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, 7 (4), e34929

Mitochondria-specific Accumulation of Amyloid β Induces Mitochondrial Dysfunction Leading to Apoptotic Cell Death

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Mitochondria-specific Accumulation of Amyloid β Induces Mitochondrial Dysfunction Leading to Apoptotic Cell Death

Moon-Yong Cha et al. PLoS One.

Abstract

Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ(1-42) in HT22 cells using Aβ(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ(1-42) accumulation, which mimics the apoptosis process in exogenous Aβ(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ(1-42) accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aβ-mediated signaling alterations.

Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts. One of the authors (HSH) is employed by a commercial company, Medifron DBT Co Ltd. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Morphological alteration of mitochondria in AβPP/PS1 mice brains and Aβ1–42-treated HT22 cell line.
A. Electron microscopic (EM) image of mitochondria in wild type and AβPP/PS1 mice (10 months, cortex, scale bar: 2 µm) B. EM image of mitochondria in vehicle and Aβ-treated HT22 cells (yellow scale bar: 2 µm, white scale bar: 1 µm) C. Immunostaining of HSP60 in Aβ1–42-treated HT22 cell line by different time period of treatment (scale bar: 20 µm).
Figure 2
Figure 2. Functional assays for mitochondria in AβPP/PS1 mice brains and Aβ1–42-treated HT22 cell line.
Four types of mitochondrial functional assays: MTT (A); ROS level (B); ATP generation (C) and TMRM intensity (D) were assessed in vehicle or 5 µM Aβ1–42-treated HT22 cells. MTT assay were measured after 24 h of Aβ treatment, other assays were after 6 h of Aβ treatment (* p<0.05, ** p<0.01).
Figure 3
Figure 3. Clathrin-mediated endocytosis blocker inhibited Aβ1–42-induced mitochondrial dysfunction.
A. Mitochondrial shapes were identified by immunostaining of HSP60 in vehicle, Aβ1–42, chlorpromazine+Aβ1–42, mouse anti-RAGE IgG+Aβ1–42 treatment in HT22 cells, respectively (Scale bar: 20 µm). B. Altered mitochondrial shapes are quantified using form factor and aspect ratio (blue: vehicle, red: chlorpromazine+Aβ1–42, green: Aβ1–42 in left graph). Four functional assessments of mitochondria are shown, including MTT (C), ROS levels (D), ATP generation (E) and TMRM intensity (F). * p<0.05, ** p<0.01, *** p<0.001 compared with vehicle, # p<0.05 compared with Aβ1–42.
Figure 4
Figure 4. Mitochondria-specific accumulation of mito Aβ1–42.
A. Western blot analysis showed the mitochondria-specific accumulation of Aβ1–42 with the presence of TOM20, mitochondrial marker and the absence of β-actin. B. EM image of mitochondria in mock and mito Aβ1–42-transfected HT22 cells (yellow scale bar: 2 µm, white scale bar: 1 µm).
Figure 5
Figure 5. Mito Aβ1–42 induces not only mitochondrial morphological alteration but also functional impairments.
A. Immunostaining of HSP60 and YFP in mock or mito Aβ1–42–transfected HT22 cells (white scale bar: 20 µm). B. Quantification of alteration in mitochondrial shape is presented as form factor and aspect ratio (** p<0.01). Four functional assessments for mitochondria are shown, including MTT (C), ROS levels (D), ATP generation (E) and TMRM staining (F). G. TMRM intensity is quantified as percent of control. * p<0.05, ** p<0.01, *** p<0.001 compared to mock, white scale bar in F: 50 µm.
Figure 6
Figure 6. Apoptotic protein expression and cellular death in both exogenous Aβ1–42 treatment and mito Aβ1–42–transfected cells.
Western blot analysis was performed in both exogenous Aβ1–42 treatment and mito Aβ1–42–transfected HT22 cells to characterize the expression level of Aβ1–42, Bcl-2, Bax using 6E10, Bcl-2 antibody and Bax antibody (A). Expression level of Bcl-2 and Bax was confirmed in mitochondrial fraction (B). Different concentrations of mito Aβ1–42 DNA constructs were used, 2 µg and 4 µg. Cytochrome C release assay (C) and calcein cell viability assay (D) were performed in vehicle-treated, exogenous Aβ1–42-treated, mock and mito Aβ1–42 transfected HT22 cells, respectively (* p<0.05, ** p<0.01 compared with vehicle, # p<0.05 compared to mock).

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