Figure 7. Cortex glial inhibition of endocytosis rescues zyd seizures.

(A-B) Behavioral analysis of HS induced seizures. (A) Cortex glial knockdown of Rab5 and EndoA rescues seizures in the zyd mutant. Inset shows effects after 1 minute of HS (N = 3 groups of 20 flies/genotype, p<0.0001 at 1 minute and 2 minutes). (B) Cortex glial conditional overexpression of Rab5^RNAi and dominant-negative Rab5 (Rab5^DN) using UAS/gal4/gal80^ts. Rearing adult flies at the restrictive temperature (>30°C) for gal80^ts allows expression of Rab5^RNAi or Rab5^DN only in adults. A significant reduction in seizures was seen after 16 hr of incubation at the restrictive temperature for gal80^ts (31°C), with only ~25% zyd/Rab5^RNAi flies showing seizures (p=0.0006), and ~40% zyd/Rab5^DN larvae (p=0.0015)/adult flies (p=0.0055) not showing seizures. (N = 3 groups of >10 flies/genotype). (C) Fluorescence images showing accumulation of Rab5::GFP puncta in zyd cortex glia relative to wildtype cortex glia. Rab5::GFP was expressed using a cortex glial-specific driver (GMR54H02-gal4; scale bar = 5 μm. n ≥ 5 animals/genotype). (D) Analysis of the number of large (>0.1 μm²) Rab5::GFP puncta in wildtype and zyd cortex glia. The number of Rab5::GFP puncta in zyd cortex glia was increased relative to wildtype (average of 4.64 ± 0.58 and 2.85 ± 0.37 puncta/ROI respectively, p=0.0088). The number of large Rab5::GFP puncta in zyd treated with 1 mM CsA for 24 hr was decreased relative to zyd (average of 3.19 ± 0.37 puncta/ROI, p=0.0378; n ≥ 25 ROIs/3 animals/genotype/treatment). (E-F) Conditional inhibition of endocytosis by cortex glial overexpression of shi^ts. (E) Schematic representation of the experimental design. Adult zyd; NP2222 >shi^ts male flies (>1 day old) were incubated at the shi^ts restrictive temperature (31°C) for 3 or 6 hr and then tested for HS-induced seizures (red arrowheads, N = 3 groups of >15 flies/time point). (F) Behavioral analysis of HS induced seizures. Left: A significant reduction in seizures is observed in flies that were incubated at 31°C for 3 hr (p=0.0283) or 6 hr (p=0.013). (N = 3 groups of >15 flies/time point). Right: zyd;NP2222 >Shi^ts flies seizures re-occur after removal from the Shi^ts restrictive temperature (25°C, N = 4 groups of 10–15 animals/time point; 12 hr: p<0.0001). Error bars are SEM, *=P < 0.05, **=P < 0.01, ***=P < 0.001, ****=P < 0.0001, Student’s t-test.

Figure 7—video 1. The response of zyd/NP2222>Rab5^RNAi flies to a 38.5°C heat-shock is shown.

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DOI: 10.7554/eLife.44186.024