B Esmaeili et al. (2004) European Worm Meeting "FUNCTIONAL ANALYSIS OF THE C. ELEGANS SMN-1 GENE, AN ORTHOLOGUE OF THE HUMAN SMN GENE DEFECTIVE IN SPINAL MUSCULAR ATROPHY"

D Sattelle, M Briese, E Burt, B Esmaeili, S Christodoulou

[European Worm Meeting, 2004]

Analysis of smn-1(ok355) deletion mutant Spinal muscular atrophy (SMA) is a common human neuromuscular disorder characterised by a progressive degeneration of the spinal motor neurons and inherited in an autosomal, recessive manner. The causative gene is SMN (Survival of Motor Neurons), with orthologues described in a number of species including C. elegans (smn-1) (1). The SMN protein functions in the assembly of ribonucleoproteins involved in RNA splicing, ribosomal biogenesis and may also be involved in transcriptional regulation. In spite of its housekeeping function it is unclear why motorneurons are especially susceptible to loss of SMN. We have characterised the phenotype of smn-1 (ok355), a null allele. The smn-1 deletion mutants arrest at larval stages with reduced pharyngeal pumping, defecation rate and movement, all of which progressively worsen leading to paralysis and death. We have analysed the morphology of smn-1(ok355) nervous system architecture using the pan-neuronal::GFP marker as well as its neuromuscular junction (NMJ) organisation using UNC-25::SNB-1GFP (synaptobrevin) and UNC-49::GFP (GABA receptor) pre/postsynaptic markers. These studies have shown that although the neuronal morphology of smn-1(ok355) mutants is unaffected the NMJ organisation is disrupted. In this respect, the worm smn-1(ok355) mutant phenotype is similar to that of Drosophila survival of motroneurone gene mutant (2). Snap-back RNAi to remove smn-1 from specific neurones. To address the importance of smn-1 in motorneurones, we are in the process of using hairpin RNAi constructs in order to silence smn-1 in ventral nerve cord cholinergic motorneuornes. Snap-back constructs Promoters/Motoneurones: unc-3/ (DA,DB,VA,VB,AS) unc-4/ (DA, VA and VCs) unc-129/ (DAs and DBs) 1. Miguel-Aliaga I. et al.(1999). Hum Mol Genet 8: 2133-43 2. Chan YB et al.(2003). Hum Mol Genet 12: 1367-76

Sleigh JN et al. (2009) UK SMA Conference in Edinburgh "Conserved genes and cellular pathways modify SMN loss-of-function defects in invertebrate models"

Walker AK, Barsby T, Kalloo G, Artavanis-Tsakonas S, Chang H C-H, Sleigh JN, Hart AC, Dimitriadi M, Harris J, Li C, Satterlee JS, Van Vactor D, Sen A

[UK SMA Conference in Edinburgh, 2009]

Spinal Muscular Atrophy (SMA) is characterised by the selective loss of lower motor neurons and manifests due to reduced function of the Survival of Motor Neuron (SMN) protein. SMN is essential for cell viability and has been implicated in a number of diverse cellular processes including spliceosome assembly and mRNA transport; however, the pathways relevant to neuromuscular dysfunction and disease pathology remain unresolved. At present, only Plastin 3 (PLS3), a broadly expressed actin-bundling/stabilising protein, has been identified as a bona fide genetic modifier of SMA in humans. Disease severity in females is dependent on PLS3 levels, but the mechanisms underlying this interaction merit further study. In order to elucidate cellular pathways relevant to SMA, a combined functional and genetic approach was implemented to highlight and verify modifiers of SMN loss-of-function defects in invertebrate models. Drosophila and C. elegans both have a single gene orthologous to human SMN, diminished function of which causes neuromuscular defects and lethality (1, 2). It was found that PLS3 orthologues of both invertebrates modify SMN loss-of-function defects, which suggests that conserved plastin-associated pathways are important for SMN function and also serves to validate the two models. Two strategies utilising RNAi were subsequently used to identify additional genetic modifiers of SMN common to both invertebrates. First, an independent C. elegans genome-wide screen identified 1 orthologue of a previously described Drosophila modifier and 4 novel genes that altered SMN developmental and/or neuromuscular defects. Secondly, C. elegans orthologues of genes identified in a Drosophila SMN modifier screen were tested for modulation of SMN loss-of-function defects. 13 of the orthologues exacerbated or ameliorated these phenotypes and were identified as modifiers crossing invertebrate species. Among these was atn-1, which has previously been implicated in plastin-associated pathways, further validating the approach. The elucidation of conserved genes, pathways and modifiers pertaining to SMA will undoubtedly improve the understanding of disease pathology and may help accelerate the future development of SMA therapies. 1) Chan YB, et al. (2003) Hum Mol Genet 12: 1367-1376. 2) Briese M, et al. (2009) Hum Mol Genet 18: 97-104.