Depletion of SMN Protein in Mesenchymal Progenitors Impairs the Development of Bone and Neuromuscular Junction in Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder characterized by the deficiency of the survival motor neuron (SMN) protein, which leads to motor neuron dysfunction and muscle atrophy. In addition to the requirement for SMN in motor neurons, recent studies suggest that SMN deficiency in peripheral tissues plays a key role in the pathogenesis of SMA. Using limb mesenchymal progenitor cells (MPCs)-specific SMN-depleted mouse models, we reveal that SMN reduction in chondrocytes and fibro-adipogenic progenitors (FAPs) derived from limb MPCs causes defects in the development of bone and neuromuscular junction (NMJ), respectively. We showed that impaired growth plate homeostasis, which causes skeletal growth defects in SMA, is due to reduced IGF signaling from chondrocytes rather than the liver. Furthermore, the reduction of SMN in FAPs resulted in abnormal NMJ maturation, altered release of neurotransmitters, and NMJ morphological defects. Transplantation of healthy FAPs rescued the morphological deterioration. Our findings highlight the significance of mesenchymal SMN in neuromusculoskeletal pathogenesis in SMA and provide insights into potential therapeutic strategies targeting mesenchymal cells for the treatment of SMA.


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The survival motor neuron (SMN) protein is a crucial component of the spliceosome complex 36 and is essential for the proper function of all cell types (Mercuri et al., 2022). Deficiency in 37 SMN protein disrupts the formation of spliceosome complexes, ultimately causing splicing 38 defects in multiple genes. Mutations in the SMN1 gene, which encodes the SMN protein,    maturation is in progress (Figure 2A). Our examination revealed the presence of NF 207 varicosities in SMN2 1-copy mutants as compared with control mice (Figure 2A and 2D).

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Additionally, the number of nerve branches was decreased and half of the total NMJs were 209 poorly arborized in SMN2 1-copy mutants ( Figure 2B-C). These presynaptic alterations are 210 specific phenotypes in neurogenic atrophy like SMA. Unlike neurogenic atrophy, physiologic 211 atrophy shows no differences in presynaptic morphology, such as nerve branching 212 (Deschenes et al., 2006). This suggests that the NMJ phenotypes observed in SMN2 1-copy 213 Smn ΔMPC mutant mice are not caused by decreased muscle size and activity resulting from 214 bone growth abnormalities. The morphology of AChR clusters shows that the mutants have 215 more immature plaque-like NMJs than the controls' pretzel-like structure ( Figure 2E).

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Therefore, these findings indicate that SMN2 1-copy Smn ΔMPC mutants exhibit NMJ maturation abnormalities common in SMA mouse models.

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Undisturbed NMJ formation in neonatal SMN2 1-copy Smn ΔMPC mutants 220 To determine whether any NMJ defects were present prior to juvenile NMJ maturation in 221 SMN2 1-copy Smn ΔMPC , we examined NMJ formation in SMN2 1-copy Smn ΔMPC mice at the 222 neonatal stage on postnatal day 3. We evaluated the AChR and nerve terminal areas to assess 223 postsynaptic and presynaptic development, respectively ( Figure 2F). Measurements of AChR 224 cluster size indicated no differences between control and SMN2 1-copy Smn ΔMPC mice 225 ( Figure 2G). However, the area of AChR covered by nerve terminals was slightly larger in 226 SMN2 1-copy Smn ΔMPC ( Figure 2H). We have no reasonable explanation for why the 227 coverage is higher in the mutant. However, there does not appear to be abnormal 228 development of the NMJ in the mutant, at least until the neonatal period. Therefore, we 229 reasoned that SMN2 1-copy Smn ΔMPC mutants began to exhibit deterioration in the NMJ 230 maturation during the juvenile stage, following the intact neonatal development of NMJ.

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Aberrant NMJ morphology in the adult SMN2 1-copy Smn ΔMPC mice 233 To evaluate the organization of NMJ after the conclusion of postnatal NMJ development, 234 considering mesenchymal SMN expression, we examined NMJ morphology in the TA muscle was quantified, demonstrating nerve terminal shrinkage ( Figure 3B). Additionally, NF ends 240 displayed more severe varicosities than at P21 and were only connected to the proximal nerve 241 by very thin NF, unlike control and SMN2 2-copy mice ( Figure 3C). Remarkably, numerous 242 presynaptic islands formed in SMN2 1-copy Smn ΔMPC mice through the merging of 243 fragmented presynapses and NF varicosity. In SMN2 1-copy mutants, AChR clusters 244 displayed fragmented grape-shaped morphology that overlapped with nerve terminals, 245 whereas control and SMN2 2-copy mice displayed pretzel-like structures ( Figure 3D). These 246 results suggest that defects in adult NMJ morphology occur when mesenchymal SMN protein 247 is reduced to the extent of the SMN2 1-copy Smn ΔMPC mutants. Zanetti 2018). mEPP, a response that occurs when spontaneously released acetylcholine binds 257 to nicotinic AChR without nerve stimulation, was measured ex vivo near the NMJs of the 258 EDL muscles in the control and SMN2 1-copy Smn ΔMPC mice ( Figure 4A). The mEPP 259 amplitude was increased in SMN2 1-copy Smn ΔMPC mice ( Figure 4B), whereas mEPP 260 frequency was comparable between the controls and mutants ( Figure 4C). The results

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indicate that the NMJ synapses of SMN2 1-copy Smn ΔMPC mice are functional and more 262 sensitive to acetylcholine compared to the controls. Next, we measured the eEPP by 263 stimulating an action potential at the peroneal nerve ( Figure 4D). Despite the increased mEPP 264 amplitude, the amplitude of eEPPs was significantly decreased in SMN2 1-copy Smn ΔMPC 265 mice ( Figure 4E). These results suggest that the nerve terminals in SMN2 1-copy Smn ΔMPC 266 mice exhibit decreased quantal content. This could be due to a decrease in vesicle release 267 probability or a reduced readily releasable pool (RRP). Notably, there was no difference in 268 the paired-pulse response, indicating normal neurotransmitter release probability ( Figure 4F).

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Taken together, these findings suggest that the presynaptic neurotransmission ability of the 270 NMJ is reduced in SMN2 1-copy Smn ΔMPC mutants.

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Disturbed nerve terminal structure in SMN2 1-copy Smn ΔMPC mice 273 To examine the NMJ ultrastructure of SMN2 1-copy Smn ΔMPC mutants, we utilized 274 transmission electron microscopy (TEM) ( Figure 5A). The density of junctional folds in 275 SMN2 1-copy Smn ΔMPC mutant specimens was comparable to that of the control ( Figure 5B).

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However, the density of synaptic vesicles was substantially elevated in the SMN2 1-copy 277 mutants ( Figure 5C). Since previous electrophysiological results suggested a decrease in 278 presynaptic neurotransmission capacity in SMN2 1-copy mutants, this could be due to 279 synaptic vesicles failing to fuse with the membrane, leading to the accumulation of vesicles 280 in the terminal. Additionally, the detachment of the nerve terminal is more frequent at the 281 NMJ of mutants ( Figure 5D). The detachment of nerve terminals observed in SMN2 1-copy 282 Smn ΔMPC mutants could have resulted in diminished presynaptic neurotransmission capacity.
terminal-specific pathological defects at the NMJ ultrastructural level.  In this paper, we elucidate the contribution of SMN depletion in mesenchymal progenitors for 303 the pathogenesis of SMA. To test this hypothesis, we generated conditional knockout mouse 304 strains to delete the Smn allele specifically in limb mesenchymal cells and carry human 305 SMN2 copies. Our research using these mouse models resulted in three major discoveries.
First, SMN deficiency in FAPs contributes to NMJ pathological defects in SMA. We 307 observed delayed NMJ maturation and varicosities in juvenile SMN2 1-copy Smn ΔMPC 308 mutant. The pathogenic NMJ phenotypes were also observed in the SMAΔ7 mutant, which is

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We observed reduced bone size and volume in juvenile SMN2 1-copy Smn ΔMPC mutant.              (G) There were no significant differences in AChR cluster size between the SMN2 1-copy control and mutant at P3 (n = 3-4 mice in each genotype; Unpaired t-test with Welch's correction