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

Identification and functional analysis of BICD2, a causal gene of autosomal dominant SMA

<p>Spinal muscular atrophies (SMAs) are characterized by degeneration of spinal motor neurons and muscle weakness. Autosomal recessive SMA is the most common form and is caused by homozygous deletions/mutations of the SMN1 gene. Additionally, dominant inheritance SMA families have been reported, for most of them the causal gene remains unknown. The starting point of the current study was a Dutch family (1) with non-progressive SMA, autosomal dominant (SMALED2; MIM #615290 AD) pattern of inheritance, unaltered SMN1, and congenital contractures. Linkage analysis and whole exome sequencing were performed and led to the identification of a heterozygous missense mutation (c.320C>T, p.Ser107Leu) in the BICD2 gene. BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D. BICD2 was sequenced in twenty additional families with SMALED2 finding additional mutations (p. Asn188Thr; p.Ala535Val; p.Thr703Met; p.Arg747Cys), and a rare variant p.90 Lys>Arg (VS frequency 0.4%) (5, 6). The patients differ in the severity of the symptoms, although some of those mutations lay on the same protein region. Overexpression of the mutant BICD2 cDNAs in HeLa cells was performed observing fragmentation of the Golgi Apparatus (GA). Fibroblast cell lines were derived from the patients carrying the mutations p.Thr703Met (severe phenotype) and p.Asn188Thr. A severe GA fragmentation was observed in the fibroblast cells from the patient p.Thr703Met suggesting a possible correlation between the grade of fragmentation and the severity of the disease. The integrity of the GA depends on the microtubule network and BICD2 is implicated in transport along microtubules. The microtubules of the fibroblasts carrying the p.Thr703Met were stained and alteration in their pattern was observed. BICD2 interacts with the small GTPase Rab6a (2); which plays an essential role in Golgi transport. Rab6a Pull Down analysis showed no alteration in the interaction of the BICD2 mutants and Rab6a. In order to understand the molecular mechanisms of the other mutations in BICD2, protein stability assays and additional interacting studies are being performed.</p> <p> </p

Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor, Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

<p>Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of He La cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.</p>