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, 105 (49), 19318-23

Amyloid-beta Overproduction Causes Abnormal Mitochondrial Dynamics via Differential Modulation of Mitochondrial Fission/Fusion Proteins

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Amyloid-beta Overproduction Causes Abnormal Mitochondrial Dynamics via Differential Modulation of Mitochondrial Fission/Fusion Proteins

Xinglong Wang et al. Proc Natl Acad Sci U S A.

Abstract

Mitochondrial dysfunction is a prominent feature of Alzheimer disease but the underlying mechanism is unclear. In this study, we investigated the effect of amyloid precursor protein (APP) and amyloid beta on mitochondrial dynamics in neurons. Confocal and electron microscopic analysis demonstrated that approximately 40% M17 cells overexpressing WT APP (APPwt M17 cells) and more than 80% M17 cells overexpressing APPswe mutant (APPswe M17 cells) displayed alterations in mitochondrial morphology and distribution. Specifically, mitochondria exhibited a fragmented structure and an abnormal distribution accumulating around the perinuclear area. These mitochondrial changes were abolished by treatment with beta-site APP-cleaving enzyme inhibitor IV. From a functional perspective, APP overexpression affected mitochondria at multiple levels, including elevating reactive oxygen species levels, decreasing mitochondrial membrane potential, and reducing ATP production, and also caused neuronal dysfunction such as differentiation deficiency upon retinoic acid treatment. At the molecular level, levels of dynamin-like protein 1 and OPA1 were significantly decreased whereas levels of Fis1 were significantly increased in APPwt and APPswe M17 cells. Notably, overexpression of dynamin-like protein 1 in these cells rescued the abnormal mitochondrial distribution and differentiation deficiency, but failed to rescue mitochondrial fragmentation and functional parameters, whereas overexpression of OPA1 rescued mitochondrial fragmentation and functional parameters, but failed to restore normal mitochondrial distribution. Overexpression of APP or Abeta-derived diffusible ligand treatment also led to mitochondrial fragmentation and reduced mitochondrial coverage in neuronal processes in differentiated primary hippocampal neurons. Based on these data, we concluded that APP, through amyloid beta production, causes an imbalance of mitochondrial fission/fusion that results in mitochondrial fragmentation and abnormal distribution, which contributes to mitochondrial and neuronal dysfunction.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
APP causes abnormal mitochondrial dynamics in M17 cells. (A) Representative immunoblot of APP. Tubulin is used as an internal loading control. (B) Representative pictures of stable M17 lines with different mitochondrial distribution patterns are shown. Green, tubulin; red, mito-DsRed2; blue, DAPi. Mitochondrial distribution (C) and morphology (D) are analyzed. At least 1,000 cells were measured for each cell line. (Scale bars, 10 μm.) *, P < 0.05, Student t test.
Fig. 2.
Fig. 2.
APP expression affects mitochondrial fusion. M17 cells were transfected with mito-Dendra2 to label mitochondria. Before photo-conversion (Pre), Dendra2 emits green fluorescence. At time 0, laser activation is applied to ROI (square box) to allow full photo-conversion, from green to red, of all of the mitochondria within the ROI. Thereafter, mitochondrial behavior in the entire cell was monitored for 60 min. Active mitochondria fission (filled arrowhead) and fusion (open arrowhead) of individual mitochondrion could be observed in the representative control cell. (Scale bars, 20 μm.)
Fig. 3.
Fig. 3.
APP expression modulates mitochondrial fission/fusion proteins. Representative immunoblot (A) and quantification analysis (B) revealed that DLP1 and OPA1 were reduced, whereas Fis1 was increased significantly in APPwt and APPswe M17 cells (*, P < 0.05).
Fig. 4.
Fig. 4.
APP causes abnormal mitochondrial dynamics in differentiated rat E18 primary hippocampal neurons. Neurons (DIV7) were co-transfected with mito-AcGFP and DsRed-tagged APP or APPswe in the absence or presence of 20 nM BACE inhibitor IV. Three days after transfection, neurons were fixed and evaluated. (A) Representative pictures of positively transfected neurons with different mitochondrial morphology and distribution pattern were shown. Red, DsRed-APP; green, mito-AcGFP. (Scale bar, 100 μm.) (B) Higher magnification pictures of the boxed area in A. (Scale bar, 20 μm.) (C and D) Mitochondria (length and coverage) in a segment of neuronal process at least 200 μm in length beginning from the cell body of neurons were analyzed. At least 20 cells were analyzed each time in three independent experiments.

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