Calpain system is altered in survival motor neuron-reduced cells from in vitro and in vivo spinal muscular atrophy models

Spinal Muscular Atrophy is clinically recognized as a progressive weakness within the trunk and proximal limbs that will lead to breathing failure and death within infants. As a neurodegenerative genetic disease, SMA is caused by loss of motor neurons, which in turn is caused by low levels of the Survival Motor Neuron (SMN) protein. The mechanism by which a ubiquitously expressed protein such as SMN is able to cause the specific death of motor neurons is highly debated and of great interest. Work presented here focuses on understanding the biological requirements of SMN and its downstream effects on the neuromuscular junction. To this end we utilize viral based gene delivery as a powerful tool to assess the effects of genes of interest in vivo. Our findings contribute to the conversation regarding whether SMA is truly a "motor neuron" disease, suggesting that astrocytes play a meaningful role in staving off SMA. Further, we investigate the domains within SMN needed to maintain its function in a mammalian system. We take a novel and challenging approach to identify a minimal domain capable of maintaining function. Finally, we demonstrate the practical use of morophological analysis of the neuromuscular junction as a means to characterize SMA pathology.

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Fluorescence Correlation Spectroscopy Reveals Survival Motor Neuron Oligomerization but No Active Transport in Motor Axons of a Zebrafish Model for Spinal Muscular Atrophy

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.639904 ◽

2021 ◽

Vol 9 ◽

Author(s):

Angela Koh ◽

Menachem Viktor Sarusie ◽

Jürgen Ohmer ◽

Utz Fischer ◽

Christoph Winkler ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Active Transport ◽

Fluorescence Correlation Spectroscopy ◽

Muscular Atrophy ◽

Correlation Spectroscopy ◽

Survival Motor Neuron ◽

Transgenic Zebrafish ◽

Fluorescence Correlation

Spinal Muscular Atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons that is caused by a deficiency in ubiquitously expressed Survival Motor Neuron (SMN) protein. Two mutually exclusive hypotheses have been discussed to explain increased motor neuron vulnerability in SMA. Reduced SMN levels have been proposed to lead to defective snRNP assembly and aberrant splicing of transcripts that are essential for motor neuron maintenance. An alternative hypothesis proposes a motor neuron-specific function for SMN in axonal transport of mRNAs and/or RNPs. To address these possibilities, we used a novel in vivo approach with fluorescence correlation spectroscopy (FCS) in transgenic zebrafish embryos to assess the subcellular dynamics of Smn in motor neuron cell bodies and axons. Using fluorescently tagged Smn we show that it exists as two freely diffusing components, a monomeric, and a complex-bound, likely oligomeric, component. This oligomer hypothesis was supported by the disappearance of the complex-bound form for a truncated Smn variant that is deficient in oligomerization and a change in its dynamics under endogenous Smn deficient conditions. Surprisingly, our FCS measurements did not provide any evidence for an active transport of Smn in axons. Instead, our in vivo observations are consistent with previous findings that SMN acts as a chaperone for the assembly of snRNP and mRNP complexes.

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Effects of Survival Motor Neuron Protein on Germ Cell Development in Mouse and Human

International Journal of Molecular Sciences ◽

10.3390/ijms22020661 ◽

2021 ◽

Vol 22 (2) ◽

pp. 661

Author(s):

Wei-Fang Chang ◽

Min Peng ◽

Jing Hsu ◽

Jie Xu ◽

Huan-Chieh Cho ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Motor Neuron ◽

Cell Biology ◽

Muscular Atrophy ◽

Primordial Germ Cell ◽

Cell Types ◽

Survival Motor Neuron

Survival motor neuron (SMN) is ubiquitously expressed in many cell types and its encoding gene, survival motor neuron 1 gene (SMN1), is highly conserved in various species. SMN is involved in the assembly of RNA spliceosomes, which are important for pre-mRNA splicing. A severe neurogenic disease, spinal muscular atrophy (SMA), is caused by the loss or mutation of SMN1 that specifically occurred in humans. We previously reported that SMN plays roles in stem cell biology in addition to its roles in neuron development. In this study, we investigated whether SMN can improve the propagation of spermatogonia stem cells (SSCs) and facilitate the spermatogenesis process. In in vitro culture, SSCs obtained from SMA model mice showed decreased growth rate accompanied by significantly reduced expression of spermatogonia marker promyelocytic leukemia zinc finger (PLZF) compared to those from heterozygous and wild-type littermates; whereas SMN overexpressed SSCs showed enhanced cell proliferation and improved potency. In vivo, the superior ability of homing and complete performance in differentiating progeny was shown in SMN overexpressed SSCs in host seminiferous tubule of transplant experiments compared to control groups. To gain insights into the roles of SMN in clinical infertility, we derived human induced pluripotent stem cells (hiPSCs) from azoospermia patients (AZ-hiPSCs) and from healthy control (ct-hiPSCs). Despite the otherwise comparable levels of hallmark iPCS markers, lower expression level of SMN1 was found in AZ-hiPSCs compared with control hiPSCs during in vitro primordial germ cell like cells (PGCLCs) differentiation. On the other hand, overexpressing hSMN1 in AZ-hiPSCs led to increased level of pluripotent markers such as OCT4 and KLF4 during PGCLC differentiation. Our work reveal novel roles of SMN in mammalian spermatogenesis and suggest new therapeutic targets for azoospermia treatment.

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SMN-primed ribosomes modulate the translation of transcripts related to Spinal Muscular Atrophy

10.1101/751701 ◽

2019 ◽

Author(s):

F Lauria ◽

P Bernabò ◽

T Tebaldi ◽

EJN Groen ◽

E Perenthaler ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Protein Loss ◽

Molecular Control ◽

Coding Sequence ◽

Rare Codons ◽

Smn Protein

AbstractThe contribution of ribosome heterogeneity and ribosome-associated factors to the molecular control of proteomes in health and disease remains enigmatic. We demonstrate that Survival Motor Neuron (SMN) protein, loss of which causes the neuromuscular disease spinal muscular atrophy (SMA), binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are positioned within the first five codons of a set of mRNAs which are enriched in IRES-like sequences in the 5’UTR and rare codons at the beginning of their coding sequence. Loss of SMN at early-stages of SMA induces translational defects in vivo, characterized by ribosome depletion in rare codons at the third and fifth position of the coding sequence. These positional defects cause ribosome depletion from mRNAs bound by SMN-primed ribosomes and translational impairment of proteins involved in motor neuron function and stability, including acetylcholinesterase. Thus, SMN plays a crucial role in the regulation of ribosome fluxes along mRNAs which encode proteins relevant to SMA pathogenesis.

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Discovery of a CNS penetrant small molecule SMN2 splicing modulator with improved tolerability for spinal muscular atrophy

Scientific Reports ◽

10.1038/s41598-020-74346-9 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Shiori Ando ◽

Shunya Suzuki ◽

Shoichi Okubo ◽

Kazuki Ohuchi ◽

Kei Takahashi ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Small Molecule ◽

Therapeutic Potential ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Tolerability Profile ◽

Loss Of Function ◽

Cryptic Exon

Abstract Spinal muscular atrophy (SMA) is a motor neuron disease, typically resulting from loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Nusinersen/SPINRAZA, a splice-switching oligonucleotide that modulates SMN2 (a paralog of SMN1) splicing and consequently increases SMN protein levels, has a therapeutic effect for SMA. Previously reported small-molecule SMN2 splicing modulators such as risdiplam/EVRYSDI and its analog SMN-C3 modulate not only the splicing of SMN2 but also that of secondary splice targets, including forkhead box protein M1 (FOXM1). Through screening SMA patient-derived fibroblasts, a novel small molecule, designated TEC-1, was identified that selectively modulates SMN2 splicing over three secondary splice targets. TEC-1 did not strongly affect the splicing of FOXM1, and unlike risdiplam, did not induce micronucleus formation. In addition, TEC-1 showed higher selectively on galactosylceramidase and huntingtin gene expression compared to previously reported compounds (e.g., SMN-C3) due to off-target effects on cryptic exon inclusion and nonsense-mediated mRNA decay. Moreover, TEC-1 significantly ameliorated the disease phenotype in an SMA murine model in vivo. Thus, TEC-1 may have promising therapeutic potential for SMA, and our study demonstrates the feasibility of RNA-targeting small-molecule drug development with an improved tolerability profile.

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