Dysregulation of Mdm2 and Mdm4 alternative splicing underlies motor neuron death in spinal muscular atrophy.

Ubiquitous deficiency in the survival motor neuron (SMN) protein causes death of motor neurons-a hallmark of the neurodegenerative disease spinal muscular atrophy (SMA)-through poorly understood mechanisms. Here, we show that the function of SMN in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) regulates alternative splicing of Mdm2 and Mdm4, two nonredundant repressors of p53. Decreased inclusion of critical Mdm2 and Mdm4 exons is most prominent in SMA motor neurons and correlates with both snRNP reduction and p53 activation in vivo. Importantly, increased skipping of Mdm2 and Mdm4 exons regulated by SMN is necessary and sufficient to synergistically elicit robust p53 activation in wild-type mice. Conversely, restoration of full-length Mdm2 and Mdm4 suppresses p53 induction and motor neuron degeneration in SMA mice. These findings reveal that loss of SMN-dependent regulation of Mdm2 and Mdm4 alternative splicing underlies p53-mediated death of motor neurons in SMA, establishing a causal link between snRNP dysfunction and neurodegeneration.

Improvement of TaC9-ABE mediated correction of human SMN2 gene.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Gene editing technology repairs the conversion of the 6th base T to C in exon 7 of the paralogous SMN2 gene, compensating for the SMN protein expression and promoting the survival and function of motor neurons. However, low editing efficiency and unintended off-target effects limit the application of this technology. Here, we optimized a TaC9-adenine base editor (ABE) system by combining Cas9 nickase with the transcription activator-like effector (TALE)-adenosine deaminase fusion protein to effectively and precisely edit SMN2 without detectable Cas9 dependent off-target effects in human cell lines. We also generated human SMA-induced pluripotent stem cells (SMA-iPSCs) through the mutation of the splice acceptor or deletion of the exon 7 of SMN1. TaC9-R10 induced 45% SMN2 T6 > C conversion in the SMA-iPSCs. The SMN2 T6 > C splice-corrected SMA-iPSCs were directionally differentiated into motor neurons, exhibiting SMN protein recovery and antiapoptosis ability. Therefore, the TaC9-ABE system with dual guides from the combination of Cas9 with TALE could be a potential therapeutic strategy for SMA with high efficacy and safety.

Analytical validation of the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis assay to diagnose spinal muscular atrophy.

OBJECTIVES: Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous deletion and compound heterozygous mutations in survival motor neuron 1 (SMN1), with severity tied to the copy number of survival motor neuron 2 (SMN2). This study aimed to develop a rapid and comprehensive method for the diagnosis of SMA. METHODS: A total of 292 children with clinically suspected SMA and 394 family members were detected by the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis (ARMS-PCR-CE) method, which targeted 19 reported mutations, and the results were compared with those in multiplex ligation-dependent probe amplification (MLPA). Individuals with identified point mutations were further confirmed by SMN1 long-range PCR and Sanger sequencing. RESULTS: A total of 202 children with SMA, 272 carriers, and 212 normal individuals were identified in this study. No difference was found in the R-value distribution of exons 7 and 8 in SMN1 and SMN2 among these cohorts, with coefficients of variation consistently below 0.08. To detect exon 7 and 8 copy numbers in SMN1 and SMN2, the ARMS-PCR-CE results were concordant with those of MLPA. Approximately 4.95 % (10/202) of the study patients had compound heterozygous mutations. CONCLUSIONS: The ARMS-PCR-CE assay is a comprehensive, rapid, and accurate diagnostic method for SMA that simultaneously detects copy numbers of exons 7 and 8 in SMN1/SMN2, as well as 19 point mutations in SMN1 and 2 enhancers in SMN2. This approach can effectively reduce the time frame for diagnosis, facilitating early intervention and preventing birth defects.

Diminished motor neuron activity driven by abnormal astrocytic EAAT1 glutamate transporter activity in spinal muscular atrophy is not fully restored after lentiviral SMN delivery.

Spinal muscular atrophy (SMA) is characterized by the loss of the lower spinal motor neurons due to survival motor neuron (SMN) deficiency. The motor neuron cell autonomous and non-cell autonomous disease mechanisms driving early glutamatergic dysfunction, a therapeutically targetable phenotype prior to motor neuron cell loss, remain unclear. Using microelectrode array analysis, we demonstrate that the secretome and cell surface proteins needed for proper synaptic modulation are likely disrupted in human SMA astrocytes and lead to diminished motor neuron activity. While healthy astrocyte conditioned media did not improve SMA motor neuron activity, SMA motor neurons robustly responded to healthy astrocyte neuromodulation in direct contact cultures. This suggests an important role of astrocyte synaptic-associated plasma membrane proteins and contact-mediated cellular interactions for proper motor neuron function in SMA. Specifically, we identified a significant reduction of the glutamate Na+ dependent excitatory amino acid transporter EAAT1 within human SMA astrocytes and SMA lumbar spinal cord tissue. The selective inhibition of EAAT1 in healthy co-cultures phenocopied the diminished neural activity observed in SMA astrocyte co-cultures. Caveolin-1, an SMN-interacting protein previously associated with local translation at the plasma membrane, was abnormally elevated in human SMA astrocytes. Although lentiviral SMN delivery to SMA astrocytes partially rescued EAAT1 expression, limited activity of healthy motor neurons was still observed in SMN-transduced SMA astrocyte co-cultures. Together, these data highlight the detrimental impact of astrocyte-mediated disease mechanisms on motor neuron function in SMA and that SMN delivery may be insufficient to fully restore astrocyte function at the synapse.

Keeping the balance: The noncoding RNA 7SK as a master regulator for neuron development and function.

The noncoding RNA 7SK is a critical regulator of transcription by adjusting the activity of the kinase complex P-TEFb. Release of P-TEFb from 7SK stimulates transcription at many genes by promoting productive elongation. Conversely, P-TEFb sequestration by 7SK inhibits transcription. Recent studies have shown that 7SK functions are particularly important for neuron development and maintenance and it can thus be hypothesized that 7SK is at the center of many signaling pathways contributing to neuron function. 7SK activates neuronal gene expression programs that are key for terminal differentiation of neurons. Proteomics studies revealed a complex protein interactome of 7SK that includes several RNA-binding proteins. Some of these novel 7SK subcomplexes exert non-canonical cytosolic functions in neurons by regulating axonal mRNA transport and fine-tuning spliceosome production in response to transcription alterations. Thus, a picture emerges according to which 7SK acts as a multi-functional RNA scaffold that is integral for neuron homeostasis.

SMN loss dysregulates microtubule-associated proteins in spinal muscular atrophy model.

Spinal muscular atrophy (SMA) is a rare neurodegenerative disease caused by the absence of survival motor neuron (SMN) protein. SMN loss results in impairments of the cytoskeleton, including microtubules and regulatory proteins. However, the contribution of microtubule-associated proteins (MAPs) to microtubule dysregulations in SMA is not fully understood. In this study, we investigated neuronal MAPs responsible for the microtubule stability and growth, including MAP1A, MAP2, MAP6, MAP7, EB1, and EB3 using an in vitro model of SMA. Decreased MAP2 and EB3 levels were found in SMN-deficient motor neuron-like cells, and EB3 protein level was also relevant to MAP1B. SMN loss leads to an increase in EB3 comet numbers at proximal neurites, indicating increased microtubule growth. Our findings suggest that SMN deficiency simultaneously causes dysregulations of several MAPs, contributing to the perturbations of microtubule dynamics in SMA.

The SMN Complex at the Crossroad between RNA Metabolism and Neurodegeneration.

In the cell, RNA exists and functions in a complex with RNA binding proteins (RBPs) that regulate each step of the RNA life cycle from transcription to degradation. Central to this regulation is the role of several molecular chaperones that ensure the correct interactions between RNA and proteins, while aiding the biogenesis of large RNA-protein complexes (ribonucleoproteins or RNPs). Accurate formation of RNPs is fundamentally important to cellular development and function, and its impairment often leads to disease. The survival motor neuron (SMN) protein exemplifies this biological paradigm. SMN is part of a multi-protein complex essential for the biogenesis of various RNPs that function in RNA metabolism. Mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy (SMA). A fundamental question in SMA biology is how selective motor system dysfunction results from reduced levels of the ubiquitously expressed SMN protein. Recent clarification of the central role of the SMN complex in RNA metabolism and a thorough characterization of animal models of SMA have significantly advanced our knowledge of the molecular basis of the disease. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP biogenesis. We discuss developments in our understanding of SMN activity as a molecular chaperone of RNPs and how disruption of SMN-dependent RNA pathways can contribute to the SMA phenotype.

Survival motor neuron protein deficiency alters microglia reactivity.

Survival motor neuron (SMN) protein deficiency results in loss of alpha motor neurons and subsequent muscle atrophy in patients with spinal muscular atrophy (SMA). Reactive microglia have been reported in SMA mice and depleting microglia rescues the number of proprioceptive synapses, suggesting a role in SMA pathology. Here, we explore the contribution of lymphocytes on microglia reactivity in SMA mice and investigate how SMN deficiency alters the reactive profile of human induced pluripotent stem cell (iPSC)-derived microglia. We show that microglia adopt a reactive morphology in spinal cords of SMA mice. Ablating lymphocytes did not alter the reactive morphology of SMA microglia and did not improve the survival or motor function of SMA mice, indicating limited impact of peripheral immune cells on the SMA phenotype. We found iPSC-derived SMA microglia adopted an amoeboid morphology and displayed a reactive transcriptome profile, increased cell migration, and enhanced phagocytic activity. Importantly, cell morphology and electrophysiological properties of motor neurons were altered when they were incubated with conditioned media from SMA microglia. Together, these data reveal that SMN-deficient microglia adopt a reactive profile and exhibit an exaggerated inflammatory response with potential impact on SMA neuropathology.

Management of Spinal Muscular Atrophy in the Adult Population.

Spinal muscular atrophy (SMA) is a group of neurodegenerative disorders resulting from the loss of spinal motor neurons. 95% of patients share a pathogenic mechanism of loss of survival motor neuron (SMN) 1 protein expression due to homozygous deletions or other mutations of the SMN1 gene, with the different phenotypes influenced by variable copy numbers of the SMN2 gene. Advances in supportive care, disease modifying treatment and novel gene therapies have led to an increase in the prevalence of SMA, with a third of SMA patients now represented by adults. Despite the growing number of adult patients, consensus on the management of SMA has focused primarily on the pediatric population. As the disease burden is vastly different in adult SMA, an approach to treatment must be tailored to their unique needs. This review will focus on the management of the adult SMA patient as they age and will discuss proper transition of care from a pediatric to adult center, including the need for continued monitoring for osteoporosis, scoliosis, malnutrition, and declining mobility and functioning. As in the pediatric population, multidisciplinary care remains the best approach to the management of adult SMA. Novel and emerging therapies such as nusinersen and risdiplam provide hope for these patients, though these medications are of uncertain efficacy in this population and require additional study.

Revisiting the role of mitochondria in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease of variable clinical severity that is caused by mutations in the survival motor neuron 1 (SMN1) gene. Despite its name, SMN is a ubiquitous protein that functions within and outside the nervous system and has multiple cellular roles in transcription, translation, and proteostatic mechanisms. Encouragingly, several SMN-directed therapies have recently reached the clinic, albeit this has highlighted the increasing need to develop combinatorial therapies for SMA to achieve full clinical efficacy. As a subcellular site of dysfunction in SMA, mitochondria represents a relevant target for a combinatorial therapy. Accordingly, we will discuss our current understanding of mitochondrial dysfunction in SMA, highlighting mitochondrial-based pathways that offer further mechanistic insights into the involvement of mitochondria in SMA. This may ultimately facilitate translational development of targeted mitochondrial therapies for SMA. Due to clinical and mechanistic overlaps, such strategies may also benefit other motor neuron diseases and related neurodegenerative disorders.

Advances and limitations for the treatment of spinal muscular atrophy.

Spinal muscular atrophy (5q-SMA; SMA), a genetic neuromuscular condition affecting spinal motor neurons, is caused by defects in both copies of the SMN1 gene that produces survival motor neuron (SMN) protein. The highly homologous SMN2 gene primarily expresses a rapidly degraded isoform of SMN protein that causes anterior horn cell degeneration, progressive motor neuron loss, skeletal muscle atrophy and weakness. Severe cases result in limited mobility and ventilatory insufficiency. Untreated SMA is the leading genetic cause of death in young children. Recently, three therapeutics that increase SMN protein levels in patients with SMA have provided incremental improvements in motor function and developmental milestones and prevented the worsening of SMA symptoms. While the therapeutic approaches with Spinraza®, Zolgensma®, and Evrysdi® have a clinically significant impact, they are not curative. For many patients, there remains a significant disease burden. A potential combination therapy under development for SMA targets myostatin, a negative regulator of muscle mass and strength. Myostatin inhibition in animal models increases muscle mass and function. Apitegromab is an investigational, fully human, monoclonal antibody that specifically binds to proforms of myostatin, promyostatin and latent myostatin, thereby inhibiting myostatin activation. A recently completed phase 2 trial demonstrated the potential clinical benefit of apitegromab by improving or stabilizing motor function in patients with Type 2 and Type 3 SMA and providing positive proof-of-concept for myostatin inhibition as a target for managing SMA. The primary goal of this manuscript is to orient physicians to the evolving landscape of SMA treatment.

Mastication in Patients with Spinal Muscular Atrophy Types 2 and 3 is Characterized by Abnormal Efficiency, Reduced Endurance, and Fatigue.

Mastication problems can have a negative impact on the intake of food and quality of life. This cross-sectional study characterizes mastication problems using clinical and instrumental assessments in patients with spinal muscular atrophy (SMA) types 2 and 3 with self-reported bulbar problems. We included 27 patients (aged 13-67 years), 18 with SMA type 2 and 9 patients with SMA type 3 (of whom three were still ambulant) and applied a questionnaire, clinical mastication tests (TOMASS and 6-min mastication test), and muscle ultrasound of the mastication muscles. Non-ambulant patients demonstrated inefficient mastication as reflected by median z scores for masticatory cycles (z = 1.8), number of swallows (z = 4.3) and time needed to finish the cracker (z = 3.4), and limited endurance of continuous mastication as demonstrated by the median z scores of the 6-min mastication test (z = - 1.5). Patients reported increased fatigue directly after the 6-min mastication test as well as 5 min after completing the test (p < 0.001; p = 0.003). Reduced maximal mouth opening was associated with mastication problems (p < 0.001). Muscle ultrasound of the mastication muscles showed an abnormal muscle structure in 90% of both ambulant and non-ambulant patients. This study aims to understand the nature and underlying mechanisms of mastication problems in patients with SMA types 2 and 3 with reported bulbar problems.

NSC Physiological Features in Spinal Muscular Atrophy: SMN Deficiency Effects on Neurogenesis.

While the U.S. Food and Drug Administration and the European Medicines Evaluation Agency have recently approved new drugs to treat spinal muscular atrophy 1 (SMA1) in young patients, they are mostly ineffective in older patients since many motor neurons have already been lost. Therefore, understanding nervous system (NS) physiology in SMA patients is essential. Consequently, studying neural stem cells (NSCs) from SMA patients is of significant interest in searching for new treatment targets that will enable researchers to identify new pharmacological approaches. However, studying NSCs in these patients is challenging since their isolation damages the NS, making it impossible with living patients. Nevertheless, it is possible to study NSCs from animal models or create them by differentiating induced pluripotent stem cells obtained from SMA patient peripheral tissues. On the other hand, therapeutic interventions such as NSCs transplantation could ameliorate SMA condition. This review summarizes current knowledge on the physiological properties of NSCs from animals and human cellular models with an SMA background converging on the molecular and neuronal circuit formation alterations of SMA fetuses and is not focused on the treatment of SMA. By understanding how SMA alters NSC physiology, we can identify new and promising interventions that could help support affected patients.

Targeted-Deletion of a Tiny Sequence via Prime Editing to Restore SMN Expression.

Spinal muscular atrophy (SMA) is a devastating autosomal recessive motor neuron disease associated with mutations in the survival motor neuron 1 (SMN1) gene, the leading genetic cause of infant mortality. A nearly identical copy gene (SMN2) is retained in almost all patients with SMA. However, SMN2 fails to prevent disease development because of its alternative splicing, leading to a lack of exon 7 in the majority of SMN2 transcripts and yielding an unstable truncated protein. Several splicing regulatory elements, including intronic splicing silencer-N1 (ISS-N1) of SMN2 have been described. In this study, targeted-deletion of ISS-N1 was achieved using prime editing (PE) in SMA patient-specific induced pluripotent stem cells (SMA-iPSCs) with a high efficiency of 7/24. FL-SMN expression was restored in the targeted-deletion iPS clones and their derived motor neurons (iMNs). Notably, the apoptosis of the iMNs, caused by the loss of SMN protein that leads to the hyperactivity of endoplasmic reticulum (ER) stress, was alleviated in targeted-deletion iPSCs derived-iMNs. Thus, this is the first study to demonstrate that the targeted-deletion of ISS-N1 via PE for restoring FL-SMN expression holds therapeutic promise for SMA.

Recent research on the treatment of spinal muscular atrophy. / è„Šé«“æ€§è‚ŒèŽç¼©ç—‡æ²»ç–—ç ”ç©¶è¿›å±•.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by progressive muscular weakness and atrophy. SMA, as an inherited disease, is the leading cause of death in infants and young children. Rapid progress has been made in the research field of SMA in recent years, and some related treatment drugs have been successfully approved for marketing. This article reviews the recent research advances in the treatment of SMA.

Dual SMN inducing therapies can rescue survival and motor unit function in symptomatic ∆7SMA mice.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by survival motor neuron (SMN) protein deficiency which results in motor neuron loss and muscle atrophy. SMA is caused by a mutation or deletion of the survival motor neuron 1 (SMN1) gene and retention of the nearly identical SMN2 gene. SMN2 contains a C to T change in exon 7 that results in exon 7 exclusion from 90% of transcripts. SMN protein lacking exon 7 is unstable and rapidly degraded. The remaining full-length transcripts from SMN2 are insufficient for normal motor neuron function leading to the development of SMA. Three different therapeutic approaches that increase full-length SMN (FL-SMN) protein production are approved for treatment of SMA patients. Studies in both animal models and humans have demonstrated increasing SMN levels prior to onset of symptoms provides the greatest therapeutic benefit. Treatment of SMA, after some motor neuron loss has occurred, is also effective but to a lesser degree. The SMN∆7 mouse model is a well characterized model of severe or type 1 SMA, dying at 14 days of age. Here we treated three groups of ∆7SMA mice starting before, roughly during, and after symptom onset to determine if combining two mechanistically distinct SMN inducing therapies could improve the therapeutic outcome both before and after motor neuron loss. We found, compared with individual therapies, that morpholino antisense oligonucleotide (ASO) directed against ISS-N1 combined with the small molecule compound RG7800 significantly increased FL-SMN transcript and protein production resulting in improved survival and weight of ∆7SMA mice. Moreover, when give late symptomatically, motor unit function was completely rescued with no loss in function at 100 days of age in the dual treatment group. We have therefore shown that this dual therapeutic approach successfully increases SMN protein and rescues motor function in symptomatic ∆7SMA mice.

Intracellular pathways involved in cell survival are deregulated in mouse and human spinal muscular atrophy motoneurons.

Spinal Muscular Atrophy (SMA) is a severe neuromuscular disorder caused by loss of the Survival Motor Neuron 1 gene (SMN1). Due to this depletion of the survival motor neuron (SMN) protein, the disease is characterized by the degeneration of spinal cord motoneurons (MNs), progressive muscular atrophy, and weakness. Nevertheless, the ultimate cellular and molecular mechanisms leading to cell loss in SMN-reduced MNs are only partially known. We have investigated the activation of apoptotic and neuronal survival pathways in several models of SMA cells. Even though the antiapoptotic proteins FAIM-L and XIAP were increased in SMA MNs, the apoptosis executioner cleaved-caspase-3 was also elevated in these cells, suggesting the activation of the apoptosis process. Analysis of the survival pathway PI3K/Akt showed that Akt phosphorylation was reduced in SMA MNs and pharmacological inhibition of PI3K diminished SMN and Gemin2 at transcriptional level in control MNs. In contrast, ERK phosphorylation was increased in cultured mouse and human SMA MNs. Our observations suggest that apoptosis is activated in SMA MNs and that Akt phosphorylation reduction may control cell degeneration, thereby regulating the transcription of Smn and other genes related to SMN function.

Thrombotic Microangiopathy Following Onasemnogene Abeparvovec for Spinal Muscular Atrophy: A Case Series.

Spinal muscular atrophy is treated with onasemnogene abeparvovec, which replaces the missing survival motor neuron 1 gene via an adeno-associated virus vector. As of July 1, 2020, we had identified 3 infants who developed thrombotic microangiopathy following onasemnogene abeparvovec. Early recognition and treatment of drug-induced thrombotic microangiopathy may lessen mortality and morbidity.

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

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.

The Importance of Digging into the Genetics of SMN Genes in the Therapeutic Scenario of Spinal Muscular Atrophy.

After 26 years of discovery of the determinant survival motor neuron 1 and the modifier survival motor neuron 2 genes (SMN1 and SMN2, respectively), three SMN-dependent specific therapies are already approved by FDA and EMA and, as a consequence, worldwide SMA patients are currently under clinical investigation and treatment. Bi-allelic pathogenic variants (mostly deletions) in SMN1 should be detected in SMA patients to confirm the disease. Determination of SMN2 copy number has been historically employed to correlate with the phenotype, predict disease evolution, stratify patients for clinical trials and to define those eligible for treatment. In view that discordant genotype-phenotype correlations are present in SMA, besides technical issues with detection of SMN2 copy number, we have hypothesized that copy number determination is only the tip of the iceberg and that more deepen studies of variants, sequencing and structures of the SMN2 genes are necessary for a better understanding of the disease as well as to investigate possible influences in treatment responses. Here, we highlight the importance of a comprehensive approach of SMN1 and SMN2 genetics with the perspective to apply for better prediction of SMA in positive neonatal screening cases and early diagnosis to start treatments.