Abstract
Mutation of Glass bottom boat, the Drosophila homologue of the bone morphogenetic protein or growth/differentiation factor (BMP/GDF) family of genes in vertebrates, has been shown to disrupt development of neuromuscular junctions (NMJ). Here we tested whether this same conclusion can be broadened to vertebrate BMP/GDF genes. This analysis was also extended to consider whether such genes are required for NMJ maintenance in post-larval stages, as this would argue that BMP genes are viable candidates for analysis in progressive neuromuscular disease. Zebrafish mutants harboring homozygous null mutations in the BMP-family gene gdf6a were raised to adulthood and assessed for neuromuscular deficits. Fish lacking gdf6a exhibited decreased endurance (∼50%, p = 0.005) compared to wild type, and this deficit progressively worsened with age. These fish also presented with significantly disrupted NMJ morphology (p = 0.009), and a lower abundance of spinal motor neurons (∼50%, p<0.001) compared to wild type. Noting the similarity of these symptoms to those of Amyotrophic Lateral Sclerosis (ALS) model mice and fish, we asked if mutations in gdf6a would enhance the phenotypes observed in the latter, i.e. in zebrafish over-expressing mutant Superoxide Dismutase 1 (SOD1). Amongst younger adult fish only bigenic fish harboring both the SOD1 transgene and gdf6a mutations, but not siblings with other combinations of these gene modifications, displayed significantly reduced endurance (75%, p<0.05) and strength/power (75%, p<0.05), as well as disrupted NMJ morphology (p<0.001) compared to wild type siblings. Bigenic fish also had lower survival rates compared to other genotypes. Thus conclusions regarding a role for BMP ligands in effecting NMJ can be extended to vertebrates, supporting conservation of mechanisms relevant to neuromuscular degenerative diseases. These conclusions synergize with past findings to argue for further analysis of GDF6 and other BMP genes as modifier loci, potentially affecting susceptibility to ALS and perhaps a broader suite of neurodegenerative diseases.
Highlights
Development, growth and stabilization of neuromuscular junctions (NMJ) in larval Drosophila require the bone morphogenetic protein (BMP) gene glass bottom boat [1]
BMP/ GDFs have been implicated in amyotrophic lateral sclerosis (ALS) progression via a separate line of inquiry, in that a Drosophila model of familial ALS8 demonstrates disrupted BMP signaling at their NMJs; mutations in VAPB cause ALS8 [9,10] and altering VapB disrupts BMP signaling at the Drosophila NMJ [11]
To assess if gdf6a2/2 embryos display motoneuron disease, we utilized sensitive assays of motoneuron disease/ALS that are deployed frequently in embryonic zebrafish, consisting of examining branching and pathfinding defects in GFP-positive primary motoneurons of transgenic fish [20,21,22,24,25,26,27]. gdf6a2/2 embryos did not display a significant increase in motoneuron pathfinding or axonopathies as determined in a transgenic background that enables sensitive detection of motoneuron morphology based on GFP fluorescence; both gdf6a2/2 and siblings had a low rate of abnormalities in this scoring system (Supplemental Figure S1; p$0.315, n$9 larvae per genotype)
Summary
Development, growth and stabilization of neuromuscular junctions (NMJ) in larval Drosophila require the bone morphogenetic protein (BMP) gene glass bottom boat (gbb) [1]. Gbb is the Drosophila homolog of a family of vertebrate genes including the BMP and growth/differentiation factor ligands (BMP/GDF Family), which is itself a sub-family of the transforming growth factor b (TGFb) genes [1,2,3] Extending their role to encompass vertebrate NMJs would embolden speculation that mutations in BMP/GDF genes can sensitize patients to progressive late-onset neuromuscular disease. The synthesis of disparate literature by several authors has recently suggested that mutations in BMP/GDFs are good candidates for sensitizing patients to amyotrophic lateral sclerosis (ALS), if not representing causal instigators of disease etiology [4,5] This suggestion was based on the aforementioned role of Gbb [1,2], and upon the requirement for the proteins constituting Gbb receptors in NMJ development [6,7,8]. A role for BMP/GDFs in other neuromuscular diseases has been proposed, including Spinal Muscular Atrophy, Hereditary Spastic Paraplegias, Multiple Sclerosis and Huntington’s Disease [4]
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