Figure 4

Figure 4

Study of GNPDA2 overexpression in zf Tau P301L transgenic embryos. (A) Experimental design used in the zebrafish model. (B) Representative pictures of 30 hpf not-injected embryos or embryos injected with cLuc and GNPDA2 mRNAs (both DsRed/mutant TAU-positive and -negative embryos) after whole-mount immunostaining with znp1/zn-1 antibodies. White asterisks indicate the position of the four axonal extensions quantified in not injected Tau− pictures. (C) Graph shows the mean ± SEM of the total axon length of the first four caudal primary motoneurons anterior to the end of the yolk extension of the indicated experimental groups. ** p < 0.01; *** p < 0.001. (D) Representative pictures of 48 hpf not-injected embryos or embryos injected with cLuc and GNPDA2 RNAs (both DsRed/mutant TAU-positive and -negative embryos) after TUNEL staining. The spinal cord area, where the number of TUNEL-positive cells was quantified, is highlighted in not injected Tau− pictures. (E) Graph shows the mean ± SEM of the number of TUNEL-positive neurons present along the spinal cord area of the indicated experimental groups. *** p < 0.001. (F) Western blot developed with anti-Tau (Phospho S396), anti-Tau (total) and anti-GNPDA2 antibodies of 48 hpf embryos from the indicated experimental groups. Ponceau S red staining corresponding to fragments used for developing pTau and total Tau are shown below each antibody-specific staining. Numbers correspond to the optical density quantification (arbitrary units) of Ser396-phosphorylated Tau with respect to the total Tau levels. Specimens in the study: not injected Tau− (zebrafish specimens with no Tau and no GNPDA2); not injected Tau+ (zebrafish specimens overexpressing human TauP301L and no GNPDA2); cLUC Tau− (zebrafish specimens with no Tau and expressing the luciferase vector); cLUC Tau− (zebrafish specimens overexpressing human TauP301L and expressing the luciferase vector); GNPDA2 Tau− (zebrafish specimens with no Tau and expressing human GNPDA2) and GNPDA2 Tau+ (zebrafish specimens overexpressing human TauP301L and expressing human GNPDA2).