A splicing silencer in SMN2 intron 6 is critical in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a fatal neuromuscular disease caused by homozygous deletions or mutations of the SMN1 gene. SMN2 is a paralogous gene of SMN1 and a modifying gene of SMA. A better understanding of how SMN2 exon 7 splicing is regulated helps discover new therapeutic targets for SMA therapy. Based on an antisense walk method to map exonic and intronic splicing silencers (ESSs and ISSs) in SMN2 exon 7 and the proximal regions of its flanking introns, we identified one ISS (ISS6-KH) at upstream of the branch point site in intron 6. By using mutagenesis-coupled RT-PCR with SMN1/2 minigenes, immunochromatography, overexpression and siRNA-knockdown, we found this ISS consists of a bipartite hnRNP A1 binding cis-element and a poly-U sequence located between the proximal hnRNP A1 binding site (UAGCUA) and the branch site. Both HuR and hnRNP C1 proteins promote exon 7 skipping through the poly-U stretch. Mutations or deletions of these motifs lead to efficient SMN2 exon 7 inclusion comparable to SMN1 gene. Furthermore, we identified an optimal antisense oligonucleotide that binds the intron six ISS and causes striking exon 7 inclusion in the SMN2 gene in patient fibroblasts and SMA mouse model. Our findings demonstrate that this novel ISS plays an important role in SMN2 exon 7 skipping and highlight a new therapeutic target for SMA therapy.

Intraocular pressure across the lifespan of Tg-MYOCY437H mice.

Transgenic C57BL/6 mice expressing human myocilinY437 (Tg-MYOCY437H) are a well-established model for primary open-angle glaucoma (POAG). While the reduced trabecular meshwork (TM) cellularity due to severe endoplasmic reticulum (ER) stress has been characterized as the etiology of this model, there is a limited understanding of how glaucomatous phenotypes evolve over the lifespan of Tg-MyocY437H mice. In this study, we compiled the model's intraocular pressure (IOP) data recorded in our laboratory from 2017 to 2023 and selected representative eyes to measure the outflow facility (Cr), a critical parameter indicating the condition of the conventional TM pathway. We found that Tg-MYOCY437H mice aged 4-12 months exhibited significantly higher IOPs than age-matched C57BL/6 mice. Notably, a decline in IOP was observed in Tg-MYOCY437H mice at 17-24 months of age, a phenomenon not attributable to the gene dosage of mutant myocilin. Measurements of the Cr of Tg-MYOCY437H mice indicated that the age-related IOP reduction was not a result of ongoing TM damage. Instead, Hematoxylin and Eosin staining, immunohistochemistry analysis, and transmission electron microscopic examination revealed that this reduction might be induced by degenerations of the non-pigmented epithelium in the ciliary body of aged Tg-MYOCY437H mice. Overall, our findings provide a comprehensive profile of mutant myocilin-induced ocular changes over the Tg-MYOCY437H mouse lifespan and suggest a specific temporal window of elevated IOP that may be ideal for experimental purposes.

A severe mouse model of spinal muscular atrophy develops early systemic inflammation.

Spinal muscular atrophy (SMA) is a fatal genetic disease, mainly affecting children. A number of recent studies show, aside from lower motor neuron degeneration and atrophy of skeletal muscles, widespread defects present in the central nervous system (CNS) and peripheral non-neuronal cell types of SMA patients and mouse models, particularly of severe forms. However, molecular mechanisms underlying the multi-organ manifestations of SMA were hardly understood. Here, using histology, flow cytometry and gene expression analysis in both messenger RNA and protein levels in various tissues, we found that a severe SMA mouse model develops systemic inflammation in early symptomatic stages. SMA mice had an enhanced intestinal permeability, resulting in microbial invasion into the circulatory system. Expression of proinflammatory cytokines was increased in all tissues and the acute phase response in the liver was activated. Systemic inflammation further mobilized glucocorticoid signaling and in turn led to dysregulation of a large set of genes, including robust upregulation of FAM107A in the spinal cord, increased expression of which has been implicated in neurodegeneration. Moreover, we show that lipopolysaccharide challenge markedly suppressed survival of motor neuron 2 exon 7 splicing in all examined peripheral and CNS tissues, resulting in global survival of motor neuron level reduction. Therefore, we identified a novel pathological mechanism in a severe SMA mouse model, which affects phenotypic severity through multiple paths and should contribute to progression of broad neuronal and non-neuronal defects.

Downregulation of Survivin contributes to cell-cycle arrest during postnatal cardiac development in a severe spinal muscular atrophy mouse model.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, characterized by progressive degeneration of spinal-cord motor neurons, leading to atrophy of skeletal muscles. However, accumulating evidence indicates that it is a multi-system disorder, particularly in its severe forms. Several studies delineated structural and functional cardiac abnormalities in SMA patients and mouse models, yet the abnormalities have been primarily attributed to autonomic dysfunction. Here, we show in a severe mouse model that its cardiomyocytes undergo G0/G1 cell-cycle arrest and enhanced apoptosis during postnatal development. Microarray and real-time RT-PCR analyses revealed that a set of genes associated with cell cycle and apoptosis were dysregulated in newborn pups. Of particular interest, the Birc5 gene, which encodes Survivin, an essential protein for heart development, was down-regulated even on pre-symptomatic postnatal day 0. Interestingly, cultured cardiomyocytes depleted of SMN recapitulated the gene expression changes including downregulation of Survivin and abnormal cell-cycle progression; and overexpression of Survivin rescued the cell-cycle defect. Finally, increasing SMN in SMA mice with a therapeutic antisense oligonucleotide improved heart pathology and recovered expression of deregulated genes. Collectively, our data demonstrate that the cardiac malfunction of the severe SMA mouse model is mainly a cell-autonomous defect, caused by widespread gene deregulation in heart tissue, particularly of Birc5, resulting in developmental abnormalities through cell-cycle arrest and apoptosis.

iPSC-derived cells stimulate ABCG2+/NES+ endogenous trabecular meshwork cell proliferation and tissue regeneration.

A major risk factor for glaucoma, the first leading cause of irreversible blindness worldwide, is the decellularisation of the trabecular meshwork (TM) in the conventional outflow pathway. Stem cell-based therapy, particularly the utilisation of induced pluripotent stem cells (iPSCs), presents an enticing potential for tissue regeneration and intraocular pressure (IOP) maintenance in glaucoma. We have previously observed that differentiated iPSCs can stimulate endogenous cell proliferation in the TM, a pivotal factor in TM regeneration and aqueous humour outflow restoration. In this study, we investigated the response of TM cells in vivo after interacting with iPSC-derived cells and identified two subpopulations responsible for this relatively long-term tissue regeneration: ATP Binding Cassette Subfamily G Member 2 (ABCG2)-positive cells and Nestin (NES)-positive cells. We further uncovered that alterations of these responsive cells are linked to ageing and different glaucoma etiologies, suggesting that ABCG2+ subpopulation decellularization could serve as a potential risk factor for TM decellularization in glaucoma. Taken together, our findings illustrated the proliferative subpopulations in the conventional outflow pathway when stimulated with iPSC-derived cells and defined them as TM precursors, which may be applied to develop novel therapeutic approaches for glaucoma.

Application of a Magnetic Platform in α6 Integrin-Positive iPSC-TM Purification.

The emergence of induced pluripotent stem cell (iPSC) technology has provided a new approach to regenerating decellularized trabecular meshwork (TM) in glaucoma. We have previously generated iPSC-derived TM (iPSC-TM) using a medium conditioned by TM cells and verified its function in tissue regeneration. Because of the heterogeneity of iPSCs and the isolated TM cells, iPSC-TM cells appear to be heterogeneous, which impedes our understanding of how the decellularized TM may be regenerated. Herein, we developed a protocol based on a magnetic-activated cell sorting (MACS) system or an immunopanning (IP) method for sorting integrin subunit alpha 6 (ITGA6)-positive iPSC-TM, an example of the iPSC-TM subpopulation. We first analyzed the purification efficiency of these two approaches by flow cytometry. In addition, we also determined cell viability by analyzing the morphologies of the purified cells. To conclude, the MACS-based purification could yield a higher ratio of ITGA6-positive iPSC-TM and maintain a relatively higher cell viability than the IP-based method, allowing for the preparation of any iPSC-TM subpopulation of interest and facilitating a better understanding of the regenerative mechanism of iPSC-based therapy.

History of development of the life-saving drug "Nusinersen" in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder with an incidence of 1/6,000-1/10,000 and is the leading fatal disease among infants. Previously, there was no effective treatment for SMA. The first effective drug, nusinersen, was approved by the US FDA in December 2016, providing hope to SMA patients worldwide. The drug was introduced in the European Union in 2017 and China in 2019 and has so far saved the lives of several patients in most parts of the world. Nusinersen are fixed sequence antisense oligonucleotides with special chemical modifications. The development of nusinersen progressed through major scientific discoveries in medicine, genetics, biology, and other disciplines, wherein several scientists have made substantial contributions. In this article, we will briefly describe the pathogenesis and therapeutic strategies of SMA, summarize the timeline of important scientific findings during the development of nusinersen in a detailed, scientific, and objective manner, and finally discuss the implications of the development of nusinersen for SMA research.

Rescue of mis-splicing of a common SLC26A4 mutant associated with sensorineural hearing loss by antisense oligonucleotides.

A wide spectrum of SLC26A4 mutations causes Pendred syndrome and enlarged vestibular aqueduct, both associated with sensorineural hearing loss (SNHL). A splice-site mutation, c.919-2A>G (A-2G), which is common in Asian populations, impairs the 3' splice site of intron 7, resulting in exon 8 skipping during pre-mRNA splicing and a subsequent frameshift that creates a premature termination codon in the following exon. Currently, there is no effective drug treatment for SHNL. For A-2G-triggered SNHL, molecules that correct mis-splicing of the mutant hold promise to treat the disease. Antisense oligonucleotides (ASOs) can promote exon inclusion when targeting specific splicing silencers. Here, we systematically screened a large number of ASOs in a minigene system and identified a few that markedly repressed exon 8 skipping. A lead ASO, which targets a heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 intronic splicing silencer (ISS) in intron 8, promoted efficient exon 8 inclusion in cultured peripheral blood mononuclear cells derived from two homozygous patients. In a partially humanized Slc26a4 A-2G mouse model, two subcutaneous injections of the ASO at 160 mg/kg significantly rescued exon 8 splicing in the liver. Our results demonstrate that the ISS-targeting ASO has therapeutic potential to treat genetic hearing loss caused by the A-2G mutation in SLC26A4.