NCALD Antisense Oligonucleotide Therapy in Addition to Nusinersen further Ameliorates Spinal Muscular Atrophy in Mice.

Spinal muscular atrophy (SMA) is a neuromuscular disease causing the most frequent genetic childhood lethality. Recently, nusinersen, an antisense oligonucleotide (ASO) that corrects SMN2 splicing and thereby increases full-length SMN protein, has been approved by the FDA and EMA for SMA therapy. However, the administration of nusinersen in severe and/or post-symptomatic SMA-affected individuals is insufficient to counteract the disease. Therefore, additional SMN-independent therapies are needed to support the function of motoneurons and neuromuscular junctions. We recently identified asymptomatic SMN1-deleted individuals who were protected against SMA by reduced expression of neurocalcin delta (NCALD). NCALD reduction is proven to be a protective modifier of SMA across species, including worm, zebrafish, and mice. Here, we identified Ncald-ASO3-out of 450 developed Ncald ASOs-as the most efficient and non-toxic ASO for the CNS, by applying a stepwise screening strategy in cortical neurons and adult and neonatal mice. In a randomized-blinded preclinical study, a single subcutaneous low-dose SMN-ASO and a single intracerebroventricular Ncald-ASO3 or control-ASO injection were presymptomatically administered in a severe SMA mouse model. NCALD reduction of >70% persisted for about 1 month. While low-dose SMN-ASO rescues multiorgan impairment, additional NCALD reduction significantly ameliorated SMA pathology including electrophysiological and histological properties of neuromuscular junctions and muscle at P21 and motoric deficits at 3 months. The present study shows the additional benefit of a combinatorial SMN-dependent and SMN-independent ASO-based therapy for SMA. This work illustrates how a modifying gene, identified in some asymptomatic individuals, helps to develop a therapy for all SMA-affected individuals.

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis.

Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca2+-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.

CHP1 reduction ameliorates spinal muscular atrophy pathology by restoring calcineurin activity and endocytosis.

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZATM, has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular junction size, impaired maturity of neuromuscular junctions and smaller muscle fibre size compared to low-dose SMN antisense oligonucleotide alone. In NSC34 cells, Chp1 knockdown tripled macropinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1 knockdown restored macropinocytosis in Smn-depleted cells by elevating calcineurin phosphatase activity. CHP1 is an inhibitor of calcineurin, which collectively dephosphorylates proteins involved in endocytosis, and is therefore crucial in synaptic vesicle endocytosis. Indeed, we found marked hyperphosphorylation of dynamin 1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1 mutant allele. Taken together, we show that CHP1 is a novel SMA modifier that directly interacts with PLS3, and that CHP1 reduction ameliorates SMA pathology by counteracting impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy.

Advances in understanding the role of disease-associated proteins in spinal muscular atrophy.

INTRODUCTION: Spinal muscular atrophy (SMA) is a neurodegenerative disorder characterized by alpha motor neuron loss in the spinal cord due to reduced survival motor neuron (SMN) protein level. While the genetic basis of SMA is well described, the specific molecular pathway underlying SMA is still not fully understood. Areas covered: This review discusses the recent advancements in understanding the molecular pathways in SMA using different omics approaches and genetic modifiers identified in both vertebrate and invertebrate systems. The findings that are summarized in this article were deduced from original articles and reviews with a particular focus on the latest advancements in the field. Expert commentary: The identification of genetic modifiers such as PLS3 and NCALD in humans or of SMA modulators such as Elavl4 (HuD), Copa, Uba1, Mapk10 (Jnk3), Nrxn2 and Tmem41b (Stasimon) in various SMA animal models improved our knowledge of impaired cellular pathways in SMA. Inspiration from modifier genes and their functions in motor neuron and neuromuscular junctions may open a new avenue for future SMA combinatorial therapies.

Accuracy of Balancing at Total Knee Surgery Using an Instrumented Tibial Trial.

BACKGROUND: Balancing is an important part of a total knee procedure, and in recent years, more emphasis has been given to quantifying the process. METHODS: During 101 total knee surgeries, initial bone cuts were made using navigation. Lateral and medial contact forces were determined throughout flexion using an instrumented tibial trial. Balancing was defined as a ratio of the medial and total force, the target being 0.5 (equal lateral and medial forces). Based on the initial values, surgical corrections were selected to achieve balancing. The most common corrections were soft tissue releases (63 incidences), including MCL, posterolateral corner, posteromedial corner, and changing tibial insert thicknesses (34 incidences). RESULTS: After final balancing, the mean ratio was 0.52 ± 0.14, between 0.35 and 0.65 being achieved in 80% of cases. In 84% of cases, only 0-2 corrections were required. The average total force on the condyles was 215 ± 86 N. CONCLUSION: Our study provides data to surgeons on the results to expect when balancing a knee, which can enhance both accuracy and consistency of the procedure.

Hyphenation of Liquid Chromatography and Trapped Ion Mobility - Mass Spectrometry for Characterization of Isomeric Phosphatidylethanolamines with Focus on N-Acylated Species.

In prior research, hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) has demonstrated applicability for characterizing regioisomers in lipidomics studies, including phosphatidylglycerols (PG) and bis(monoacyl)glycerophosphates (BMP). However, there are other lipid regioisomers, such as phosphatidylethanolamines (PE) and lyso-N-acyl-PE (LNAPE), that have not been studied as extensively. Therefore, hyphenated mass spectrometric methods are needed to investigate PE and LNAPE regioisomers individually. The asymmetric structure of LNAPE favors isomeric species, which can result in coelution and chimeric MS/MS spectra. One way to address the challenge of chimeric MS/MS spectra is through mobility-resolved fragmentation using trapped ion mobility spectrometry (TIMS). Therefore, we developed a multidimensional HILIC-TIMS-MS/MS approach for the structural characterization of isomeric phosphatidylethanolamines in both negative and positive ionization modes. The study revealed the complementary fragmentation pattern and ion mobility behavior of LNAPE in both ionization modes, which was confirmed by a self-synthesized LNAPE standard. With this knowledge, a distinction of regioisomeric PE and LNAPE was achieved in human plasma samples. Furthermore, regioisomeric LNAPE species containing at least one unsaturated fatty acid were noted to exhibit a change in collision cross-section in positive ionization mode, leading to a lipid characterization with respect to fatty acyl positional level. Similar mobility behavior was also observed for the biological LNAPE precursor N-acyl-PE (NAPE). Application of this approach to plasma and cereal samples demonstrated its effectiveness in regioisomeric LNAPE and NAPE species' elucidation.

Determination of Polar Lipids in Wheat and Oat by a Complementary Approach of Hydrophilic Interaction Liquid Chromatography and Reversed-Phase High-Performance Liquid Chromatography Hyphenated with High-Resolution Mass Spectrometry.

Cereals contain lipids that fulfill important physiological roles and are associated with stress in the plant. However, many of the specific biological roles of lipids are yet unknown. Comprehensive analysis of these polar lipid categories in whole grain wheat and oat, cereals highly relevant also in nutrition, was performed. Hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with high-resolution mass spectrometry using electrospray ionization in both positive and negative ionization mode was used. Exploiting the different separation mechanisms, HILIC was used as a screening method for straightforward lipid class assignment and enabled differentiation of isomeric lipid classes, like phosphatidylethanolamine and lyso-N-acylphosphatidylethanolamine, while RP-HPLC facilitated separation of constitutional isomers. In combination with data-dependent MS/MS experiments, 67 lipid species belonging to nine polar lipid classes could be identified. Furthermore, with both ionization modes, fatty acyl chains directly connected to the lipid headgroups could be assigned. This work focused on the four lipid classes N-acylphosphatidylethanolamines, acyl-monogalactosyldiacylglycerols, digalactosyldiacylglycerols, and monogalactosyldiacylglycerols as they were less studied in detail in the past. Applying the complementary approach, the relative lipid species compositions in these lipid classes was investigated in detail.

Cardiogenic Shock Due to Atrial Arrhythmia as the Initial Presentation of Transthyretin Cardiac Amyloidosis.

Atrial arrhythmias are common in transthyretin cardiac amyloidosis (ATTR-CA), with a prevalence of ≤80%. They are often not well tolerated. We describe 3 patients with decompensated heart failure and cardiogenic shock precipitated by atrial arrhythmias who ultimately received diagnoses of ATTR-CA. (Level of Difficulty: Intermediate.).

Neurocalcin Delta Knockout Impairs Adult Neurogenesis Whereas Half Reduction Is Not Pathological.

Neurocalcin delta (NCALD) is a brain-enriched neuronal calcium sensor and its reduction acts protective against spinal muscular atrophy (SMA). However, the physiological function of NCALD and implications of NCALD reduction are still elusive. Here, we analyzed the ubiquitous Ncald knockout in homozygous (Ncald KO/KO) and heterozygous (Ncald KO/WT) mice to unravel the physiological role of NCALD in the brain and to study whether 50% NCALD reduction is a safe option for SMA therapy. We found that Ncald KO/KO but not Ncald KO/WT mice exhibit significant changes in the hippocampal morphology, likely due to impaired generation and migration of newborn neurons in the dentate gyrus (DG). To understand the mechanism behind, we studied the NCALD interactome and identified mitogen-activated protein kinase kinase kinase 10 (MAP3K10) as a novel NCALD interacting partner. MAP3K10 is an upstream activating kinase of c-Jun N-terminal kinase (JNK), which regulates adult neurogenesis. Strikingly, the JNK activation was significantly upregulated in the Ncald KO/KO brains. Contrary, neither adult neurogenesis nor JNK activation were altered by heterozygous Ncald deletion. Taken together, our study identifies a novel link between NCALD and adult neurogenesis in the hippocampus, possibly via a MAP3K10-JNK pathway and emphasizes the safety of using NCALD reduction as a therapeutic option for SMA.

Biallelic CHP1 mutation causes human autosomal recessive ataxia by impairing NHE1 function.

OBJECTIVE: To ascertain the genetic and functional basis of complex autosomal recessive cerebellar ataxia (ARCA) presented by 2 siblings of a consanguineous family characterized by motor neuropathy, cerebellar atrophy, spastic paraparesis, intellectual disability, and slow ocular saccades. METHODS: Combined whole-genome linkage analysis, whole-exome sequencing, and focused screening for identification of potential causative genes were performed. Assessment of the functional consequences of the mutation on protein function via subcellular fractionation, size-exclusion chromatography, and fluorescence microscopy were done. A zebrafish model, using Morpholinos, was generated to study the pathogenic effect of the mutation in vivo. RESULTS: We identified a biallelic 3-bp deletion (p.K19del) in CHP1 that cosegregates with the disease. Neither focused screening for CHP1 variants in 2 cohorts (ARCA: N = 319 and NeurOmics: N = 657) nor interrogating GeneMatcher yielded additional variants, thus revealing the scarcity of CHP1 mutations. We show that mutant CHP1 fails to integrate into functional protein complexes and is prone to aggregation, thereby leading to diminished levels of soluble CHP1 and reduced membrane targeting of NHE1, a major Na+/H+ exchanger implicated in syndromic ataxia-deafness. Chp1 deficiency in zebrafish, resembling the affected individuals, led to movement defects, cerebellar hypoplasia, and motor axon abnormalities, which were ameliorated by coinjection with wild-type, but not mutant, human CHP1 messenger RNA. CONCLUSIONS: Collectively, our results identified CHP1 as a novel ataxia-causative gene in humans, further expanding the spectrum of ARCA-associated loci, and corroborated the crucial role of NHE1 within the pathogenesis of these disorders.

Investigational therapies for the treatment of spinal muscular atrophy.

INTRODUCTION: Currently, there is no cure available for the hereditary neurodegenerative disease proximal spinal muscular atrophy (SMA), which is the number one genetic killer in early childhood. However, growing knowledge of SMA pathophysiology has opened new avenues for potential therapeutic interventions. AREAS COVERED: This review summarizes a variety of investigational therapeutic approaches for SMA. Focusing on the current state-of-the-art applications, the authors discuss the outcome of the first clinical interventions and compare the first results from the newest strategies. The achievements of the investigational drugs highlighted in this article were deduced from original articles, pharmaceutical company press releases and clinical trial results. EXPERT OPINION: Nearly two decades after the discovery of the disease causing gene survival motor neuron 1, many therapeutic options for SMA have been developed, some of which made it to clinical trials but could not prove their promising experimental results. Recently, big research efforts from academia, government and the pharmaceutical industry have led to the development of highly promising compounds that are currently in clinical trials, and which could lead to feasible treatment options in the future.