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

Reviewer #1 (Public Review):

Summary:
The manuscript by Hann et. al examines the role of survival motor neuron protein (SMN) in lateral plate mesoderm-derived cells using the Prrx1Cre to elucidate how changing cell-specific SMN levels coordinate aspects of the spinal muscular atrophy (SMA) pathology. SMN has generally been studied in neuronal cells, and this is one of the first insights into non-neuronal cells that may contribute to SMA disease. The authors generated 3 mouse lines: a Prrx1;Smnf/f conditional null mouse, as well as, single and double copy Prrx1;Smnf/f;SMN2 mice carrying either one or two copies of a human SMN2 transgene. First, the bone development and growth of all three were assessed; the conditional null Smn mutation was lethal shortly after birth, while the SMN2 2-copy mutant did not exhibit bone growth phenotypes. Meanwhile, single-copy SMN2 mutant mice showed reduced size and shorter limbs with shorter proliferative and hypertrophic chondrocyte zones. The authors suggested that this was cell autonomous by assessing the expression of extrinsic factors known to modulate proliferation/differentiation of growth plate chondrocytes. After assessing bone phenotypes, the authors transitioned to the assessments of neuromuscular junction (NMJ) phenotypes, since there are documented neuromuscular impairments in SMA and the Prrx1Cre transgene is expressed in muscle-associated fibro-adipogenic progenitors (FAPs). Neonatal NMJ development was unchanged in mutant mice with two copies of SMN2 , but adult single-copy SMN2 mutant mice had abnormal NMJ morphology, altered presynaptic neurotransmission, and problematic nerve terminal structure. Finally, the authors sought to assess the ability to rescue NMJ phenotypes via FAP cell transplantation and showed wild-type FAPs were able to reduce pre/postsynaptic fragmentation and neurofilament varicosities.

Strengths:
The conditional genetic approaches are novel and interestingly demonstrate the potential for chondrocyte and fibro-adipogenic progenitor-specific contributions to the SMA pathology.

The characterizations of the neuromuscular and NMJ phenotypes are relatively strong.

The data strongly suggest a non-neuronal contribution to SMA, which indicates a need for further mechanistic (cellular and molecular) studies to better understand SMA.

Weaknesses:
The skeletal analyses are not rigorous and likely do not get to the core of how SMN regulates bone development.

The overall work is descriptive and lacks convincing mechanisms.

Additional experimentation is likely needed to fully justify the conclusions.

Reviewer #2 (Public Review):

Summary:
Sang-Hyeon et al. laid out a compelling rationale to explore the role of the SMN protein in mesenchymal cells, to determine whether SMN deficiency there could be a pathologic mechanism of SMA. They crossed Smnf7/f7 mice with Prrx1Cre mice to produce SmnΔMPC mice where exon 7 was specifically deleted and thus SMN protein was eliminated in limb mesenchymal progenitor cells (MPCs). To demonstrate gene dosage-dependence of phenotypes, SmnΔMPC mice were crossed with transgenic mice expressing human SMN2 to produce SmnΔMPC mice with different copies of SMN2 (0, 1, or 2). The paper provides genetic evidence that SMN in mesenchymal cells regulates the development of bones and neuromuscular junctions. Genetic data were convincing and revealed novel functions of SMN.

Strengths:
Overall, the paper provided genetic evidence that SMN deficiency in mesenchymal cells caused abnormalities in bones and NMJs, revealing novel functions of SMN and leading to future experiments. As far as genetics is concerned, the data were convincing (except for the rescue experiment, see below); the conclusions are important.

Weaknesses:
The paper seemed to be descriptive in nature and could be improved with more experiments to investigate underlying mechanisms. In addition, some data appeared to be contradicting or difficult to explain. The rescue data were not convincing.

Reviewer #3 (Public Review):

Summary:
SMN expression in non-neuronal cells, particularly in limb mesenchymal progenitors is essential for the proper growth of chondrocytes and the formation of adult NMJ junctions.

Strengths:
The authors show copy numbers of smndelta7 in MPC influence NMJ structure.

Weaknesses:
Functional recovery by FAP transplantation is not complete. Mesenchymal progenitors are heterogeneous, and how heterogeneity influences this study is not clear. Part of the main findings to show the importance of SMN expression in non-neuronal cells is partly published by the same group (Kim et al., JCI Insight 2022). In the study, the authors used Dpp4(+) cells. The difference between the current study and the previous study is not so clear.