Dissociation of tau toxicity and phosphorylation: role of GSK-3beta, MARK and Cdk5 in a Drosophila model

Abstract

Hyperphosphorylation of tau at multiple sites has been implicated in the formation of neurofibrillary tangles in Alzheimer's disease; however, the relationship between toxicity and phosphorylation of tau has not been clearly elucidated. Putative tau kinases that play a role in such phosphorylation events include the proline-directed kinases glycogen synthase kinase-3beta (GSK-3beta) and cyclin-dependent kinase 5 (Cdk5), as well as nonproline-directed kinases such as microtubule affinity-regulating kinase (MARK)/PAR-1; however, whether the cascade of events linking tau phosphorylation and neurodegeneration involves sequential action of kinases as opposed to parallel pathways is still a matter of controversy. Here, we employed a well-characterized Drosophila model of tauopathy to investigate the interdependence of tau kinases in regulating the phosphorylation and toxicity of tau in vivo. We found that tau mutants resistant to phosphorylation by MARK/PAR-1 were indeed less toxic than wild-type tau; however, this was not due to their resistance to phosphorylation by GSK-3beta/Shaggy. On the contrary, a tau mutant resistant to phosphorylation by GSK-3beta/Shaggy retained substantial toxicity and was found to have increased affinity for microtubules compared with wild-type tau. The fly homologs of Cdk5/p35 did not have major effects on tau toxicity or phosphorylation in this model. These data suggest that, in addition to tau phosphorylation, microtubule binding plays a crucial role in the regulation of tau toxicity when misexpressed. These data have important implications for the understanding and interpretation of animal models of tauopathy.

Figure 1.

Misexpression of PAR-1 (68) using GMR-GAL4 produces a rough eye with the disruption of retinal architecture, whereas Sgg (63) misexpression produces a relatively normal eye. (A–C) SEM images. The normal-eye phenotype observed using the GMR-GAL4 driver (A) is disrupted in eyes misexpressing PAR-1 (B) but not in eyes misexpressing Shaggy (C). (D–F) Retinal whole mounts stained with phalloidin-TRITC to identify rhabdomeres of photoreceptor neurons (single tangential confocal sections). At this apical section, normally, clusters of seven photoreceptors within each ommatidium form a characteristic chevron-shaped structure (D). Full genotypes: (i) w¹¹¹⁸, GMR-GAL4/+ (A and D); (ii) w¹¹¹⁸, GMR-GAL4/+, UAS-PAR-1/+ (B and E); (iii) w¹¹¹⁸, GMR-GAL4/+, UAS-Sgg/+ (C and F). Scale bars: 100 µm (A–C); 10 µM (D–F).

Figure 2.

Schematic representation of the tau constructs used. Tau^S2A was constructed by mutating two serine residues (S262 and S356) to alanine; these are the phosphoepitopes recognized by the 12E8 antibody. Tau^S11A has 11 serine and threonine residues mutated to alanine. The epitopes recognized by phosphorylation-dependent antibodies, e.g. AT8, AT180, PHF1 and AT100, are shown. GMR-GAL4 driver was used to drive combinations of UAS-Tau^wt, UAS-Tau^S2A and UAS-Tau^S11A along with PAR1 and Shaggy. gl drivers were also used for the same purpose.

Figure 3.

The retinal phenotype of wild-type tau (Tau^wt) under the control of GMR-GAL4 is more severe than that of S2A tau. (A and B) SEM images. The mild rough-eye phenotype produced by Tau^wt (A) is not observed with Tau^S2A (B). Scale bars: 100 µm. (C and D) Confocal images of adult retina stained with TRITC-phalloidin (red). Ommatidial disorganization and cell loss are apparent in the eyes overexpressing the Tau^wt transgene (C) compared with eyes expressing Tau^S2A (D), which displays a largely normal trapezoidal array of rhabdomeres. Genotypes: (i) GMR-GAL4/+, UAS-Tau^wt/+ (A and C); (ii) GMR-GAL4/+, UAS-Tau^S2A/+ (B and D). (E) Immunoblot using T46 demonstrates that several independently derived Tau^wt and Tau^S2A lines express comparable amounts of total tau protein. β-Tubulin-loading control for total protein in head extracts is shown below. Scale bars: 100 µm (A and B); 10 µm (C and D).

Figure 4.

S2A is less toxic than wild-type Tau and relatively resistant to PAR-1-induced phosphorylation. (A–D) SEM images. The rough-eye phenotype of transgenics misexpressing Tau^wt (A) is severely enhanced when coexpressed with PAR-1 (B), whereas the phenotype of Tau^S2A (C) is only mildly enhanced by PAR-1 overexpression (D). (E–H) Phalloidin-TRITC staining of whole-mount retina. Tau^S2A (G) is less toxic than Tau^wt (E), and the enhancement in response to PAR-1 is less severe in Tau^S2A (H) compared with Tau^wt (F). Genotypes: (i) GMR-GAL4/+, UAS-Tau^wt/+ (A and E); (ii) GMR-GAL4/+, UAS-Tau^wt/+, UAS-PAR-1/+ (B and F); (iii) GMR-GAL4/+, UAS-Tau^S2A/+ (C and G); (iv) GMR-GAL4/+, UAS-Tau^S2A/+, UAS-PAR-1 (D and H). Scale bars: 100 µm (A–D); 10 µm (E–H). (I) Immunoblot analysis with 12E8 detects stronger immunoreactivity with Tau^wt compared with Tau^S2A. (J) Histograms representing relative phosphorylation level changes after the coexpression of PAR-1 and Shaggy. Results shown are derived from densitometric analysis of three separate blots. Each bar represents mean ± SEM (n = 3). *P < 0.05; **P < 0.01 (ANOVA with the Newman–Keuls post hoc comparison).

Figure 5.

Tau^S2A is relatively resistant to Shaggy-induced toxicity but not phosphorylation. (A–D) SEM images. The rough-eye phenotype of Tau^wt (A) is enhanced by the coexpression of Shaggy (B). The normal eye phenotype of TauS2A (C) remains unchanged when coexpressed with Shaggy (D). (E–H) Phalloidin staining. Shaggy expression enhances the phenotype of Tau^wt (F) but not Tau^S2A (H). Genotypes: (i) GMR-GAL4/+, UAS-Tau^wt/+ (A and E); (ii) GMR-GAL4/+, UAS-Tau^wt/+, UAS-Sgg/+ (B and F); (iii) GMR-GAL4/+, UAS-Tau^S2A/+ (C and G); (iv) GMR-GAL4/+, UAS-Tau^S2A/+, UAS-Sgg (D and H). Scale bars: 100 µm (A–D); 10 µm (E–H). (I) Immunoblot analysis shows that priming of tau by PAR-1 is not required for subsequent phosphorylation by overexpressed Sgg. The magnitude of the increase in phosphorylation at the AT8 and PHF-1 epitopes when stimulated by Sgg is greater for Tau^S2A than for Tau^wt. (J) Histograms show relative effects on phosphorylation derived from densitometric analysis of three separate blots. Each bar represents mean ± SEM (n = 3). *P < 0.05; **P < 0.01 (ANOVA with the Newman–Keuls post hoc comparison).

Figure 6.

Tau^S11A retains toxicity despite being resistant to phosphorylation by Sgg. (A–C) and (G–I) SEM images. Direct fusion of gl-Tau^wt (A–C) and gl-Tau^S11A (G–I) phenotypes is shown. Tau^S11A retains toxicity as evidenced by a severe rough-eye phenotype with disordered ommatidial morphology (G). However, the degree of enhancement by Sgg (I) is less than that observed with wild-type Tau (C). Scale bar: 100 µm. (D–F) and (J–L) TRITC-phalloidin staining indicates that Tau^S11A (J) retains some toxic effects on ommatidial morphology, although the response to Shaggy (L) is blunted compared with Tau^wt. Scale bars: 100 µm (A–C and G–I); 10 µm (D–F and J–L). Genotypes: (i) GMR-GAL4/+, gl-Tau^wt/+ (A and D); (ii) GMR-GAL4/+, gl-Tau^wt/+, UAS-PAR-1/+ (B and E); (iii) GMR-GAL4/+, gl-Tau^wt/+, UAS-Sgg/+ (C and F); (iv) GMR-GAL4/+, gl-Tau^S11A/+ (G and J); (v) GMR-GAL4/+, gl-Tau^S11A/+, UAS-PAR-1/+ (H and K); (vi) GMR-GAL4/+, gl-Tau^S11A/+, UAS-Sgg/+(I and L). (M) Immunoblot analysis showing decreased basal and induced phosphorylation at AT8, PHF1, AT180 and AT100 phosphoepitopes for Tau^S11A compared with Tau^wt. Results obtained using the direct fusion gl-Tau construct in combination with GMR-GAL4 driving UAS-kinases in this figure differ somewhat from those in which GMR-GAL4 drives both tau and kinases owing to less amplification of Tau expression in the former case, i.e. greater tau expression is achieved when glass drives GAL4 expression, which drives Tau. This difference results in a lower ratio of Tau to kinase when gl-Tau is used.

Figure 7.

Toxicity of S11A and lack of effects of S2A tau correlate not with the formation of sarcosyl-soluble and -insoluble tau fractions, but with microtubule affinity. (A) Sarcosyl extracts of wild-type, S2A and S11A tau probed with T46 (total tau) and PHF-1 (a pathological phosphoepitope). Upper panel, input tau for the sarcosyl extraction; middle panel, sarcosyl-soluble and -insoluble fractions probed with T46 (total tau); lower panel, pellets and supernatants probed with PHF-1 (a pathological phosphoepitope). Tau^S11A does not form any soluble or insoluble aggregates compared with wild-type tau, whereas for Tau^S2A, the amount of insoluble material is much less compared with wild-type Tau. (B) Microtubule-binding assays. Upper panel, input tau for the microtubule-binding studies; middle panel, free or microtubule-bound tau. Compared with wild-type tau, Tau^S2A associates with microtubules somewhat less avidly, whereas Tau^S11A shows a markedly increased affinity for microtubules. T46 blotting indicates that the levels of input tau were the same for all three genotypes in both sarcosyl extraction and microtubule-binding experiments. All experiments used UAS constructs in trans to GMR-GAL4. Histograms represent the relative levels of free and bound tau in the supernatant (S) and pellet (P) fractions, respectively, for the three different transgenics. Each bar represents mean ± SEM (n = 3). *P < 0.05 for Tau^wt and Tau^S2A, and **P < 0.001 for Tau^S11A. (One-way ANOVA with the Newman–Keul’s post hoc comparison).