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. 2010 Aug 1;19(15):2947-57.
doi: 10.1093/hmg/ddq200. Epub 2010 May 12.

Tau Ser262 phosphorylation is critical for Abeta42-induced tau toxicity in a transgenic Drosophila model of Alzheimer's disease

Koichi Iijima  1 , Anthony Gatt, Kanae Iijima-Ando
Affiliations

Tau Ser262 phosphorylation is critical for Abeta42-induced tau toxicity in a transgenic Drosophila model of Alzheimer's disease

Koichi Iijima et al. Hum Mol Genet. .

Abstract

The amyloid-beta 42 (Abeta42) peptide has been suggested to promote tau phosphorylation and toxicity in Alzheimer's disease (AD) pathogenesis; however, the underlying mechanisms are not fully understood. Using transgenic Drosophila expressing both human Abeta42 and tau, we show here that tau phosphorylation at Ser262 plays a critical role in Abeta42-induced tau toxicity. Co-expression of Abeta42 increased tau phosphorylation at AD-related sites including Ser262, and enhanced tau-induced neurodegeneration. In contrast, formation of either sarkosyl-insoluble tau or paired helical filaments was not induced by Abeta42. Co-expression of Abeta42 and tau carrying the non-phosphorylatable Ser262Ala mutation did not cause neurodegeneration, suggesting that the Ser262 phosphorylation site is required for the pathogenic interaction between Abeta42 and tau. We have recently reported that the DNA damage-activated Checkpoint kinase 2 (Chk2) phosphorylates tau at Ser262 and enhances tau toxicity in a transgenic Drosophila model. We detected that expression of Chk2, as well as a number of genes involved in DNA repair pathways, was increased in the Abeta42 fly brains. The induction of a DNA repair response is protective against Abeta42 toxicity, since blocking the function of the tumor suppressor p53, a key transcription factor for the induction of DNA repair genes, in neurons exacerbated Abeta42-induced neuronal dysfunction. Our results demonstrate that tau phosphorylation at Ser262 is crucial for Abeta42-induced tau toxicity in vivo, and suggest a new model of AD progression in which activation of DNA repair pathways is protective against Abeta42 toxicity but may trigger tau phosphorylation and toxicity in AD pathogenesis.

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Figures

Figure 1.
Figure 1.
Enhancement of human tau-induced neurodegeneration by Aβ42 in fly eyes and brains. (A–I) Enhancement of tau-induced retinal degeneration by Aβ42. External eyes (A–D) and internal retinal sections (E–H) from females (1 dae). Internal degeneration is apparent from the thickness of the retina, indicated by the double-headed arrows. (A and E) Eyes from control flies carrying the pan-retinal gmr-GAL4 driver only. (B and F) Expression of human tau in the eye reduces eye size and retinal thickness. (C and G) Eyes co-expressing human tau and Aβ42 have smaller eyes and thinner retinas than flies expressing tau alone. (D and H) Expression of Aβ42 alone does not change eye size or retinal thickness. (I) Retinal thickness was quantified and shown as a ratio relative to the control. Similar results were obtained from two independent Aβ42 transgenic fly lines (Aβ42#1 and Aβ42#2) [mean ± SD, n = 6–8, * and **P < 0.05 (Student's t-test)]. (J–N) Enhancement of human tau-induced defects in brain structures by co-expression of Aβ42. Sections containing the calyx neuropil of the mushroom body in brains of 10 dae male flies. (J) Control flies carrying the pan-neuronal elav-GAL4 driver only. The calyx is indicated by an arrowhead and hatched line. (K) Flies expressing tau have a smaller calyx or occasionally lack the calyx. (L) Flies co-expressing tau and Aβ42 do not have the calyx. (M) Flies expressing Aβ42 alone have a normal calyx structure. (N) The percentage of the hemispheres with the calyx neuropil in each genotype.
Figure 2.
Figure 2.
Increase of human tau phosphorylation at Ser202, Thr231 and Ser262 by Aβ42 in fly eyes and brains. (A) Schematic representation of the structure of tau. Positions of the phosphorylation sites tested in this study are shown. Gray boxes, four repeats of the microtubule-binding domain. (B and C) Fly heads expressing human tau alone (tau) or with Aβ42 (tau + Aβ42) driven by the pan-retinal gmr-GAL4 at 1 dae (B) or pan-neuronal elav-GAL4 at 25 dae (C) were subjected to western blotting with anti-tau (total tau), anti-phospho-tau (pS202, pT231 and pS262) and anti-Aβ42 antibodies. Flies carrying the driver only were used as the negative control (control). The phosphorylation levels in the eye and brain of flies co-expressing tau and Aβ42 (tau + Aβ42) are shown as a ratio relative to that in flies expressing tau alone (tau). Representative blots are shown. Asterisks indicate significant differences from tau alone (tau) [n = 4 or 5, *P < 0.05 (Student's t-test)]. (D) Co-expression of Aβ42 did not increase sarkosyl-insoluble tau in the fly brain. Western blotting of sarkosyl-soluble and -insoluble fractions of head extracts from flies expressing tau alone (tau), or tau and Aβ42 (tau + Aβ42), driven by the pan-neuronal elav-GAL4 driver. Head extracts from flies expressing Aβ42 alone (Aβ42) was used as a negative control. Flies are at 35 dae.
Figure 3.
Figure 3.
The Ser262 phosphorylation site of tau is critical for the pathogenic interaction between Aβ42 and tau. (A) Effect of S262A mutation on tau phosphorylation at Ser202 and Thr231. Western blot of head lysates of 1 dae females with anti-tau, and anti-phospho-tau antibodies (pS202 and pT231). Control: flies carrying the pan-retinal gmr-Gal4 driver only. Phosphorylation of S262A tau at Ser202 or Thr231 is shown as a ratio relative to that of wild-type tau. Asterisks indicate significant differences from wild-type tau [n = 3 or 4, *P < 0.05 (Student's t-test)]. (B and C) Co-expression of Aβ42 and S262A tau using the pan-retinal gmr-GAL4 driver did not cause eye degeneration. External eyes of 1 dae females expressing S262A tau (B, S262A tau) or S262A tau with Aβ42 (C, Aβ42 + S262A tau). (D–F) Co-expression of Aβ42 and S262A tau using the pan-neuronal elav-GAL4 driver did not cause structural defects in the brains. Sections containing the calyx neuropil of the mushroom body (indicated by arrowheads and hatched line) in brains of 10 dae male flies. (D) Flies expressing S262A tau. (E) Flies co-expressing Aβ42 and S262A tau. (F) The percentage of the hemispheres with the calyx neuropil in each genotype.
Figure 4.
Figure 4.
Aβ42-induced increases in expression of Chk2 and a number of DNA repair pathway genes in fly brains. mRNA levels in heads from flies (25 dae) expressing Aβ42 or Aβ42 with the familial Aβ42Arc driven by the pan-neuronal elav-GAL4 driver are shown as ratios relative to controls (flies carrying the elav-Gal4 driver only) [mean ± SD, n = 5, *P < 0.05 (Student's t-test)]. Relative mRNA levels of genes encoding Drosophila homologs of Chk2, and genes involved in DNA repair pathways (Ku70, Rad50, Rad54, Ligase 4, nbs, RfC40, PCNA and mtSSB), are increased in both Aβ42 and Aβ42Arc fly brains.
Figure 5.
Figure 5.
Enhancement of Aβ42-induced locomotor defects by neuronal expression of dominant negative forms of p53. The average percentage of flies at the top (white), middle (light gray) or bottom (dark gray) of assay vials is shown (mean ± SD, n = 5). (A) The effect of overexpression of DN-p53-259H or DN-p53-Ct by the pan-neuronal elav-GAL4 on Aβ42-induced locomotor defects in flies at 7, 19 and 29 dae. Asterisks indicate the significant differences in the percentage of flies that stayed at the bottom [*P < 0.05 (Student's t-test)]. (B) In the absence of Aβ42, overexpression of DN-p53-259H or DN-p53-Ct does not cause any locomotor defects even at 36 dae.
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