The Toll-->NFkappaB signaling pathway mediates the neuropathological effects of the human Alzheimer's Abeta42 polypeptide in Drosophila

Abstract

Alzheimer's (AD) is a progressive neurodegenerative disease that afflicts a significant fraction of older individuals. Although a proteolytic product of the Amyloid precursor protein, the Alphabeta42 polypeptide, has been directly implicated in the disease, the genes and biological pathways that are deployed during the process of Alphabeta42 induced neurodegeneration are not well understood and remain controversial. To identify genes and pathways that mediated Alphabeta42 induced neurodegeneration we took advantage of a Drosophila model for AD disease in which ectopically expressed human Alphabeta42 polypeptide induces cell death and tissue degeneration in the compound eye. One of the genes identified in our genetic screen is Toll (Tl). It encodes the receptor for the highly conserved Tl-->NFkB innate immunity/inflammatory pathway and is a fly homolog of the mammalian Interleukin-1 (Ilk-1) receptor. We found that Tl loss-of-function mutations dominantly suppress the neuropathological effects of the Alphabeta42 polypeptide while gain-of-function mutations that increase receptor activity dominantly enhance them. Furthermore, we present evidence demonstrating that Tl and key downstream components of the innate immunity/inflammatory pathway play a central role in mediating the neuropathological activities of Alphabeta42. We show that the deleterious effects of Alphabeta42 can be suppressed by genetic manipulations of the Tl-->NFkB pathway that downregulate signal transduction. Conversely, manipulations that upregulate signal transduction exacerbate the deleterious effects of Abeta42. Since postmortem studies have shown that the Ilk-1-->NFkB innate immunity pathway is substantially upregulated in the brains of AD patients, the demonstration that the Tl-->NFkB signaling actively promotes the process of Alphabeta42 induced cell death and tissue degeneration in flies points to possible therapeutic targets and strategies.

Figure 1. Aβ42 induced rough eye phenotype is sensitive to Toll activity.

WT: Wild type flies. Aβ: pGMR-Aβ42 transgenic flies. In the experiments shown in this figure, the flies are hemizygous for the pGMR-Aβ42 transgene. Panel A: WT: eye of a wild type fly. Aβ/+: eye of a wild type fly hemizygous for the pGMR-Aβ42 transgene. Panels B through F show sibling pGMR-Aβ42 transgene flies that are either wild type Tl or heterozygous for the indicated Tl allele. Panel B: Aβ/+: hemizygous for the Aβ transgene; Aβ/Tl^r4: heterozygous for the LOF allele Tl^r4. Panel C: Aβ/+: hemizygous for the Aβ transgene; Aβ/Tl^r3: heterozygous for the LOF allele Tl^r4. Panel D: Aβ/+: hemizygous for the Aβ transgene; Aβ/Tl^KG: heterozygous for the LOF allele Tl ^KG03609. Panel E: Aβ/+: hemizygous for the Aβ transgene; Aβ/Tl^rk: heterozygous for the LOF allele Tl ^rK343. Panel F: Aβ/+: hemizygous for the Aβ transgene; Aβ/Tl³: heterozygous for the GOF allele Tl ^r3.

Figure 2. The Drosophila Toll→Dorsal/Dif and mammalian Interleukin Receptor→NFκB innate immunity pathways.

Diagram of the Tl→NFκB signaling pathways in Drosophila and mammals. See text for details.

Figure 3. Components of the Toll-NFκB signaling pathway modulate Aβ42 polypetide induced neurodegeneration.

Panels A–F: In the experiments shown in this figure all flies are siblings that are hemizygous for the pGMR-Aβ42 transgene. The fly on the left side of each panel is a wild type sib, while the fly on the right side of each panel is a sib that is heterozygous for the indicated mutation in the Toll-NFκB signaling pathway mutant. Panel A: dl¹. Panel B: dl⁴. Panel C: dl⁸. Panel D: pll². Panel E: pll⁷. Panel F: tub.

Figure 4. Dorsal mediates Aβ42 dependent neurodegeneration of the eye.

Panels A–E: In the experiments shown in this figure all flies are siblings that are homozygous for the pGMR-Aβ42 transgene. The fly on the left side of each panel is a wild type sib, while the fly on the right side of each panel is a sib that is either heterozygous or homozygous for the indicated dl mutation. Panel A: dl¹/+. Panel B: dl⁴/+ Panel C: dl⁸/+ Panel D: dl¹/dl¹. Panel E: dl⁴/dl⁴. Panel F: In this experiment both flies are hemizygous for the pGMR-Aβ42 transgene. The fly on the left is wild type, while the fly on the right is homozygous dl ^`. Arrows in panels A and E point to ommatidia with dead or dying cells.

Figure 5. Cactus protein expression is induced by the Aβ42 polypeptide.

Panel A. Extracts were prepared from the heads of 10 wild type flies (WT) or 10 flies that are homozygous for the pGMR-Aβ42 transgene (Aβ). After gel electrophoresis and blotting, the blots were probed with Cactus and Snf (Sans fille—the fly U1A/U2B″ snRNP protein) antibodies. The upper panel is Cactus, while the lower panel is the Snf loading control. Two independent experiments are shown. Quantitation indicates that the level of Cactus protein is about 3.5–5 fold higher in the heads of flies carrying the transgene. Panel B. Extracts were prepared from heads of single wild type or pGMR-Aβ42 transgenic flies. After gel electrophoresis and blotting the blots were probed with Cactus (and Snf: not shown) antibodies. Quantitation indicates that the level of Cactus protein in heads of individual transgenic flies was between 3–8 fold higher than in wild type.

Figure 6. Reducing Tl activity partially ameliorates the reduction in lifespan induced by expressing the Aβ42 polypeptide in the CNS.

Filled circles: UAS:Aβ42/elav-GAL4 transgenic flies. Open boxes: UAS:Aβ42/elav-GAL4 transgene flies heterozygous for Tl⁴. Ovals: elav-GAL4 transgenic flies. Statistical tests using the Log Rank and Wilcoxon tests from the Lifetest Procedure both gave a Chi-Square of <0.0001 for the difference between the life span of UAS:Aβ42/elav-GAL4 flies and of UAS:Aβ42/elav-GAL4; Tl⁴/+ flies. Though small differences in life span (2–3 days or about 10%) were observed for two other Tl mutations and for mutations in tub and pll, the sample size was not large enough to be statistically significant.