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

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

<p>The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology.</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

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

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

Phosphokinase Antibody Arrays on Dendron-Coated Surface

<div><p>Monitoring protein phosphorylation at the cellular level is important to understand the intracellular signaling. Among the phosphoproteomics methods, phosphokinase antibody arrays have emerged as preferred tools to measure well-characterized phosphorylation in the intracellular signaling. Here, we present a dendron-coated phosphokinase antibody array (DPA) in which the antibodies are immobilized on a dendron-coated glass slide. Self-assembly of conically shaped dendrons well-controlled in size and structure resulted in precisely controlled lateral spacing between the immobilized phosphosite-specific antibodies, leading to minimized steric hindrance and improved antigen-antibody binding kinetics. These features increased sensitivity, selectivity, and reproducibility in measured amounts of protein phosphorylation. To demonstrate the utility of the DPA, we generated the phosphorylation profiles of brain tissue samples obtained from Alzheimer's disease (AD) model mice. The analysis of the profiles revealed signaling pathways deregulated during the course of AD progression.</p></div

A network model describing perturbed signaling pathways in the AD brain.

<p>The nodes are arranged based on the signaling pathways in which the nine DPSs are involved (see text for the nine DPSs used for the network modeling). The node (center) and boundary colors represent the increase (red) and decrease (green) in phosphorylation measured by the DPA in AD samples at two and six months, respectively, compared to Control. The nodes corresponding to the four DPSs whose differential phosphorylation was confirmed by western blotting (STAT3, RelA, PKCδ/θ and Akt1) were denoted by large nodes. The arrows represent activation while the inhibition symbols represent inactivation. The dashed lines indicate interactions obtained from Alzpathway, and the solid lines indicate protein-protein interactions collected from BIND, HPRD, and BioGrid databases.</p

Application of DPA to AD brain tissues.

<p><b>A</b>. Experimental scheme for phosphorylation profiling from AD brain tissues obtained from normal (Control) and AD mouse model (AD) at the ages of 2 and 6 months. <b>B</b>. Boxplots of the four DPSs whose alterations in AD, compared to control, were confirmed by Western blotting. ***P<0.001 from ANOVA followed by post-hoc tests with Bonferroni correction. <b>C</b>. Results of Western blotting of the four DPSs. Up-regulation of STAT3(Y705) and RelA(S534) at 6 months, and down-regulation of PKCδ/θ(S643/676) at 2 months and Akt1(S473) at 6 months were confirmed in independent samples (n = 3). Data are normalized to the β-actin abundance and presented as percentage changes from the control group. Data are shown as means ± SEM. *P<0.05 from ANOVA followed by Tukey's leat significant difference post-hoc tests.</p