Altered mitochondrial function in fibroblast cell lines derived from disease carriers of spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive childhood-onset neuromuscular disease with a carrier frequency of ~1:50. Mitochondrial abnormalities are widespread in patients with SMA. Disease carriers for SMA (i.e., the parents of patients with SMA) are viewed as asymptomatic for SMA disease. As far as we are aware, mitochondria have not been previously examined in SMA carriers, yet as they are maternally inherited, mitochondrial function in SMA carriers has putative implications for disease pathogenesis. METHODS: Fibroblast cell lines derived from SMA carriers and controls were obtained from two different sources and cultured under standard conditions. The mitochondrial membrane potential, reactive oxygen species (ROS) production, citrate synthase activity, and bioenergetic analysis were examined as measures of mitochondrial function. The mitochondrial genome was also sequenced in a subset of the fibroblast cell lines to identify any mitochondrial DNA variants. RESULTS: Here, we show a depolarized mitochondrial membrane potential, increased levels of reactive oxygen species, and reduced citrate synthase activity in SMA carriers compared with controls. A likely pathogenic variant in the MT-CO3 gene (which encodes subunit III of cytochrome c oxidase) was also identified in a paternal carrier. CONCLUSIONS: This study was conducted as a preliminary investigation of mitochondrial function in SMA carriers. Our findings suggest that disease carriers of SMA show differences in mitochondrial function, indicative of a subclinical mitochondrial phenotype. Further investigation in a larger sample set is warranted.

Spinal muscular atrophy (SMA) is a disease that mostly affects children in which the muscles become weaker over time, and often leads to death in untreated individuals. It is caused by a defective gene that children often inherit from their parents. The parents of children with SMA are known as disease carriers if they do not show any symptoms of SMA themselves. We studied skin cells from the parents of people with SMA and found changes in a component of the cells called the mitochondria. These changes are not normally present in healthy individuals. Further work is needed to fully understand the implications of our findings for those with SMA and their parents.

IgM anti-GM2 antibodies in patients with multifocal motor neuropathy target Schwann cells and are associated with early onset.

BACKGROUND: Multifocal motor neuropathy (MMN) is a rare, chronic immune-mediated polyneuropathy characterized by asymmetric distal limb weakness. An important feature of MMN is the presence of IgM antibodies against gangliosides, in particular GM1 and less often GM2. Antibodies against GM1 bind to motor neurons (MNs) and cause damage through complement activation. The involvement of Schwann cells (SCs), expressing GM1 and GM2, in the pathogenesis of MMN is unknown. METHODS: Combining the data of our 2007 and 2015 combined cross-sectional and follow-up studies in Dutch patients with MMN, we evaluated the presence of IgM antibodies against GM1 and GM2 in serum from 124 patients with MMN and investigated their binding to SCs and complement-activating properties. We also assessed the relation of IgM binding and complement deposition with clinical characteristics. RESULTS: Thirteen out of 124 patients (10%) had a positive ELISA titer for IgM anti-GM2. Age at onset of symptoms was significantly lower in MMN patients with anti-GM2 IgM. IgM binding to SCs correlated with IgM anti-GM2 titers. We found no correlation between IgM anti-GM2 titers and MN binding or with IgM anti-GM1 titers. IgM binding to SCs decreased upon pre-incubation of serum with soluble GM2, but not with soluble GM1. IgM anti-GM2 binding to SCs correlated with complement activation, as reflected by increased C3 fixation on SCs and C5a formation in the supernatant. CONCLUSION: Circulating IgM anti-GM2 antibodies define a subgroup of patients with MMN that has an earlier onset of disease. These antibodies probably target SCs specifically and activate complement, similarly as IgM anti-GM1 on MNs. Our data indicate that complement activation by IgM antibodies bound to SCs and MNs underlies MMN pathology.

A 21-bp deletion in the complement regulator CD55 promotor region is associated with multifocal motor neuropathy and its disease course.

BACKGROUND AND AIMS: To further substantiate the role of antibody-mediated complement activation in multifocal motor neuropathy (MMN) immunopathology, we investigated the distribution of promotor polymorphisms of genes encoding the membrane-bound complement regulators CD46, CD55, and CD59 in patients with MMN and controls, and evaluated their association with disease course. METHODS: We used Sanger sequencing to genotype five common polymorphisms in the promotor regions of CD46, CD55, and CD59 in 133 patients with MMN and 380 controls. We correlated each polymorphism to clinical parameters. RESULTS: The genotype frequencies of rs28371582, a 21-bp deletion in the CD55 promotor region, were altered in patients with MMN as compared to controls (p .009; Del/Del genotype 16.8% vs. 7.7%, p .005, odds ratio: 2.43 [1.27-4.58]), and patients carrying this deletion had a more favorable disease course (mean difference 0.26 Medical Research Council [MRC] points/year; 95% confidence interval [CI]: 0.040-0.490, p .019). The presence of CD59 rs141385724 was associated with less severe pre-diagnostic disease course (mean difference 0.940 MRC point/year; 95% CI: 0.083-1.80, p .032). INTERPRETATION: MMN susceptibility is associated with a 21-bp deletion in the CD55 promotor region (rs2871582), which is associated with lower CD55 expression. Patients carrying this deletion may have a more favorable long-term disease outcome. Taken together, these results point out the relevance of the pre-C5 level of the complement cascade in the inflammatory processes underlying MMN.

Neurofilament light protein as a biomarker for spinal muscular atrophy: a review and reference ranges.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, characterized by progressive neuromuscular degeneration resulting from mutations in the survival motor neuron (SMN1) gene. The availability of disease-modifying therapies for SMA therapies highlights the pressing need for easily accessible and cost-effective blood biomarkers to monitor treatment response and for better disease management. Additionally, the wide implementation of newborn genetic screening programs in Western countries enables presymptomatic diagnosis of SMA and immediate treatment administration. However, the absence of monitoring and prognostic blood biomarkers for neurodegeneration in SMA hinders effective disease management. Neurofilament light protein (NfL) is a promising biomarker of neuroaxonal damage in SMA and reflects disease progression in children with SMA undergoing treatment. Recently, the European Medicines Agency issued a letter of support endorsing the potential utilization of NfL as a biomarker of pediatric neurological diseases, including SMA. Within this review, we comprehensively assess the potential applications of NfL as a monitoring biomarker for disease severity and treatment response in pediatric-onset SMA. We provide reference ranges for normal levels of serum based NfL in neurologically healthy children aged 0-18 years. These reference ranges enable accurate interpretation of NfL levels in children and can accelerate the implementation of NfL into clinical practice.

Innovating spinal muscular atrophy models in the therapeutic era.

Spinal muscular atrophy (SMA) is a severe, monogenetic, neuromuscular disease. A thorough understanding of its genetic cause and the availability of robust models has led to the development and approval of three gene-targeting therapies. This is a unique and exciting development for the field of neuromuscular diseases, many of which remain untreatable. The development of therapies for SMA not only opens the door to future therapeutic possibilities for other genetic neuromuscular diseases, but also informs us about the limitations of such treatments. For example, treatment response varies widely and, for many patients, significant disability remains. Currently available SMA models best recapitulate the severe types of SMA, and these models are genetically and phenotypically more homogeneous than patients. Furthermore, treating patients is leading to a shift in phenotypes with increased variability in SMA clinical presentation. Therefore, there is a need to generate model systems that better reflect these developments. Here, we will first discuss current animal models of SMA and their limitations. Next, we will discuss the characteristics required to future-proof models to assist the field in the development of additional, novel therapies for SMA.

Multifocal motor neuropathy is not associated with altered innate immune responses to endotoxin.

OBJECTIVE: Antibody- and complement-mediated peripheral nerve inflammation are central in the pathogenesis of MMN. Here, we studied innate immune responses to endotoxin in patients with MMN and controls to further our understanding of MMN risk factors and disease modifiers. METHODS: We stimulated whole blood of 52 patients with MMN and 24 controls with endotoxin and collected plasma. With a multiplex assay, we determined levels of the immunoregulating proteins IL-1RA, IL-1ß, IL-6, IL-10, IL-21, TNF-α, IL-8 and CD40L in unstimulated and LPS-stimulated plasma. We compared baseline and stimulated protein levels between patients and controls and correlated concentrations to clinical parameters. RESULTS: Protein level changes after stimulation were comparable between groups (p > 0.05). IL-1RA, IL-1ß, IL-6 and IL-21 baseline concentrations showed a positive correlation with monthly IVIg dosage (all corrected p-values < 0.016). Patients with anti-GM1 IgM antibodies showed a more pronounced IL-21 increase after stimulation (p 0.048). CONCLUSIONS: Altered endotoxin-induced innate immune responses are unlikely to be a susceptibility factor for MMN.

Population-based assessment of nusinersen efficacy in children with spinal muscular atrophy: a 3-year follow-up study.

Nusinersen (Spinraza®) improves survival of infants with hereditary proximal spinal muscular atrophy and motor function in children up to 12 years. Population-based assessments of treatment efficacy are limited and confined to select cohorts of patients. We performed a nationwide, population-based, single-centre cohort study in children with spinal muscular atrophy younger than 9.5 years at start of treatment in line with reimbursement criteria in the Netherlands. We assessed age-relevant motor function scores, the need for tube feeding, hours of ventilatory support and documented adverse events. We used linear mixed modelling to assess treatment effects. We compared motor function during treatment with natural history data and to individual trajectories of muscle strength and motor function before the start of treatment. We included 71 out of 72 Dutch children who were treated (median age 54 months; range 0-117) and followed them for a median of 38 months (range 5-52). We observed improvement of motor function in 72% and stabilization in another 18% of the symptomatic children, which differed from the natural disease course in a matched cohort of which we had previously collected natural history data. Longitudinal analysis showed that motor function improved up to a median of 24 months (range 12-30) of treatment after which it stabilized. Shorter disease duration at start of treatment resulted in better treatment efficacy (P < 0.01). Sixteen children (23%) achieved new motor milestones. Bulbar and respiratory function did not improve significantly during treatment. In 15 patients from whom treatment-naïve data were available, the pre-treatment trajectory of motor function decline changed to stabilization or improvement after the start of treatment. We documented 82 adverse events after 934 injections (9%) in 45 patients. None of the adverse events led to treatment discontinuation. Intrathecal nusinersen treatment is safe and improves or stabilizes motor function in 90% of young children with spinal muscular atrophy types 1c-3a. We did not observe improvement of respiratory and bulbar functions.

The mitochondrial protein Sideroflexin 3 (SFXN3) influences neurodegeneration pathways in vivo.

Synapses are a primary pathological target in neurodegenerative diseases. Identifying therapeutic targets at the synapse could delay progression of numerous conditions. The mitochondrial protein SFXN3 is a neuronally enriched protein expressed in synaptic terminals and regulated by key synaptic proteins, including α-synuclein. We first show that SFXN3 uses the carrier import pathway to insert into the inner mitochondrial membrane. Using high-resolution proteomics on Sfxn3-KO mice synapses, we then demonstrate that SFXN3 influences proteins and pathways associated with neurodegeneration and cell death (including CSPα and Caspase-3), as well as neurological conditions (including Parkinson's disease and Alzheimer's disease). Overexpression of SFXN3 orthologues in Drosophila models of Parkinson's disease significantly reduced dopaminergic neuron loss. In contrast, the loss of SFXN3 was insufficient to trigger neurodegeneration in mice, indicating an anti- rather than pro-neurodegeneration role for SFXN3. Taken together, these results suggest a potential role for SFXN3 in the regulation of neurodegeneration pathways.

SMN1 Duplications Are Associated With Progressive Muscular Atrophy, but Not With Multifocal Motor Neuropathy and Primary Lateral Sclerosis.

OBJECTIVE: To assess the association between copy number (CN) variation in the survival motor neuron (SMN) locus and multifocal motor neuropathy (MMN), progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS) susceptibility and to determine the association of SMN1 and SMN2 CN with MMN, PMA, and PLS disease course. METHODS: In this monocenter study, we used multiplex ligation-dependent probe amplification to determine SMN1 and SMN2 CN in Dutch patients with MMN, PMA, and PLS and controls. We stratified clinical parameters for SMN1 and SMN2 CN. We analyzed SMN1 and SMN2 exons 1-6, intron 6, and exon 8 CN to study the genetic architecture of SMN1 duplications. RESULTS: SMN1 and SMN2 CN were determined in 132 patients with MMN, 150 patients with PMA, 104 patients with PLS, and 956 control subjects. MMN and PLS were not associated with CN variation in SMN1 or SMN2. By contrast, patients with PMA more often than controls carried SMN1 duplications (≥3 SMN1 copies, 12.0% vs 5.0%, odds ratio 2.69 (1.43-4.91), p 0.0020). SMN1 and SMN2 CN status was not associated with MMN, PLS, or PMA disease course. In case of SMN1 exon 7 duplications, exons 1-6, exon 8, and introns 6 and 7 were also duplicated, suggesting full SMN1 duplications. CONCLUSIONS: SMN1 duplications are associated with PMA, but not with PLS and MMN. SMN1 duplications in PMA are balanced duplications. The results of this study highlight the primary effect of altered SMN CN on lower motor neurons.

Intragenic and structural variation in the SMN locus and clinical variability in spinal muscular atrophy.

Clinical severity and treatment response vary significantly between patients with spinal muscular atrophy. The approval of therapies and the emergence of neonatal screening programmes urgently require a more detailed understanding of the genetic variants that underlie this clinical heterogeneity. We systematically investigated genetic variation other than SMN2 copy number in the SMN locus. Data were collected through our single-centre, population-based study on spinal muscular atrophy in the Netherlands, including 286 children and adults with spinal muscular atrophy Types 1-4, including 56 patients from 25 families with multiple siblings with spinal muscular atrophy. We combined multiplex ligation-dependent probe amplification, Sanger sequencing, multiplexed targeted resequencing and digital droplet polymerase chain reaction to determine sequence and expression variation in the SMN locus. SMN1, SMN2 and NAIP gene copy number were determined by multiplex ligation-dependent probe amplification. SMN2 gene variant analysis was performed using Sanger sequencing and RNA expression analysis of SMN by droplet digital polymerase chain reaction. We identified SMN1-SMN2 hybrid genes in 10% of spinal muscular atrophy patients, including partial gene deletions, duplications or conversions within SMN1 and SMN2 genes. This indicates that SMN2 copies can vary structurally between patients, implicating an important novel level of genetic variability in spinal muscular atrophy. Sequence analysis revealed six exonic and four intronic SMN2 variants, which were associated with disease severity in individual cases. There are no indications that NAIP1 gene copy number or sequence variants add value in addition to SMN2 copies in predicting the clinical phenotype in individual patients with spinal muscular atrophy. Importantly, 95% of spinal muscular atrophy siblings in our study had equal SMN2 copy numbers and structural changes (e.g. hybrid genes), but 60% presented with a different spinal muscular atrophy type, indicating the likely presence of further inter- and intragenic variabilities inside as well as outside the SMN locus. SMN2 gene copies can be structurally different, resulting in inter- and intra-individual differences in the composition of SMN1 and SMN2 gene copies. This adds another layer of complexity to the genetics that underlie spinal muscular atrophy and should be considered in current genetic diagnosis and counselling practices.

Pre-natal manifestation of systemic developmental abnormalities in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in survival motor neuron 1 (SMN1). SMN-restoring therapies have recently emerged; however, preclinical and clinical studies revealed a limited therapeutic time window and systemic aspects of the disease. This raises a fundamental question of whether SMA has presymptomatic, developmental components to disease pathogenesis. We have addressed this by combining micro-computed tomography (µCT) and comparative proteomics to examine systemic pre-symptomatic changes in a prenatal mouse model of SMA. Quantitative µCT analyses revealed that SMA embryos were significantly smaller than littermate controls, indicative of general developmental delay. More specifically, cardiac ventricles were smaller in SMA hearts, whilst liver and brain remained unaffected. In order to explore the molecular consequences of SMN depletion during development, we generated comprehensive, high-resolution, proteomic profiles of neuronal and non-neuronal organs in SMA mouse embryos. Significant molecular perturbations were observed in all organs examined, highlighting tissue-specific prenatal molecular phenotypes in SMA. Together, our data demonstrate considerable systemic changes at an early, presymptomatic stage in SMA mice, revealing a significant developmental component to SMA pathogenesis.

Analysis of FUS, PFN2, TDP-43, and PLS3 as potential disease severity modifiers in spinal muscular atrophy.

OBJECTIVE: To investigate mutations in genes that are potential modifiers of spinal muscular atrophy (SMA) severity. METHODS: We performed a hypothesis-based search into the presence of variants in fused in sarcoma (FUS), transactive response DNA-binding protein 43 (TDP-43), plastin 3 (PLS3), and profilin 2 (PFN2) in a cohort of 153 patients with SMA types 1-4, including 19 families. Variants were detected with targeted next-generation sequencing and confirmed with Sanger sequencing. Functional effects of the identified variants were analyzed in silico and for PLS3, by analyzing expression levels in peripheral blood. RESULTS: We identified 2 exonic variants in FUS exons 5 and 6 (p.R216C and p.S135N) in 2 unrelated patients, but clinical effects were not evident. We identified 8 intronic variants in PLS3 in 33 patients. Five PLS3 variants (c.1511+82T>C; c.748+130 G>A; c.367+182C>T; c.891-25T>C (rs145269469); c.1355+17A>G (rs150802596)) potentially alter exonic splice silencer or exonic splice enhancer sites. The variant c.367+182C>T, but not RNA expression levels, corresponded with a more severe phenotype in 1 family. However, this variant or level of PLS3 expression did not consistently correspond with a milder or more severe phenotype in other families or the overall cohort. We found 3 heterozygous, intronic variants in PFN2 and TDP-43 with no correlation with clinical phenotype or effects on splicing. CONCLUSIONS: PLS3 and FUS sequence variants do not modify SMA severity at the population level. Specific variants in individual patients or families do not consistently correlate with disease severity.

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

Molecular Mechanisms Underlying Sensory-Motor Circuit Dysfunction in SMA.

Activation of skeletal muscle in response to acetylcholine release from the neuromuscular junction triggered by motor neuron firing forms the basis of all mammalian locomotion. Intricate feedback and control mechanisms, both from within the central nervous system and from sensory organs in the periphery, provide essential inputs that regulate and finetune motor neuron activity. Interestingly, in motor neuron diseases, such as spinal muscular atrophy (SMA), pathological studies in patients have identified alterations in multiple parts of the sensory-motor system. This has stimulated significant research efforts across a range of different animal models of SMA in order to understand these defects and their contribution to disease pathogenesis. Several recent studies have demonstrated that defects in sensory components of the sensory-motor system contribute to dysfunction of motor neurons early in the pathogenic process. In this review, we provide an overview of these findings, with a specific focus on studies that have provided mechanistic insights into the molecular processes that underlie dysfunction of the sensory-motor system in SMA. These findings highlight the role that cell types other than motor neurons play in SMA pathogenesis, and reinforce the need for therapeutic interventions that target and rescue the wide array of defects that occur in SMA.

Active Ribosome Profiling with RiboLace.

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy.

Deafferentation of motor neurons as a result of defective sensory-motor connectivity is a critical early event in the pathogenesis of spinal muscular atrophy, but the underlying molecular pathways remain unknown. We show that restoration of ubiquitin-like modifier-activating enzyme 1 (UBA1) was sufficient to correct sensory-motor connectivity in the spinal cord of mice with spinal muscular atrophy. Aminoacyl-tRNA synthetases, including GARS, were identified as downstream targets of UBA1. Regulation of GARS by UBA1 occurred via a non-canonical pathway independent of ubiquitylation. Dysregulation of UBA1/GARS pathways in spinal muscular atrophy mice disrupted sensory neuron fate, phenocopying GARS-dependent defects associated with Charcot-Marie-Tooth disease. Sensory neuron fate was corrected following restoration of UBA1 expression and UBA1/GARS pathways in spinal muscular atrophy mice. We conclude that defective sensory motor connectivity in spinal muscular atrophy results from perturbations in a UBA1/GARS pathway that modulates sensory neuron fate, thereby highlighting significant molecular and phenotypic overlap between spinal muscular atrophy and Charcot-Marie-Tooth disease.

riboWaltz: Optimization of ribosome P-site positioning in ribosome profiling data.

Ribosome profiling is a powerful technique used to study translation at the genome-wide level, generating unique information concerning ribosome positions along RNAs. Optimal localization of ribosomes requires the proper identification of the ribosome P-site in each ribosome protected fragment, a crucial step to determine the trinucleotide periodicity of translating ribosomes, and draw correct conclusions concerning where ribosomes are located. To determine the P-site within ribosome footprints at nucleotide resolution, the precise estimation of its offset with respect to the protected fragment is necessary. Here we present riboWaltz, an R package for calculation of optimal P-site offsets, diagnostic analysis and visual inspection of ribosome profiling data. Compared to existing tools, riboWaltz shows improved accuracies for P-site estimation and neat ribosome positioning in multiple case studies. riboWaltz was implemented in R and is available as an R package at https://github.com/LabTranslationalArchitectomics/RiboWaltz.

Whole blood transcriptome analysis in amyotrophic lateral sclerosis: A biomarker study.

The biological pathways involved in amyotrophic lateral sclerosis (ALS) remain elusive and diagnostic decision-making can be challenging. Gene expression studies are valuable in overcoming such challenges since they can shed light on differentially regulated pathways and may ultimately identify valuable biomarkers. This two-stage transcriptome-wide study, including 397 ALS patients and 645 control subjects, identified 2,943 differentially expressed transcripts predominantly involved in RNA binding and intracellular transport. When batch effects between the two stages were overcome, three different models (support vector machines, nearest shrunken centroids, and LASSO) discriminated ALS patients from control subjects in the validation stage with high accuracy. The models' accuracy reduced considerably when discriminating ALS from diseases that mimic ALS clinically (N = 75), nor could it predict survival. We here show that whole blood transcriptome profiles are able to reveal biological processes involved in ALS. Also, this study shows that using these profiles to differentiate between ALS and mimic syndromes will be challenging, even when taking batch effects in transcriptome data into account.