Physiological Features of the Neural Stem Cells Obtained from an Animal Model of Spinal Muscular Atrophy and Their Response to Antioxidant Curcumin.

The most prevalent rare genetic disease affecting young individuals is spinal muscular atrophy (SMA), which is caused by a loss-of-function mutation in the telomeric gene survival motor neuron (SMN) 1. The high heterogeneity of the SMA pathophysiology is determined by the number of copies of SMN2, a separate centromeric gene that can transcribe for the same protein, although it is expressed at a slower rate. SMA affects motor neurons. However, a variety of different tissues and organs may also be affected depending on the severity of the condition. Novel pharmacological treatments, such as Spinraza, Onasemnogene abeparvovec-xioi, and Evrysdi, are considered to be disease modifiers because their use can change the phenotypes of the patients. Since oxidative stress has been reported in SMA-affected cells, we studied the impact of antioxidant therapy on neural stem cells (NSCs) that have the potential to differentiate into motor neurons. Antioxidants can act through various pathways; for example, some of them exert their function through nuclear factor (erythroid-derived 2)-like 2 (NRF2). We found that curcumin is able to induce positive effects in healthy and SMA-affected NSCs by activating the nuclear translocation of NRF2, which may use a different mechanism than canonical redox regulation through the antioxidant-response elements and the production of antioxidant molecules.

Beyond Motor Neurons in Spinal Muscular Atrophy: A Focus on Neuromuscular Junction.

5q-Spinal muscular atrophy (5q-SMA) is one of the most common neuromuscular diseases due to homozygous mutations in the SMN1 gene. This leads to a loss of function of the SMN1 gene, which in the end determines lower motor neuron degeneration. Since the generation of the first mouse models of SMA neuropathology, a complex degenerative involvement of the neuromuscular junction and peripheral axons of motor nerves, alongside lower motor neurons, has been described. The involvement of the neuromuscular junction in determining disease symptoms offers a possible parallel therapeutic target. This narrative review aims at providing an overview of the current knowledge about the pathogenesis and significance of neuromuscular junction dysfunction in SMA, circulating biomarkers, outcome measures and available or developing therapeutic approaches.

Mutant androgen receptor induces neurite loss and senescence independently of ARE binding in a neuronal model of SBMA.

Spinal and bulbar muscular atrophy (SBMA) is a slowly progressing neuromuscular disease caused by a polyglutamine (polyQ)-encoding CAG trinucleotide repeat expansion in the androgen receptor (AR) gene, leading to AR aggregation, lower motor neuron death, and muscle atrophy. AR is a ligand-activated transcription factor that regulates neuronal architecture and promotes axon regeneration; however, whether AR transcriptional functions contribute to disease pathogenesis is not fully understood. Using a differentiated PC12 cell model of SBMA, we identified dysfunction of polyQ-expanded AR in its regulation of neurite growth and maintenance. Specifically, we found that in the presence of androgens, polyQ-expanded AR inhibited neurite outgrowth, induced neurite retraction, and inhibited neurite regrowth. This dysfunction was independent of polyQ-expanded AR transcriptional activity at androgen response elements (ARE). We further showed that the formation of polyQ-expanded AR intranuclear inclusions promoted neurite retraction, which coincided with reduced expression of the neuronal differentiation marker ß-III-Tubulin. Finally, we revealed that cell death is not the primary outcome for cells undergoing neurite retraction; rather, these cells become senescent. Our findings reveal that mechanisms independent of AR canonical transcriptional activity underly neurite defects in a cell model of SBMA and identify senescence as a pathway implicated in this pathology. These findings suggest that in the absence of a role for AR canonical transcriptional activity in the SBMA pathologies described here, the development of SBMA therapeutics that preserve this activity may be desirable. This approach may be broadly applicable to other polyglutamine diseases such as Huntington's disease and spinocerebellar ataxias.

Effect of E134K pathogenic mutation of SMN protein on SMN-SmD1 interaction, with implication in spinal muscular atrophy: A molecular dynamics study.

Spinal muscular atrophy (SMA) is a disease that results from mutations in the Survival of Motor Neuron (SMN) gene 1, leading to muscle atrophy due to motor neurons degeneration. SMN plays a crucial role in the assembly of spliceosomal small nuclear ribonucleoprotein complexes via binding to the arginine-glycine rich C-terminal tails of Sm proteins recognized by SMN Tudor domain. E134K Tudor mutation, cause of the more severe type I SMA, compromises the SMN-Sm interaction without a perturbation of the domain fold. By molecular dynamics simulations, we investigated the mechanism of Tudor-SmD1 interaction, and the effects on it of E134K mutation. It was observed that E134 is crucial to catch the positive dimethylated arginines (DMRs) of the SmD1 tail that, wrapping around the acidic Tudor surface, enters a central DMR into an aromatic cage. The flexible cage residue Y130 must be blocked from the wrapped tail to assure a stable binding. The charge inversion in E134K mutation causes the loss of a critical anchor point, disfavoring the tail wrapping and leaving Y130 free to swing, leading to DMR detachments and exposition of the C-terminal region of the tail. This could suggest new hypotheses regarding a possible autoimmune response by anti-Sm autoantibodies.

What could be the function of the spinal muscular atrophy-causing protein SMN in macrophages?

Spinal Muscular Atrophy (SMA), a neurodegenerative disorder, extends its impact beyond the nervous system. The central protein implicated in SMA, Survival Motor Neuron (SMN) protein, is ubiquitously expressed and functions in fundamental processes such as alternative splicing, translation, cytoskeletal dynamics and signaling. These processes are relevant for all cellular systems, including cells of the immune system such as macrophages. Macrophages are capable of modulating their splicing, cytoskeleton and expression profile in order to fulfil their role in tissue homeostasis and defense. However, less is known about impairment or dysfunction of macrophages lacking SMN and the subsequent impact on the immune system of SMA patients. We aimed to review the potential overlaps between SMN functions and macrophage mechanisms highlighting the need for future research, as well as the current state of research addressing the role of macrophages in SMA.

From data to discovery: AI-guided analysis of disease-relevant molecules in spinal muscular atrophy (SMA).

Spinal Muscular Atrophy is caused by partial loss of survival of motoneuron (SMN) protein expression. The numerous interaction partners and mechanisms influenced by SMN loss result in a complex disease. Current treatments restore SMN protein levels to a certain extent, but do not cure all symptoms. The prolonged survival of patients creates an increasing need for a better understanding of SMA. Although many SMN-protein interactions, dysregulated pathways, and organ phenotypes are known, the connections among them remain largely unexplored. Monogenic diseases are ideal examples for the exploration of cause-and-effect relationships to create a network describing the disease-context. Machine learning tools can utilize such knowledge to analyze similarities between disease-relevant molecules and molecules not described in the disease so far. We used an artificial intelligence-based algorithm to predict new genes of interest. The transcriptional regulation of 8 out of 13 molecules selected from the predicted set were successfully validated in an SMA mouse model. This bioinformatic approach, using the given experimental knowledge for relevance predictions, enhances efficient targeted research in SMA and potentially in other disease settings.

Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes.

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2 (hereinafter referred to as SMN1/2), produce multiple circular RNAs (circRNAs), including C2A-2B-3-4 that encompasses early exons 2A, 2B, 3 and 4. C2A-2B-3-4 is a universally and abundantly expressed circRNA of SMN1/2. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A-2B-3-4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4172 genes) by C2A-2B-3-4. About half of the affected genes by C2A-2B-3-4 remained unaffected by L2A-2B-3-4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/2. These findings underscore the unique role of the structural context of C2A-2B-3-4 in gene regulation. A surprisingly high number of upregulated genes by C2A-2B-3-4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/2 transcripts, C2A-2B-3-4 and L2A-2B-3-4 upregulated and downregulated SMN1/2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A-2B-3-4 with very limited overlap with those affected by L2A-2B-3-4. Independent validations confirmed the effect of C2A-2B-3-4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A-2B-3-4, and expands our understanding of functions of SMN1/2 genes.

Systemic LPS Administration Stimulates the Activation of Non-Neuronal Cells in an Experimental Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by deficiency of the survival motor neuron (SMN) protein. Although SMA is a genetic disease, environmental factors contribute to disease progression. Common pathogen components such as lipopolysaccharides (LPS) are considered significant contributors to inflammation and have been associated with muscle atrophy, which is considered a hallmark of SMA. In this study, we used the SMNΔ7 experimental mouse model of SMA to scrutinize the effect of systemic LPS administration, a strong pro-inflammatory stimulus, on disease outcome. Systemic LPS administration promoted a reduction in SMN expression levels in CNS, peripheral lymphoid organs, and skeletal muscles. Moreover, peripheral tissues were more vulnerable to LPS-induced damage compared to CNS tissues. Furthermore, systemic LPS administration resulted in a profound increase in microglia and astrocytes with reactive phenotypes in the CNS of SMNΔ7 mice. In conclusion, we hereby show for the first time that systemic LPS administration, although it may not precipitate alterations in terms of deficits of motor functions in a mouse model of SMA, it may, however, lead to a reduction in the SMN protein expression levels in the skeletal muscles and the CNS, thus promoting synapse damage and glial cells' reactive phenotype.

Molecular Mechanisms of Medicinal Plant Securinega suffruticosa-derived Compound Securinine against Spinal Muscular Atrophy based on Network Pharmacology and Experimental Verification.

BACKGROUND: Spinal Muscular Atrophy (SMA) is a severe motor neuronal disorder with high morbidity and mortality. Securinine has shown the potential to treat SMA; however, its anti-SMA role remains unclear. OBJECTIVE: This study aims to reveal the anti-SMA mechanisms of securinine. METHODS: Securinine-associated targets were acquired from Herbal Ingredients' Targets (HIT), Similarity Ensemble Approach (SEA), and SuperPred. SMA-associated targets were obtained from GeneCards and Dis- GeNET. Protein-protein Interaction (PPI) network was constructed using GeneMANIA, and hug targets were screened using cytoHubba. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using ClusterProfifiler. Molecular docking was conducted using Pymol and Auto- Dock. In vitro assays were used to verify the anti-SMA effects of securinine. RESULTS: Twenty-six intersection targets of securinine and SMA were obtained. HDAC1, HDAC2, TOP2A, PIK3R1, PRMT5, JAK2, HSP90AB1, TERT, PTGS2, and PAX8 were the core targets in PPI network. GO analysis demonstrated that the intersecting targets were implicated in the regulation of proteins, steroid hormones, histone deacetylases, and DNA transcription. KEGG analysis, pathway-pathway, and hub target-pathway networks revealed that securinine might treat SMA through TNF, JAK-STAT, Ras, and PI3K-Akt pathways. Securinine had a favorable binding affinity with HDAC1, HSP90AB, JAK2, PRMT5, PTGS2, and TERT. Securinine rescued viability suppression, mitochondria damage, and SMN loss in the SMA cell model. Furthermore, securinine increased HDAC1 and PRMT5 expression, decreased PTGS2 expression, suppressed the JAK2-STAT3 pathway, and promoted the PI3K-Akt pathway. CONCLUSION: Securinine might alleviate SMA by elevating HDAC1 and PRMT5 expression and reducing PTGS2 via JAK2-STAT3 suppression and PI3K-Akt activation.

The SMN-ribosome interplay: a new opportunity for Spinal Muscular Atrophy therapies.

The underlying cause of Spinal Muscular Atrophy (SMA) is in the reduction of survival motor neuron (SMN) protein levels due to mutations in the SMN1 gene. The specific effects of SMN protein loss and the resulting pathological alterations are not fully understood. Given the crucial roles of the SMN protein in snRNP biogenesis and its interactions with ribosomes and translation-related proteins and mRNAs, a decrease in SMN levels below a specific threshold in SMA is expected to affect translational control of gene expression. This review covers both direct and indirect SMN interactions across various translation-related cellular compartments and processes, spanning from ribosome biogenesis to local translation and beyond. Additionally, it aims to outline deficiencies and alterations in translation observed in SMA models and patients, while also discussing the implications of the relationship between SMN protein and the translation machinery within the context of current and future therapies.

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 cell (MPC)-specific SMN-depleted mouse models, we reveal that SMN reduction in limb MPCs causes defects in the development of bone and neuromuscular junction (NMJ). Specifically, these mice exhibited impaired growth plate homeostasis and reduced insulin-like growth factor (IGF) signaling from chondrocytes, rather than from the liver. Furthermore, the reduction of SMN in fibro-adipogenic progenitors (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 of SMA and provide insights into potential therapeutic strategies targeting mesenchymal cells for the treatment of SMA.

A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

SMN deficiency perturbs monoamine neurotransmitter metabolism in spinal muscular atrophy.

Beyond motor neuron degeneration, homozygous mutations in the survival motor neuron 1 (SMN1) gene cause multiorgan and metabolic defects in patients with spinal muscular atrophy (SMA). However, the precise biochemical features of these alterations and the age of onset in the brain and peripheral organs remain unclear. Using untargeted NMR-based metabolomics in SMA mice, we identify cerebral and hepatic abnormalities related to energy homeostasis pathways and amino acid metabolism, emerging already at postnatal day 3 (P3) in the liver. Through HPLC, we find that SMN deficiency induces a drop in cerebral norepinephrine levels in overt symptomatic SMA mice at P11, affecting the mRNA and protein expression of key genes regulating monoamine metabolism, including aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DßH) and monoamine oxidase A (MAO-A). In support of the translational value of our preclinical observations, we also discovered that SMN upregulation increases cerebrospinal fluid norepinephrine concentration in Nusinersen-treated SMA1 patients. Our findings highlight a previously unrecognized harmful influence of low SMN levels on the expression of critical enzymes involved in monoamine metabolism, suggesting that SMN-inducing therapies may modulate catecholamine neurotransmission. These results may also be relevant for setting therapeutic approaches to counteract peripheral metabolic defects in SMA.

Differential effect of Fas activation on spinal muscular atrophy motoneuron death and induction of axonal growth.

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the most common motoneuron diseases affecting adults and infants, respectively. ALS and SMA are both characterized by the selective degeneration of motoneurons. Although different in their genetic etiology, growing evidence indicates that they share molecular and cellular pathogenic signatures that constitute potential common therapeutic targets. We previously described a motoneuron-specific death pathway elicited by the Fas death receptor, whereby vulnerable ALS motoneurons show an exacerbated sensitivity to Fas activation. However, the mechanisms that drive the loss of SMA motoneurons remains poorly understood. Here, we describe an in vitro model of SMA-associated degeneration using primary motoneurons derived from Smn2B/- SMA mice and show that Fas activation selectively triggers death of the proximal motoneurons. Fas-induced death of SMA motoneurons has the molecular signature of the motoneuron-selective Fas death pathway that requires activation of p38 kinase, caspase-8, -9 and -3 as well as upregulation of collapsin response mediator protein 4 (CRMP4). In addition, Rho-associated Kinase (ROCK) is required for Fas recruitment. Remarkably, we found that exogenous activation of Fas also promotes axonal elongation in both wildtype and SMA motoneurons. Axon outgrowth of motoneurons promoted by Fas requires the activity of ERK, ROCK and caspases. This work defines a dual role of Fas signaling in motoneurons that can elicit distinct responses from cell death to axonal growth.

Mitigating aberrant Cdk5 activation alleviates mitochondrial defects and motor neuron disease symptoms in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the top genetic cause of infant mortality, is characterized by motor neuron degeneration. Mechanisms underlying SMA pathogenesis remain largely unknown. Here, we report that the activity of cyclin-dependent kinase 5 (Cdk5) and the conversion of its activating subunit p35 to the more potent activator p25 are significantly up-regulated in mouse models and human induced pluripotent stem cell (iPSC) models of SMA. The increase of Cdk5 activity occurs before the onset of SMA phenotypes, suggesting that it may be an initiator of the disease. Importantly, aberrant Cdk5 activation causes mitochondrial defects and motor neuron degeneration, as the genetic knockout of p35 in an SMA mouse model rescues mitochondrial transport and fragmentation defects, and alleviates SMA phenotypes including motor neuron hyperexcitability, loss of excitatory synapses, neuromuscular junction denervation, and motor neuron degeneration. Inhibition of the Cdk5 signaling pathway reduces the degeneration of motor neurons derived from SMA mice and human SMA iPSCs. Altogether, our studies reveal a critical role for the aberrant activation of Cdk5 in SMA pathogenesis and suggest a potential target for therapeutic intervention.

Nucleolar reorganization after cellular stress is orchestrated by SMN shuttling between nuclear compartments.

Spinal muscular atrophy is an autosomal recessive neuromuscular disease caused by mutations in the multifunctional protein Survival of Motor Neuron, or SMN. Within the nucleus, SMN localizes to Cajal bodies, which are associated with nucleoli, nuclear organelles dedicated to the first steps of ribosome biogenesis. The highly organized structure of the nucleolus can be dynamically altered by genotoxic agents. RNAP1, Fibrillarin, and nucleolar DNA are exported to the periphery of the nucleolus after genotoxic stress and, once DNA repair is fully completed, the organization of the nucleolus is restored. We find that SMN is required for the restoration of the nucleolar structure after genotoxic stress. During DNA repair, SMN shuttles from the Cajal bodies to the nucleolus. This shuttling is important for nucleolar homeostasis and relies on the presence of Coilin and the activity of PRMT1.

Analysis of Free Circulating Messenger Ribonucleic Acids in Serum Samples from Late-Onset Spinal Muscular Atrophy Patients Using nCounter NanoString Technology.

5q-related Spinal muscular atrophy (SMA) is a hereditary multi-systemic disorder leading to progressive muscle atrophy and weakness caused by the degeneration of spinal motor neurons (MNs) in the ventral horn of the spinal cord. Three SMN-enhancing drugs for SMA treatment are available. However, even if these drugs are highly effective when administrated early, several patients do not benefit sufficiently or remain non-responders, e.g., adults suffering from late-onset SMA and starting their therapy at advanced disease stages characterized by long-standing irreversible loss of MNs. Therefore, it is important to identify additional molecular targets to expand therapeutic strategies for SMA treatment and establish prognostic biomarkers related to the treatment response. Using high-throughput nCounter NanoString technology, we analyzed serum samples of late-onset SMA type 2 and type 3 patients before and six months under nusinersen treatment. Four genes (AMIGO1, CA2, CCL5, TLR2) were significantly altered in their transcript counts in the serum of patients, where differential expression patterns were dependent on SMA subtype and treatment response, assessed with outcome scales. No changes in gene expression were observed six months after nusinersen treatment, compared to healthy controls. These alterations in the transcription of four genes in SMA patients qualified those genes as potential SMN-independent therapeutic targets to complement current SMN-enhancing therapies.

NRF2 has a splicing regulatory function involving the survival of motor neuron (SMN) in non-small cell lung cancer.

The nuclear factor erythroid 2-like 2 (NFE2L2; NRF2) signaling pathway is frequently deregulated in human cancers. The critical functions of NRF2, other than its transcriptional activation, in cancers remain largely unknown. Here, we uncovered a previously unrecognized role of NRF2 in the regulation of RNA splicing. Global splicing analysis revealed that NRF2 knockdown in non-small cell lung cancer (NSCLC) A549 cells altered 839 alternative splicing (AS) events in 485 genes. Mechanistic studies demonstrated that NRF2 transcriptionally regulated SMN mRNA expression by binding to two antioxidant response elements in the SMN1 promoter. Post-transcriptionally, NRF2 was physically associated with the SMN protein. The Neh2 domain of NRF2, as well as the YG box and the region encoded by exon 7 of SMN, were required for their interaction. NRF2 formed a complex with SMN and Gemin2 in nuclear gems and Cajal bodies. Furthermore, the NRF2-SMN interaction regulated RNA splicing by expressing SMN in NRF2-knockout HeLa cells, reverting some of the altered RNA splicing. Moreover, SMN overexpression was significantly associated with alterations in the NRF2 pathway in patients with lung squamous cell carcinoma from The Cancer Genome Atlas. Taken together, our findings suggest a novel therapeutic strategy for cancers involving an aberrant NRF2 pathway.

Multi-omics profiling of CSF from spinal muscular atrophy type 3 patients after nusinersen treatment: a 2-year follow-up multicenter retrospective study.

Spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by mutations in the SMN1 gene resulting in reduced levels of the SMN protein. Nusinersen, the first antisense oligonucleotide (ASO) approved for SMA treatment, binds to the SMN2 gene, paralogue to SMN1, and mediates the translation of a functional SMN protein. Here, we used longitudinal high-resolution mass spectrometry (MS) to assess both global proteome and metabolome in cerebrospinal fluid (CSF) from ten SMA type 3 patients, with the aim of identifying novel readouts of pharmacodynamic/response to treatment and predictive markers of treatment response. Patients had a median age of 33.5 [29.5; 38.25] years, and 80% of them were ambulant at time of the enrolment, with a median HFMSE score of 37.5 [25.75; 50.75]. Untargeted CSF proteome and metabolome were measured using high-resolution MS (nLC-HRMS) on CSF samples obtained before treatment (T0) and after 2 years of follow-up (T22). A total of 26 proteins were found to be differentially expressed between T0 and T22 upon VSN normalization and LIMMA differential analysis, accounting for paired replica. Notably, key markers of the insulin-growth factor signaling pathway were upregulated after treatment together with selective modulation of key transcription regulators. Using CombiROC multimarker signature analysis, we suggest that detecting a reduction of SEMA6A and an increase of COL1A2 and GRIA4 might reflect therapeutic efficacy of nusinersen. Longitudinal metabolome profiling, analyzed with paired t-Test, showed a significant shift for some aminoacid utilization induced by treatment, whereas other metabolites were largely unchanged. Together, these data suggest perturbation upon nusinersen treatment still sustained after 22 months of follow-up and confirm the utility of CSF multi-omic profiling as pharmacodynamic biomarker for SMA type 3. Nonetheless, validation studies are needed to confirm this evidence in a larger sample size and to further dissect combined markers of response to treatment.

Early Splicing Complexes and Human Disease.

Over the last decade, our understanding of spliceosome structure and function has significantly improved, refining the study of the impact of dysregulated splicing on human disease. As a result, targeted splicing therapeutics have been developed, treating various diseases including spinal muscular atrophy and Duchenne muscular dystrophy. These advancements are very promising and emphasize the critical role of proper splicing in maintaining human health. Herein, we provide an overview of the current information on the composition and assembly of early splicing complexes-commitment complex and pre-spliceosome-and their association with human disease.