Neurocalcin delta (NCALD) knockout impairs adult neurogenesis whereas half reduction is a safe therapeutic option for spinal muscular atrophy

Spinal muscular atrophy (SMA) is the second most common recessive monogenic disease characterized by loss of motor neurons and impaired neuromuscular junctions which leads to proximal muscles atrophy. SMA is primarily caused by mutation/loss of survival of motor neuron 1 (SMN1) gene encoding the SMN protein. Interestingly, humans carry additionally SMN2, a copy gene of SMN1 which is relatively new in evolutionary scale of genes and the SMA disease severity is inversely correlated with the copy number of the SMN2 gene. In the recent year, SMA therapeutics achieved a landmark with FDA and EMA approval of the splice modifier molecule named Nusinersen, which is an SMN dependent therapy as it elevates the SMN protein level derived from the SMN2 transcript. However, SMN dependent therapies have not been sufficient to fully cure the SMA, especially in most severe SMA type I patients which may carry even a single allele of SMN2. Therefore, a combinatorial therapy which includes SMN independent strategies would be remarkably beneficial. Certain modifier genes, which upon differential expression can protect against SMA are most relevant and potential candidates to be studied in context of combinatorial therapies. Recently, reduction of a neuronal calcium sensor, Neurocalcin delta (NCALD) significantly ameliorated major SMA symptoms: reduced motor neuron axon length, neuromuscular junction size, muscle fibre size, impaired endocytosis and motor functions in various SMA model systems. Considering these results, reduction of NCALD was proposed for SMA combinatorial therapeutics. However, depending upon the physiological functions of NCALD in various cellular processes, reduction of NCALD may potentially incur certain side effects. NCALD is a member of the brain-enriched neuronal calcium sensor sub-family Visinin like proteins (VILIPs). However, till date only few NCALD functions have been explored based on the functions of its close homologues VILIP1, hippocalcin and guanylate cyclase activating protein (GCAP). Therefore, in this study we first investigated non redundant physiological functions of NCALD by characterizing Ncald knockout (NcaldKO/KO) mice; then referring to the phenotypes of NcaldKO/KO mice we further analysed heterozygous (NcaldKO/WT) mice to verify any potential physiological defects that may arise at 50% NCALD reduction which has been proposed as a potential SMA therapeutic option. Significant changes were observed in the gross brain morphology of adult NcaldKO/KO mice (4 month-old) with bigger ventricles, thinner cortex and smaller hippocampus including significantly reduced length of dentate gyrus sub granular zone. These morphological defects could arise either due to progressive neurodegeneration or impaired brain development/ maturation. Therefore, we investigated major hallmarks of progressive neurodegeneration like loss of mature neurons, astrogliosis, reduced dendritic branching or exacerbation of the gross morphological changes with aging. However, we did not find any evidence of progressive neurodegeneration. On other hand, we found very low NCALD levels in embryonic brain (E16) as well as at birth (P1), however a significant increase was observed at P10, which implies relevance of NCALD during postnatal brain development/ maturation. Therefore, we further investigated generation of doublecortin (DCX) positive new-born granule cells in the dentate gyrus (DG) as well as myelination, two major processes of postnatal brain maturation. We did not observe any changes in DCX positive neurons at P14 and only a tendency of reduction at P30 in NcaldKO/KO brain compared to controls. However, 4-month old adult NcaldKO/KO animals exhibit significantly reduced amount of DCX cells in DG. Loss of DCX positive cells in adult brain is widely accepted as a loss of adult neurogenesis. Furthermore, we investigated the mechanism behind the observed defects in NcaldKO/KO brain by investigating the NCALD interactome using mass spectrometry analysis and as co-immunoprecipitation. We identified a novel NCALD interacting partner named mitogen-activated protein kinase kinase kinase 10 (MAP3K10). MAP3K10 is an upstream kinase in c-Jun N-terminal kinase (JNK) pathway. Therefore we analysed the JNK activation in NcaldKO/KO brain and found significantly upregulated pJNK levels compared to controls. Contrary to these observations, adult NcaldKO/WT brain analysis showed neither JNK activation nor loss DCX positive cells, thereby ruling out the potential side effects of NCALD reduction in adult neurogenesis. Conclusively, this is the first study to report the effect of NCALD deletion on DCX positive neuron generation, which is a widely accepted as adult neurogenesis marker and to identify MAP3K10 as a novel interacting partner of NCALD; additionally it reports on the safety of using NCALD reduction for SMA combinatorial therapy

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 (NcaldKO/KO) and heterozygous (NcaldKO/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 NcaldKO/KO but not NcaldKO/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 NcaldKO/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

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

This document is the Accepted Manuscript version of the following article: Riessland et al., 'Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis', The American Journal of Human Genetics, Vol. 100 (2): 297-315, first published online 26 January 2017. The final, published version is available online at doi: http://dx.doi.org/10.1016/j.ajhg.2017.01.005 © 2017 American Society of Human Genetics.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 Ca(2+)-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.Peer reviewedFinal Accepted Versio

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 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