± 5.6; n = 4) mice did not show a significant difference from those on age-matched controls (31.6 ± 3.3; n = 4), the amplitudes of the evoked muscle potentials were reduced (SMA, 4.3 ± 5.5, n = 4; normals, 15.07 ± 8.9, n = 4). These results indicate that demyelination of nerves is not a feature of SMA. Instead, it is likely that defects in neuronal axons contribute to SMA pathology in mildly affected mice.
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Motor neuron degeneration in type III SMA mice
To assess motor neuron degeneration in 3.5-mo-old SMN2;Smn-/- type III SMA mice heterozygous for the A2G transgene, transverse sections of the spinal lumbar region (unpublished data) and coronal sections of the facial nucleus (Fig. 6, A and B) were stained with cresyl echt violet and the morphology and numbers of the cells determined. The most obvious difference between the SMA mice and age-matched controls is a significant decrease in the numbers of motor neurons in the former. In the lumbar spinal cord, SMA mice have 29% fewer motor neurons than age-matched Smn+/- controls, while in the facial nucleus there is a 19% loss of motor neurons (Table I; Fig. 6, A and B). Previously, we showed a significant loss of motor neurons in the spinal cord and brain stem of 5-d-old severe SMA mice (Monani et al., 2000). To determine whether motor neuron loss occurs this early in type III SMA mice, 5-d-old SMN2;Smn-/- animals heterozygous for the A2G transgene were killed and motor neuron cell bodies in the facial nucleus counted. We found no evidence of motor neuron loss at this age in type III SMA mice (unpublished data). This is consistent with our previous finding that motor neuron loss is a late event in SMA. To further examine motor neuron degeneration, we also conducted ventral root counts on our type III SMA mice. Mice perfused with 4% paraformaldehyde, 1% glutaraldehyde were used to isolate the ventral roots from the L1L5 lumbar spinal cord region. Semi-thin sections were cut, stained with toluidene blue (Fig. 6, C and D), and at least four different fields examined to quantitatively determine the number of motor axons in the roots of diseased mice and an age-matched control. Two striking differences were observed. First, type III SMA mice have fewer myelinated axons (Table II). The loss in motor axons correlates with the loss of motor neuron cell bodies in the spinal cord. Second, many of the remaining axons in the diseased mice are shriveled and exhibit signs of Wallerian degeneration (Fig. 6 D).
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Discussion |
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It is intriguing to consider how the A2G mutation on an SMN2;Smn-/- background results in a mild SMA phenotype. The mutation is the most 5' one reported so far. It is neither in the oligomerization domain in exon 6 nor in exon 2b. It is not in the Sm protein binding domain nor does it affect SIP-1 binding (unpublished data). Yet, we have shown that the A2G mutation does disrupt self-association and affects SmN binding presumably by disrupting the formation of SMN oligomers. A to G mutations have been shown to profoundly affect -helices (Lyu et al., 1990; Chakrabartty et al., 1991). It is not unlikely that the mutation disrupts the three-dimensional structure of the protein enough to affect the oligomerization domains in exon 2b and/or exon 6 as well as the Sm binding domain. Although the A2G protein retains some level of self-association activity, it is possible this deficiency results in that mutant's inability to form higher order complexes necessary for SMN function and interactions with protein partners. This could explain why SMN A2G does not rescue Smn-/- embryonic lethality. If, however, full-length SMN serves as a scaffold, then low levels of the protein might promote the formation of higher order FL-SMN:SMN A2G oligomers with an enhanced ability to bind other interacting proteins such as SmN. Our data show that SMN A2G binds with a higher affinity to FL-SMN than does
7 SMN. FL-SMN:SMN A2G complexes would therefore be more stable than FL-SMN:
7 SMN ones. Furthermore, it has been shown that protein levels in patients with missense mutations correlate with the disease phenotype (Lefebvre et al., 1997). These data and our observations lead us to a plausible molecular model of SMA (Fig. 7) in which SMN forms a number of complexes with different partners. These partners compete for the same sites on SMN, binding being determined by their relative affinities for SMN. In the presence of low levels of full-length SMN, low affinity binding partners are out-competed by high affinity proteins. It is possible that the low affinity binding partners are selectively found in motor neurons and are unable to form functional complexes in SMA patients due to the low SMN levels. Loss or disruption of the low affinity complex in motor neurons would result in the degeneration of these, but not other, tissues. Our model is consistent with the observation of increased SMN levels and a mild phenotype in mice carrying the A2G missense mutation on a severe SMA genetic background.
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Materials and methods |
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In vitro binding assays
SMN A2G, FL-SMN, and 7 SMN cDNAs were subcloned into the vector pGEX-3X (Amersham Biosciences). GST fusion proteins were prepared and purified using glutathione-linked agarose beads as previously described (Lorson et al., 1998). To prepare 35S-labeled SMN proteins, the T7 transcription/translation (TnT) rabbit reticulocyte lysate system (Promega) was used. The protocol was essentially the same as that described by Lorson et al. (1998) except that densitometry was performed using a Phosphorimager (Molecular Dynamics, Inc.)
RT-PCR, Southern, and Western analysis
To determine the expression of the mutant A2G transgene, Smn+/+ carrying the transgene but no SMN2 were killed by cervical dislocation and polyA+ RNA was isolated using the QuickPrep Micro mRNA purification kit (Amersham Biosciences) according to the manufacturer's recommendations. After treating the RNA with DNase I to get rid of contaminating genomic DNA, 100 ng was reverse transcribed and amplified using the following human SMN-specific primers in exons 1 and 4, respectively: Ex1F, 5'-GCGGCGGCAGTGGTGGCGGC-3', and Ex4R, 5'-TGGAGCAGATTTGGGCTTGA-3'. Native murine Smn and the knockout allele were followed by Southern blot analysis after Bam HI digestion of tail DNA using a probe in Smn intron 1. The probe was radioactively labeled and hybridized to the filter containing the DNA according to standard methods. The filter was washed at high stringency and then exposed to Hyperfilm MP (Amersham Biosciences) with an intensifying screen. Copy number of the SMN A2G transgene was determined by Southern blot analysis of DNA from SMN A2G;Smn+/+ animals. An SMN exon 68 cDNA fragment and a murine Smn intron 1 probe of approximately equal length and GC content were radioactively labeled and simultaneously hybridized to the blot. The blot was washed and exposed to film as described above. Intensities of bands corresponding to the transgene were then determined using a Shimadzu 9000 CS scanner. Copy number was determined by comparing the intensities of these bands to those of the single-copy murine Smn gene in each animal. For Western blot analysis, 100 mg of tissue from 5-d-old mice was dissolved in blending buffer (10% SDS, 62.5 mM Tris, pH 6.8, 5 mM EDTA). 2550 µg of protein was mixed with an equal volume of sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 10% 2 ME, 0.4 mg bromophenol blue) and electrophoresed on a 12.5% polyacrylamide gel. Samples were transferred to Immobilon P (Millipore) as previously described (Coovert et al., 1997). The blot was blocked in 5% milk powder, 0.5% BSA in TBS-Tween for 2 h, and then incubated for 1 h with anti-SMN primary antibody (MANSMA2). Bound primary antibody was detected by HRP-conjugated secondary antibody followed by chemiluminescence (Amersham Biosciences). The blots were then stripped and reprobed with a ß-actin antibody to control for loading amounts.
Histology and immunocytochemistry
For hematoxylin-eosin stains and spinal cord immunocytochemistry, adult type III SMA mice and normal controls were killed by CO2 gas, transcardially perfused briefly with cold 1x PBS, and the muscle and spinal cord tissue dissected out, mounted on wooden blocks, and flash frozen in liquid nitrogencooled isopentane. 1012-µm thick transverse muscle sections were cut, mounted on Superfrost (Fisher Scientific) slides, and then air dried before processing. Sections were fixed in 100% alcohol (30 s), rinsed in tap water, and stained in hematoxylin (Sigma-Aldrich) for 30 s followed by a second rinse in tap water. They were then stained in eosin (Sigma-Aldrich) for 20 s, rinsed in tap water, dehydrated in alcohol, and cleared in xylene before mounting in Permount (Sigma-Aldrich) for light microscopy. Immunocytochemistry on spinal cord sections was essentially performed as described previously (Monani et al., 2000). For motor neuron studies and ventral root counts, mice were transcardially perfused with 1x PBS followed by 4% paraformaldehyde, 1% glutaraldehyde. Ventral roots, lumbar spinal cord, and the brain stem were post-fixed for 24 h in the same solution. The brain stem and spinal cord were then embedded in paraffin according to conventional procedures. Serial sections (6 µm) prepared with a rotary microtome (American Optical Instruments) were mounted on glass slides and Nissl's stained as previously described (Sendtner et al., 1990). Ventral roots were post-fixed in 1% osmium tetroxide (1 h), rinsed in 0.1 M phosphate buffer (pH 7.3), and then dehydrated. They were then embedded in Spurr resin and 0.5-µm sections were cut on a Reichert Ultracut E microtome. Sections were stained with toluidine blue and visualized by light microscopy.
To look for axonal sprouting in type III SMA mice, animals were killed by cervical dislocation and the intercostals, gastrocnemius, and triceps muscles were dissected out. Muscle tissue was immersed in 3% disodium EDTA for 20 s, mounted on wooden blocks, and flash frozen in liquid nitrogencooled isopentane. 50-µm thick longitudanal sections were cut in a cryostat and placed on Superfrost (Fisher Scientific) slides. A drop of 3% disodium EDTA was immediately placed on the sections to prevent muscle contracture and the sections were allowed to air dry. Cholinesterase and axonal staining were performed as described previously (Pestronk and Drachman, 1978), except that sections were stained in a 10% rather than 20% silver nitrate solution for 10 min. We found this reduces the extent of muscle fiber shrinkage and separation and better preserves the integrity of intramuscular nerves. To determine the extent of sprouting, synapses in ten 50-µm thick sections from the gastrocnemius (medial and lateral parts) and triceps were examined. The 500-µm portion included the middle of the muscle and sections on either side. Synapses were systematically scored for those with or without sprouts. Terminal and nodal sprouts were combined and expressed as a percentage of the total readable synapses. Approximately 400 synapses in each muscle were scored.
Electrophysiological studies
46-mo-old mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (60 mg/kg) and xylazine (3.5 mg/kg). They were maintained at 34°C under anesthesia using isofluorine. For EMG testing, a monopolar needle (26 gauge; Electrode store) was used. Intramuscular potentials were recorded on an EMG instrument (model Neuropack Four mini [MEB-5304K]; Nihon Kohden Corp.). Muscle EMG testing was grouped into the following sites: proximal thoracic limb muscles, proximal pelvic limb muscles, distal thoracic limb muscles, distal pelvic limb muscles, cervical epaxial muscles, and remaining epaxial muscles. The minimum criteria used to define the presence of spontaneous activity was the persistence of an abnormal waveform (>3 s) after complete cessation of needle movement that was reproducible with redirection of the EMG needle. MNCV recordings of the tibial nerve were performed with an Evoked Potential Measuring System. Subdermal stimulating electrodes (10-mm platinum safelead electrodes, model F-E2; Grass Instruments) were placed at the sciatic notch (proximal stimulation site) and the head of the fibula (distal stimulation site) for the sciatic nerve (upper tibial) evaluation. The distal portion of the tibial nerve was evaluated by placing subdermal stimulating electrodes at the head of the fibula (proximal stimulation site) and the ankle (distal stimulation site). Percutaneous intramuscular recording and reference electrodes (26 gauge, 40 x 0.45-mm monopolar needles; Dantec Neurosupplies) were placed on the extensor digitorum brevis muscle of the lateral toe. A ground subcutaneous electrode was placed over the ankle. Stimulation was performed with a single square wave pulse of 0.2-ms duration at 1 Hz with a current of 1015 mA. Reading parameters for the M wave are an analysis time of 10 ms, sensitivity of 10 mV, and band filter of 503,000 Hz. Motor nerve conduction was determined by the formula Velocity (ms) = distance/time. Distance was measured using a flexible tape between the cathodes of each stimulation site.
Online supplemental material
The supplemental material (Figs. S1 and S2) is available online at http://www.jcb.org/cgi/content/full/jcb.200208079/DC1. Fig. S1 shows survival curves for mild SMA mice, and Fig. S2 shows EMG results in the muscles indicated.
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Footnotes |
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U.R. Monani and M.T. Pastore contributed equally to this work.
* Abbreviations used in this paper: EMG, electromyograph; MNCV, motor nerve conduction velocity; MUNE, motor unit number estimation; SMA, spinal muscular atrophy; SMN, survival of motor neurons.
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Acknowledgments |
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This study was supported by Families of SMA (FSMA), the Madison Fund, National Institutes of Health grants NS 38650 to A.H.M. Burghes and NS 40275 to E.J. Androphy. M. Sendtner is supported by a grant from the Deutsche Forschungsgemeinschaft (To61/8-4). U. Monani and C. Andreassi are the recipients of a development grant from the Muscular Dystrophy Association of America and a postdoctoral fellowship from FSMA, respectively.
Submitted: 13 August 2002
Revised: 27 November 2002
Accepted: 2 December 2002
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