Temperature-sensitive spinal muscular atrophy-causing point mutations lead to SMN instability, locomotor defects and premature lethality in Drosophila

Abstract

Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising from homozygous deletion or mutation of the survival motor neuron 1 (SMN1) gene. SMN protein expressed from a paralogous gene, SMN2, is the primary genetic modifier of SMA; small changes in overall SMN levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that regulate SMN protein stability should lead to better therapeutic outcomes. Here, we show that SMA patient-derived missense mutations in the Drosophila SMN Tudor domain exhibit a pronounced temperature sensitivity that affects organismal viability, larval locomotor function and adult longevity. These disease-related phenotypes are domain specific and result from decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN levels during various stages of Drosophila development. Owing to a large maternal contribution of mRNA and protein, Smn is not expressed zygotically during embryogenesis. Interestingly, we find that only baseline levels of SMN are required during larval stages, whereas high levels of the protein are required during pupation. This previously uncharacterized period of elevated SMN expression, during which the majority of adult tissues are formed and differentiated, could be an important and translationally relevant developmental stage in which to study SMN function. Taken together, these findings illustrate a novel in vivo role for the SMN Tudor domain in maintaining SMN homeostasis and highlight the necessity for high SMN levels at crucial developmental time points that are conserved from Drosophila to humans.

Keywords: Drosophila models of human disease; SMN protein; Spinal muscular atrophy; Tudor domain.

Fig. 1.

Effects of temperature limits on viability of SMN patient-derived missense mutant lines. (A) Diagram of the SMN protein showing orthologous SMA patient-derived missense mutations in Drosophila SMN. Schematic of Drosophila SMN transgene construct, which includes an N-terminal 3X-FLAG tag. Note that the construct also contains a 1.7 kb upstream sequence that includes the native Smn promoter and a 0.4 kb downstream flanking region (not shown). (B,C) Viability assays for the wild-type and mutant Smn transgenic lines, measured as the fraction of larvae reaching the pupal (B) and adult (C) stages when raised at either the standard culturing temperature of 25°C (green), at 18°C (light blue) or at 29°C (red). X7^+/– animals are heterozygotes that contain one endogenous copy of Smn, whereas WT are animals that contain one wild-type copy of the Flag-Smn transgene in an otherwise null background. Mutants are organized by their location within the three functional domains of SMN: Gemin2, Tudor or YG box. The number of larvae for each genotype-temperature combination ranged from 100 to 420 animals. Larvae were split into vials of ∼50 animals and viability of each vial was measured as a separate replicate (displayed as data points). Error bars represent mean ±95% c.i. Adjusted P-value was calculated using two-way ANOVA and Tukey's multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001. Sample size (number of larvae): X7^+/− 18°C (500), 25°C (500), 29°C (500); WT 18°C (139), 25°C (400), 29°C (394); D20V 18°C (128), 25°C (400), 29°C (200); F70S 18°C (150), 25°C (400), 29°C (130); V72G 18°C (113), 25°C (400), 29°C (300); G73R 18°C (122), 25°C (400), 29°C (400); I93F 18°C (144), 25°C (400), 29°C (150); Y107C 18°C (131), 25°C (400), 29°C (150); T205I 18°C (123), 25°C (400), 29°C (420); Y208C 18°C (100), 25°C (400), 29°C (150); G210C 18°C (100), 25°C (400), 29°C (150); G210V 18°C (121), 25°C (400), 29°C (394).

Fig. 2.

Effects of small temperature changes on SMN Tudor mutant viability and locomotor function. (A,B) Viability assay of flies expressing a wild-type Smn transgene, one of four Tudor domain mutations (F70S, V72G, G73R or I93F) or a YG box domain mutation (T205I). Viability is measured as the fraction of larvae that reach the pupal (A) and adult (B) stages, while being raised at either 25°C (green), a slightly cooler temperature (22°C, blue) or a slightly warmer temperature (27°C, orange). The number of larvae for each experimental group ranged from 150 to 400 animals. Larvae were split into vials of ∼50 and the viability of each vial was treated as a separate replicate. The 25°C viability data are the same data from Fig. 1C,D and are included for ease of comparison. Error bars represent mean±95% c.i. Adjusted P-values calculated using Tukey's multiple comparisons test: *P<0.05, **P<0.01, ***P<0.001. (C) Locomotion assays of wandering 3rd instar larvae of the same genotypes described in A and B raised at 22°C (blue) or 27°C (orange). A total of 30-35 larvae were assayed per condition. Locomotion was measured in body lengths per second (BLPS), which measures the larva's speed in relationship to its body size. Error bars represent mean±95% c.i. P-values were calculated using Student's t-test: ns, not significant (P >0.05); ** P<0.01, *** P<0.001. For A and B, sample size (number of larvae): WT 22°C (150), 25°C (400), 27°C (350); F70S 22°C (150), 25°C (400), 27°C (350); V72G 22°C (150), 25°C (400), 27°C (300); G73R 22°C (150), 25°C (400), 27°C (350); I93F 22°C (150), 25°C (400), 27°C (350); T205I 22°C (150), 25°C (400), 27°C (350). (C) Sample size (number of larvae): WT 22°C (32), 27°C (30); F70S 22°C (30), 27°C (30); V72G 22°C (30), 27°C (30); G73R 22°C (31), 27°C (30); I93F 22°C (31), 27°C (30); T205I 22°C (31), 27°C (30).

Fig. 3.

Effects of small temperature changes on SMN protein levels in Tudor domain mutants. (A) Representative western blots of wandering 3rd instar larvae from wild type, Tudor domain mutants and YG box domain mutants (T205I) raised at 22°C (blue), 25°C (green) or 27°C (orange) (eight to 12 larvae per sample). Protein was visualized using an HRP-conjugated primary antibody that recognizes the 3X-FLAG tag on the N terminus of the SMN transgenic construct. (B) Quantification of western blot biological replicates represented in A. Each sample contained ten wandering 3rd instar larvae, each genotype-temperature combination contains seven to nine samples. Total protein was used as a loading control to standardize SMN levels (see Fig. S1). SMN levels for each sample were normalized to the ‘WT 25’ SMN protein levels for their respective replicate. Error bars represent mean±95% c.i. The adjusted P-value was calculated using two-way ANOVA and Tukey's multiple comparisons test: *P<0.05, **P<0.01, ***P<0.001. Sample size (replicates): WT 22°C (9), 25°C (9), 27°C (9); F70S 22°C (9), 25°C (9), 27°C (9); V72G 22°C (9), 25°C (7), 27°C (9); G73R 22°C (8), 25°C (8), 27°C (9); I93F 22°C (8), 25°C (8), 27°C (8); T205I 22°C (9), 25°C (7), 27°C (9).

Fig. 4.

Cycloheximide experiments measure SMN protein stability in F70S Tudor domain mutants. (A) Schematic of CHX experiment, showing treatment types, time points, and treatment temperature. (B) Representative western blots of WT and F70S mutant wandering 3rd instar larvae in the presence of CHX over 12 h, ten dissected larvae per sample. The control treatment contained Schneider's medium, while the CHX treatment contained Schneider's medium and CHX. All blots show FLAG-SMN levels, total protein used for standardization not shown. (C) Quantification of western blots represented in B; three to nine samples were analyzed per condition. ‘Control’ represents the medium-only treatment; ‘CHX’ represents the medium and CHX treatment. All SMN protein levels were standardized using total protein and relative to ‘0 h’ SMN levels of each genotype. (D,E) Protein structure model of Drosophila SMN Tudor domain with bound dimethylated arginine (red-white-blue stick model, upper left) (generation of the model is described in the Materials and Methods section). (D) Wild-type SMN Tudor domain with residues of interest highlighted by ball-and-stick structures (F70 orange, V72 magenta, G73 green, I93 blue). (E) Same structure as D, but with I93F overlay (silver, black arrow). Steric clash is shown by red circles. This is the most common of four conformations for I93F, but all four conformations experience steric clash. Error bars represent mean±95% c.i. Adjusted P-values calculated using Tukey's multiple comparisons test: **P<0.01, ***P<0.001. Sample size (replicates): WT control 0 h (9), 4 h (4), 8 h (4), 12 h (4); F70S control 0 h (9), 4 h (4), 8 h (4), 12 h (4); WT CHX 0 h (9), 4 h (8), 8 h (8), 12 h (3); F70S CHX 0 h (9), 4 h (9), 8 h (8), 12 h (7).

Fig. 5.

Non-permissive-to-permissive temperature-shift viability assays during early larval development. (A) Proteomic analysis of SMN levels throughout Drosophila developmental stages, data mined from Casas-Vila et al. (2017) via FlyBase (https://flybase.org/). (B) FAIRE and RNA sequence analysis of the Drosophila Smn genomic region over embryonic development and in larval imaginal discs, data mined from McKay and Lieb (2013). (C) Transcriptomic analysis of SMN mRNA levels throughout Drosophila developmental stages, data mined from Graveley et al., 2011 via FlyBase (https://flybase.org/). (D) Workflow schematic of non-permissive-to-permissive temperature-shift experiments, switching larvae from 29°C to 22°C at 1 or 2 DPE. (E,F) Pupation (E) and eclosion (F) percentages of wild-type, Tudor domain mutant and YG box mutant (T205I) larvae raised exclusively at 22°C (blue), switched from 22°C to 29°C at L1 stage (yellow), switched from 22°C to 29°C at L2 stage (orange) or raised exclusively at 29°C (red) (150-420 larvae per genotype, 50 larvae per biological replicate). Error bars represent mean±95% c.i. Adjusted P-values calculated using Tukey's multiple comparisons test: ns, not significant (P>0.05); *P<0.05, **P<0.01, ***P<0.001). Sample size (number of larvae): WT 22°C (150), L1 (167), L2 (192), 29°C (394); F70S 22°C (150), L1 (233), L2 (255), 29°C (130); V72G 22°C (150), L1 (300), L2 (350), 29°C (290); G73R 22°C (150), L1 (278), L2 (293), 29°C (400); I93F 22°C (150), L1 (300), L2 (300), 29°C (150); T205I 22°C (150), L1 (345), L2 (300), 29°C (420).

Fig. 6.

Permissive-to-non-permissive temperature-shift viability assays during late larval and pupal development. (A) Workflow schematic of permissive-to-non-permissive temperature-shift experiments, switching wandering 3rd instar (W3) larvae from 22°C to 29°C. (B,C) Pupation (B) and eclosion (C) percentages of wild-type, Tudor domain mutant and YG box mutant (T205I) larvae raised exclusively at 22°C (blue), switched from 22°C to 29°C at W3 stage (teal) or raised exclusively at 29°C (red); 150-420 larvae per genotype, ∼50 larvae per biological replicate. Error bars represent mean±95% c.i. Adjusted P-values calculated using two-way ANOVA and Tukey's multiple comparisons test: ns, not significant (P>0.05); *P<0.05, **P<0.01, ***P<0.001. Sample size (number of larvae): WT 22°C (150), W3 (220), 29°C (394); F70S 22°C (150), W3 (230), 29°C (130); V72G 22°C (150), W3 (176), 29°C (290); G73R 22°C (150), W3 (221), 29°C (400); I93F 22°C (150), W3 (206), 29°C (150); T205I 22°C (150), W3 (232), 29°C (420).

Fig. 7.

Adult longevity of select SMN Tudor domain mutants at permissive and non-permissive temperatures. (A,B) Survival plots of WT and Tudor domain mutant adult flies at 29°C (A) or 25°C (B). The flies were raised at 25°C (WT, G73R, I93F) or 22°C (F70S) prior to eclosion and then moved to the experimental temperature <24 h post-eclosion. The number of adults for each group ranges from 150 to 600. Live adults were counted every 2 days. Error bars represent mean±95% c.i. WT, black; F70S, green; G73R, yellow; I93F, purple. (C) Average time to reach 10% survival for adults at 29°C (red) and 25°C (green). Error bars represent mean±95% c.i. (D) Comparing relative longevity (10% survival 29°C/10% survival 25°C). Error bars represent mean±95% c.i. (E) Survival plot of WT (black) and F70S mutant (green) adult flies raised and maintained at 22°C. (F) Average time to reach 10% survival for adults raised at 22°C (blue) and 29°C (red). Error bars represent mean±95% c.i. (G) relative survival (10% survival 29°C/10% survival 22°C). Error bars represent mean±95% c.i. P-values for 10% survival were calculated using two-way ANOVA with Sidak's multiple comparisons test: ***P<0.001. P-values for longevity ratios were calculated using one-way ANOVA and Student's t-test: ns, not significant (P>0.05); ***P<0.001. Sample size (number of adults): WT 22°C (326), 25°C (289), 29°C (292); F70S 22°C (385), 25°C (578), 29°C (586); G73R 25°C (94), 29°C (105); I93F 25°C (196), 29°C (209).