Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress

Abstract

Many nervous system pathologies are associated with increased levels of apolipoprotein D (ApoD), a lipocalin also expressed during normal development and aging. An ApoD homologous gene in Drosophila, Glial Lazarillo, regulates resistance to stress, and neurodegeneration in the aging brain. Here we study for the first time the protective potential of ApoD in a vertebrate model organism. Loss of mouse ApoD function increases the sensitivity to oxidative stress and the levels of brain lipid peroxidation, and impairs locomotor and learning abilities. Human ApoD overexpression in the mouse brain produces opposite effects, increasing survival and preventing the raise of brain lipid peroxides after oxidant treatment. These observations, together with its transcriptional up-regulation in the brain upon oxidative insult, identify ApoD as an acute response protein with a protective and therefore beneficial function mediated by the control of peroxidated lipids.

Figure 1. Alterations in behavioral output in ApoD-KO and HApoD-Tg mice

(A) Open field test on locomotor exploratory behavior in ApoD-KO mice. Both horizontal (number of squares visited) and vertical (number of rearings) activities are decreased in the ApoD-KO mice, while an anxiety-related behavior (exploration of the center of the 1 m² arena) is not altered. N= 12 mice/genotype (6th backcross generation onto C57BL/6 background). Unpaired two-sided Student’s t-test, *p < 0.05, **p < 0.01. (B) Open field test in HApoD-Tg mice. Vertical activity increase in HApoD-Tg mice (left panel), while horizontal activity differences are not significant (right panel). A smaller arena was used in this case, and the anxiety parameter was not measured. WT: N = 16; HApoD-Tg: N = 13 (11th backcross generation onto C57BL/6 background). Unpaired two-sided Student’s t-test, *p < 0.05. (C) Motor learning abilities were tested as the increment of the Rotarod test score over a 2 hours interval, and is represented normalized to the score of the first test. ApoD-KO mice show a lower learning ability under normal conditions. N= 14 mice/genotype (11th backcross generation onto C57BL/6 background). Sign non-parametric test, *p < 0.05. (D) Barnes maze test on spatial learning. ApoD-KO mice fail to increase their rate of success in finding a safe escape hole, while wild type mice learn and remember using distal spatial clues. Mice were tested daily for 70 days. Results from each five consecutive daily sessions were combined in blocks. N= 6 mice/genotype (5th backcross generation onto C57BL/6 background). ANOVA test, p < 0.0001. (A–D) Data are represented as mean ± SD.

Figure 2. Oxidative stress-compromised survival is decreased in the absence of ApoD and increased when human ApoD is over-expressed in the mouse brain

(A) Survival analysis of mice treated daily with the ROS generator paraquat (15 mg/kg of body weight; Protocol A). KO and WT genotypes in C57BL/6 background (8th backcross generation) are compared. N= 11 mice/genotype. The absence of ApoD shortens the mice survival (Gehan-test, p = 0.026; Cox’s F-test, p = 0.028). A decrease of 26% in mean lifespan and 56% in maximum lifespan is observed. The survival phenotype is stable after changing the genetic background (Balb/C, 8th outcross generation) with 11% decrease in mean lifespan and 9% in maximum lifespan. N= 12 mice/genotype. Gehan-test, p = 0.031; Cox’s F-test, p = 0.043 (curve not shown). (B and C) Survival analysis of WT and transgenic (HApoD-Tg) mice treated with single PQ doses of 30 (B) or 50 (C) mg/kg of body weight (Protocol B). Death occurrence was recorded up to 10 days. Surviving animals were sacrificed at day 10. Data was fitted to a Gompertz function. (B) Over-expression of ApoD increases the mice survival. WT: N = 16; HApoD-Tg: N = 13. Gehan-test, p = 0.029; Cox’s F-test, p = 0.043. (C) Over-expression of ApoD also protects mice at higher doses of paraquat. WT: N = 12; HApoD-Tg: N = 9. Gehan-test, p = 0.045; Cox’s F-test, p = 0.033. All transgenic mice are backcrossed on the C57Bl/6 background (11th generation).

Figure 3. The endogenous mouse ApoD is transiently up-regulated by exposure to PQ while the human transgene is expressed constitutively in the brain

(A) Northern blot analysis of mouse ApoD mRNA shows a transient increase in response to a single injection of paraquat (30 mg/kg; Protocol B). Quantification of mRNA expression by band densitometry. Values were normalized with the GAPDH gene. Error bars represent SD (N=3). (B) Mouse ApoD is specifically up-regulated in the brain upon acute PQ treatment (3 hours after a single PQ dose; 30mg/kg; Protocol B). No induction is observed in liver or lung. The GAPDH gene was used as a control. (C) Northern blot analysis shows a comparable expression of the mouse ApoD endogenous mRNA in the brain of WT and HApoD-Tg mice 3 hours after a single PQ dose (30mg/kg; Protocol B), and the absence of response to PQ of the human transgene. The GAPDH gene was used as a control.

Figure 4. Loss of ApoD function specifically alters lipid peroxidation in the brain and an over-dose of human ApoD prevents their accumulation upon oxidative insult

(A) Oxidation of proteins (Carbonyls-ELISA assay) and lipids (TBARS assay) in control conditions (sham injection of PBS). Lipid peroxidation, and not protein carbonylation, is increased in the ApoD-KO brains, while lipid peroxidation in the lung remains unchanged. (B) Brain oxidation status assayed upon acute (3 hours, Protocol B) or chronic (2 weeks low-dose, Protocol C) exposure to paraquat. ApoD-KO mouse brain show a higher level of lipid peroxidation while protein carbonylation levels do not change with genotype. (A and B) N= 7 mice/genotype/sex (11th backcross generation onto C57BL/6 background). Data are represented as mean ± SD normalized to the wild-type value. Unpaired two-sided Student’s t-test, *p < 0.05, **p < 0.01 ***p < 0.001. (C) Analysis of lipid peroxidation two weeks after PQ treatment (30 mg/kg of body weight). Human ApoD expression prevents the rise of oxidized lipids accumulation in the brain. WT: N = 16; HApoD-Tg: N = 13 (11th backcross generation onto C57BL/6 background). Unpaired two-sided Student’s t-test, ***p < 0.001. Data are represented as mean ± SD normalized with respect to the control values (sham injection of PBS).