Accumulation of amyloid precursor protein in the mitochondrial import channels of human Alzheimer's disease brain is associated with mitochondrial dysfunction

Abstract

Mitochondrial dysfunction is one of the major intracellular lesions of Alzheimer's disease (AD). However, the causative factors involved in the mitochondrial dysfunction in human AD are not well understood. Here we report that nonglycosylated full-length and C-terminal truncated amyloid precursor protein (APP) accumulates exclusively in the protein import channels of mitochondria of human AD brains but not in age-matched controls. Furthermore, in AD brains, mitochondrially associated APP formed stable approximately 480 kDa complexes with the translocase of the outer mitochondrial membrane 40 (TOM40) import channel and a super complex of approximately 620 kDa with both mitochondrial TOM40 and the translocase of the inner mitochondrial membrane 23 (TIM23) import channel TIM23 in an "N(in mitochondria)-C(out cytoplasm)" orientation. Accumulation of APP across mitochondrial import channels, which varied with the severity of AD, inhibited the entry of nuclear-encoded cytochrome c oxidase subunits IV and Vb proteins, which was associated with decreased cytochrome c oxidase activity and increased levels of H2O2. Regional distribution of mitochondrial APP showed higher levels in AD-vulnerable brain regions, such as the frontal cortex, hippocampus, and amygdala. Mitochondrial accumulation of APP was also observed in the cholinergic, dopaminergic, GABAergic, and glutamatergic neuronal types in the category III AD brains. The levels of translocationally arrested mitochondrial APP directly correlated with mitochondrial dysfunction. Moreover, apolipoprotein genotype analysis revealed that AD subjects with the E3/E4 alleles had the highest content of mitochondrial APP. Collectively, these results suggest that abnormal accumulation of APP across mitochondrial import channels, causing mitochondrial dysfunction, is a hallmark of human AD pathology.

Figure 1.

Mitochondrial targeting of APP in human frontal cortex of AD and NC brains. Immunoblot analysis of marker proteins for synaptosomal (S.S), mitochondrial (Mito.), and PM fractions from frontal cortex of AD and NC using antibodies to synaptophysin (S.P) (A), TIM44 (B), and Na⁺/K⁺ ATPase (C). Levels of APP in mitochondrial and PM fractions of AD and NC brains (frontal cortex) by immunoblot using APP Nt Ab (D) and Aβ Ab (F). E, Quantitation of immunoblot shown in D. G and H indicate the levels of TIM44 and Na⁺/K⁺ ATPase proteins in Mito and PM fractions by western immunoblotting using antibodies to TIM44 and Na⁺/K⁺ ATPase, respectively. Fifty micrograms of protein of each subcellular fraction were used in all cases. I, Immunoblot analysis of glycosidase-treated mitochondrial and PM fractions (100 μg each) from frontal cortex of AD16 brain using APP Nt Ab. M.W, Molecular weights. *p < 0.05 compared with AD1 PM fractions. Mean ± SEM calculated from three separate blots.

Figure 2.

Topology of mitochondrial APP in the frontal cortex of AD brain. Limited trypsin treatment, followed by Western immunoblot analysis of mitochondrial (Mito) and PM fractions (100 μg) was performed using APP Nt Ab (A) and Aβ Ab (B) from AD16 frontal cortex. Immunoelectron microscopy of AD1 (C) and NC15 (D) frontal cortex (BM10 area) using APP Nt Ab, TOM40 Ab, and Aβ Ab. M.W, Molecular weights; M, mitochondrion; arrow 1, Aβ containing COOH terminus of APP (5 nm gold particle); arrow 2, TOM40 (20 nm gold particle); arrow 3, NH₂ terminus of APP (10 nm gold particle). E, Western immunoblot analysis of Aβ in the mitochondrial fractions (50 μg) isolated from AD1 and NC17 frontal cortex. Aβstd, Synthetic Aβ peptide standard.

Figure 3.

Characterization of APP-associated mitochondrial outer and inner membrane translocase complexes and the protein import efficiency of mitochondria from frontal cortex of AD brains. A, BN-PAGE-coupled Western immunoblot analysis of high-molecular-weight complexes in mitochondrial (Mito) and microsomal (Micro) fractions (200 μg) of AD and NC brains was performed using APP Nt Ab. B, Lower (480 kDa) and upper (620 kDa) complexes of AD mitochondria were quantitated by using Quantity One software from three independent experiments. M.W, Molecular weights. *p < 0.05 compared with AD1 480 kDa band; **p < 0.05 compared with AD1 620 band. BN-PAGE-coupled immunoblot analysis of AD and NC mitochondrial TOM and TIM complexes using TOM40 Ab (C) and TIM23 Ab (D), respectively. @, Free TOM40 complex; &, free TIM23 complex.

Figure 4.

Levels of cytochrome c oxidase (A), H₂O₂ (B), and import of cytochrome c oxidase subunits Vb (C) and IV (D) in the isolated mitochondria from frontal cortex of AD and NC brains were measured as described in Materials and Methods using 150 μg of trypsin per milliliter of reaction (+). im, Immature; m, mature. E, Mitochondrially imported radioactive bands in C and D were quantified using a Phosphoimager. In A, B, and E (n = 3), *p < 0.05 compared with NC. Error bars indicate SEM.

Figure 5.

Levels of mitochondrial APP in various regions of AD brains carrying multiple ApoE genotypes: APP distribution in various brain regions of mitochondrial fractions (50 μg) were evaluated by immunoblot analysis using APP Nt in AD1 (A), AD11 (C), and AD16 (E) subjects. Densitometric analysis of A, C, and E are represented in B, D, and F, respectively (in each case, n = 3). *p < 0.05 compared with PC. G, Content of mitochondrial marker TIM44 in AD16 brain was analyzed by immunoblot analysis using antibodies to TIM44. H, Effect of ApoE genotyping on mitochondrial APP levels in AD brains by Western immunoblotting using APP Nt Ab. APP levels were represented as arbitrary absorbance units. I, Quantitation of the blot presented in H (n = 3). *p < 0.05 compared with E3/E3 genotype (AD1). Error bars indicate SEM. BG, Basal ganglia; CE, cerebellum; OC, occipital cortex; TC, temporal cortex; TH, thalamus.

Figure 6.

Confocal immunofluorescence microscopy analysis of mitochondrial APP in cholinergic neurons. Frontal cortex (BM10 area) tissue sections from NC17 (A–C) and AD16 (F–H) were triple immunostained with APP Nt Ab (A, F), TOM40 Ab (B, G), and ChAT Ab (C, H). Staining patterns (A–C, F–H) were developed with appropriate secondary antibodies conjugated to Alexa dyes. D and E represent overlay patterns of A + B and A + B + C, respectively, in the NC brain. In the AD brain, I and J represent overlays of F + G and F + G + H, respectively. Scale bars, 15 μm.

Figure 7.

Mitochondrial accumulation of APP in noncholinergic neurons. Confocal immunofluorescence analysis of triple-stained sections from AD16 (A, C, E) and NC17 (B, D, F) brains were performed as described in Materials and Methods. A and D were from basal ganglia (striatum), and B, C, E, and F were from frontal cortex (BM10 area). Green and red staining patterns in A–F brain represent APP and mitochondrial marker TOM40, respectively. Blue staining in A,D, B,E, and C,F represent tyrosine hydroxylase (TH), glutamate (Glu), and GAD patterns, respectively. Brown–yellow staining in A–C of AD brain represent colocalization of green staining (APP) with red staining (mitochondrial TOM40). Scale bars, 15 μm.

Figure 8.

Correlation between mitochondrial APP versus MTT and cytochrome c oxidase in AD brains. Total APP levels in mitochondria (Mito) were estimated by quantitative APP ELISA in the frontal cortex, hippocampus, and amygdala of all 20 AD subjects. Scatter plots of mitochondrial APP versus mitochondrial functions measured by MTT activity were generated for FC (A), HP (B), and AG (C). Similarly, scatter plots of mitochondrial APP versus cytochrome c oxidase for FC (D), HP (E), and AG (F). Regression analysis was performed using Origin 7.5 software.