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. 2024 Jan 4;111(1):96–118. doi: 10.1016/j.ajhg.2023.12.004

Figure 4.

Figure 4

elav-GAL4-mediated neuronal overexpression of PPFIA3 variants result in climbing defect, bang sensitivity, and neuromuscular junction (NMJ) bouton loss

(A) elav-GAL4 mediated neuronal expression of PPFIA3 p.Arg39Cys, PPFIA3 p.Ala315Ser, PPFIA3 p.Arg415Trp, and PPFIA3 p.Arg784Trp result in impaired motor coordination on the climbing assay compared to PPFIA3 WT and empty control flies. Crosses were set and maintained at 25°C. Behavioral testing was conducted at 20°C–21°C with both sexes.

(B) elav-GAL4-mediated neuronal expression of PPFIA3 p.Arg39Cys, PPFIA3 p.Ala315Ser, and PPFIA3 p.Arg415Trp have bang sensitivity with delayed recovery from vortexing compared to PPFIA3 WT and UAS-empty control flies. Crosses were set and maintained at 25°C. Behavioral test was conducted at 20°C–21°C with both sexes.

(C) elav-GAL4-mediated neuronal overexpression of PPFIA3 variants result in NMJ bouton loss without a significant change in NMJ length.

(i) Model depicting the method for visualizing the NMJ in fruit fly third-instar larva.

(ii) Representative images of third-instar larval NMJs of each genotype including elav GAL4>UAS empty, elav GAL4>PPFIA3 WT, p.Arg39Cys, p.Arg415Trp, p.Trp546Cys, and p.Arg784Trp are shown. Horseradish peroxidase (HRP) is a pan-neuronal marker (green) and Brp (Bruchpilot) is an active zone marker (magenta). Scale bar is 24 μm.

(iii) Quantification of total number of boutons in the muscle 6/7 (abdominal segment 3) NMJ show that PPFIA3 p.Arg39Cys and p.Arg415Trp result in bouton loss compared to PPFIA3 WT and empty control. In contrast, PPFIA3 p.Trp546Cys and p.Arg784Trp show no alteration in bouton numbers.

(iv) Quantification of total NMJ length in each genotype is shown, and there is no significant difference between PPFIA3 WT, variants, and UAS-empty control. Crosses were set and maintained at 25°C. Statistical analysis conducted with one-way ANOVA and Tukey’s post-hoc analysis. Data shown as mean ± SEM with the sample size of total number of quantified NMJs shown above the bars. Significance shown as ∗∗p < 0.01, ∗∗∗p < 0.001. Non-significance shown as ns.