Embryonic Rat Spinal Cord Research Articles

Development of inhibition by ephrin-A5 on outgrowth of embryonic spinal motor neurites.

The spinal motor pool maps systematically onto the surface of muscles. This map is detectable in rat embryonic muscles, and is partially restored after reinnervation. Recent evidence shows that either overexpression or deletion of the ephrin-A5 gene significantly disrupts the map, suggesting that ephrin-A5 plays a critical role in the formation of this topography. Several studies have demonstrated that ephrin-A5 is a repulsive molecule in the nervous system, including the neuromuscular system. To examine the development of sensitivity of ventral spinal axons to this inhibitory ligand, slices of E11 to E15 embryonic rat spinal cords were cocultured with membranes derived from ephrin-A5-expressing cell lines. We detected a progressive expression of inhibition by ephrin-A5 between E11 and E15. By E15, rostral and caudal spinal neurites showed clear differences in responsiveness to the ephrin-A5 ligand. Further, we found that at this age caudal neurites are more sensitive to changes of ephrin-A5 concentration along a gradient. In addition, growth cones of caudal, more than rostral, neurites tended to assume a collapsed shape in the presence of the ligand. These results demonstrate a progressive development of sensitivity to ephrin-A5, and suggest a divergence in this sensitivity between rostral and caudal spinal cord neurites. These results provide further insight into how subtle rostrocaudal differences in the development of sensitivity to ephrin-A5 may explain, in part, neuromuscular topography.

Responsiveness to neurturin of subpopulations of embryonic rat spinal motoneuron does not correlate with expression of GFRα1 or GFRα2

Glial cell-line derived neurotrophic factor (GDNF) and its relative neurturin (NTN) are both potent trophic factors for motoneurons. They exert their biological effects by activating the RET tyrosine kinase in the presence of a GPI-linked coreceptor, either GFRα1 (considered to be the favored coreceptor for GDNF) or GFRα2 (the preferred NTN coreceptor). By whole-mount in situ hybridization on embryonic rat spinal cord, we demonstrate that, whereas Ret is expressed by nearly all motoneurons, Gfra1 and Gfra2 exhibit complementary and sometimes overlapping patterns of expression. In the brachial and sacral regions, the majority of motoneurons express Gfra1 but only a minority express Gfra2. Accordingly, most motoneurons purified from each region are kept alive in culture by GDNF. However, brachial motoneurons respond poorly to NTN, whereas NTN maintains as many sacral motoneurons as does GDNF. Thus, spinal motoneurons are highly heterogeneous in their expression of receptors for neurotrophic factors of the GDNF family, but their differing responses to NTN are not correlated with expression levels of Gfra1 or Gfra2. © 2001 Wiley-Liss, Inc.

On the possible role of muscle in the pathogenesis of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common human inherited disease characterized by degeneration of motoneurons and muscular atrophy. SMA results from deletions or mutations of the SMN (survival motor neuron) gene. A nerve-muscle coculture model, consisting of human muscle cells innervated by rat embryonic spinal cord explants, was used to study the pathogenesis of SMA. Previous studies have shown that myotubes formed by fusion of satellite muscle cells from patients with SMA I or SMA II (but not SMA III) underwent a characteristic degeneration 1-3 weeks after innervation. To correlate this cellular study with a molecular approach, we used reverse transcriptase-polymerase chain reaction (RT-PCR), and showed that SMN mRNAs were expressed throughout the fusion of normal satellite muscle cells with two peaks, the first appearing prior to the onset of fusion and the second one or two days before innervation. When satellite muscle cells from patients with SMA I or II were used, only the first peak was observed. Because in these cases the SMN telomeric gene (SMNtel) is deleted, it was concluded that the contribution of SMNtel-dependent mRNAs to the second peak is predominant in normal myogenesis and involved in maturation of myotubes. In addition, diseased satellite muscle cells did not fuse at the same rate as normal satellite muscle cells. Studies on myf-5, a muscle specific transcription factor family, showed that its expression was impaired during the fusion of satellite muscle cells from patients with SMA I or II compared with normal satellite muscle cells. Taken together, these observations suggest that (a) there is a muscle specific expression pattern of SMN, and (b) SMN probably plays a crucial role in maintenance of a functional motor unit, by allowing muscle cells to correctly differentiate and to allow motoneuron survival.

Responsiveness to neurturin of subpopulations of embryonic rat spinal motoneuron does not correlate with expression of GFR alpha 1 or GFR alpha 2.

Glial cell-line derived neurotrophic factor (GDNF) and its relative neurturin (NTN) are both potent trophic factors for motoneurons. They exert their biological effects by activating the RET tyrosine kinase in the presence of a GPI-linked coreceptor, either GFR alpha 1 (considered to be the favored coreceptor for GDNF) or GFR alpha 2 (the preferred NTN coreceptor). By whole-mount in situ hybridization on embryonic rat spinal cord, we demonstrate that, whereas Ret is expressed by nearly all motoneurons, Gfra1 and Gfra2 exhibit complementary and sometimes overlapping patterns of expression. In the brachial and sacral regions, the majority of motoneurons express Gfra1 but only a minority express Gfra2. Accordingly, most motoneurons purified from each region are kept alive in culture by GDNF. However, brachial motoneurons respond poorly to NTN, whereas NTN maintains as many sacral motoneurons as does GDNF. Thus, spinal motoneurons are highly heterogeneous in their expression of receptors for neurotrophic factors of the GDNF family, but their differing responses to NTN are not correlated with expression levels of Gfra1 or Gfra2.

L1 and GAD65 are expressed on dorsal commissural axons in embryonic rat spinal cord

Using immunocytochemical methods, the cell adhesion molecule L1 was detected on axons crossing in the dorsal commissure of developing rat spinal cord. Immunoreactive axons were found in locations similar to fiber bundles illustrated by Ramón y Cajal and designated the anterior, middle and posterior bundles of the dorsal commissure. L1-immunoreactive dorsal commissural axons were first observed on embryonic day 17 (E17), appeared more numerous by E19, and remained detectable in early postnatal ages. The massive middle axon bundles extended bilaterally from the dorsolateral funiculi towards the midline and crossed in the central part of the commissure. In horizontal sections, bundles of L1-labeled middle axons were observed to traverse the dorsal commissure in a periodic pattern along the entire rostrocaudal extent of the spinal cord. Bundles of glutamic acid decarboxylase (GAD65)-positive axons were detected crossing in the middle and posterior regions of the dorsal commissure between E17 and E20. Results from double-labeling experiments demonstrated that GAD65-positive fibers were embedded in larger bundles of L1-labeled axons and that some dorsal commissural axons were double-labeled. To determine if there were axons crossing in the dorsal commissure that did not express L1, double-labeling experiments were conducted using neurofilament and L1 antibodies. Results indicated that bundles of axons identified with anti-neurofilament antibodies were also L1-positive, whereas individually coursing axons within the commissure were L1-negative. The predominance of L1 on fiber bundles traversing the dorsal commissure adds to the growing evidence that this molecule may play a role in axon outgrowth and fasciculation.

In vitro induction of neuronal apoptosis by anti-Fas antibody-containing sera from amyotrophic lateral sclerosis patients

Sera from 26% of patients with sporadic amyotrophic lateral sclerosis (ALS) induced in vitro apoptosis of a human neuroblastoma cell line, as detected by two methods, and most contained anti-Fas autoantibodies. In contrast, Alzheimer sera (studied as controls) very rarely induced apoptosis and did not contain detectable anti-Fas antibodies. Soluble Fas-ligand levels in ALS sera were not different from those in normal sera, except for slightly higher levels in a single case. In mixed cultures of rat embryonic brain and spinal cord cells, ALS sera (and agonistic anti-Fas monoclonal antibodies and soluble Fas-ligand) induced the apoptosis of a subpopulation of neurons. These neurons were motoneurons on the basis of staining with the monoclonal antibody SMI 32 and Fas expression was restricted to these SMI 32-positive neurons. These data are compatible with the hypothesis of the participation of an autoimmune mechanism possibly related to anti-Fas autoantibodies in certain ALS patients.

Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord

Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague–Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague–Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured spinal cord.

Cell Types Required to Efficiently Innervate Human Muscle Cells in Vitro

Previous studies carried out in our laboratory have shown that myofibers formed by fusion of muscle satellite cells from donors with spinal muscular atrophy (SMA) type I or II undergo a characteristic degeneration 1.5–3 weeks after innervation with rat embryonic spinal cord explants. The only cells responsible for degeneration of innervated cocultures are SMA muscle satellite cells. In order to study the kinetics of nerve and muscle cell degeneration in nerve–muscle cocultures implicating SMA muscle cells, we attempted to simplify the nervous component of the coculture and identify the nerve cell types necessary for a successful innervation. We demonstrate here that motoneurons alone were unable to innervate myotubes. However, when three cell types (motoneurons, sensory neurons, and Schwann cells) were added onto a reconstituted muscular component consisting of cloned muscle satellite cells and cloned muscular fibroblasts, myotubes contracted, indicating that functional neuromuscular junctions were formed. We concluded that the three cell types were required for a successful innervation. Moreover, we studied the effects of culture medium conditioned by different combinations of nerve cells on innervation; we observed that physical contacts among sensory neurons, motoneurons, and myotubes are required for a successful innervation; in contrast Schwann cells can be replaced by a Schwann-cell-conditioned medium, indicating that these cells produce a putative soluble “innervation-promoting factor.” Obviously such a reconstituted system does not reflect the in vivo situation but it allows the formation of functional motor synapses and could therefore allow us to elucidate neuromuscular disease pathogenesis, especially that of spinal muscular atrophy.

Heparin blocks functional innervation of cultured human muscle by rat motor nerve

In vitro innervated human muscle is the only experimental model to study synaptogenesis of the neuromuscular junction in humans. Cultured human muscle never contracts spontaneously but will if innervated and therefore is a suitable model to study the effects of specific neural factors on the formation of functional neuromuscular contacts. Here, we tested the hypothesis that nerve derived factor agrin is essential for the formation of functional synapses between human myotubes and motoneurons growing from the explant of embryonic rat spinal cord. Agrin actions were blocked by heparin and the formation of functional neuromuscular contacts was quantitated. At a heparin concentration of 25 microg/ml, the number of functional contacts was significantly reduced. At higher concentrations, formation of such contacts was blocked completely. Except at the highest heparin concentrations (150 microg/ml) neuronal outgrowth was normal indicating that blockade of neuromuscular junction formation was not due to neuronal dysfunction. Our results are in accord with the concept that binding of neural agrin to the synaptic basal lamina is essential for the formation of functional neuromuscular junctions in the human muscle.

Ectopic Adenoviral Vector-Directed Expression of Sema3A in Organotypic Spinal Cord Explants Inhibits Growth of Primary Sensory Afferents

Sema3A (Sema III, SemD, collapsin-1) can induce neuronal growth cone collapse and axon repulsion of distinct neuronal populations. To study Sema3A function in patterning afferent projections into the developing spinal cord, we employed the recombinant adenoviral vector technique in embryonic rat spinal cord slices. Virus solution was injected in the dorsal aspect of organotypic spinal cord cultures with segmentally attached dorsal root ganglia (sc-DRG). In cultures grown in the presence of nerve growth factor (NGF), injected either with the control virus AdCMVLacZ or with vehicle only, afferent innervation patterns were similar to those of control. However, unilateral injection of AdCMVSema3A/AdCMVLacZ in sc-DRG slices revealed a strong inhibitory effect on NGF-dependent sensory afferent growth. Ectopic Sema3A in the dorsal spinal cord, the target area of NGF-responsive DRG fibers in vivo, created an exclusion zone for these fibers and as a result they failed to reach and innervate their appropriate target zones. Taken together, gain of Sema3A function in the dorsal aspect of sc-DRG cultures revealed a dominant inhibitory effect on NGF-dependent, nociceptive sensory DRG afferents, an observation in line with the model proposed by E. K. Messersmith et al. (1995, Neuron 14, 949–959), suggesting that Sema3A secreted by spinal cord cells can act to repel central sensory fibers during the formation of lamina-specific connections in the spinal cord.

FGF9: a motoneuron survival factor expressed by medial thoracic and sacral motoneurons.

In the nervous system, fibroblast growth factor-9 (FGF9) is produced mainly by neurons. By whole-mount in situ hybridization, on embryonic rat spinal cord, we observed Fgf9 expression in a subpopulation of motoneurons located in the thoracic and sacral regions of the median motor column that innervate the axial muscles. Furthermore, FGF9 prevented death of purified rat and chicken motoneurons in culture in the same concentration range as FGF2. The targets of FGF9 are more restricted than that of the other FGFs, however, because conversely to FGF1 or FGF2, FGF9 had only weak or inexistent survival effects on chicken ciliary neurons or rat DRG. FGF9 may therefore play a role as an autocrine/paracrine survival factor for motoneurons.

Large-scale identification of differentially expressed genes during neurogenesis.

We report here a modified mRNA differential display method and its application for the analysis of differential gene expression in NGF-treated PC12 cells and in embryonic rat spinal cord. The optimized protocol is based on low fidelity priming of multiple cDNAs followed by high fidelity amplification. In PC12 cells induction by nerve growth factor (NGF) altered the expression of 4% of the 466 transcripts evaluated. During neurogenesis of the spinal cord we found that 30% of the 288 examined products changed. The differential expression of the characterized genes was confirmed by independent quantitative PCR. We conclude this method is suitable for the identification of increases and decreases of mRNA levels and allows the discovery of differentially expressed unknown transcripts.

Neuroprotective effect of cyclic GMP against radical-induced toxicity in cultured spinal motor neurons.

We have previously reported that nitric oxide-related cyclic guanosine-3',5'-monophosphate (GMP) protected spinal nonmotor neurons, but not motor neurons against chronic glutamate-induced toxicity, which is associated with selective motor neuronal death after glutamate stress. In this report, we investigated the effect of cyclic GMP against reactive oxygen species (ROS)-induced toxicity in cultured neurons from embryonic rat spinal cords. Pretreatment with a cGMP analogue, 8-bromoguanosine monophosphate (8br-cGMP), for 12-24 hours protected both spinal motor neurons and nonmotor neurons against injury induced by either hydrogen peroxide (H(2)O(2)), or a glutathione depletor, L-buthionine-[S,R]-sulfoximine (BSO). This protective effect was reversed by coadministration with the cGMP-dependent protein kinase (PKG) inhibitor Arg-Lys-Arg-Ala-Arg-Lys-Glu. Interestingly, when cultures were exposed to BSO for 24 hours to allow irreversible inhibition of glutathione synthesis, 8br-cGMP protected only nonmotor neurons. Our results indicate that cGMP attenuates oxidative injury to cultured spinal neurons, in a mechanism associated with glutathione synthesis.

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord.

Intracellular pH was measured with the pH-sensitive fluorescent probe BCECF in spinal cord neurones cultured from rat embryos. At an external pH of 7.3, the average steady-state pHi was 7.18 +/- 0.03 (SEM, n = 97) and 7.02 +/- 0.01 (n = 221) in HEPES-buffered and in bicarbonate-buffered medium, respectively. In both external media, pHi was strongly dependent on external pH (pHe). In HEPES-buffered medium, pHi recovery following an acid load induced by transient application of ammonium required external Na+ and was inhibited by amiloride, indicating the presence of a Na+/H+ exchange. Na(+)- and HCO3(-)-dependent, DIDS-sensitive alkalinizing mechanisms also contributed to pHi regulation in CO2/bicarbonate-buffered medium. The presence of an electrogenic Na(+)-HCO3- cotransporter was confirmed by the alkalinizing effect of KCl application. The fact that pHi is lower in CO2/bicarbonate- than in HEPES-buffered medium and the alkalinization observed upon suppression of external Cl- suggest that the acidifying Cl-/HCO3- transporter plays an important role in defining pHi.

Expression and subcellular localization of two isoforms of the survival motor neuron protein in different cell types.

The survival motor neuron (SMN) gene is deleted or mutated in over 98% of spinal muscular atrophy patients who show specific motoneuron loss. By performing transfection experiments with rat smn cDNA, we show that two isoforms of SMN with Mr of 32 kDa and 35 kDa are produced by the same cDNA. In cultured motoneurons, both forms colocalize in coiled bodies and not in GEMS bodies as shown for HeLa cells. Subcellular fractionation of cells acutely dissociated from rat embryonic ventral spinal cord shows that the two SMN isoforms have a different subcellular localization, namely, that the 32 kDa isoform is enriched in the cytosol, whereas the 35 kDa isoform is segregating in the microsomal fraction. We show that the 35 kDa isoform of SMN is part of an insoluble complex but is absent from the cytoplasmic membranes and from the mitochondria. Immunostaining studies show that neither SMN isoform colocalizes with Bcl-2, the mitochondrial antiapoptotic protein suggested to bind to SMN in HeLa cells. Our results show that the isoforms of SMN protein have different subcellular localization and may therefore play independent biological roles. Moreover, the absence of colocalization of SMN with Bcl-2 in motoneurons suggests that some of the interactors of SMN may vary depending on the cell type, and this underscores the importance of identifying motoneuron-specific SMN interactors.

Heparin blocks functional innervation of cultured human muscle by rat motor nerve.

In vitro innervated human muscle is the only experimental model to study synaptogenesis of the neuromuscular junction in humans. Cultured human muscle never contracts spontaneously but will if innervated and therefore is a suitable model to study the effects of specific neural factors on the formation of functional neuromuscular contacts. Here, we tested the hypothesis that nerve derived factor agrin is essential for the formation of functional synapses between human myotubes and motoneurons growing from the explant of embryonic rat spinal cord. Agrin actions were blocked by heparin and the formation of functional neuromuscular contacts was quantitated. At a heparin concentration of 25 μg/ml, the number of functional contacts was significantly reduced. At higher concentrations, formation of such contacts was blocked completely. Except at the highest heparin concentrations (150 μg/ml) neuronal outgrowth was normal indicating that blockade of neuromuscular junction formation was not due to neuronal dysfunction. Our results are in accord with the concept that binding of neural agrin to the synaptic basal lamina is essential for the formation of functional neuromuscular junctions in the human muscle.

Nerve growth factor (NGF) induces motoneuron apoptosis in rat embryonic spinal cord in vitro.

Recent studies have demonstrated that nerve growth factor (NGF) induces apoptosis of several cell types in the central nervous system through its low-affinity p75 neurotrophin receptor (p75NTR). To test the effect of NGF on embryonic motoneuron survival, we developed an organotypic culture system which allowed the in vitro development of intact embryonic rat spinal cords. In our system, neural tubes were taken and cultured at E13, just before the onset of physiological motoneuron death. After 2 days in vitro (DIV), motoneurons underwent apoptosis over a time-course similar to that in vivo. In this system, the addition of NGF (200 ng/mL) for 2 days enhanced the number of apoptotic motoneurons by 37%. This pro-apoptotic effect was completely reversed by the blocking anti-p75NTR (REX) antibody which inhibits NGF binding to p75NTR. Other neurotrophins, e.g. brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5) did not have any effect, while glial cell-derived neurotrophic factor (GDNF) promoted motoneuron survival. Anti-BDNF blocking antibodies enhanced motoneuron death indicating that endogenous BDNF promotes motoneuron survival in explants. Our results demonstrate, for the first time, that NGF can induce embryonic motoneuron apoptosis through its receptor p75NTR.

Excitotoxic death of a subset of embryonic rat motor neurons in vitro.

We have used cultures of purified embryonic rat spinal cord motor neurons to study the neurotoxic effects of prolonged ionotropic glutamate receptor activation. NMDA and non-NMDA glutamate receptor agonists kill a maximum of 40% of the motor neurons in a concentration- and time-dependent manner, which can be blocked by receptor subtype-specific antagonists. Subunit-specific antibodies stain all of the motor neurons with approximately the same intensity and for the same repertoire of subunits, suggesting that the survival of the nonvulnerable population is unlikely to be due to the lack of glutamate receptor expression. Extracellular Ca2+ is required for excitotoxicity, and the route of entry initiated by activation of non-NMDA, but not NMDA, receptors is L-type Ca2+ channels. Ca2+ imaging of motor neurons after application of specific glutamate receptor agonists reveals a sustained rise in intracellular Ca2+ that is present to a similar degree in most motor neurons, and can be blocked by appropriate receptor/channel antagonists. Although the lethal effects of glutamate receptor agonists are seen in only a subset of cultured motor neurons, the basis of this selectivity is unlikely to be simply the glutamate receptor phenotype or the level/pattern of rise in agonist-evoked intracellular Ca2+.

Mechanism of selective motor neuronal death after exposure of spinal cord to glutamate: involvement of glutamate-induced nitric oxide in motor neuron toxicity and nonmotor neuron protection.

In this study, we analyzed the mechanism of selective motor neuronal death, a characteristic of amyotrophic lateral sclerosis, using embryonic rat spinal cord culture. When dissociated cultures were exposed to low-level glutamate (Glu) coadministered with the Glu transporter inhibitor L-trans-pyrrolidine-2,4-decarboxylate (PDC) for 24 hours, motor neurons were selectively injured through N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. Nitric oxide synthase (NOS) inhibitors attenuated this toxicity, and long-acting nitric oxide (NO) donors damaged motor neurons selectively. Nonmotor neurons survived after exposure to low-dose Glu/PDC, but Glu-induced toxicity was potentiated by coadministration of an NO-dependent guanylyl cyclase inhibitor. In addition, 8-bromo-cyclic GMP, a soluble cyclic GMP analogue, rescued nonmotor neurons, but not motor neurons, exposed to high-dose Glu/PDC. Twenty-four hours' incubation with PDC elevated the number of neuronal NOS-immunoreactive neurons by about twofold compared with controls, and a double-staining study, using the motor neuron marker SMI32, revealed that most of them were nonmotor neurons. These findings suggest that selective motor neuronal death caused by chronic low-level exposure to Glu is mediated by the formation of NO in nonmotor neurons, which inversely protects nonmotor neurons through the guanylyl cyclase-cyclic GMP cascade. Induction of neuronal NOS in nonmotor neurons might enhance both the toxicity of motor neurons and the protection of nonmotor neurons, which could explain the pathology of amyotrophic lateral sclerosis.

Muscle could be the therapeutic target in SMA treatment.

A nerve-muscle coculture model (human muscle cells innervated by embryonic rat spinal cord) was used to explore the pathogenesis of spinal muscular atrophy (SMA). Previous studies showed that myofibers from donors with SMA type I or SMA type II (but not SMA type III) undergo a characteristic degeneration 1-3 weeks after innervation (Braun et al. [1995] Lancet 345:694-695). To determine which cells are involved in degeneration, we cloned satellite cells and fibroblasts derived from muscle biopsies of normal (healthy) donors and donors with SMA. We show that fibroblasts are required for successful innervation, that fibroblasts from normal and SMA donors contribute equally well to the establishment of cocultures, and that only SMA satellite cells are responsible for the degeneration of innervated cocultures. We succeeded in preventing the degeneration of cloned satellite cells from SMA donors by adding 50% cloned satellite cells from normal donors to the culture to make heteromyotubes. In mixed cocultures, after innervation, we did not observe degeneration. This result suggests that survival of the cocultures depends on a message derived from the muscle cells. Consequently, we propose that therapeutic approaches for SMA that could repair (or compensate for) the genetic defect in muscle cells (which are otherwise much more accessible for gene therapy than neurons) might prevent motoneuron degeneration. The role of muscle cells in the establishment and the degeneration of neuromuscular junctions deserves further attention and investigation.