Motor defects in a Drosophila model for spinal muscular atrophy result from SMN depletion during early neurogenesis

Abstract

Spinal muscular atrophy (SMA) is the most common autosomal recessive neurodegenerative disease, and is characterised by spinal motor neuron loss, impaired motor function and, often, premature death. Mutations and deletions in the widely expressed survival motor neuron 1 (SMN1) gene cause SMA; however, the mechanisms underlying the selectivity of motor neuron degeneration are not well understood. Although SMA is degenerative in nature, SMN function during embryonic and early postnatal development appears to be essential for motor neuron survival in animal models and humans. Notwithstanding, how developmental defects contribute to the subversion of postnatal and adult motor function remains elusive. Here, in a Drosophila SMA model, we show that neurodevelopmental defects precede gross locomotor dysfunction in larvae. Furthermore, to specifically address the relevance of SMN during neurogenesis and in neurogenic cell types, we show that SMN knockdown using neuroblast-specific and pan-neuronal drivers, but not differentiated neuron or glial cell drivers, impairs adult motor function. Using targeted knockdown, we further restricted SMN manipulation in neuroblasts to a defined time window. Our aim was to express specifically in the neuronal progenitor cell types that have not formed synapses, and thus a time that precedes neuromuscular junction formation and maturation. By restoring SMN levels in these distinct neuronal population, we partially rescue the larval locomotor defects of Smn mutants. Finally, combinatorial SMN knockdown in immature and mature neurons synergistically enhances the locomotor and survival phenotypes. Our in-vivo study is the first to directly rescue the motor defects of an SMA model by expressing Smn in an identifiable population of Drosophila neuroblasts and developing neurons, highlighting that neuronal sensitivity to SMN loss may arise before synapse establishment and nerve cell maturation.

Fig 1. Neurodevelopmental defects precede gross locomotor dysfunction in Smn mutants.

(A) The Drosophila developmental stages. Drosophila go through two major rounds of neurogenesis during embryogenesis, in which the larval nervous system is set up, and during the larval stage, in which the adult neurons and glia are formed (these mature during metamorphosis and early adult life); (B) Smn^x7/Smn^A embryos were collected and the number of hatched larvae scored for each genotype and normalised to numbers observed from control Smn^x7/+. Fewer Smn^x7/Smn^A larvae hatched from embryos at 25°C and 20°C (error bars [SEM] represent three experiments, each with n > 90; ***P < 0.001; Kruskal–Wallis test with Dunn’s multiple comparisons); (C) Smn^x7/Smn^A larvae survived for a median of 3 and 4 days in a developmentally immature state when kept at 25°C and 20°C, respectively (error bars [SEM] represent three experiments, each with n > 60, *P < 0.05 Mantel-Cox). (D) confocal images of 5-ethynyl-2’-deoxyuridine (EdU) incorporation in Smn^x7/+ and smn^x7/smn^A trans-heterozygous larvae aged 5 days; (E) counts of EdU-containing foci in the thoracic ganglion over 72 h for larvae kept at 25°C and 20°C. At both temperatures, Smn^x7/Smn^A failed to show an increase in EdU incorporation. (**P < 0.01; ***P < 0.001, n = 15 per genotype; Kruskal–Wallis test with Dunn’s multiple comparisons); (F and G) body wall contractions were scored at 0, 24 and 48 h after hatching over a 1-min period at (F) 25°C and (G) 20°C. Smn^x7/Smn^A larvae underwent significant contraction defects at 48 h at both 25°C (***P < 0.001; Kruskal–Wallis test n = 36) and 20°C (**P < 0.01; Kruskal–Wallis test n = 36). All error bars [SEM]Scale bar = 20 μm.

Fig 2. Survival motor neuron (Smn) knockdown in neurogenic cell types leads to larval developmental defects and locomotor dysfunction.

(A) Pros-GAL4 driven expression of membrane-bound CD8-green fluorescent protein (GFP) in the larval central nervous system. GFP expression is observed in the post-embryonic neuroblasts and their immature daughter cells; (B) Pros-GAL4 expression in the adult ventral nerve cord. No neurons within the thoracic ganglion show visible expression. Only a small number of neurons, which reside in the abdominal ganglion within the ventral nerve cord of the adult, expressed Pros-GAL4; (C) GAL4 nervous system expression patterns detailing the neuronal and glial cell type expression patterns; (D–E) SMN was knocked-down (UAS-SMN-RNAi^N4) pan neuronally (Elav-GAL4 and nSyb-GAL4) predominantly in motor neurons (D42-GAL4 and OK371-GAL4), cholinergic neurons (Cha-GAL4), neuroblasts and undifferentiated daughter cells (Pros-GAL4 and Insc-GAL4), pan-glia (Repo-GAL4) and in the larval fat body (CG-GAL4); (D) body wall contractions were scored at 48 h, with significant differences observed with Elav-GAL4, Pros-GAL4 and Insc-GAL4 driven UAS-SMN-RNAi^N4 (**P < 0.01, ***P < 0.00, Kruskal–Wallis test with Dunn’s multiple Comparisons; n > 20); (E) day of pupariation formation (three experiments each with n > 50; Kruskal–Wallis test with Dunn’s multiple comparisons) data showing that Insc-GAL4 and Pros-GAL4 SMN knockdown leads to a delay in time to pupariation; All error bars [SEM]. Scale bar = 10 μm.

Fig 3. Survival motor neuron (Smn) knockdown in neurogenic cell types leads to adult motor dysfunction.

Flies were tested for motor activity, (A) and (B), and flight ability (C) and (D) at 2 days (A) and (C), and 8 Days (B) and (D). Drosophila activity was detected in adult flies over 1 day using the Trikinetics activity monitors in controlled conditions; (A and B) only Pros-GAL4/UAS-SMN-RNAi^N4 and Insc-GAL4/UAS-SMN-RNAi^N4 progressively declined in activity over 2 (F; **P < 0.01, n = 20; Kruskal–Wallis test with Dunn’s multiple comparisons) and 8 days (G; ***P < 0.001; n = 20; Kruskal–Wallis test with Dunn’s multiple comparisons); (C and D) Pros-GAL4; UAS-SMN-RNAi^N4 and Insc-GAL4/UAS-SMN-RNAi^N4 flies showed a significant reduction in flight ability, with more flies residing at the bottom of the chamber, over 2 (F; *P < 0.05; **P < 0.01; n = 40; Kruskal–Wallis test with Dunn’s multiple comparisons) and 8 days (G; ***P < 0.001; n = 40; Kruskal–Wallis test with Dunn’s multiple comparisons). All error bars [SEM].

Fig 4. Adult motor defects persist with developmentally targeted spatiotemporal survival motor neuron (SMN) knockdown.

(A) The GAL80^TS system was used to eliminate any adult GAL4 expression. Larvae were reared at 29°C (GAL80^TS is inactive; GAL4 is active) and then switched to 19°C (GAL80^TS is active; GAL4 is repressed) during pupation; (B and C) two non-overlapping RNAi constructs were used (SMN-RNAi^N4 and SMN-RNAi^C25) for flight and activity defects. Both (B) activity (SMN-RNAi^N4_, ***P < 0.001; SMN-RNAi^C25, **P < 0.01, n = 20, Kruskal–Wallis test with Dunn’s multiple comparisons) and (C) flight defects (SMN-RNAi^N4, ***P < 0.001; SMN-RNAi^C25, **P < 0.01, n = 40; Kruskal–Wallis test with Dunn’s multiple comparisons) were detected using this method.

Fig 5. Restoration of survival motor neuron (SMN) in neurogenic cell types rescues the motor phenotypes in Smn mutant larvae.

For rescue studies, both the classical binary GAL4 system (A) and the GAL80^TS system (A’) were used. For targeting, a temperature sensitive GAL80 (GAL80^TS) represses GAL4 at 19°C but becomes inactive at 29°C. Embryos were reared for 24 h at 29°C, during which GAL4 is expressed, then switched to 19°C to eliminate expression. Expression of SMN using Insc-GAL4 rescue the embryonic attrition seen in SMN mutants with both the (B) binary and (C) TARGET GAL80^TS GAL4 systems (***P < 0.001, three experiments for each genotype, each with n = 60; Kruskal–Wallis test with Dunn’s multiple comparisons); (D) Insc-GAL4/UAS-SMN; Smn^A/Smn^x7 larvae show significant rescue of locomotor activity at 72 h compared with mutant Insc-GAL4; Smn^A/Smn^x7 (***P < 0.001, n = 15, Kruskal–Wallis test with Dunn’s multiple comparisons); (E) larval survival was extended from a median of 3 days to a median of 7 days (three experiments for each genotype, each with n > 30; ***P < 0.001; Mantel-Cox); (F) Tub-GAL80^TS; Insc-GAL4/UAS-SMN; Smn^A/Smn^x7 larvae display a significant rescue of motor function at 72 h, compared with controls (***P < 0.001, n = 20, Kruskal–Wallis test with Dunn’s multiple comparisons); (G) larval survival was extended from a median of 4 days to a median of 8 days (three experiments for each genotype, each with n > 30; ***P < 0.001; Mantel-Cox). All error bars [SEM].

Fig 6. Dual knockdown of survival motor neuron (SMN) in neuroblasts and differentiated neurons synergistically enhances the SMA model phenotypes.

(A) GAL4 nervous system expression patterns detailing the driver type and single and double-driver combinations used to knock down Smn. For negative controls Elav-GAL4 + Insc-GAL4 and nSyb-GAL4 + Insc-GAL4 were used. Elav-GAL4, Pros-GAL4 and Insc-GAL4 driven UAS-SMN-RNAi^N4 were used as positive controls and compared with Elav-GAL4 + Insc-GAL4 and nSyb-GAL4 + Insc-GAL4 driven UAS-SMN-RNAi^N4; (B) the genotypes were assessed for locomotor dysfunction at 72 h after hatching. Elav-GAL4 + Insc-GAL4 and nSyb-GAL4 + Insc-GAL4 driven UAS-SMN-RNAi^N4 underwent reduced peristaltic contractions compared with negative and positive controls (***P < 0.001, n >20, Kruskal–Wallis test with Dunn’s multiple comparisons); (C) fly hatching number was then assessed to analyse survival to adulthood. Elav-GAL4 + Insc-GAL4 and nSyb-GAL4 + Insc-GAL4 driven UAS-SMN-RNAi^N4 survived compared with negative and positive controls (***P < 0.001; three experiments, each with n > 50; Kruskal–Wallis test with Dunn’s multiple comparisons). All error bars [SEM].