Survival Motor Neuron Research Articles

Improved therapeutic approach for spinal muscular atrophy via ubiquitination-resistant survival motor neuron variant.

Zolgensma is a gene-replacement therapy that has led to a promising treatment for spinal muscular atrophy (SMA). However, clinical trials of Zolgensma have raised two major concerns: insufficient therapeutic effects and adverse events. In a recent clinical trial, 30% of patients failed to achieve motor milestones despite pre-symptomatic treatment. In addition, more than 20% of patients showed hepatotoxicity due to excessive virus dosage, even after the administration of an immunosuppressant. Here, we aimed to test whether a ubiquitination-resistant variant of survival motor neuron (SMN), SMNK186R, has improved therapeutic effects for SMA compared with wild-type SMN (SMNWT). A severe SMA mouse model, SMA type 1.5 (Smn-/-; SMN2+/+; SMN∆7+/-) mice, was used to compare the differences in therapeutic efficacy between AAV9-SMNWT and AAV9-SMNK186R. All animals were injected within Postnatal Day (P) 1 through a facial vein or cerebral ventricle. AAV9-SMNK186R-treated mice showed increased lifespan, body weight, motor neuron number, muscle weight and functional improvement in motor functions as compared with AAV9-SMNWT-treated mice. Lifespan increased by more than 10-fold in AAV9-SMNK186R-treated mice (144.8±26.11days) as compared with AAV9-SMNWT-treated mice (26.8±1.41days). AAV9-SMNK186R-treated mice showed an ascending weight pattern, unlike AAV9-SMNWT-treated mice, which only gained weight until P20 up to 5g on average. Several motor function tests showed the improved therapeutic efficacy of SMNK186R. In the negative geotaxis test, AAV9-SMNK186R-treated mice turned their bodies in an upward direction successfully, unlike AAV9-SMNWT-treated mice, which failed to turn upwards from around P23. Hind limb clasping phenotype was rarely observed in AAV9-SMNK186R-treated mice, unlike AAV9-SMNWT-treated mice that showed clasping phenotype for more than 20 out of 30s. At this point, the number of motor neurons (1.5-fold) and the size of myofibers (2.1-fold) were significantly increased in AAV9-SMNK186R-treated mice compared with AAV9-SMNWT-treated mice without prominent neurotoxicity. AAV9-SMNK186R had fewer liver defects compared with AAV9-SMNWT, as judged by increased proliferation of hepatocytes (P<0.0001) and insulin-like growth factor-1 production (P<0.0001). Especially, low-dose AAV9-SMNK186R (nine-fold) also reduced clasping time compared with SMNWT. SMNK186R will provide improved therapeutic efficacy in patients with severe SMA with insufficient therapeutic efficacy. Low-dose treatment of SMA patients with AAV9-SMNK186R can reduce the adverse events of Zolgensma. Collectively, SMNK186R has value as a new treatment for SMA that improves treatment effectiveness and reduces adverse events simultaneously.

Whole-genome sequencing in prenatally detected congenital malformations: prospective cohort study in clinical setting.

To investigate the diagnostic yield of trio whole-genome sequencing (WGS) in fetuses with various congenital malformations referred to a tertiary center for prenatal diagnosis. In this prospective study, 50 pregnancies with different congenital malformations, negative for trisomies and causative copy-number variants, were analyzed further with fetal-parental trio WGS analysis. Parents were eligible for inclusion if they accepted further investigation following the detection of isolated or multiple malformations on prenatal ultrasound. Cases with isolated increased nuchal translucency, gamete donation or multiple pregnancy were excluded. WGS with the Illumina Inc. 30× polymerase-chain-reaction-free short-read sequencing included analysis of single-nucleotide variants, insertions and deletions, structural variants, short tandem repeats and copy-number identification of SMN1 and SMN2 genes. A molecular diagnosis was achieved in 13/50 (26%) cases. Causative sequence variants were identified in 12 genes: FGFR3 (n = 2), ACTA1 (n = 1), CDH2 (n = 1), COL1A2 (n = 1), DHCR7 (n = 1), EYA1 (n = 1), FBXO11 (n = 1), FRAS1 (n = 1), L1CAM (n = 1), OFD1 (n = 1), PDHA1 (n = 1) and SOX9 (n = 1). The phenotypes of the cases were divided into different groups, with the following diagnostic yields: skeletal malformation (4/9 (44%)), multisystem malformation (3/7 (43%)), central nervous system malformation (5/15 (33%)) and thoracic malformation (1/10 (10%)). Additionally, two cases carried variants that were considered potentially clinically relevant, even though they were assessed as variants of uncertain significance, according to the guidelines provided by the American College of Medical Genetics and Genomics. Overall, we identified a causative or potentially clinically relevant variant in 15/50 (30%) cases. We demonstrate a diagnostic yield of 26% with clinical WGS in prenatally detected congenital malformations. This study emphasizes the benefits that WGS can bring to the diagnosis of fetal structural anomalies. It is important to note that causative chromosomal aberrations were excluded from our cohort before WGS. As chromosomal aberrations are a well-known cause of prenatally detected congenital malformations, future studies using WGS as a primary diagnostic test, including assessment of chromosomal aberrations, may show that the detection rate exceeds the diagnostic yield of this study. WGS can add clinically relevant information, explaining the underlying cause of the fetal anomaly, which will provide information concerning the specific prognosis of the condition, as well as estimate the risk of recurrence. A genetic diagnosis can also provide more reproductive choice for future pregnancies. © 2024 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

The effects of hyaluronan and proteoglycan link protein 1 (HAPLN1) in ameliorating spinal cord injury mediated by Nrf2.

Excessive inflammatory response and oxidative stress (OS) play an important role in the pathogenesis of spinal cord injury (SCI). Balance of inflammation and prevention of OS have been considered an effective strategy for the treatment of SCI. Hyaluronan and proteoglycan link protein 1 (HAPLN1), also known as cartilage link protein, has displayed a wide range of biological and physiological functions in different types of tissues and cells. However, whether HAPLN1 regulates inflammation and OS during SCI is unknown. Therefore, we aimed to examine whether HAPLN1 can have a protective effect on SCI. In this study, both in vitro and in vivo SCI models were established. Nissl staining and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays were used. Western blotting and enzyme-linked immunosorbent assay were employed to assess the expression of proteins. Our results demonstrate that the administration of HAPLN1 promoted the recovery of motor neurons after SCI by increasing the Basso mouse scale score, increasing the numbers of motor neurons, and preventing apoptosis of spinal cord cells. Additionally, HAPLN1 mitigated OS in spinal cord tissue after SCI by increasing the content of superoxide dismutase SOD and the activity of glutathione peroxidase but reducing the levels of malondialdehyde. Importantly, we found that HAPLN1 stimulated the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway and stimulated the expression of heme oxygenase-1 and nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1, which mediated the attenuation of HAPLN1 in activation of the NOD-like receptor protein 3 (NLRP3) inflammasome by reducing the levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin-1β. Correspondingly, in vitro experiments show that the presence of HAPLN1 suppressed the NLRP3 inflammasome and prevented cell injury against H2O2 in PC12 cells. These effects were mediated by the Nrf2/ARE pathway, and inhibition of Nrf2 with ML385 abolished the beneficial effects of HAPLN1. Based on these findings, we conclude that HAPLN1 inhibits the NLRP3 inflammasome through the stimulation of the Nrf2/ARE pathway, thereby suppressing neuroinflammation, enhancing motor neuronal survival, and improving the recovery of nerve function after SCI.

Early spinal muscular atrophy treatment following newborn screening: A 20-month review of the first Italian regional experience.

Mandatory newborn screening (NBS) for spinal muscular atrophy (SMA) was implemented for the first time in Italy at the end of 2021, allowing the identification and treatment of patients at an asymptomatic stage. DNA samples extracted from dried blood spot (DBS) from newborns in Apulia region were analysed for SMA screening by using a real-time PCR-based assay. Infants harbouring homozygous deletion of SMN1 exon 7 confirmed by diagnostic molecular tests underwent clinical and neurophysiological assessment and received a timely treatment. Over the first 20 months since regional NBS introduction, four out of 42,492 (0.009%) screened children were found to carry a homozygous deletion in the exon 7 of SMN1 gene, with an annual incidence of 1:10,623. No false negatives were present. Median age at diagnosis was 7 days and median age at treatment was 20.5 days. Three of them had two copies of SMN2 and received gene therapy, while the one with three SMN2 copies was treated with nusinersen. All but one were asymptomatic at birth, showed no clinical signs of disease after a maximum follow-up of 16 months and reached motor milestones appropriate with their age. The minimum interval between diagnosis and the treatment initiation was 9 days. The timely administration of disease-modifying therapies prevented presymptomatic subjects to develop disease symptoms. Mandatory NBS for SMA should be implemented on a national scale.

Analysis of spinal muscular atrophy carrier screening results in 32,416 pregnant women and 7,231 prepregnant women.

Spinal muscular atrophy (SMA) is an autosomal recessive disease that is one of the most common in childhood neuromuscular disorders. Our screenings are more meaningful programs in preventing birth defects, providing a significant resource for healthcare professionals, genetic counselors, and policymakers involved in designing strategies to prevent and manage SMA. We screened 39,647 participants from 2020 to the present by quantitative real-time PCR, including 7,231 pre-pregnancy participants and 32,416 pregnancy participants, to detect the presence of SMN1 gene EX7 and EX8 deletion in the DNA samples provided by the subjects. To validate the accuracy of our findings, we also utilized the Multiplex Ligation-dependent Probe Amplification (MLPA) to confirm the reliability of screening results obtained by quantitative real-time PCR. Among the 39,647 participants who were screened, 726 participants were the carriers of SMN1. The overall carrier rate was calculated to be 1.83% (95% confidence interval: 0.86-2.8%). After undergoing screening, a total of 592 pregnancy carriers were provided with genetic counseling and only 503 of their spouses (84.97, 95% confidence interval: 82.09-87.85%) voluntarily underwent SMA screening. This study provides crucial insights into the prevalence and distribution of SMA carriers among the female population. The identification of 726 asymptomatic carriers highlights the necessity of comprehensive screening programs to identify at-risk individuals and ensure appropriate interventions are in place to minimize the impact of SMA-related conditions.

Screening of Spinal Muscular Atrophy Carriers and Prenatal Diagnosis in Pregnant Women in Yancheng, China.

Spinal muscular atrophy (SMA) is a neuromuscular disorder with an autosomal recessive inheritance pattern. Patients with severe symptoms may suffer respiratory failure, leading to death. The homozygous deletion of exon 7 in the SMN1 gene accounts for nearly 95% of all cases. Population carrier screening for SMA and prenatal diagnosis by amniocentesis for high-risk couples can assist in identifying the risk of fetal disease. We provided the SMA carrier screening process to 55,447 pregnant women in Yancheng from October 2020 to December 2022. Among them, 8185 participated in this process, with a participation rate of around 14.76% (95% CI 14.47-15.06%). Quantitative real-time polymerase chain reaction (qPCR) was used to detect deletions of SMN1 exons 7 and 8 (E7, E8) in screened pregnant women. 127 were identified as carriers (111 cases of E7 and E8 heterozygous deletions, 15 cases of E7 heterozygous deletions, and 1 case of E7 heterozygous deletions and E8 homozygous deletions), resulting in a carrying rate of around 1.55% (95% CI 1.30-1.84%). After genetic counseling, 114 spouses of pregnant women who tested positive underwent SMA carrier screening; three of them were screened as SMA carriers. Multiplexed ligation-dependent probe amplification (MLPA) was used for the prenatal diagnosis of the fetuses of high-risk couples. Two of them exhibited two copies of SMN1 exon 7 (normal), and the pregnancy was continued; one exhibited no copies of SMN1 exon 7 and exon 8 (SMA patient), and the pregnancy was terminated. Analyzing SMN1 mutations in Yancheng and provide clinical evidence for SMA genetic counseling and birth defect prevention. Interventional prenatal diagnosis for high-risk families can promote informed reproductive selection and prepare for the fetus's early treatment.

Administration of adipose-derived stem cells extracellular vesicles in a murine model of spinal muscular atrophy: effects of a new potential therapeutic strategy

BackgroundSpinal Muscular Atrophy (SMA) is an autosomal-recessive neuromuscular disease affecting children. It is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene resulting in lower motor neuron (MN) degeneration followed by motor impairment, progressive skeletal muscle paralysis and respiratory failure. In addition to the already existing therapies, a possible combinatorial strategy could be represented by the use of adipose-derived mesenchymal stem cells (ASCs) that can be obtained easily and in large amounts from adipose tissue. Their efficacy seems to be correlated to their paracrine activity and the production of soluble factors released through extracellular vesicles (EVs). EVs are important mediators of intercellular communication with a diameter between 30 and 100 nm. Their use in other neurodegenerative disorders showed a neuroprotective effect thanks to the release of their content, especially proteins, miRNAs and mRNAs.MethodsIn this study, we evaluated the effect of EVs isolated from ASCs (ASC-EVs) in the SMNΔ7 mice, a severe SMA model. With this purpose, we performed two administrations of ASC-EVs (0.5 µg) in SMA pups via intracerebroventricular injections at post-natal day 3 (P3) and P6. We then assessed the treatment efficacy by behavioural test from P2 to P10 and histological analyses at P10.ResultsThe results showed positive effects of ASC-EVs on the disease progression, with improved motor performance and a significant delay in spinal MN degeneration of treated animals. ASC-EVs could also reduce the apoptotic activation (cleaved Caspase-3) and modulate the neuroinflammation with an observed decreased glial activation in lumbar spinal cord, while at peripheral level ASC-EVs could only partially limit the muscular atrophy and fiber denervation.ConclusionsOur results could encourage the use of ASC-EVs as a therapeutic combinatorial treatment for SMA, bypassing the controversial use of stem cells.

Role of Facial Nerve Reconstruction With Anastomosis and Polyglycolic Acid Tube in Accelerating Functional Recovery After Axotomy in the Rat Facial Nucleus.

Facial nerve injuries stem from trauma or tumor surgery, triggering neurodegeneration and neuronal cell death in the facial nucleus, consequently inducing irreversible nerve paralysis. Following facial nerve transection, glial cells are activated and undergo proliferation, facilitating motor neuron survival, repair, and regeneration. Clinical approaches, including nerve anastomosis and hypoglossal nerve grafting, require delicate microscopic techniques. Recent advancements involve nerve reconstruction using polyglycolic acid (PGA) tubes, which yield nerve function improvement. However, the central pathophysiological effects of these procedures remain unclear. Therefore, using PGA tubes, we evaluated neurodegeneration and microglial inflammatory response in rats after facial nerve transection. Facial nerve functions were evaluated using vibrissae and blink reflex scores. In the end-to-end anastomosis and PGA tube reconstruction groups, a partial improvement in facial motor function was observed, with increased nerve fiber survival in the former. Approximately 90% of neurons survived in both groups, wherein gliosis exhibited increased microglial activation compared to that in the transection group. These results indicate that PGA tube-assisted nerve reconstruction post-facial nerve transection, although inferior to end-to-end anastomosis, improved certain functions and prevented neuronal cell death. Furthermore, the prolonged inflammatory response in the facial nerve nucleus underscored the correlation between neuronal function and survival and microglia.

The dynamic of changes of pNFH levels in the CSF compared with the motor scales’ scores during three years of nusinersen treatment in children with spinal muscular atrophy types 2 and 3

Abstract: Neurofilaments are crucial in neuronal cytoskeleton formation, influencing axonal growth and impulse modulation. This study focuses on understanding the dynamics of the phos-phorylated neurofilament heavy subunit (pNFH) in pediatric spinal muscular atrophy (SMA) pa-tients undergoing Nusinersen treatment. The presence of five neurofilament types, particularly pNFH, is explored as a potential biomarker. SMA, an autosomal recessive disease impacting motor neurons, is characterized by disease severity linked to the number of SMN2 gene copies. Approved drugs, including Nusinersen, have demonstrated efficacy in enhancing motor activity. Methods: A retrospective analysis was conducted on 18 pediatric SMA patients treated with Nusinersen from October 2018 to July 2023. Cerebrospinal fluid (CSF) samples were utilized to assess pNFH levels. Motor scales were employed to evaluate performance, focusing on patients with varying SMN2 gene copies. Results: Following the initiation of Nusinersen treatment, a substantial decrease in pNFH levels was observed in CSF samples. Motor scales indicated improved performance, partic-ularly in patients with more SMN2 copies. However, the correlation between pNFH levels and motor improvement was not strongly evident, suggesting a limited role as a prognostic indicator within this timeframe. Conclusion: Nusinersen effectively reduced pNFH levels in pediatric SMA patients, showcasing promising outcomes in motor function. However, the predictive value of pNFH remains inconclusive, emphasizing the need for further research. Study limitations, including the rarity of SMA, the absence of a control group, and the disease's dynamic nature over time, should be considered when interpreting these findings.

Carrier Screening and Diagnosis for Spinal Muscular Atrophy Using Droplet Digital PCR Versus MLPA: Analytical Validation and Early Test Outcome.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular life-threatening disorder. Owing to high carrier frequency, population-wide SMA screening to quantify the copy number of SMN gene is recommended by American College of Medical Genetics and Genomics. An accurate, reliable, short runaround time and cost-effective method may be helpful in mass population screening for SMA. Methods: Multiplex ligation-dependent probe amplification (MLPA) is a gold standard to estimate the copy number variation (CNV) for SMN1 and SMN2 genes. In this study, we validated droplet digital polymerase chain reaction (ddPCR) for the determination of CNV for both SMN1 and SMN2 exon 7 for a diagnostic purpose. In total, 66 clinical samples were tested using ddPCR, and results were compared with the MLPA as a reference test. Results: For all samples, CNV for SMN1 and SMN2 exon 7 was consentaneous between ddPCR and MLPA test results (κ = 1.000, p < 0.0001). In addition, ddPCR also showed a significant acceptable degree of test repeatability, coefficient of variation < 4%. Conclusion: ddPCR is expected to be utilitarian for CNV detection for carrier screening and diagnosis of SMA. ddPCR test results for CNV detection for SMN1/SMN2 exon 7 are concordant with the gold standard. ddPCR is a more cost-effective and time-saving diagnostic test for SMA than MLPA. Furthermore, it can be used for population-wide carrier screening for SMA.

An Automated Imaging-Based Screen for Genetic Modulators of ER Organisation in Cultured Human Cells.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of mono-genetic inherited neurological disorders, whose primary manifestation is the disruption of the pyramidal system, observed as a progressive impaired gait and leg spasticity in patients. Despite the large list of genes linked to this group, which exceeds 80 loci, the number of cellular functions which the gene products engage is relatively limited, among which endoplasmic reticulum (ER) morphogenesis appears central. Mutations in genes encoding ER-shaping proteins are the most common cause of HSP, highlighting the importance of correct ER organisation for long motor neuron survival. However, a major bottleneck in the study of ER morphology is the current lack of quantitative methods, with most studies to date reporting, instead, on qualitative changes. Here, we describe and apply a quantitative image-based screen to identify genetic modifiers of ER organisation using a mammalian cell culture system. An analysis reveals significant quantitative changes in tubular ER and dense sheet ER organisation caused by the siRNA-mediated knockdown of HSP-causing genes ATL1 and RTN2. This screen constitutes the first attempt to examine ER distribution in cells in an automated and high-content manner and to detect genes which impact ER organisation.

Real-world evidence: Risdiplam in a patient with spinal muscular atrophy type I with a novel splicing mutation and one SMN2 copy.

Spinal muscular atrophy (SMA), which results from the deletion or/and mutation in the SMN1 gene, is an autosomal recessive neuromuscular disorder that leads to weakness and muscle atrophy. SMN2 is a paralogous gene of SMN1. SMN2 copy number affects the severity of SMA, but its role in patients treated with disease modifying therapies is unclear. The most appropriate individualized treatment for SMA has not yet been determined. Here, we reported a case of SMA type I with normal breathing and swallowing function. We genetically confirmed that this patient had a compound heterozygous variant: one deleted SMN1 allele and a novel splice mutation c.628-3T>G in the retained allele, with one SMN2 copy. Patient-derived sequencing of 4 SMN1 cDNA clones showed that this intronic single transversion mutation results in an alternative exon (e)5 3' splice site, which leads to an additional 2 nucleotides (AG) at the 5' end of e5, thereby explaining why the patient with only one copy of SMN2 had a mild clinical phenotype. Additionally, a minigene assay of wild type and mutant SMN1 in HEK293T cells also demonstrated that this transversion mutation induced e5 skipping. Considering treatment cost and goals of avoiding pain caused by injections and starting treatment as early as possible, risdiplam was prescribed for this patient. However, the patient showed remarkable clinical improvements after treatment with risdiplam for 7months despite carrying only one copy of SMN2. This study is the first report on the treatment of risdiplam in a patient with one SMN2 copy in a real-world setting. These findings expand the mutation spectrum of SMA and provide accurate genetic counseling information, as well as clarify the molecular mechanism of careful genotype-phenotype correlation of the patient.

Postnatal survival of phrenic motor neurons is promoted by BDNF/TrkB.FL signaling.

The number of motor neurons (MNs) declines precipitously during the final trimester before birth. Thereafter, the number of MNs remains relatively stable, with their connections to skeletal muscle dependent on neurotrophins, including brain-derived neurotrophic factor (BDNF) signaling through its high-affinity full-length tropomyosin-related kinase receptor subtype B (TrkB.FL) receptor. As a genetic knockout of BDNF leads to extensive MN loss and postnatal death within 1-2 days after birth, we tested the hypothesis that postnatal inhibition of BDNF/TrkB.FL signaling is important for postnatal phrenic MN (PhMN) survival. In the present study, we used a 1NMPP1-sensitive TrkBF616A mutant mouse to evaluate the effects of inhibition of TrkB kinase activity on phrenic MN (PhMN) numbers and diaphragm muscle (DIAm) fiber cross-sectional area (CSA). Pups were exposed to 1NMPP1 or vehicle (DMSO) from birth to 21 days old (weaning) via the mother's ingestion in the drinking water. Following weaning, the right phrenic nerve was exposed in the neck and the proximal end dipped in a rhodamine solution to retrogradely label PhMNs. After 24 h, the cervical spinal cord and DIAm were excised. Labeled PhMNs were imaged using confocal microscopy, whereas DIAm strips were frozen at ∼1.5× resting length, cryosectioned, and stained with hematoxylin and eosin to assess CSA. We observed an ∼34% reduction in PhMN numbers and increased primary dendrite numbers in 1NMPP1-treated TrkBF616A mice. The distribution of PhMN size (somal surface area) DIAm fiber cross-sectional areas did not differ. We conclude that survival of PhMNs during early postnatal development is sensitive to BDNF/TrkB.FL signaling.NEW & NOTEWORTHY During early postnatal development, BDNF/TrkB signaling promotes PhMN survival. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact PhMN size. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact the number or CSA of DIAm fibers.

Diagnosis of Challenging Spinal Muscular Atrophy Cases with Long-Read Sequencing

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disorder primarily caused by the deletion or mutation of the Survival Motor Neuron 1 (SMN1) gene. This study assesses the diagnostic potential of Long-Read Sequencing (LRS) in three SMA patients. For Patient 1, who has a heterozygous SMN1 deletion, LRS unveiled a missense mutation in SMN1 exon 5. In Patient 2, an Alu/Alu-mediated rearrangement covering the SMN1 promoter and exon 1 was identified through a blend of Multiplex Ligation-Dependent Probe Amplification (MLPA), LRS, and Gap-PCR. The third patient, born to a consanguineous family, bore four copies of hybrid SMN genes. LRS determined the genomic structures, indicating two distinct hybrids of SMN2 exon 7-SMN1 exon 8. However, a discrepancy was found between the SMN1-SMN2 ratio interpretations by LRS (0:2) and MLPA (0:4), which suggested a limitation of LRS in SMA diagnosis. In conclusion, this newly adapted long PCR-based third-generation sequencing introduces an additional avenue for SMA diagnosis.

Alteration of LARGE1 abundance in patients and a mouse model of 5q-associated spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by recessive pathogenic variants affecting the survival of motor neuron (SMN1) gene (localized on 5q). In consequence, cells lack expression of the corresponding protein. This pathophysiological condition is clinically associated with motor neuron (MN) degeneration leading to severe muscular atrophy. Additionally, vulnerability of other cellular populations and tissues including skeletal muscle has been demonstrated. Although the therapeutic options for SMA have considerably changed, treatment responses may differ thus underlining the persistent need for validated biomarkers. To address this need and to identify novel marker proteins for SMA, we performed unbiased proteomic profiling on cerebrospinal fluid derived (CSF) from genetically proven SMA type 1–3 cases and afterwards performed ELISA studies on CSF and serum samples to validate the potential of a novel biomarker candidates in both body fluids. To further decipher the pathophysiological impact of this biomarker, immunofluorescence studies were carried out on spinal cord and skeletal muscle derived from a 5q-SMA mouse model. Proteomics revealed increase of LARGE1 in CSF derived from adult patients showing a clinical response upon treatment with nusinersen. Moreover, LARGE1 levels were validated in CSF samples of further SMA patients (type 1–3) by ELISA. These studies also unveiled a distinguishment between groups in improvement of motor skills: adult patients do present with lowered level per se at baseline visit while no elevation upon treatment in the pediatric cohort can be observed. ELISA-based studies of serum samples showed no changes in the pediatric cohort but unraveled elevated level in adult patients responding to future intervention with nusinersen, while non-responders did not show a significant increase. Additional immunofluorescence studies of LARGE1 in MN and skeletal muscle of a SMA type 3 mouse model revealed an increase of LARGE1 during disease progression. Our combined data unraveled LARGE1 as a protein dysregulated in serum and CSF of SMA-patients (and in MN and skeletal muscle of SMA mice) holding the potential to serve as a disease marker for SMA and enabling to differentiate between patients responding and non-responding to therapy with nusinersen.

Alleviating Severe Cytoskeletal Destruction of Spinal Motor Neurons: Another Effect of Docosahexaenoic Acid in Spinal Cord Injury.

Spinal cord injury (SCI) treatment remains a major challenge. Spinal motor neurons (MNs) are seriously injured in the early stage after SCI, but this has not received sufficient attention. Oxidative stress is known to play a crucial role in SCI pathology. Our studies demonstrated that oxidative stress can cause severe damage to the cytoskeleton of spinal MNs. Docosahexaenoic acid (DHA) has been shown to have beneficial effects on SCI, but the mechanism remains unclear, and no study has investigated the effect of DHA on oxidative stress-induced spinal MN injury. Here, we investigated the effect of DHA on spinal MN injury through in vivo and in vitro experiments, focusing on the cytoskeleton. We found that DHA not only promoted spinal MN survival but, more importantly, alleviated the severe cytoskeletal destruction of these neurons induced by oxidative stress in vitro and in mice with SCI in vivo. In addition, the mechanisms involved were investigated and elucidated. These results not only suggested a beneficial role of DHA in spinal MN cytoskeletal destruction caused by oxidative stress and SCI but also indicated the important role of the spinal MN cytoskeleton in the recovery of motor function after SCI. Our study provides new insights for the formulation of SCI treatment.

Optimized MLPA workflow for spinal muscular atrophy diagnosis: identification of a novel variant, NC_000005.10:g.(70919941_70927324)del in isolated exon 1 of SMN1 gene through long-range PCR

BackgroundSpinal muscular atrophy (SMA) is a rare autosomal recessive hereditary neuromuscular disease caused by survival motor neuron 1 (SMN1) gene deletion or mutation. Homozygous deletions of exon 7 in SMN1 result in 95% of SMA cases, while the remaining 5% are caused by other pathogenic variants of SMN1.MethodsWe analyzed two SMA-suspected cases that were collected, with no SMN1 gene deletion and point mutation in whole-exome sequencing. Exon 1 deletion of the SMN gene was detected using Multiplex ligation-dependent probe amplification (MLPA) P021. We used long-range polymerase chain reaction (PCR) to isolate the SMN1 template, optimized-MLPA P021 for copy number variation (CNV) analysis within SMN1 only, and validated the findings via third-generation sequencing.ResultsTwo unrelated families shared a genotype with one copy of exon 7 and a novel variant, g.70919941_70927324del, in isolated exon 1 of the SMN1 gene. Case F1-II.1 demonstrated no exon 1 but retained other exons, whereas F2-II.1 had an exon 1 deletion in a single SMN1 gene. The read coverage in the third-generation sequencing results of both F1-II.1 and F2-II.1 revealed a deletion of approximately 7.3 kb in the 5’ region of SMN1. The first nucleotide in the sequence data aligned to the 7385 bp of NG_008691.1.ConclusionRemarkably, two proband families demonstrated identical SMN1 exon 1 breakpoint sites, hinting at a potential novel mutation hotspot in Chinese SMA, expanding the variation spectrum of the SMN1 gene and corroborating the specificity of isolated exon 1 deletion in SMA pathogenesis. The optimized-MLPA P021 determined a novel variant (g.70919941_70927324del) in isolated exon 1 of the SMN1 gene based on long-range PCR, enabling efficient and affordable detection of SMN gene variations in patients with SMA, providing new insight into SMA diagnosis to SMN1 deficiency and an optimized workflow for single exon CNV testing of the SMN gene.

VEGF expression disparities in brainstem motor neurons of the SOD1G93A ALS model: Correlations with neuronal vulnerability

Amyotrophic lateral sclerosis (ALS) is a rare neuromuscular disease characterized by severe muscle weakness mainly due to degeneration and death of motor neurons. A peculiarity of the neurodegenerative processes is the variable susceptibility among distinct neuronal populations, exemplified by the contrasting resilience of motor neurons innervating the ocular motor system and the more vulnerable facial and hypoglossal motor neurons. The crucial role of vascular endothelial growth factor (VEGF) as a neuroprotective factor in the nervous system is well-established since a deficit of VEGF has been related to motoneuronal degeneration. In this study, we investigated the survival of ocular, facial, and hypoglossal motor neurons utilizing the murine SOD1G93A ALS model at various stages of the disease. Our primary objective was to determine whether the survival of the different brainstem motor neurons was linked to disparate VEGF expression levels in resilient and susceptible motor neurons throughout neurodegeneration. Our findings revealed a selective loss of motor neurons exclusively within the vulnerable nuclei. Furthermore, a significantly higher level of VEGF was detected in the more resistant motor neurons, the extraocular ones. We also examined whether TDP-43 dynamics in the brainstem motor neuron of SOD mice was altered. Our data suggests that the increased VEGF levels observed in extraocular motor neurons may potentially underlie their resistance during the neurodegenerative processes in ALS in a TDP-43-independent manner. Our work might help to better understand the underlying mechanisms of selective vulnerability of motor neurons in ALS.

КЛИНИЧЕСКИЙ СЛУЧАЙ БОЛЕЗНИ КУГЕЛЬБЕРГА-ВЕЛАНДЕРА (СПИНАЛЬНОЙ МЫШЕЧНОЙ АТРОФИИ III ТИПА)

Background. Differential diagnosis of neuromuscular diseases is one of the most difficult areas in neurology. Molecular genetic research in these patients is of particular importance. The article presents a clinical case of 5q spinal muscular atrophy type III (Kugelberg – Welander disease), confirmed by molecular genetic testing. Description of the clinical case. The article presents the clinical observation of a patient with an initial diagnosis of Erb – Roth myopathy. A genetic study was carried out. The analysis of the presence of exon 7 of the SMN1/SMN2 genes showed the absence of a signal corresponding to exon 7 of the SMN1 gene. A search for deletions in the SMN1 gene showed the homozygous deletion of exons 7–8 of the SMN1 gene in the proband. Homozygous deletion of exons 7–8 of the SMN1 gene causes 5q proximal spinal muscular atrophy. As a result of determining the num-ber of copies of the SMN1 and SMN2 genes, 0 copies of exons 7–8 of the SMN1 gene and 4 copies of exons 7–8 of the SMN2 gene were registered, which confirms the diagnosis of 5q spinal muscular atrophy type III. Conclusion. Adult patients suffering from neuromuscular diseases are recommended to undergo confirmatory DNA diagnostics to prescribe pathogenetic treatment, which increases their chances of survival. In young people with a clinical diagnosis of neuromuscular disease, genetic testing is required to clarify the diagnosis and further treatment tactics.

Risdiplam in Spinal Muscular Atrophy: Safety Profile and Use Through The Early Access to Medicine Scheme for the Paediatric Cohort in Great Britain.

Spinal muscular atrophy (SMA) is a progressive neuromuscular disease caused by mutations in Survival motor neuron 1 (SMN1) gene, leading to reduction in survival motor neuron protein (SMN), key for motor neuron survival and function in the brainstem and spinal cord. Risdiplam is an orally administered SMN2-splicing modifier which increases production of functional SMN protein. Risdiplam was offered in the UK under early access to medicines scheme (EAMS) to SMA type 1 and 2 patients aged 2 months and older, not suitable for authorised treatments from September 2020 to December 2021. To describe the largest paediatric European real-world set of data on patients' characteristics and short-term safety for risdiplam in Great Britain through EAMS. We collated data from SMA REACH UK a national clinical and research network for all patients enrolled onto EAMS and assessed all submitted adverse events. Of the 92 patients; 78% were Type 2 SMA, mean age 10.9 years, range 0-17 years. 56 were treatment naïve, 33 previously treated; of these 25 had received nusinersen, 3 previous treatment unknown. Sixty adverse events (AEs) were reported occurring in 34 patients. The commonest were respiratory tract infections and gastrointestinal disturbance. Four life-threatening events were reported with 2 deaths and permanent cessation of risdiplam in 3 patients.Overall, 38/60 AEs were considered unrelated to risdiplam, 10/60 related to risdiplam and for 12/60 causality not specified. This study found a safety profile similar to clinical trials with no new safety concerns identified. With the restricted eligibility of onasemnogene abeparvovec and complications of nusinersen administration, EAMS allowed access or continued treatment to naïve patients or patients no longer suitable for approved medications. Collection of longitudinal data for this complex population is needed, to provide greater insights into risdiplam's role in addressing patients' needs into the future.