Human ApoD, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in Drosophila

Abstract

Apolipoprotein D (ApoD) expression increases in several neurological disorders and in spinal cord injury. We provide a report of a physiological role for human ApoD (hApoD): Flies overexpressing hApoD are long-lived and protected against stress conditions associated with aging and neurodegeneration, including hyperoxia, dietary paraquat, and heat stress. We show that the fly ortholog, Glial Lazarillo, is strongly up-regulated in response to these extrinsic stresses and also can protect in vitro-cultured cells in situations modeling Alzheimer's disease (AD) and Parkinson's disease (PD). In adult flies, hApoD overexpression reduces age-associated lipid peroxide accumulation, suggesting a proximal mechanism of action. Similar data obtained in the mouse [Ganfornina, M.D., et al., (2008) Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress. Aging Cell 10.1111/j.1474-9726.2008.00395.] as well as in plants (Charron et al., personal communication) suggest that ApoD and its orthologs play an evolutionarily conserved role in response to stress, possibly managing or preventing lipid peroxidation.

Fig. 1.

Generation of two hApoD transgenic lines by using the UAS/GAL4 system. (A) RT-PCR shows hApoD driven by Da in each strain compared with control strains. (B) Horizontal cryosection through Da/UAS-hApoD1 shows strong, ubiquitous green VFP signal (the red color is due to cuticle autofluorescence). Normal signal is seen in UAS-hApoD1/+ control flies. (C) Horizontal cryosection through Da/UAS-hApoD2, which carries the HA-tag. Green fluorescence (FITC) reveals the presence of hApoD, stained by HA antibody, in most tissues (less intense than the VFP marker).

Fig. 2.

Overexpression of hApoD extends normal lifespan of Drosophila. Survival was recorded on standard food in normoxia at 25°C. (A) Da/UAS-hApoD1 flies had a 40% longer median lifespan than UAS-hApoD1/+ controls (P < 0.001). Maximum lifespan also was improved by 20%. (B) Da/UAS-hApoD2 flies had a 41% longer median lifespan than UAS-hApoD2/+ controls (P < 0.001). Maximum lifespan also was improved by 19%.

Fig. 3.

Overexpression of hApoD protects against hyperoxia in Drosophila. Survival was recorded on standard food in 100% O₂ at 25°C. (A) Da/UAS-hApoD1 flies had a 25% longer median lifespan than UAS-hApoD1/+ controls (P < 0.001). Maximum survival also was improved by 20%. (B) Da/UAS-hApoD2 flies had a 20% longer median survival than UAS-hApoD2/+ controls (P < 0.001). Maximum survival also was improved by 15%.

Fig. 4.

hApoD overexpression reduces lipid peroxide accumulation in old Drosophila. Adult flies were maintained on standard food at 25°C. (A) Da/UAS-hApoD1 flies did not display any significant increase in lipid peroxide burden at 40 days of age, whereas control flies saw this burden increase 2-fold during the same period (***, P < 0.005, t test). (B) Similar results were found with the Da/UAS-hApoD2 flies compared with controls (***, P < 0.005, t test).

Fig. 5.

Induction of GLaz mRNA in wild-type flies (CS) by extrinsic stress. Data represent fold increase in GLaz mRNA levels compared with flies in control conditions, assessed by qRT-PCR (t = 0, normoxia, standard food). (A) On 5% sucrose/20 mM paraquat, demonstrating a dramatic up-regulation of GLaz mRNA. Induction peaked at 7-fold after 2 h and decreased back to 3-fold after 12 h (control: t = 0, no paraquat). (B) In 100% O₂ on standard fly food. GLaz mRNA induction peaked at 12-fold after 2 h and stabilized at 6-fold after 10 h. (C) At 37°C on standard fly food. GLaz mRNA was induced 4.5-fold at 2 h and remained up-regulated 2-fold after 4 h. During the time frame of each experiment, no death occurred. All values were normalized by using housekeeping genes rp49 and TBP as references (see Materials and Methods). Values are averages of three independent experiments ± SEM. Student's t test performed on the Ct values yielded P values (***, P < 0.005).

Fig. 6.

GLaz protects Drosophila S2 cells against death induced by paraquat and Aβ42. (A) Anti-HA Western blot showing that GLaz-HA is expressed under the actin promoter in Drosophila S2 cells and secreted into the medium. (B) S2 cells were transfected for 48 h and then exposed to 10 mM paraquat or 1 μM Aβ42 for 24 h and assayed for viability. Cells transfected with a control plamid (actin promoter alone, pAc) showed low survival when exposed to paraquat or Aβ42 (35% and 53%, respectively). GLaz overexpression by pAc-GLaz was sufficient to protect the cells against the stress of both paraquat and Aβ42 (94% and 95% viability, respectively, after 24 h; ***, P < 0.005, t test).