Clathrin-mediated endocytosis blocker inhibited Aβ_1–42-induced mitochondrial dysfunction.

<p>A. Mitochondrial shapes were identified by immunostaining of HSP60 in vehicle, Aβ_1–42, chlorpromazine+Aβ_1–42, mouse anti-RAGE IgG+Aβ_1–42 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+Aβ_1–42, green: Aβ_1–42 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 Aβ_1–42.</p

Morphological alteration of mitochondria in AβPP/PS1 mice brains and Aβ_1–42-treated HT22 cell line.

<p>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 Aβ_1–42-treated HT22 cell line by different time period of treatment (scale bar: 20 µm).</p

Apoptotic protein expression and cellular death in both exogenous Aβ_1–42 treatment and mito Aβ_1–42–transfected cells.

<p>Western blot analysis was performed in both exogenous Aβ_1–42 treatment and mito Aβ_1–42–transfected HT22 cells to characterize the expression level of Aβ_1–42, 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 Aβ_1–42 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 Aβ_1–42-treated, mock and mito Aβ_1–42 transfected HT22 cells, respectively (* p<0.05, ** p<0.01 compared with vehicle, # p<0.05 compared to mock).</p

Mito Aβ_1–42 induces not only mitochondrial morphological alteration but also functional impairments.

<p>A. Immunostaining of HSP60 and YFP in mock or mito Aβ_1–42–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.</p

Mitochondria-specific accumulation of mito Aβ_1–42.

<p>A. Western blot analysis showed the mitochondria-specific accumulation of Aβ_1–42 with the presence of TOM20, mitochondrial marker and the absence of β-actin. B. EM image of mitochondria in mock and mito Aβ_1–42-transfected HT22 cells (yellow scale bar: 2 µm, white scale bar: 1 µm).</p

Functional assays for mitochondria in AβPP/PS1 mice brains and Aβ_1–42-treated HT22 cell line.

<p>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 Aβ_1–42-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).</p