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Human Molecular Genetics Advance Access originally published online on July 21, 2004
Human Molecular Genetics 2004 13(18):2031-2042; doi:10.1093/hmg/ddh222
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Human Molecular Genetics, Vol. 13, No. 18 © Oxford University Press 2004; all rights reserved

Characterization of Ighmbp2 in motor neurons and implications for the pathomechanism in a mouse model of human spinal muscular atrophy with respiratory distress type 1 (SMARD1)

Katja Grohmann1,3, Wilfried Rossoll1, Igor Kobsar2, Bettina Holtmann1, Sibylle Jablonka1, Carsten Wessig2, Gisela Stoltenburg-Didinger4, Utz Fischer5, Christoph Hübner3, Rudolf Martini2 and Michael Sendtner1,*

1Institute for Clinical Neurobiology and 2Department of Neurology, University of Wuerzburg, D-97080 Wuerzburg, Germany, 3Department of Neuropediatrics and 4Institute for Neuropathology, Charité University Medical Center, D-13353 Berlin, Germany and 5Institute of Biochemistry, University of Wuerzburg, D-97074 Wuerzburg, Germany

Received May 5, 2004; Revised June 30, 2004; Accepted July 8, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is caused by recessive mutations of the IGHMBP2 gene. The role of IGHMBP2 (immunoglobulin µ-binding protein 2) in the pathomechanism of motor neuron disease is unknown. We have generated antibodies against Ighmbp2 and showed that low levels of Ighmbp2 immunoreactivity are present in the nucleus of spinal motor neurons and high levels in cell bodies, axons and growth cones. Ighmbp2 protein levels are strongly reduced in neuromuscular degeneration (nmd) mice, the mouse model of SMARD1. Mutant mice show severe motor neuron degeneration before first clinical symptoms become apparent. The loss of motor neuron cell bodies in lumbar spinal cord is followed by axonal degeneration in corresponding nerves such as the femoral quadriceps and sciatic nerve and loss of axon terminals at motor endplates. Motor neuron degeneration and clinical symptoms then slowly progress until the mice die at the age of 3–4 months. In addition, myopathic changes seem to contribute to muscle weakness and especially to respiratory failure, which is characteristic of the disorder in humans. Cultured motor neurons from embryonic nmd mice did not show any abnormality with respect to survival, axonal growth or growth cone size, thus differing from motor neurons derived from, e.g. Smn (survival motor neuron) deficient mice, the model of spinal muscular atrophy (SMA). Our data suggest that the pathomechanism in SMARD1 is clearly distinct from other motor neuron diseases such as classic SMA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive motor neuron disease which affects infants. Patients present with respiratory distress due to diaphragmatic paralysis and progressive muscle weakness with predominantly distal lower limb muscle involvement (1). These clinical features distinguish SMARD1 from classic spinal muscular atrophy type 1 (SMA1). In SMA1 patients, weakness occurs primarily in proximal muscle groups and inefficient respiration develops because of intercostal muscle atrophy (2). Unlike classic SMA, which is caused by mutations in the telomeric survival motor neuron (SMN) gene on human chromosome 5q13 (3), SMARD1 results from mutations in the gene encoding IGHMBP2 (immunoglobulin µ-binding protein 2) on chromosome 11q13 (1,4–6). The cellular function of IGHMBP2 is so far not well understood. Several groups have identified IGHMBP2 and its orthologs as a transcriptional activator (7–11) or repressor (12) in various contexts. DNA helicase activity of IGHMBP2 was demonstrated for partially double-stranded DNA substrates (13,14). Computer analysis of the 993 amino acids IGHMBP2 sequence predicts four main domains, separated by low complexity regions. These domains are a DEXDc domain with an AAA-type ATPase domain, a single-stranded nucleic acid-binding R3H domain and a zinc finger motif (15). The DEAD/H box-like motif of IGHMBP2 is typical for RNA helicases and its sequence is most closely related to that of a group of helicases designated superfamily 1 (13,16,17). RNA helicases are known to be involved in a variety of cellular functions e.g. transcription, translation, splicing, nuclear export, ribosome biogenesis and nonsense-mediated mRNA decay. Recently, the solution structure of the R3H domain from IGHMBP2 was determined and it has been proposed to not only bind ss-DNA or RNA, but also to provide a surface area that could be involved in yet unknown protein–protein interactions (18).

Substantial progress has been made in the understanding of the pathogenesis of SMA and other forms of motor neuron disease. Defects in RNA metabolism can contribute to motor neuron degeneration and a specific vulnerability of lower motor neurons to defects in RNA metabolism has been suggested (19). Not only defects in the assembly of spliceosomal U snRNPs (20,21), but also defects in axonal transport or processing of ß-actin mRNA in motor neurons (22) are discussed in the context of classic SMA. The gene causing dominant Charcot–Marie–Tooth disease 2D and distal SMA type V has been identified as the glycyl tRNA synthetase gene (23). Recently, mutations in the RNA helicase Senataxin have been described in ataxia-ocular apraxia 2 (AOA2) (17) and amyotrophic lateral sclerosis type 4 (ALS4) (24). Therefore, it is tempting to speculate that a function of IGHMBP2 in RNA metabolism is important for the maintenance of the peripheral nervous system.

Nothing is known so far about the distribution of IGHMBP2 in developing and postnatal motor neurons. Moreover, it is not clear whether the disease starts in axons, as recently demonstrated in mouse models of motor neuron diseases such as the pmn mutant or the dynamitin overexpressing mouse (25–27). To address these questions, we investigated the developmental expression and subcellular distribution of Ighmbp2 in mouse motor neurons. We found that the protein is located not only in the nucleus but also, predominantly, in the cytoplasm and axons, suggesting that its cellular role in motor neurons goes beyond a function as a DNA helicase, which cooperates in the regulation of gene transcription in the nucleus. We also assessed spinal motor neuron degeneration as well as axonal loss and axon pathology in motor and sensory nerves of the neuromuscular degeneration (nmd) mouse mutant, the corresponding mouse model of SMARD1 (28–30), and correlated motor neuron pathology with the clinical features of the mutants. We found that motor neuron loss is prominent in nmd mice even before first symptoms of the disease occur and that motor neuron cell death occurs without prior axon or motor endplate degeneration. We could not detect axonal growth defects in cultured embryonic nmd mutant motor neurons. These data suggest that the pathomechanism of SMARD1 clearly differs from that of SMA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Developmental expression and subcellular distributionof Ighmbp2 protein in motor neurons
In order to characterize the role of Ighmbp2 in motor neurons, we generated an antiserum against a peptide corresponding to the most C-terminal 19 amino acids of the protein. The specificity of the antiserum was confirmed by western blot analysis using extracts from HEK 293 cells transfected with an expression plasmid containing HA-tagged full-length Ighmbp2 as a positive control and by staining of tissue extracts with pre-immune serum and antiserum after pre-absorption with the corresponding peptide as negative controls. Spinal cord extracts from wild-type C57BL/6 mice were prepared at different stages of pre- and postnatal development and subjected to western blot analysis. Ighmbp2 protein was detected as a band of ~110 kDa, which corresponds to the full-length protein with 993 amino acids (Fig. 1A, {alpha}-peptide). High levels of Ighmbp2 were detected during embryonic and early postnatal development. Between postnatal day 10 and 21 a strong reduction of Ighmbp2 protein levels was observed in spinal cord extracts (Fig. 1A). At embryonic day 15 (E15), full-length Ighmbp2 was expressed in all tissues from wild-type mice examined with highest levels in brain, spinal cord and muscle and a lower level in lung. A very weak signal was observed in extracts from heart and liver (Fig. 1B).



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  		Figure 1. Expression and distribution of Ighmbp2. (A, B) Western blot probed with Ighmbp2 antiserum raised against a C-terminal peptide ({alpha}-peptide). (A) Spinal cord extracts of wild-type mice show highest expression levels of Ighmbp2 protein between embryonic day 15 (E15) and postnatal day 10. The second row indicates the 55–60 kDa immunoreactive band. (B) Tissue specific expression of Ighmbp2 protein in wild-type (wt) and nmd mutant (mut) mice at E 15. The second row indicates the 55–60 kDa band. Anti-actin was used as control for equal loading. (C) Western blot probed with anti-recombinant IGHMBP2 antibodies ({alpha}-recomb.) (D–I) Subcellular distribution of Ighmbp2 in cultured motor neurons (D–F), in lumbar spinal cord (G, H) and sciatic nerve section (I) from wild-type mice using the Ighmbp2 peptide-antiserum. The arrows indicate the growth cones. (J) Staining of a cross section of lumbar spinal cord with pre-immune serum, (K) with pre-absorbed Ighmbp2 peptide antiserum and (L) only with secondary antibody. (M–O) Staining of cultured motor neurons of wild-type (wt) and nmd mutant (mut) mouse embryos (M, N) and of adult wild-type spinal cord section (O) with anti-recombinant IGHMBP2 antibodies.

 
The nmd mutant mice harbour a mutation in intron 4 of the Ighmbp2 gene leading to the creation of a cryptic splice donor site, which results in 20% normally spliced and 80% aberrantly spliced transcripts. The aberrantly spliced mRNA includes 23 additional nucleotides and a premature stop codon (29). The resulting protein is predicted to be truncated at the C-terminus with a molecular weight of 21 kDa. When we investigated protein extracts from nmd mice at E15, the 110 kDa band corresponding to full-length Ighmbp2 was strongly reduced (Fig. 1B). Because our antiserum was raised against the C-terminus, we could not detect the predicted nmd specific truncated Ighmbp2 isoform.

In extracts of both adult wild-type mice and nmd mutant mice, a prominent second band was detectable at 55–60 kDa. This band was hardly detectable at E15 but increased in strength after birth (Fig. 1A and B). It appeared specific as it was not detectable when the antiserum was pre-adsorbed with the corresponding peptide or staining was performed with pre-immune serum (Fig. 1B). Moreover, the same band was also detected by antibodies raised against recombinant full-length IGHMBP2 protein (Fig. 1C, {alpha}-recomb.). This suggests that a 55–60 kDa form of Ighmbp2 in brain extracts of wild-type and nmd mutant mice including the C-terminus may be expressed either from an alternative start codon or it could be a result of alternative splicing. To screen for alternatively spliced transcripts, RT–PCR with a C-terminal reverse primer from exon 15 in combination with forward primers from exons 1, 8 and 12 was used to amplify and sequence Ighmbp2 cDNA from nmd mutant and control mice. Although we identified alternatively spliced transcripts, none can result in the putative shorter isoform detected with our anti-C-terminal peptide antiserum (data not shown). Several ATGs which could give rise to alternative start codons can be found e.g. in exon 9. However, there is no difference in the intensity of the 55–60 kDa band between wild-type and nmd mutant mice, suggesting that the presence of this protein does not correlate with the clinical phenotype in nmd mice.

We have also investigated the distribution of Ighmbp2 in tissue sections of mouse spinal cord, sciatic nerve and in cultured motor neurons from 13.5-day-old mouse embryos. Using the peptide antiserum, Ighmbp2 immunoreactivity was found predominantly in the cytoplasm of cultured motor neurons from wild-type mouse embryos. The nucleus was also weakly stained but the nucleolus was spared. Staining was also detectable in axon-like processes and growth cones (Fig. 1D–F). In the lumbar spinal cord of adult wild-type mice Ighmbp2 immunoreactivity was detectable in particular in motor neuron cell bodies. The nucleus was weakly stained but no Ighmbp2 immunoreactivity was found in the nucleolus (Fig. 1G and H). We further investigated whether Ighmbp2 was present in axons. A strong signal became apparent in large diameter axons of the sciatic nerve (Fig. 1I). Specificity of the staining was tested in parallel spinal cord sections with pre-immune serum (Fig. 1J), pre-absorbed peptide antiserum (Fig. 1K) and by staining with only the secondary antibody (Fig. 1L). Staining of cultured motor neurons and spinal cord sections of nmd mice with our peptide antiserum revealed a mild reduction in intensity (data not shown). This may be explained by the high expression of the 55–60 kDa band both in wild-type and nmd mutant mice. Similar results were obtained with the antibodies against recombinant full-length IGHMBP2 (Fig. 1M–O). Staining of cultured motor neurons and spinal cord motor neurons of wild-type mice (Fig. 1M and O) revealed the same subcellular distribution of Ighmbp2. Again, cultured motor neurons of nmd mice showed a mild decrease in Ighmbp2 immunoreactivity (Fig. 1N).

Comparative analysis of motor neuron loss anddevelopment of clinical symptoms in nmd mutant mice, a mouse model of SMARD1
Starting at an age of 3 weeks, first clinical symptoms became apparent in nmd mice on a C57BL/6 genetic background. Subsequently, they rapidly developed muscle weakness that started in the hindlimbs and proceeded to generalized weakness of limb and trunk muscles, as reported earlier (28–30). Suspended by the tail, mutant mice were no longer able to spread their hindlimbs and toes and to pull themselves up from the ground (Fig. 2A). Mutant mice were severely paralyzed at an age of 5 weeks but most animals survived up to 14 weeks. At an age of 5 weeks, all mutant mice failed in the rotarod test and could not stay on the accelerating wheel for longer than a few seconds (P=0.0001) (Fig. 2B).



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  		Figure 2. Phenotype of nmd mice. (A) Suspended by the tail, nmd mice (right) are not able to spread toes and hindlimbs and to pull themselves up from the ground, compared to wild-type littermates (left) at the age of 14 weeks. (B) Rotarod test of wild-type (white bar) and mutant mice (black bar). (C) Motor neuron numbers in lumbar spinal cord of wild-type (white bars) and mutant mice (black bars) at different ages. n, Number of wild-type or nmd mutant mice investigated at a particular age; ns, not significant. ***P<0.001 (two-tailed unpaired t-test). (D–G) Nissl-staining of motor neurons in lumbar spinal cord from 5-week-old wild-type (D) and nmd mice (E–G). Degenerating motor neurons are marked with arrows.

 
Prior to the onset of clinical symptoms at an age of 3 weeks, a severe loss of spinal motor neurons was observed (Fig. 2C). No significant difference in motor neuron number in lumbar spinal cord was observed in 5-day-old nmd mutants compared with littermate controls. At the age of 10 days, the number of motor neuron cell bodies was already reduced to 63% in comparison to wild-type controls (P=0.0002). No further reduction of motor neuron number was observed between 10-day- and 3-week-old nmd mice. Subsequently, motor neuron loss was progressive towards final stages of the disease with a reduction to 41% at the age of 5 weeks (P=0.0003). At 12–14 weeks of age only 28% of motor neuron cell bodies were found in lumbar spinal cord in comparison with wild-type littermates (P=0.0004) (Fig. 2C). The cytoplasm of degenerating spinal motor neurons in nmd mice was characterized by severely reduced Nissl structure, which was particularly apparent in 5-week-old mice (Fig. 2E–G). The nucleolus was preserved and the diameter of the nucleus did not differ between wild-type (17.82±2.021 µm, n=35) and nmd mutant mice (17.64±1.964 µm, n=45).

Analysis of axonal degeneration in nmd mutant mice
We have also searched for axonal alterations during the course of the disease. For analysing histopathological changes in peripheral nerves, we investigated the sciatic, femoral quadriceps and femoral saphenous nerves in wild-type and nmd mutant mice at the age of 10 days and 3, 5 and 12–14 weeks by both light and electron microscopic techniques (Fig. 3A–F). In peripheral nerves of 10-day-old mutants, most of the larger caliber axons were surrounded by a developing myelin sheath, which is typical for peripheral nerves of wild-type littermates (Fig. 3A and B). There was no detectable delay in myelination between wild-type mice and mutants. However, in contrast to nerves of wild-type mice, axonopathic changes were frequently seen in sciatic and femoral quadriceps nerves. They manifested as abnormal cytoskeleton (increased density or absence) or vacuolization of axons (Fig. 3B, E and F). The axonopathic changes were mostly seen in axons undergoing myelination or in myelinated axons. The corresponding Schwann cells often showed features reminiscent of secondary myelin degeneration as seen under lesioning conditions or in models for primary axonopathy (31). In addition, axonopathic changes were also seen in prospective myelinated axons of larger caliber that were still organized in bundles (data not shown). At the age of 3 months and older, axonopathic changes were still seen in the mutants, but not in the wild-type control mice (Fig. 3C and D). Interestingly, these hallmarks were more frequent at 10 days and 3 weeks than at 12–14 weeks (Fig. 3G). An additional pathological feature seen at all ages was the occurrence of Schwann cells (with or without myelin structures) completely devoid of axons, suggesting axonal degeneration (Fig. 3E). At 14 weeks, the leading pathological hallmarks were abundant Schwann cell processes devoid of myelin and axons, being reminiscent of bands of Büngner seen at longterm denervation conditions (Fig. 3D).



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  		Figure 3. Axonal pathology in nmd mice. (A, B) Light microscopic and (C–F) electron microscopic analysis of (A–D, F) femoral quadriceps nerves and (E) sciatic nerve of wild-type (wt) and nmd mutant (mut) mice at the age of 10 days and 14 weeks. Quantification of pathological hallmarks of (G) axon degeneration and (H) numbers of myelinated axons in femoral quadriceps nerves in wild-type and mutant mice. (A, B) In both wild-type (A) and mutant mice (B) most axons are myelinated at the age of 10 days. In the mutant mice, there are axonopathic changes suggestive of axonal degeneration as indicated by arrowheads and presented in more detail in (E) and (F). v, Blood vessels. (C, D) The major pathological alterations in quadriceps nerves of 14-week-old mutant mice (D) are Schwann cell processes devoid of axons and reminiscent of bands of Büngner (circles). Black arrows indicate non-myelinated nerve fibers which are found in both wild-type (C) and mutant mice (D).(E, F) Higher power electron microscopy of features indicative of axonal degeneration in sciatic (E) and quadriceps (F) nerves of 10-day-old mutant mice. In (E), axonal loss is reflected by the presence of an ‘empty’ myelin sheath undergoing degeneration (dMy). In (F), degenerating myelin (dMy), unusually electron dense axoplasm (arrow) and a vacuolized axon (Vac) are visible. (G) Axonopathic changes are particularly frequent at younger ages, whereas at older age, they are less frequent. (H) Number of myelinated axons in quadriceps nerves of wild-type (white bars) and mutant mice (black bars). Note constant decrease of axon numbers in the mutants. n, number of wild-type or nmd mutant mice investigated at a particular age. ns, not significant; **P<0.01; ***P<0.001 (unpaired two-tailed t-test).

 
As a next step, we quantified axons in femoral quadriceps nerves in nmd mutants and wild-type control mice (Fig. 3H). Femoral quadriceps nerves of adult wild-type mice usually contain ~550 myelinated axons (31) from which ~50% are motor axons (I. Kobsar, M. Samsam and R. Martini, unpublished data) corroborating values obtained in the same nerve in rat (32). In 10-day-old wild-type mice, the number of myelinated axons appears lower, as not all prospective myelinated axons have achieved a 1 : 1 ratio with Schwann cells and can, thus, not be identified as myelinated fibers (33). When comparing the number of axons in mutant and wild-type mice at 10 days, there was a statistically non-significant trend towards a reduced number of axons in nmd mutant mice (Fig. 3H). From postnatal week 3 onwards, axon numbers in femoral quadriceps nerves of nmd mutant mice significantly decreased with age and showed a reduction by 40% at 12–14 weeks (P<0.0001) (Fig. 3H). In order to investigate whether sensory axons are prone to degenerate in the mutants, we investigated the myelinated axons of femoral saphenous nerves, which are exclusively sensory (34). No significant axonal loss was observed in femoral saphenous nerves of 12–14-week-old nmd mice compared with wild-type controls (n=3 wild-type: 765.3±30.5 axons; n=3 mutants: 734.7±33.56 axons, P=0.3065). These data reveal that in nmd mutants a substantial number of axons, presumably motor axons, degenerate in sciatic and femoral nerves reflecting the motor neuron degeneration observed in spinal cord (Fig. 2C).

Correlation of axonal loss and electrophysiological alterations in nmd mice
We further performed electrophysiological studies in wild-type and nmd mutant mice at the age of 5 weeks, when the mutants were already severely paralytic. The nmd mice showed a statistically significant decrease in the amplitude of the compound motor action potential (CMAP) (P=0.0087 for distal, P=0.0154 for proximal amplitude) that correlated with axonal loss in sciatic and tibial nerves (Fig. 4A–C). There was no significant difference (P=0.4851) in motor nerve conduction velocity between wild-type and nmd mice suggesting absence of a myelinopathy (data not shown). Electromyography revealed spontaneous activities in muscles from 5-week-old nmd mice, which were more pronounced in gastrocnemius than in quadriceps muscle (Fig. 4E) indicating motor axon loss.



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  		Figure 4. Motor nerve conduction studies and EMG in 5-week-old wild-type and nmd mutant mice. (A) nmd mice show a significant reduction in distal and proximal amplitude (black bars) of compound muscle action potentials (CMAP) in comparison to wild-type mice (white bars). *P<0.05; **P<0.01. n, number of wild-type or nmd mutant mice investigated. In (B), CMAP of a wild-type mouse after distal stimulation is shown. In contrast, nmd mice show a markedly reduced amplitude (C) suggesting loss of motor axons (original recordings, note different scale). Latency and shape of the CMAP are normal. (D) EMG recording of a wild-type mouse. (E) Denervation is indicated by spontaneous activity of the gastrocnemius muscle seen in the nmd mice.

 
Survival and axon growth is not disturbed in cultured nmd mutant motor neurons
To investigate a potential early embryonic defect in survival or axonal outgrowth of motor neurons we isolated lumbar spinal motor neurons from wild-type and nmd mice at E13.5 and cultured them for 5–6 days in the presence and in the absence of neurotrophic factors. Cultured motor neurons from nmd mutant mice did not show differences in survival compared to wild-type controls (survival with BDNF/CNTF: n=3 wild-type mice, 56.33±4.041% of originally plated cells; n=3 nmd mutant mice, 59.00±4.583%, P=0.4978. Survival without BDNF/CNTF: n=3 wild-type mice, 19.00±5.568% of originally plated cells; n=3 nmd mutant mice, 14.33±4.933%, P=0.3383). Axons of cultured wild-type motor neurons did not differ in length (n=150, length 511.8±301.8 µm) compared to axons of nmd mutant motor neurons (n=150, length 512.1±264.8 µm, P=0.9928). Measuring the size of the growth cone areas did not reveal significant differences (P=0.6117) in growth cone size between wild-type (25.51±11.16 µm2, n=50) and nmd mutant (26.82±14.39 µm2, n=50) motor neurons.

Degeneration of motor endplates is not detectable before onset of motor neuron degeneration in the spinal cord
As Ighmbp2 immunoreactivity is present at relatively high levels in growth cones of developing motor neurons (Fig. 1D–F), we also investigated motor endplates in wild-type and nmd mutant mice. At postnatal day 10, some motor endplates that lack the axon terminal could be detected in quadriceps and gastrocnemius muscles of nmd mice (Fig. 5A', A'', D' and D''). The number of denervated motor endplates in mutants increased dramatically towards final stages of the disease corresponding to motor neuron degeneration (Fig. 5B'–C'' and E'–F''). In the denervated motor endplates, subsynaptic clefts and folds disappeared and/or did not develop. Surprisingly, we found motor endplates in the diaphragm of 14-week-old nmd mice well preserved (Fig. 5G' and G''), which corresponded to preservation of axons in the phrenic nerve up to 14 weeks of age (n=3 wild-type mice: 225.0±2.0 axons, n=3 mutant mice, 225.3±3.215 axons, P=0.8862).



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  		Figure 5. Alterations in motor endplates and muscle fibers of nmd mice. Motor endplates from quadriceps (A–C'') and gastrocnemius (D–F'') muscle of 10-day-, 3-week- and 14-week-old wild-type (A–F) and nmd (A'–F'') mice. Endplates of nmd mice lack the axon terminal and the subsynaptic clefts and folds show degeneration. Red: anti-phospho-neurofilament SMI31, green: Alexa FluorTM 488 conjugated alpha-bungarotoxin. (G–G'') No loss of axon terminals is found in motor endplates from diaphragm specimens of nmd mice at the age of 14 weeks (G', G''). (H') Area of myofiber necrosis (arrow) in H&E staining of the diaphragm from an 8-week-old nmd mouse. (I') At 14 weeks of age muscle fibers with central nuclei are abundant (arrows). (I) This is not found in diaphragm specimen of wild-type control mice of the same age. (J', K') In quadriceps muscle specimen from 8- and 14-week-old nmd mice, neurogenic atrophy of muscle fibers is found with replacement by adipose tissue (asterisks) in 14-week-old nmd mice. Central nuclei pointing towards myopathic changes in quadriceps muscle fibers are also present (arrows).

 
Children with infantile SMARD1 develop a severe respiratory distress already during their first 13 months of life (1). Clinically, nmd mice do not develop severe respiratory distress at 5 weeks when paralysis of hindlimbs is prominent, but at late stages of the disease. Therefore, we investigated the diaphragm of 8- and 14-week-old nmd mice and detected myopathic changes in diaphragmatic muscle fibers. We found areas with necrotic myofibers in the diaphragm of 8-week-old nmd mutant mice (Fig. 5H'). In the diaphragm of 14-week-old nmd mice, muscle fibers with central nuclei were abundant indicating regeneration (Fig. 5I'). This points towards a further important role of Ighmbp2 in muscle fiber maintenance. We also investigated the quadriceps muscle in nmd mice of 8 and 14 weeks of age. Again, myofibers with central nuclei were found suggesting a myopathic component in addition to a severe neurogenic muscular atrophy with massive muscle fiber atrophy and replacement by adipose tissue (Fig. 5J' and K').


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SMARD1 resembles classic SMA1 in many parameters, and unambiguous diagnosis is possible since the underlying gene defect in the IGHMBP2 gene has been discovered and genetic diagnosis became available (1,5,6). The cellular role of IGHMBP2 in the peripheral nervous system is unclear, but published data suggest that the protein might play a role in DNA and/or RNA metabolism. Defects in RNA metabolism are also thought to be involved in the pathomechanism of classic SMA, which is caused by mutations in the SMN1 gene (3). SMN plays an essential role in the assembly of spliceosomal U snRNPs (20,21) but recent data suggest that the SMN gene product plays an additional motor neuron specific role. Animal models for SMA have suggested that axonal degeneration and specific structural defects at the axon terminals (22,35–37) might contribute to the disease, and current research focuses on the question whether the protein is also involved in axon specific functions such as RNA transport and/or translational control (22,38,39). This raises the question whether SMARD1 is caused by a defect that primarily affects motor axons and endplates or motor neuron cell bodies. It was not known so far, whether loss of motor neuron cell bodies is a consequence of the degeneration of motor endplates and axons or whether cell death is the primary feature and SMARD1 only becomes apparent after a significant number of motor neurons have been lost. Another major question for understanding the pathophysiology of SMARD1 is whether the disease is caused by disturbance or loss of a general cellular function (which is particularly important for motor neurons) or whether a motor neuron specific function is affected.

In order to address these questions we have studied the distribution of the Ighmbp2 protein in motor neurons and characterized survival and axon growth of isolated embryonic motor neurons from a mouse model of SMARD1, the nmd mouse (28–30). In addition, we compared the time course of clinical symptoms and motor neuron degeneration. We found that Ighmbp2 protein is present at high levels in the cytoplasm including axons and growth cones of motor neurons and at low levels in the nucleus suggesting a role of Ighmbp2 besides its nuclear function that has not yet been characterized. While nmd mutant mice still have normal muscle strength at postnatal day 10, loss of motor neuron cell bodies in lumbar spinal cord is already prominent. This indicates that motor neuron cell death is an early event during the disease and occurs even before first clinical symptoms become apparent. For comparison, transgenic mice with two or three copies of the human SMN2 gene on a mouse Smn knockout background do not show significant motor neuron loss at birth although these mice already appear severely paralyzed and die within few days (40). Muscle weakness in nmd mutant mice starts in the hindlimbs. It rapidly progresses to a generalized weakness of limb and trunk muscles between postnatal weeks 3 and 5. Then, progression of the disease slows down in nmd mutant mice. This is also reflected by the number of surviving spinal motor neurons. At postnatal day 10, 37% of motor neurons are lost in the lumbar spinal cord and at 12–14 weeks of age, the loss increases to >70%.

To evaluate the corresponding loss of motor axons, we selected the quadriceps nerve that contains ~550 myelinated axons (31). At 10 days after birth, there is no significant loss of myelinated axons in the femoral quadriceps nerve but 1–5% of myelinated axons unequivocally show ultrastructural hallmarks of axon degeneration. In this context it is important to note that in the quadriceps nerve only ~50% of all axons are motor axons. Since cutaneous sensory neurons do not degenerate in the nmd mouse, it appears plausible that muscle afferents do also not degenerate. Based on these assumptions, the majority of degenerating axons in the femoral quadriceps nerve probably belongs to motor neurons. Therefore doubling the values from the total axonal counts might reflect the rough values for degenerating motor axons. Thus, up to ~10% of motor axons show hallmarks of axon degeneration in the quadriceps nerve. The same animals exhibit a 37% loss of motor neuron cell bodies in the lumbar spinal cord. This suggests that the motor neuron degeneration starts at the spinal cord level and that the corresponding features of degeneration at the level of axons only ‘travel’ along the axon, a view being in line with observations in Wallerian degeneration (41). At 12–14 weeks, ~70% of motor neurons have degenerated, whereas ~40% of the total number of quadriceps nerve axons (i.e. presumably 60–80% of motor axons) are lost. This suggests that the rate of motor neuron degeneration is low at older ages resulting in a closer correlation of motor neuron cell body loss in lumbar spinal cord and subsequent axonopathy.

So far it is not clear why paralysis of the diaphragm appears so early and as a prominent feature in infantile SMARD1 patients. Clinically, nmd mice resemble patients with a milder form of the disease (42) as the respiratory distress is only detectable at late stages. This correlates with a type of mutation that still can give rise to low but significant levels of Ighmbp2 protein. Surprisingly, even at late stages of the disease, in 14-week-old nmd mice, no significant reduction in axon numbers of phrenic nerves was observed. Therefore, we searched for a defect in the diaphragm itself. Indeed, diaphragm specimens of 8-week-old nmd mice showed abundant myopathic alterations. In 14-week-old nmd mice, central nuclei and myofiber regeneration were prominent. These findings point towards an additional role of Ighmbp2 in the maintenance of muscle fibers. This hypothesis is supported by a recent study in which motor neuron degeneration in nmd mice is rescued by neuron specific expression of Ighmbp2. The rescue uncovered a cardiac and skeletal myopathy and nmd mouse mutants died from dilated cardiomyopathy and congestive heart failure (30).

Our data suggest important differences in the pathophysiology of SMARD1 and classic SMA. In cultured motor neurons from SMA mouse models, axon growth is disturbed and axon terminals show severe pathology both in cell culture (22) and in vivo (35). In contrast, motor neurons from nmd mice appear normal in cell culture. On the other hand, in nmd mice motor neuron cell death is much more prominent in early stages of the disease in comparison with SMA mouse models. The early cell death of motor neurons, which precedes the degeneration of motor endplates and axons indicates that the Ighmbp2 protein is necessary for survival of these neurons. It is still unclear why the disease primarily affects motor neurons. However, the role of Ighmbp2 in the maintenance of muscle fibers, which becomes particularly apparent in the diaphragm and heart of nmd mutant mice could aggravate paralysis so that the development of treatment should not only focus on the rescue of motor neurons.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice, rotarod performance test and electrophysiological studies
B6·BKS-lghmbp2nmd-2J/+ mice were obtained from Jackson Laboratories and backcrossed to the C57BL/6 genetic background. Mice heterozygous for the nmd2J mutation were intercrossed and homozygous mutant mice and wild-type littermates were used for analyses. Mice were genotyped as described (29). All animal manipulations were performed in accordance with institutional guidelines and permissions. For determination of muscle weakness a rotarod test was performed. Three nmd mice and four wild-type littermates at the age of 5 weeks were put three times on an accelerating rotarod and their ability to remain on the rotating rod was recorded. For motor nerve conduction and electromyographic studies three nmd mice and four wild-type littermates at the age of 5 weeks were deeply anaesthezised with fentanyl/fluanisone (Hypnorm). The compound motor action potential (CMAP) was recorded with two needle electrodes in the foot muscles after supramaximal distal stimulation of the tibial nerve at the ankle and proximal stimulation of the sciatic nerve at the sciatic notch. EMG was recorded from gastrocnemius and quadriceps muscle and assessed for spontaneous activity.

Generation of polyclonal antisera against Ighmbp2
The peptide antiserum ({alpha}-peptide) was generated by immunization of rabbits with 750 µg of a peptide corresponding to the most C-terminal 19 amino acids of the Ighmbp2 protein in complete (first immunization) and incomplete (boost injections) Freund's adjuvant (Sigma). Anti-IGHMBP2 antibodies ({alpha}-recomb.) were raised against recombinant human GST-tagged full-length protein. The antiserum was affinity-purified on a column containing His-tagged IGHMBP2 as described (43).

Histological and immunohistochemical analyses
Mice were deeply anaesthezised with 80 µg/g body weight Ketanest and 8 µg/g body weight Rompun and transcardially perfused with 4% PFA (paraformaldehyde) in phosphate buffer for 15 min. Lumbar spinal cord was prepared from 5- and 10-day-old and 3-, 5- and 14-week-old animals. Sciatic nerves were prepared from 14-week-old animals and frozen in Tissue-Tek (Sakura). Paraffin serial sections (12.5 µm) of lumbar spinal cord were processed for Nissl-staining and quantification of motor neuron numbers at the light microscope as described (44). Vibratome sections (70 µm) of lumbar spinal cord and 10 µm cryotome sections of sciatic nerve from wild-type animals were washed with 1x PBS, blocked with 10% BSA in 1x PBS and 0.3% Triton X-100 for 1 h. Sections were then incubated either with polyclonal Ighmbp2 peptide antiserum, with pre-immune serum, with antiserum after pre-absorption with the corresponding Ighmbp2 peptide (dilution 1 : 1000–1 : 2000 in 1x PBS, 1% BSA and 0.1% Triton X-100) or with anti-recombinant IGHMBP2 antibodies (1 : 100) for 2 h and Cy3-conjugated secondary antibody (Dianova, 1 : 200) for 1.5 h at RT. For light and electron microscopic analyses of sciatic, phrenic and femoral quadriceps and saphenous nerves, 10-day-old 3-, 5-, and 12–14-week-old mice were perfused with 4% PFA, 2% glutaraldehyde in 0.1 M cacodylate buffer. Nerves were processed for semithin (0.5 µm) and ultrathin sections (80 nm) according to standard protocols and examined by light (Axioplan 2, Zeiss) and electron microscopy (EM 10B, Zeiss) (45). Quantitative analysis of axons in femoral quadriceps, saphenous nerves and phrenic nerves has been performed at light and electron microscopic level as described earlier (46,47). In femoral quadriceps nerves, we assessed the number of myelinated axons or, at developmental stages, of axons undergoing myelination. As signs of axonal pathology, we considered profiles with altered axonal cytoskeleton (absent cytoskeleton or abnormally increased density) or axons containing vacuoles that covered >75% of axonal surface.

For examination of motor endplates, native specimens of quadriceps and gastrocnemius muscles were obtained from 10-day-old and 3- and 14-week-old mice and of the diaphragm from 14-week-old mice. Teased muscle fibers were fixed in 2% PFA in phosphate buffer for 3 h, rinsed in 1x PBS and blocked overnight in 10% BSA and 0.3% Triton X-100 in 1x PBS. Specimens were then probed with Alexa FluorTM 488-conjugated alpha-bungarotoxin (Molecular Probes) for 30 min to stain the subsynaptic apparatus, washed with 1x PBS for 5 h, and incubated with anti-phospho-neurofilament SMI31 monoclonal antibody (Sternberger Monoclonals) (first antibody overnight) and second antibody (Cy3-conjugated, Dianova, 1 : 200, 1.5 h) for visualization of the pre-synapse. Sections were mounted in DABCO and examined under a confocal laser microscope (Leica TCS).

For haematoxylin and eosin (H&E) staining, diaphragm and quadriceps muscles were prepared from 8- and 14-week-old nmd and wild-type mice. Specimens were frozen in Tissue-Tek (Sakura), 10 µm sections were cut with a cryotome (Leica CM3050 S) and processed for H&E staining according to standard protocols.

Motor neuron cultures and immunocytochemistry of cultured motor neurons
Isolation and culture of mouse embryonic motor neurons was performed essentially as described (48). Lumbar spinal cords were prepared from mouse embryos at E13.5, transferred into 180 µl HBSS buffer and kept on ice. An aliquot of 20 µl of 0.05% trypsin was added and samples were incubated at 37°C for 15 min. After addition of trypsin inhibitor (0.1%) spinal cords were transferred into saline solution (30 mM KCl, 0.8% NaCl), triturated and the cell suspension layered on the top of 10% metrizamide solution (Sigma). Tubes were then centrifuged at 400g for 20 min. The motor neuron fraction was transferred into Neurobasal medium (Invitrogen) containing 500 µM glutamax (Invitrogen), 10% horse serum (Linaris) and 2% B-27 supplement. Motor neurons were plated on L-polyornithine and laminin coated cover slips at a density of 3000 cells/cm2 in four-well culture dishes (Greiner) and were grown at 37°C in a 5% CO2-atmosphere for 5–6 days. The culture medium contained 10 ng/ml BDNF and 10 ng/ml CNTF (Amgen) except for parallel negative controls without neurotrophic factors. Motor neurons were fixed with 4% PFA followed by cold acetone for 5 min and blocked with 10% BSA and 0.3% Triton X-100 in 1x PBS for 1 h. Cells were incubated with the following primary antibodies overnight at 4°C: anti-Ighmbp2 peptide antiserum (1 : 1000), anti-recombinant IGHMBP2 antibodies (1 : 100), rabbit polyclonal against phospho tau for staining of axons (Sigma, 1 µg/ml), mouse monoclonal against MAP-2 for neurites (Chemicon, 1 : 1000) and actin for growth cones (Chemicon, 1 : 200). Cells were washed with 1x PBS and incubated with Cy2- and Cy3-conjugated secondary antibodies (Dianova, 1 : 200) for 1.5 h at RT. Coverslips were mounted in Mowiol and fluorescence was visualized under the Leica confocal laser microscope.

Data analysis
For quantification of axon lengths and growth cone areas, images of immunolabelled motor neurons were obtained by confocal microscopy. In total, 150 axons and 50 growth cone areas of wild-type and nmd mutant embryos were quantified with Scion Image software. These data as well as the data from electrophysiological studies and from motor neuron and axon counts were subjected to statistical analysis with GraphPad Prism software. Group differences were evaluated by use of an unpaired, two-tailed t-test with similar variances in the compared groups. Statistical significance was denoted for P<0.05 (*P<0.05; **P<0.01; ***P<0.001). All data are reported as mean±SD.

Western blot analysis
Frozen spinal cord specimen from wild-type mice at E15 and E18 and postnatal day 5, 10, as well as at 3, 5 and 14 weeks of age were homogenized in RIPA-buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitor cocktail tablets (Roche) and the protein content was measured using Bradford assay (Bio-Rad). In addition, tissue specimens from brain, spinal cord, heart, lung, liver and muscle from wild-type and nmd mice at E15 were prepared. An amount of 30 µg of total protein was boiled in Laemmli buffer for 5 min and separated on a 5–15% gradient SDS–PAGE gel. After electrophoresis, proteins were transferred on a nitrocellulose membrane, stained with Ponceau S solution, blocked in 5% milk in 1x TBS-T and incubated with anti-Ighmbp2 peptide antiserum (dilution 1 : 2000 in 5% milk/1x TBS-T) or anti-recombinant IGHMBP2 antibodies (1 : 100) and anti-rabbit IgG conjugated to horseradish peroxidase. HA-tagged Ighmbp2 was cloned by RT–PCR into pcDNA3 (Invitrogen). Cell extracts from HEK 293 cells were prepared 48 h after transfection with the expression plasmid for full-length HA-tagged Ighmbp2 (Lipofectamine 2000, Invitrogen). The immune complexes were detected using chemiluminescent detection reagents. The nitrocellulose membrane was stripped and reprobed with anti-actin monoclonal antibody for equal loading control.

RT–PCR
cDNA was reverse transcribed from RNA isolated from brain tissue (Trizol, BioRad) of adult nmd mutant and control wild-type mice (Superscript III, Invitrogen). A PCR primer derived from the C-terminus in combination with a primer from exon 1, 8 or 12 was used to amplify Ighmbp2. PCR products were re-amplified and sequenced (ABI 373, Applied Biosystems).


   ACKNOWLEDGEMENTS
 
We wish to thank Silke Clemens, Paraskevi Zisimopoulou and Hans Hilmar Göbel for help and discussions and Beate Christ, Karin Urlaub and Heinrich Blazyca for technical assistance. This work has been supported by grants from the Deutsche Forschungsgemeinschaft (GR 1837/1-1 and SFB 581).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +49 93120149771; Fax: +49 93120149788; Email: sendtner_m{at}klinik.uni-wuerzburg.de


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