Figure 8. Inhibition of the JNK pathway rescues synaptic defects in shot-tau mutant neurons.

(A) Primary Drosophila neurons at 2 DIV, obtained from embryos of the following genotypes: wildtype (wt), elav-Gal4 driven expression of UAS-hep^ac (UAS-hep^ac), tau^MR22 (tau^-/-), wnd² (wnd^-/-), tau^-/- with elav-Gal4 driven expression of UAS-puc (tau^-/- UAS-puc), tau^MR22 kay² (tau^-/- kay^-/-) and shot³ tau^MR22 wnd² (shot^-/- tau^-/- wnd^-/-), all stained with antibodies against Tubulin (tub, magenta) and Syt (green). Insets correspond to emboxed areas and show a magnified view of the Syt staining. (B) Quantification of experiments in A, shown as the number of Syt puncta normalised to wildtype (number of assessed neurons is shown in the bars, ***P_MW<0.001; **P_MW<0.01; *P_MW<0.05; ns, not significant P_MW>0.05). (C) Inter-segmental motornerves in the dorsal area of wildtype and shot³ mutant embryos at late stage 16, stained against FasII (magenta) and Syt (green); insets correspond to emboxed areas and show a magnified view of the most dorsal nascent NMJs stained for Syt; note the rescue of Syt localisation if Wnd is absent in tau-shot mutant background. (D) Quantification of the experiments in C, measured as the average intensity of Syt normalised to wt (number of assessed NMJs is shown in the bars; ***P_MW<0.001; *P_MW<0.01). (E) A region of Drosophila adult brains including the medulla; UAS-nSyb-GFP is expressed in dorsal cluster neurons using atonal-Gal4, either alone (control), together with tau^RNAi and shot^RNAi (tau^RNAi shot^RNAi) or together with tau^RNAi, shot^RNAi and UAS-bsk^DN. Brains are shown at 2–6 days (young) and 26–30 days at 29°C after eclosion (old); GFP-labelled synapses are decreased in old brains with shot-tau knock-down when compared to controls, and this effect is rescued by the expression of Bsk^DN. (F) Quantification of experiments in E, shown as number of GFP-labelled synapses in old specimen per mean number of GFP-labelled synapses in young specimens of the respective genotype (number of analysed brains is indicated in the bars; ***P_MW<0.001; **P_MW<0.01). Scale bars: 5 μm in A, 10 μm in C and 40 μm in E. A statistics summary of the data shown here is available in Figure 8—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.14694.031

Figure 8—figure supplement 1. Attenuation of the JNK pathway rescues aberrant Unc-104 localisation in shot-tau mutant neurons in culture.

(A) Primary Drosophila neurons at 2 DIV, obtained from wildtype (wt) and shot³ tau^MR22 wnd²mutant embryos (shot^-/- tau^-/- wnd^-/-), stained with antibodies against pan-neuronal HRP (magenta) and Unc-104 (green); Unc-104 in distal axon segments (emboxed and magnified in insets) is enriched in wildtype and in shot^-/- tau^-/- wnd^-/-mutant neurons (chevrons indicate neuronal somata). This is in contrast to shot-tau mutant neurons, in which Unc-104 in distal axon segments is much weaker (for reference see Figure 5D). (B) Data were quantified as average intensity of Unc-104 at the distal end of the axon divided by the average intensity at the soma (**P_MW<0.01). Scale bars: 5 μm in A. A statistics summary of the data shown here is available in Figure 8—figure supplement 1—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.14694.033

Figure 8—figure supplement 2. Attenuation of the JNK pathway rescue aberrant unc-104 localisation in shot-tau mutant embryos.

(A) The dorsal peripheral nervous system (PNS) and the central nervous system (CNS) of wildtype wildtype, shot³ tau^MR22 and shot³ tau^MR22 wnd²mutant embryos (shot^-/- tau^-/- wnd^-/-) at late stage 16 stained with FasII (red), Unc-104 (green) and elav (blue) for the PNS panels and FasII (magenta) and Unc-104 (green) in the CNS panels. Note the stark decrease of Unc-104 at the end of motor nerves (open versus white arrow heads) and the unusual accumulations of Unc-104 in the cell bodies of sensory neurons as well as in the CNS cortex in shot-tau embryos (open versus white arrows). This is in contrast to shot^-/- tau^-/- wnd^-/-mutant neurons in which Unc-104 is increased at the end of motor nerves and decreased in the cell bodies of sensory neurons as well as in the CNS cortex. Scale bars: 15 μm in PNS panels and 35 μm in CNS panels.

DOI: http://dx.doi.org/10.7554/eLife.14694.035