(A) Quantification of TUNEL-positive neurons in the brains of 10-day-old flies expressing different tau isoforms revealed significantly higher neurotoxicity in tau^R406W (***P < 0.001) and tau^E14 (***P < 0.001) transgenic flies compared with controls and tau^WT-expressing flies. Neurodegeneration was significantly higher in tau^E14- than in tau^R406W-expressing flies (P < 0.001). (B and C) The brains of 10-day-old control (B) and tau^E14-expressing (C) flies were immunostained with a phosphorylated (active) JNK-specific antibody, which accumulated in areas of neurodegeneration (arrows). Scale bar: 20 μm. Genotypes (A–C) are as follows: control, elav-GAL4/+; tau^WT, elav-GAL4/+, UAS-tau^WT/+; tau^R406W, elav-GAL4/+, UAS-tau^R406W/+; and tau^E14, elav-GAL4/+, UAS-tau^E14(26)/+. (D and E) puc-lacZ gene expression revealed JNK pathway activation in tau transgenic flies. Brains of transgenic flies expressing the puc-lacZ transgene alone (D) or in combination with tau^E14 (E) were immunostained for β-gal (arrows). Scale bar: 20 μm. (F) JNK pathway activation correlated with the extent of neurotoxicity induced by different tau transgenes. Quantification of β-gal–positive cells in 10-day-old puc-lacZ transgenic fly brains identified significant increases in JNK pathway activation in animals that also expressed tau^R406W (**P < 0.01) or tau^E14 (***P < 0.001) compared with tau^WT-expressing flies or controls. (G–L) JNK pathway activation was detected in neurons. β-gal–positive cells (green fluorescence) often coexpressed the neuronal cell marker elav (red fluorescence) (G–I) and did not colocalize with repo-positive glia (red fluorescence) (J–L). Arrowheads mark the locations of β-gal–positive cells. Scale bars: 10 μm. Genotypes (D–L) are as follows: control, elav-GAL4/+, puc-lacZ/+; tau^WT, elav-GAL4/+, UAS-tau^WT/puc-lacZ; tau^R406W, elav-GAL4/+, UAS-tau^R406W/puc-lacZ; and tau^E14, elav-GAL4/+, UAS-tau^E14(26)/puc-lacZ.