Temperature sensitive SMA-causing point mutations lead to SMN instability, locomotor defects, and premature lethality inDrosophila

Abstract

27Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising 28 from homozygous deletion or mutation of the SMN1 gene. SMN protein expressed from a 29 paralogous gene, SMN2, is the primary genetic modifier of SMA; small changes in overall SMN 30 levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that 31 regulate SMN protein stability should lead to better therapeutic outcomes. Here, we show that 32 SMA patient-derived missense mutations in the Drosophila SMN Tudor domain exhibit a 33 pronounced temperature sensitivity that affects organismal viability, larval locomotor function, 34 and adult longevity. These disease-related phenotypes are domain-specific and result from 35 decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN 36 levels during various stages of Drosophila development. Due to a large maternal contribution of 37 mRNA and protein, Smn is not expressed zygotically during embryogenesis. Interestingly, we 38 find that only baseline levels of SMN are required during larval stages, whereas high levels of 39 protein are required during pupation. This previously uncharacterized period of elevated SMN 40 expression, during which the majority of adult tissues are formed and differentiated, could be an 41 important and translationally relevant developmental stage in which to study SMN function. 42 Altogether, these findings illustrate a novel in vivo role for the SMN Tudor domain in maintaining 43 SMN homeostasis and highlight the necessity for high SMN levels at critical developmental 44 timepoints that is conserved from Drosophila to humans. 4546 47 126 127 RESULTS 128 SMN Tudor domain mutants (TDMs) are temperature-sensitive 129 Previous work using SMA patient-derived Smn missense mutations, modeled in the fly, has 130 produced robust and reproducible findings. However, in one or two instances, we noticed 131 inconsistencies in the overall viability of a given fly line that could not be attributed to normal 132 biological noise. For example, in Praveen et al. (2014), the Smn F70S mutation line (hereafter F70S) 133 displayed a relatively mild phenotype, with an eclosion frequency similar to that of the Smn WT 134 transgene (hereafter WT). In contrast, Spring et al. (2019) reported a rather severe viability defect 135 for this same F70S line. The husbandry conditions used in each study were slightly different; the 136 experiments in the earlier work were performed at room temperature (~22°C) whereas in the 137 subsequent experiments, animals were kept at a constant 25°C. In addition, we qualitatively 138 157 SMN Tudor domain mutants display SMA-related phenotypes in response to small changes 158 in temperature 159 We next examined the effect of minor changes in temperature by raising animals at 22°C and 160 27°C. The SMN WT and T205I YG box domain mutant lines were used as controls. Although the 161 (2015). Spinal Muscular Atrophy: Review of a Child Onset Disease. Br. J. Med. Med. Res. 595 6, 647-66...

Chaperoning the chaperones: Proteomic analysis of the SMN complex reveals conserved and etiologic connections to the proteostasis network

“…Furthermore, the stocks are ideal for carrying out proteomics because we do not have to perform crosses to obtain animals of the desired genotype and we are able to collect large quantities of material. We chose to use embryos because they contain a wide-variety of cell types and they naturally express ~100x the amount of SMN protein present in larval or adult stages [36].…”

“…For example, the human G279C allele (G210C in fly) is reported to be slightly hyper-oligomeric [27], whereas D44V (D20V in fly) is reported to reduce binding affinity to Gem2 [52]. The two Tudor domain (Tud) mutations (G73R and I93F) are known to cause temperature-sensitive misfolding [36,53] and are plausibly expected to interfere with binding to Sm client proteins. Therefore, we generated a heatmap of fold-change ratios for various SMN alleles versus that of the WT (Fig.…”

“…Importantly, expression of a misfolded protein is known to change the profile of HSP binding partners [101]. We recently identified a subset of SMA-causing missense mutations in the Tudor domain of SMN (G73R, I93F, V72G and F70S) that are temperature-sensitive [36]. In response to relatively small increases in culturing temperature (e.g.…”

“…In flies, SMN is maternally provided and its levels remain high throughout embryogenesis, dropping to basal levels during the three larval stages. It rises again during pupation (metamorphosis), only to fall back again to basal levels upon eclosion as adults [36,88]. In mice, depletion of SMN protein later in development has relatively little consequence compared to early depletion [89].…”

“…To begin to address these questions, we decided to search beyond the Gemins for additional SMN binding partners. Over the past decade, we have developed Drosophila as model system to understand SMN function using an allelic series of transgenic flies expressing SMA-causing missense mutations that recapitulates the full range of phenotypic severity seen in human patients [22,27,[30][31][32][33][34][35][36][37] Here, we carried out affinity purification, coupled with mass spectrometry (AP-MS), to identify novel SMN binding partners present in embryonic lysates of wildtype and hypomorphic SMA animal models. Bioinformatic analyses of the hits, along with comparisons to proximity labeling data from human cells revealed connections to at least two other major chaperone systems.…”