Beta-amyloid mediated nitration of manganese superoxide dismutase: implication for oxidative stress in a APPNLH/NLH X PS-1P264L/P264L double knock-in mouse model of Alzheimer's disease

Abstract

Alzheimer's disease is a multifactorial, progressive, age-related neurodegenerative disease. In familial Alzheimer's disease, Abeta is excessively produced and deposited because of mutations in the amyloid precursor protein, presenilin-1, and presenilin-2 genes. Here, we generated a double homozygous knock-in mouse model that incorporates the Swedish familial Alzheimer's disease mutations and converts mouse Abeta to the human sequence in amyloid precursor protein and had the P264L familial Alzheimer's disease mutation in presenilin-1. We observed Abeta deposition in double knock-in mice beginning at 6 months as well as an increase in the levels of insoluble Abeta1-40/1-42. Brain homogenates from 3-, 6-, 9-, 12-, and 14-month-old mice showed that protein levels of manganese superoxide dismutase (MnSOD) were unchanged in the double knock-in mice compared to controls. Genotype-associated increases in nitrotyrosine levels were observed. Protein immunoprecipitation revealed MnSOD as a target of this nitration. Although the levels of MnSOD protein did not change, MnSOD activity and mitochondrial respiration decreased in knock-in mice, suggesting compromised mitochondrial function. The compromised activity of MnSOD, a primary antioxidant enzyme protecting mitochondria, may explain mitochondrial dysfunction and provide the missing link between Abeta-induced oxidative stress and Alzheimer's disease.

Figure 1

Sections of frontal cortex from APP/PS-1 mice immunostained with 10D-5 antibody for Aβ. A: Three-month-old animal showing no amyloid immunostaining in cortex. B: Six-month-old mouse showing rare small deposits of Aβ (arrows). C: Nine-month-old mouse showing increased Aβ deposits. D: Twelve-month-old mouse demonstrating numerous variable size deposits of Aβ. E: Fourteen-month-old mouse showing numerous diffuse Aβ deposits in cortex. F: Fourteen-month-old mouse demonstrating many Aβ deposits in cortex in parenchymal and leptomeningeal vessels (arrows). Original magnifications, ×100.

Figure 2

Levels of Aβ1-40 and 1-42 in APP/PS-1 mice. Aβ1-40 and Aβ1-42 levels were measured by ELISA from different groups (n = 5 per group) as described in the Materials and Methods section. Both species of Aβ showed an increasing trend in the levels associated with age (R² = 0.8072 for Aβ1-40 and R² = 0.702 for Aβ1-42).

Figure 3

Representative (out of three) immunoblot (A) and densitometry analysis (B) showing the levels of MnSOD. Protein (30 μg) extracted from brain specimens of APP/PS-1 knock-in and WT mice were run on 12.5% SDS-polyacrylamide gel electrophoresis. Protein levels of MnSOD did not show significant age- and genotype-associated alterations. β-Actin was used to normalize protein loading. HO, APP/PS-1.

Figure 4

Immunoprecipitation of nitrotyrosine with MnSOD. Isolated mitochondrial protein (200 μg) was precipitated with polyclonal nitrotyrosine antibody and analyzed by Western blotting using MnSOD antibody. A: Nitrotyrosine co-immunoprecipitated with MnSOD showed that MnSOD is nitrated. HO, APP/PS-1; positive control, brain homogenate + peroxynitrite (2 μmol/L), provided by Dr. Timothy R. Miller, Graduate Center for Toxicology, University of Kentucky, Lexington, KY; pellet, pellet of the isolated mitochondrial protein (200 μg) precipitated with preimmune IgG, analyzed by Western blotting using MnSOD antibody; supernatant of the isolated mitochondrial protein (200 μg) precipitated with preimmune serum, analyzed by Western blotting using MnSOD antibody. B: Densitometric analysis and subsequent statistical analysis by two-way analysis of variance showed significant differences (*P < 0.01) when compared between genotypes. C: MnSOD activity in the brain specimen was measured by the NBT-bathocuproine sulfonate reduction inhibition method. Statistical analysis by two-way analysis of variance showed significant age- and genotype-dependent decreases (P < 0.0001) in activity of MnSOD. APP/PS-1 mice, at all ages, showed significant decreases (*P < 0.05) in MnSOD activity when compared with age-matched WT mice. WT mice at 12 and 14 months showed significant decrease (**P < 0.05) in MnSOD activity when compared with 3-month-old WT mice. Immunoprecipitation and MnSOD activity was performed in three sets of animals.

Figure 5

Decline in mitochondrial respiration via complex I in APP/PS-1 mice. Oxygen consumption was measured using a Clark-type electrode oxygraph. RCR was calculated as the ratios between state 3 and state 2 respirations. APP/PS-1 mice at 9 and 12 months of age showed a significant decrease (*P < 0.01) in mitochondrial respiration when compared to its age-matched WT mice and a significant decrease (**P < 0.001) compared to 3-month-old mice of both genotypes. Statistical analysis: two-way analysis of variance followed by Newman-Keuls multiple comparisons test.