Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling

Abstract

Parkinson's disease (PD) is the most common movement disorder characterized by dopaminergic dysfunction and degeneration. The cause of most PD cases is unknown, although postmortem studies have implicated the involvement of oxidative stress. The identification of familial PD-associated genes offers the opportunity to study mechanisms of PD pathogenesis in model organisms. Here, we show that DJ-1A, a Drosophila homologue of the familial PD-associated gene DJ-1, plays an essential role in oxidative stress response and neuronal maintenance. Inhibition of DJ-1A function through RNA interference (RNAi) results in cellular accumulation of reactive oxygen species, organismal hypersensitivity to oxidative stress, and dysfunction and degeneration of dopaminergic and photoreceptor neurons. To identify other genes that may interact with DJ-1A in regulating cell survival, we performed genetic interaction studies and identified components of the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway as specific modulators of DJ-1A RNAi-induced neurodegeneration. PI3K signaling suppresses DJ-1A RNAi phenotypes at least in part by reducing cellular reactive oxygen species levels. Consistent with the genetic interaction results, we also found reduced phosphorylation of Akt in DJ-1A RNAi animals, indicating an impairment of PI3K/Akt signaling by DJ-1A down-regulation. Together with recent findings in mammalian systems, these results implicate impairments of PI3K/Akt signaling and oxidative stress response in DJ-1-associated disease pathogenesis. We also observed impairment of PI3K/Akt signaling in the fly parkin model of PD, hinting at a common molecular event in the pathogenesis of PD. Manipulation of PI3K/Akt signaling may therefore offer therapeutic benefits for the treatment of PD.

Fig. 1.

Inhibition of DJ-1A expression by RNAi leads to photoreceptor neuron loss and eye degeneration. (A) Quantitative RT/PCR analysis of DJ-1A mRNA level after RNAi. DJ-1B and RP49 serve as controls. (B) Western blot analysis of DJ-1A protein level after RNAi. DJ-1B and actin serve as controls. (C-H) SEM images of GMR-GAL4/+ (C), GMR-GAL4>UAS-DJ-1A-RNAi (D), GMR-GAL4/Df(2R)CX1 (E), and GMR-GAL4>UAS-DJ-1A-RNAi/Df(2R)CX1 (F) eyes. Df(2R)CX1 is a chromosomal deficiency that deletes DJ-1A. G and H are magnified views of C and D, respectively. (I and J) Staining of photoreceptor neurons in GMR-GAL4/+ (I), and GMR-GAL4>UAS-DJ-1A-RNAi (J) eyes. Arrows in J mark ommatidia with photoreceptor loss. (K-N) Rescue of DJ-1A RNAi phenotypes by overexpressing DJ-1A or human DJ-1. All flies are homozygous for a recombinant GMR-GAL4;UAS-DJ-1A-RNAi chromosome and thus have a stronger phenotype than the one shown in B. In addition, the flies coexpress UAS-GFP (L), UAS-DJ-1A (M), UAS-hDJ-1 (N), or no other transgene (K).

Fig. 2.

Dopaminergic defects in DJ-1A RNAi flies. (A and B) TH immunostaining of DMC dopaminergic neurons in 35-day-old control Ddc-GAL4/+ (A) and Ddc-GAL4>DJ-1A RNAi (B) male flies. Sections containing most of the DMC dopaminergic neurons are shown. (C) Quantification of TH⁺ neurons in the DMC of control flies and DJ-1A RNAi flies directed with TH-GAL4, Ddc-GAL4, or elav-GAL4 drivers. The difference in cell count between 1-day-old and 35-day-old DJ-1A RNAi flies is significant. ^*, P < 0.01 in Student's t test. (D) Quantification of head DA levels in TH-GAL4/+ and TH-GAL4>DJ-1A RNAi flies. ^*, P < 0.01 in Student's t test.

Fig. 3.

DJ-1A RNAi leads to ROS accumulation and hypersensitivity to oxidative stress. (A and B) Comparison of survival curves of elav-GAL4/+ flies with elav-GAL4>DJ-1A RNAi flies that are treated with 1% H2O2 (A) or 100 mM 3-AT (B). (C and D) DCFH-DA staining of cultured Da-GAL4/+ (C) and Da-GAL4>DJ-1A RNAi (D) neurons. (Upper) Fluorescent DCFH-DA staining in green. (Lower) Black and white images of the neuronal culture being analyzed. (E) Recombinant DJ-1A protein exhibits detectable in vitro H₂O₂ scavenging activity, whereas the control proteins BSA, GST, and PAR-1 have no such activity.

Fig. 4.

Modification of DJ-1A RNAi phenotypes by altered expression of genes in the PI3K/Akt pathway. (A-D) SEM eye images of DJ-1A RNAi flies coexpressing UAS-Akt (A), UAS-PI3K Dp110 (B), UAS-PI3K Dp110DN (C), or UAS-PTEN (D). (E-H) SEM eye images of human tauV337M transgenic flies coexpressing UAS-GFP (E), UAS-PI3K Dp110 (F), UAS-PI3K Dp110DN (G), or UAS-PTEN (H). (I-L) SEM eye images of flies expressing UAS-Akt (I), UAS-PI3K Dp110 (J), UAS-PI3K Dp110DN (K), or UAS-PTEN (L) transgenes alone. GMR-GAL4 was used to direct UAS transgene expression in all panels.

Fig. 5.

Modification of DJ-1A RNAi-induced dopaminergic phenotype by altered expression of PI3K/Akt pathway genes and Western blot analysis showing reduced Akt phosphorylation after DJ-1A or Parkin down-regulation. (A) Quantification of TH⁺ DA neurons in the DMC of Ddc-GAL4/+ control flies, DJ-1A RNAi flies, PI3K or PI3K DN single overexpression flies, and DJ-1A RNAi flies coexpressing PI3K or PI3K DN transgenes. ^*, P < 0.01 in Student's t test. Ddc-GAL4 was used to direct transgene expression. (B and C) Western blot analysis of fly head extracts prepared from Da-GAL4/+ and Da-GAL4>DJ-1A RNAi (B) or Da-GAL4/+ and Da-GAL4>dParkin RNAi (C) genotyped flies. Blots were probed with anti-phospho-Akt and anti-Akt antibodies, respectively.