Modeling Spinal Muscular Atrophy in Drosophila

Abstract

Spinal Muscular Atrophy (SMA), a recessive hereditary neurodegenerative disease in humans, has been linked to mutations in the survival motor neuron (SMN) gene. SMA patients display early onset lethality coupled with motor neuron loss and skeletal muscle atrophy. We used Drosophila, which encodes a single SMN ortholog, survival motor neuron (Smn), to model SMA, since reduction of Smn function leads to defects that mimic the SMA pathology in humans. Here we show that a normal neuromuscular junction (NMJ) structure depends on SMN expression and that SMN concentrates in the post-synaptic NMJ regions. We conducted a screen for genetic modifiers of an Smn phenotype using the Exelixis collection of transposon-induced mutations, which affects approximately 50% of the Drosophila genome. This screen resulted in the recovery of 27 modifiers, thereby expanding the genetic circuitry of Smn to include several genes not previously known to be associated with this locus. Among the identified modifiers was wishful thinking (wit), a type II BMP receptor, which was shown to alter the Smn NMJ phenotype. Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype. The NMJ defects caused by loss of Smn function can be ameliorated by increasing BMP signals, suggesting that increased BMP activity in SMA patients may help to alleviate symptoms of the disease. These results confirm that our genetic approach is likely to identify bona fide modulators of SMN activity, especially regarding its role at the neuromuscular junction, and as a consequence, may identify putative SMA therapeutic targets.

Genetic circuitry of Survival motor neuron , the gene underlying spinal muscular atrophy

“…We examined several Smn-RNAi strains under the control of the yeast upstream activation sequence (UAS) to identify a hypomorphic Smn allele that could be used to model SMA in Drosophila more faithfully than alleles that completely abolish Smn function. We identified a transgenic strain, UAS-Smn-RNAi FL26B (FL26B), that displays a less severe phenotype than the allele used in our previous screen (12). Specifically, expression of FL26B under the control of tubulinGAL4 (tubGAL4::FL26B) results in late pupal lethality whereby ∼50% of the pupae reach a more mature (pigmented) developmental stage before death than their less mature, unpigmented siblings (Fig.…”

“…The larval phenotype includes neuromuscular junction (NMJ) abnormalities that are reminiscent of those associated with the human disease, rendering this invertebrate organism an excellent system to model SMN biology (9)(10)(11). We previously described a genetic screen for modifiers of the lethal phenotype resulting from a complete lossof-function Smn allele (12). This screen, although it probed half of the Drosophila genome, identified only a relatively small number of genes that affected the NMJ phenotype associated with Smn loss of function (12).…”

“…We previously described a genetic screen for modifiers of the lethal phenotype resulting from a complete lossof-function Smn allele (12). This screen, although it probed half of the Drosophila genome, identified only a relatively small number of genes that affected the NMJ phenotype associated with Smn loss of function (12). In particular, it did not identify genes involved in snRNP biogenesis, the molecular functionality that is most clearly associated with SMN.…”

Parallel Genetic and Proteomic Screens Identify Msps as a CLASP–Abl Pathway Interactor in Drosophila

“…While a number of screens have already made use of the Df collection (Bonds et al 2007) or the transposon insertion collection (Kankel et al 2007;Chang et al 2008;Shalaby et al 2009) individually, the logistics of obtaining and screening all of the 16,000 lines is not feasible for many labs. Thus, we applied a hierarchical strategy for our screen ( Figure 1A) that would take advantage of both collections and also would assay LOF vs. neomorphic effects.…”

“…The collection of deletions (Dfs; Parks et al 2004) was used for the primary screen, while the collection of transposon insertions (Thibault et al 2004) was utilized for the secondary screens. The use of these strains for genome-wide genetic interaction screening has been previously described (Kankel et al 2007;Chang et al 2008;Shalaby et al 2009). To confirm candidate interactors, all interacting transposons were crossed in at least two separate experiments to GMR-GAL4, UAS-CLASP-GFP and eye phenotypes were examined and imaged.…”