Deregulation of the Egfr/Ras signaling pathway induces age-related brain degeneration in the Drosophila mutant vap

Abstract

Ras signaling has been shown to play an important role in promoting cell survival in many different tissues. Here we show that upregulation of Ras activity in adult Drosophila neurons induces neuronal cell death, as evident from the phenotype of vacuolar peduncle (vap) mutants defective in the Drosophila RasGAP gene, which encodes a Ras GTPase-activating protein. These mutants show age-related brain degeneration that is dependent on activation of the EGF receptor signaling pathway in adult neurons, leading to autophagic cell death (cell death type 2). These results provide the first evidence for a requirement of Egf receptor activity in differentiated adult Drosophila neurons and show that a delicate balance of Ras activity is essential for the survival of adult neurons.

Figure 1

Age-related brain degeneration in vap mutants. Toluidin blue staining of horizontal semithin plastic sections from adult flies (A-D). In 1-day-old vap¹ flies (B) no apparent difference can be found when compared with wild type (A). Seven-day-old vap¹ flies (C) show vacuolization in the optic lobe (ol) and central brain (cb). In 14-day-old vap¹ flies (D), the stronger spongiform appearance of the brain reveals that the degenerative phenotype in vap flies is age related. The ultrastructural analysis (E–H; G is a magnification from F) shows that neurons of 1-day-old vap¹ flies accumulate autolysosomes (F and arrow in G), vacuoles containing whorls of membranous material (F and arrowhead in G), empty vacuoles or vacuoles still containing some material (F and asterisk in G); all signs of autophagic cell death that are absent in wild-type neurons (E). In 7-day-old vap flies dying neurons show a digested cytoplasm, whereas the nucleus (n) remains intact (H). The asterisk in B indicates the medula cortex, the region where the EM pictures were taken from. Scale bars, 50 μm in (A), 2 μm in (E) and 0.5 μm in (G). re (retina), la (lamina), me (medula), lo (lobula complex), cb (central brain).

Figure 2

Molecular characterization of the vap mutant alleles. (A) Molecular map of the RasGAP gene showing the mutations and the expected mutant gene products of the different vap alleles. In vap², a deletion generates the aberrant splicing event shown with a dashed line. P, PstI; E, EcoRI; H, HindIII; B, BamHI. (B) Western blot analysis of the vap mutants. Equal amounts of protein were loaded in all lanes. Hwt, wild-type adult heads; Lwt, 3rd instar wild-type larvae. For the analysis of the mutants, protein extracts from 3rd instar larvae were used. In vap¹ no signal could be detected. In vap² a 100-kDa mutant protein results from the aberrant splicing induced by the deletion. The transposon insertion in vap³ causes a reduction in the expression of RasGAP protein.

Figure 3

Neuronal expression of RasGAP. In situ hybridization of RasGAP RNA on cryosections from wild-type flies. (A) Control, using a sense RNA probe. (B) RasGAP is expressed in the entire brain cortex, suggesting widespread expression in cortical cell bodies as detected by using an antisense RNA probe. (C) Magnification of the neuropil areas of the central complex shows no expression of vap in neuropil glial cells. (D) Localization of the neuropil glia cells distribution around the neuropil area of the central complex visualized by autofluorescence. (E) β-Galactosidase detection in the enhancer-trap line vap³. (F) Anti-ELAV stainings of wild-type heads of the same cortical area as shown in E. Scale bar: (A) 100 μm; and (C) 10 μm. (f): fan shaped body; (e) ellipsoid body.

Figure 4

Functional specificity of RasGAP. Horizontal paraffin sections of adult fly heads were stained with an antibody (ab49) against Csp to visualize the neuropiles. The degenerative phenotype of 7-day-old vap¹ flies (A) can be rescued by expressing the RasGAP cDNA in neurons (B). This phenotype cannot be rescued by the pan-neural expression of the GTPase–activating protein Gap1 (C). The genotypes are vap¹ (A), vap¹;UAS-RasGAP/+;elav-GAL4/+ (B) and vap¹;UAS-Gap1/+;elav-GAL4/+ (C). Scale bar is 50 μm.

Figure 5

vap interacts with members of the Egfr/Ras pathway. In all panels flies were 18-day-old except in E and F, which were aged for 6 days. (A) Wild-type. The degenerative phenotype of 18-day-old vap² flies (B) can be enhanced by reducing the dose of sprouty (vap²;sty^S73/+) (C and J). The overexpression of rhomboid (vap²;hs-rho/+) (D and J), Egfr (vap²;hs-DER/+) (E, the arrow shows the area of degeneration) or the pan-neural expression of Ras (vap²;UAS-Ras1/+;appl-GAL4/+) (F and J) also enhances the phenotype. Reduction in the dose of drk (vap²;drk^k02401/+) (G and J) or Egfr (vap²;Egfr^top1p02/+) (H and J) suppresses the phenotype. The suppression of the phenotype can be also achieved by reducing the dose of Egfr in adulthood (vap²; Egfr^ts1a/+) (I, J). re: retina, la: lamina, me: medula, loc: lobula complex, cb: central brain. Scale bar is 50 μm.

Figure 6

Deregulation of MAPK in vap mutants. (A) Western blot analysis of larval (3rd instar) protein extracts of wild-type and vap¹ flies carrying a heat shock–inducible Egfr construct. After 1-h heat shock, the level but not the duration of the MAPK activation (rl) is deregulated in vap mutants compared with wild-type flies (w¹¹¹⁸). Detection of the cysteine-string protein (csp) was used as loading control. (B) Quantification of three independent experiments.