Journal: PLoS ONE

Article Title: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

doi: 10.1371/journal.pone.0147470

Figure Lengend Snippet: The Aβ 1–42 species used for panning and counter selection during mirror image phage display were analyzed via DGC followed by Tris-Tricine-SDS-PAGE and silver staining. The SEC peak corresponding with Aβ 1–42 monomers (A) presents Aβ 1–42 content only in fractions 1 to 2 of a DGC gradient and therefore represents exclusively monomeric Aβ 1–42 . The SEC peak corresponding with Aβ 1–42 oligomers (B) presents mainly Aβ 1–42 in fractions 4 to 6, which is in accordance with oligomeric Aβ 1–42 species. The preparation of fibrillary Aβ 1–42 resulted in monomeric and fibrillary Aβ 1–42 content as seen in fractions 1 to 2 and 13 to 14 (C). Using only the pooled fractions 12 to 14 ensured that no monomeric Aβ 1–42 species were used as counterselective agent.

Article Snippet: N-terminally biotinylated and non-biotinylated D-enantiomeric Aβ 1–42 was purchased from JPT (Berlin, Germany), N-terminal biotinylated L-enantiomeric Aβ 1–42 was purchased from AnaSpec (Fremont, CA, USA) and L-enantiomeric Aβ 1–42 was acquired from Bachem (Bubendorf, Switzerland).

Techniques: Selection, SDS Page, Silver Staining

Journal: PLoS ONE

Article Title: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

doi: 10.1371/journal.pone.0147470

Figure Lengend Snippet: The immobilization efficiency of different Aβ 1–42 species was analyzed by the binding affinity of Aβ 1–42 specific antibody 6E10 to immobilized Aβ 1–42 on each plate used for single phage ELISA. The Aβ 1–42 specific antibody 6E10 was added to wells coated with 150 ng Aβ 1–42 monomers (red) or 150 ng Aβ 1–42 oligomers and fibrils (1:1; green) or to wells only coated with streptavidin (blue). Transformation of substrate by the secondary antibody-conjugated HRP was measured at 450 nm.

Article Snippet: N-terminally biotinylated and non-biotinylated D-enantiomeric Aβ 1–42 was purchased from JPT (Berlin, Germany), N-terminal biotinylated L-enantiomeric Aβ 1–42 was purchased from AnaSpec (Fremont, CA, USA) and L-enantiomeric Aβ 1–42 was acquired from Bachem (Bubendorf, Switzerland).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: PLoS ONE

Article Title: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

doi: 10.1371/journal.pone.0147470

Figure Lengend Snippet: The relative binding affinity of single phage clones from mirror image phage display (clone numbers 6.xx) and a previously conducted mirror image phage display (clone numbers 5.xx) to SEC-derived biotinylated Aβ 1–42 monomers, oligomers and fibrils as well as the non-coated wells was analyzed. The M13 phage-specific antibody was used for detection. Transformation of substrate by the antibody-conjugated HRP was measured at 450 nm. Amplified single phage clones were added to wells coated with 150 ng Aβ 1–42 monomers (red) or 150 ng Aβ 1–42 oligomers and fibrils (1:1; green) or to wells only coated with streptavidin (blue). Cross reactivity of the M13 phage-specific antibody was tested in an approach without addition of phages. After background subtraction of the anti M13 antibody values, the values for phage to Aβ 1–42 oligomer/fibril binding were normalized to the values of phage to Aβ 1–42 monomer binding according to the outcome of coating efficiency controls with the Aβ specific antibody 6E10.

Article Snippet: N-terminally biotinylated and non-biotinylated D-enantiomeric Aβ 1–42 was purchased from JPT (Berlin, Germany), N-terminal biotinylated L-enantiomeric Aβ 1–42 was purchased from AnaSpec (Fremont, CA, USA) and L-enantiomeric Aβ 1–42 was acquired from Bachem (Bubendorf, Switzerland).

Techniques: Binding Assay, Clone Assay, Derivative Assay, Transformation Assay, Amplification

Journal: PLoS ONE

Article Title: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

doi: 10.1371/journal.pone.0147470

Figure Lengend Snippet: After incubation of 10 μM pretreated Aβ 1–42 without (left picture) and with 10 μM Mosd1 (right picture) for 24 hours at room temperature, samples were spotted onto a formvar/carbon coated copper grid and stained with 1% aqueous uranyl acetate. Samples were analyzed with a Libra 120 TEM operating at 120 kV. Scale bar presents 0.25 μm.

Article Snippet: N-terminally biotinylated and non-biotinylated D-enantiomeric Aβ 1–42 was purchased from JPT (Berlin, Germany), N-terminal biotinylated L-enantiomeric Aβ 1–42 was purchased from AnaSpec (Fremont, CA, USA) and L-enantiomeric Aβ 1–42 was acquired from Bachem (Bubendorf, Switzerland).

Techniques: Incubation, Staining

Journal: PLoS ONE

Article Title: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

doi: 10.1371/journal.pone.0147470

Figure Lengend Snippet: Silver staining of SDS gels after incubation of 80 μM Aβ 1–42 for 4.5 hours at RT and 600 rpm and additional coincubation for 40 minutes with 0 (A) / 10 (B) / 20 (C) / 40 (D) / 80 μM (E) Mosd1 followed by density gradient centrifugation for three hours at 4°C at 259,000 x g. The bands display the signal for Aβ 1–42 (4.5 kDa).

Article Snippet: N-terminally biotinylated and non-biotinylated D-enantiomeric Aβ 1–42 was purchased from JPT (Berlin, Germany), N-terminal biotinylated L-enantiomeric Aβ 1–42 was purchased from AnaSpec (Fremont, CA, USA) and L-enantiomeric Aβ 1–42 was acquired from Bachem (Bubendorf, Switzerland).

Techniques: Silver Staining, Incubation, Gradient Centrifugation

Journal: PLoS ONE

Article Title: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

doi: 10.1371/journal.pone.0147470

Figure Lengend Snippet: Concentrations of Aβ 1–42 in each DGC fraction were determined quantitatively via RP-HPLC. Samples were loaded to a Zorbax 300SB-C8 column connected to a 1260 Infinity HPLC system. Separation of the samples was achieved by elevated column temperature (80°C) and an isocratic mobile phase of 30% acetonitrile/0.1% TFA in water. The averaged concentration of Aβ 1–42 from three independent experiments (with standard deviation) is plotted against the obtained fractions F1 to F15 of different incubation approaches of Aβ 1–42 without or with Mosd1. Shown in red are the concentrations of fractions from 80 μM Aβ 1–42 incubated without Mosd1. The following columns represent the Aβ 1–42 concentrations in the fractions from 80 μM Aβ 1–42 samples coincubated with increasing concentrations of Mosd1 (10 μM = light blue; 20 μM = dark blue; 40 μM = light green; 80 μM = dark green).

Article Snippet: N-terminally biotinylated and non-biotinylated D-enantiomeric Aβ 1–42 was purchased from JPT (Berlin, Germany), N-terminal biotinylated L-enantiomeric Aβ 1–42 was purchased from AnaSpec (Fremont, CA, USA) and L-enantiomeric Aβ 1–42 was acquired from Bachem (Bubendorf, Switzerland).

Techniques: Concentration Assay, Standard Deviation, Incubation

Journal: PLoS ONE

Article Title: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

doi: 10.1371/journal.pone.0147470

Figure Lengend Snippet: Seedless Aβ 1–42 was incubated alone (black) or together with fibrillary Aβ 1–42 seeds previously incubated with (green) or without (red) a fivefold molar excess of Mosd1. ThT (20 μM) was added to each sample in order to measure fibrillar content. The data were fitted with an asymmetric five parameter fit. The (A) amplitude of relative fluorescence (RFU) of fibrillated seedless Aβ 1–42 served as 100% to which the other values were normalized. The (B) half-life (t 1/2 ), displaying the point in time, when half of the maximum ThT signal (i.e. fibrillary content) was reached. Statistical significance was determined by one-way ANOVA. Error bars display SEM. ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.

Article Snippet: N-terminally biotinylated and non-biotinylated D-enantiomeric Aβ 1–42 was purchased from JPT (Berlin, Germany), N-terminal biotinylated L-enantiomeric Aβ 1–42 was purchased from AnaSpec (Fremont, CA, USA) and L-enantiomeric Aβ 1–42 was acquired from Bachem (Bubendorf, Switzerland).

Techniques: Incubation, Fluorescence

Journal: PLoS ONE

Article Title: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

doi: 10.1371/journal.pone.0147470

Figure Lengend Snippet: Aβ 1–42 and Mosd1 were tested for their influence on PC-12 cell viability by MTT reduction assay. Cell viability (in percent) is plotted against different treatment conditions. Adherently grown PC-12 cells were incubated 24 hours with medium (black) or 0.1% TritonX-100 (yellow) as controls for viable cells and cytotoxicity, respectively. Additionally, cells incubated for 24 hours with 1 μM Aβ 1–42 (red), Aβ 1–42 + Mosd1 1:1 (light blue) and 1:0.5 (dark blue), respectively. The green bar corresponds to cells incubated with 1 μM Mosd1 for 24 hours. Viability was analyzed by subsequent incubation with MTT substrate for four hours. After solubilization, absorbance was measured at 570 nm. The averages and standard deviations of absorbance values from five independently performed experiments were calculated and normalized to untreated cells (medium). Statistical significance was tested with Mann-Whitney U test. ***: p ≤ 0.001.

Article Snippet: N-terminally biotinylated and non-biotinylated D-enantiomeric Aβ 1–42 was purchased from JPT (Berlin, Germany), N-terminal biotinylated L-enantiomeric Aβ 1–42 was purchased from AnaSpec (Fremont, CA, USA) and L-enantiomeric Aβ 1–42 was acquired from Bachem (Bubendorf, Switzerland).

Techniques: MTT Reduction Assay, Incubation, MANN-WHITNEY

Journal: Biomedicines

Article Title: Metals in Alzheimer’s Disease

doi: 10.3390/biomedicines11041161

Figure Lengend Snippet: Correlation between metals and CSF protein AD biomarkers.

Article Snippet: [ ] , CSF , Cu intake , Aβ 1–42 , t-tau, p-tau 181 , Cu intake did not affect the t-tau and p-tau 181 levels, but the Aβ 1–42 levels decreased by 30% in the placebo group and only by 10% in the verum group , AD (68) , 68.

Techniques: Clinical Proteomics, Control, Comparison

Journal: Biophysical Journal

Article Title: Single-Molecule Imaging Reveals A β 42:A β 40 Ratio-Dependent Oligomer Growth on Neuronal Processes

doi: 10.1016/j.bpj.2012.12.051

Figure Lengend Snippet: Amyloid-β(1–40) binds to primary rat hippocampal neurites with high affinity. Very densely bound oligomers are visible on neurites treated for 10 min with 50 nM HL647Aβ40 (top left image, scale bar 10 μm). Single oligomers are detected on neurites treated with 1 nM HL647Aβ40 (middle image, scale bar 5 μm), but not on neurites treated with 1 nM unlabeled Aβ40 as a control (bottom image, scale bar 5 μm). Analysis of 20 frames of neurites in each 1 nM-treated sample (right plot) yielded 237 single particles in HL647Aβ40-treated neurites (black bars), but only two particles with intensity equivalent to monomers in unlabeled Aβ40-treated neurites (hatched bar).

Article Snippet: Peptide preparation Synthetic unlabeled amyloid- β (1–40), N-terminally HiLyte Fluor 647-labeled amyloid- β (1–40), and (1–42) (HL647A β 40 and 42, respectively), and HiLyte Fluor 647 hydrazide were obtained from Anaspec (Freemont, CA).

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy *

doi: 10.1074/jbc.M115.691493

Figure Lengend Snippet: Aβ directly interacts with VDAC1 and enhances its channel conductance. A, VDAC1 was reconstituted into a PLB, and currents through the VDAC1 channel in response to voltage steps from 0 to −10 or +10 mV were recorded before and 5 min after the addition of Aβ (2 μm) incubated 5 days at 37 °C. A representative of three independent experiments is shown. B, the effect of Aβ (incubated 5 days at 37 °C) on VDAC1 conductance as a function of voltage from 60 mV to −60 mV was tested. The average steady-state conductance at a given voltage was normalized to the conductance at 10 mV. The recordings were taken before (●) and 5 min after (○) the addition of Aβ (2 μm). C, similar experiment as in A, except that non-incubated Aβ was used. D, similar experiment as in B, except that non-incubated Aβ was used. E, the effect of Aβ (2 μm) (incubated for 5 days at 37 °C) on the conductance of PLB, in response to voltage step from 0 to 10 mV, was tested with the currents recorded before and 15 min after the addition of Aβ. A representative of three independent experiments is shown. F, purified VDAC1 was fluorescently labeled using the NanoTemper blue protein-labeling kit according to the manufacturer's instructions. VDAC1 (100 nm) was incubated for 20 min with Aβ (1.2–100 μm), and thermophoresis was measured using the Monolith-NT115 apparatus. A Kd of 50 μm was determined. A representative of three independent experiments is shown. ΔF Norm, change in normalized fluorescence (G, similar experiment as in F was carried using a label-free MST system where intrinsic VDAC1 tryptophan fluorescence provided the analyzed signal. A Kd of 16.6 μm was determined for this interaction. A representative of three independent experiments is shown.

Article Snippet: Celluspots peptide arrays were obtained from INTAVIS Bioanalytical Instruments (Koln, Germany). . Amyloid β (Aβ residues 1–42) and VDAC1 N-terminal ( 1 MAVPPTYADLGKSARDVFTKGYGFGL 26 ) peptides were synthesized by GL Biochem (Shanghai, China).

Techniques: Incubation, Purification, Labeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy *

doi: 10.1074/jbc.M115.691493

Figure Lengend Snippet: Aβ interacts with VDAC1-based peptides. A, VDAC1 N-Ter peptide interaction with Aβ, as analyzed by SPR. Aβ, immobilized onto a GLC sensor surface, was exposed to the VDAC1 N-Ter peptide (0–400 μm), and the interaction was monitored using the ProteOn imaging system and related software tools. The two lines of each color represent the obtained results and fitted curves. B, Aβ interaction with the VDAC1 N-Ter peptide as analyzed using the heterogeneous ligand interaction model, where one analyte binds two different ligand species, yielding a Kd1 value of 2.5 μm and a Kd2 value of 900 μm. RU, response units.

Article Snippet: Celluspots peptide arrays were obtained from INTAVIS Bioanalytical Instruments (Koln, Germany). . Amyloid β (Aβ residues 1–42) and VDAC1 N-terminal ( 1 MAVPPTYADLGKSARDVFTKGYGFGL 26 ) peptides were synthesized by GL Biochem (Shanghai, China).

Techniques: Imaging, Software

Journal: The Journal of Biological Chemistry

Article Title: The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy *

doi: 10.1074/jbc.M115.691493

Figure Lengend Snippet: The VDAC1 N-Ter peptide prevents Aβ from binding and entering cells and protects against Aβ-induced apoptosis. A, PC12 cells were treated with different concentrations of Aβ with (●) or without (○) the VDAC1 N-Ter peptide (20 μm) for 48 h, when cell viability was assessed by the XTT assay. A representative of three independent experiments is presented. B, PC12 cells were treated with different concentrations of the VDAC1 N-Ter peptide and a constant concentration (30 μm) of Aβ for 48 h when cell viability was assessed by the XTT assay. A representative of three independent experiments is presented. C, the VDAC1 N-Ter peptide protects against Aβ-induced apoptosis in SH-SY5Y cells. SH-SY5Y cells were incubated with Aβ (10, 20 μm) in the absence or presence of the VDAC1 N-Ter peptide (20 μm) for 48 h, when aliquots were stained with acridine orange and ethidium bromide as described (30). Confocal images are shown, with early apoptotic stage cells reflected by membrane blebbing (arrows). Late apoptotic stage is indicated by arrowheads. Bar = 10 μm. D, quantification of the results obtained in C. The analysis was carried out by counting live and late apoptotic cells from a total of 120 to 160 cells in each sample.

Article Snippet: Celluspots peptide arrays were obtained from INTAVIS Bioanalytical Instruments (Koln, Germany). . Amyloid β (Aβ residues 1–42) and VDAC1 N-terminal ( 1 MAVPPTYADLGKSARDVFTKGYGFGL 26 ) peptides were synthesized by GL Biochem (Shanghai, China).

Techniques: Binding Assay, XTT Assay, Concentration Assay, Incubation, Staining, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy *

doi: 10.1074/jbc.M115.691493

Figure Lengend Snippet: Cells incubated with N-terminal peptide show decreased Aβ cell penetration. SH-SY5Y cells were incubated with Aβ (10 μm) in the absence or presence of the VDAC1 N-Ter peptide (20 μm) for 6 h, then cells were fixed and incubated with anti-Aβ antibodies followed by secondary Cy3-conjugated anti-rabbit antibodies (shown in red) and anti-Cyto c antibodies followed by secondary Cy2 conjugated anti-mouse antibodies (shown in green). Cells were also stained with DAPI (blue) and visualized by confocal microscopy. The VDAC1 N-Ter peptide significantly reduced Aβ attachment and cell entry. The third and fifth columns represent enlargements of the marked areas. Bar = 10 μm.

Article Snippet: Celluspots peptide arrays were obtained from INTAVIS Bioanalytical Instruments (Koln, Germany). . Amyloid β (Aβ residues 1–42) and VDAC1 N-terminal ( 1 MAVPPTYADLGKSARDVFTKGYGFGL 26 ) peptides were synthesized by GL Biochem (Shanghai, China).

Techniques: Incubation, Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy *

doi: 10.1074/jbc.M115.691493

Figure Lengend Snippet: Cells with low levels of VDAC1 show decreased Aβ cell penetration and cytotoxicity. SH-SY5Y cells were transfected with VDAC1-siRNA (50 nm) or not transfected, and 24 h later the cells were incubated with 20 μm Aβ for 48 h. A and B, SH-SY5Y cells transfected with 50 nm of scrambled (Scr) siRNA or VDAC1-siRNA, and 24 h post-transfection cells were treated with different concentrations of Aβ for 48 h. A, cell viability was assessed by the XTT assay for (Scr) siRNA (●)- or VDAC1-siRNA (○)-treated cells The data represent the mean ± S.D. (n = 3). The inset shows Western blot analysis of VDAC1 protein levels in SH-SY5Y cells transfected with Scr- or VDAC1-siRNA. RU indicates relative units, showing a decrease of 74% in VDAC1 level in cells treated with VDAC1-siRNA. B, a similar experiment as in A was carried out, and cell death rate was determined using propidium iodide staining for Scr-siRNA (black)- or VDAC1-siRNA (gray)-treated cells.). C, cells were fixed and incubated with anti-Aβ antibodies followed by secondary Cy3-conjugated anti-rabbit antibodies (shown in red) and anti-Cyto c antibodies followed by secondary Cy2 conjugated anti-mouse antibodies (shown in green). Cells were also stained with DAPI (blue) and visualized by confocal microscopy. The third and fifth columns represent enlargements of the marked areas. Bar = 10 μm. The lack of Aβ staining in siRNA-treated cells may indicate the Aβ inability of to cross the cell membrane.

Article Snippet: Celluspots peptide arrays were obtained from INTAVIS Bioanalytical Instruments (Koln, Germany). . Amyloid β (Aβ residues 1–42) and VDAC1 N-terminal ( 1 MAVPPTYADLGKSARDVFTKGYGFGL 26 ) peptides were synthesized by GL Biochem (Shanghai, China).

Techniques: Transfection, Incubation, XTT Assay, Western Blot, Staining, Confocal Microscopy, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy *

doi: 10.1074/jbc.M115.691493

Figure Lengend Snippet: VDAC1 targeted to the plasma membrane induces cell death and enhances Aβ-mediated apoptosis. A, SH-SY5Y cells were transfected with an mVDAC1-GFP-CAAX-encoding plasmid (green). Forty-eight hours later, the cells were fixed, and the nuclei were stained with DAPI (blue) and visualized by confocal microscopy. Bar = 20 μm. B, enlargement of the marked area in A. C–F, SH-SY5Y cells were transfected with an empty plasmid or a mVDAC1-CAAX-expressing plasmid. Twenty-four hours later, the cells were incubated for 48 h with or without Aβ, then cells were stained with propidium iodide (PI) and analyzed by flow cytometry using ec800 software. C, Western blot analysis of VDAC1 protein levels in SH-SY5Y cells transfected with plasmid mVDAC1-CAAX using long and low exposure times. Arrows indicate exogenous and mVDAC1-CAAX-fused proteins. D and E, representative flow cytometry results for control and mVDAC1-CAAX-expressing cells are shown. F, quantitation of the FACS analysis of apoptosis induced by incubation with Aβ (5 and 10 μm) of cells transfected with the empty plasmid (gray) or the mVDAC1-CAAX plasmid (black).

Article Snippet: Celluspots peptide arrays were obtained from INTAVIS Bioanalytical Instruments (Koln, Germany). . Amyloid β (Aβ residues 1–42) and VDAC1 N-terminal ( 1 MAVPPTYADLGKSARDVFTKGYGFGL 26 ) peptides were synthesized by GL Biochem (Shanghai, China).

Techniques: Clinical Proteomics, Membrane, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Expressing, Incubation, Flow Cytometry, Software, Western Blot, Control, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy *

doi: 10.1074/jbc.M115.691493

Figure Lengend Snippet: Aβ induces hexokinase detachment, VDAC1 oligomerization, and cytochrome c release. A, SH-SY5Y cells were transfected to express HK-I-GFP (green). Twenty-four hours later the cells were incubated without (Aa) or with Aβ (5 μm (Ab) or 10 μm (Ac)) for 16 h. The cells were then fixed and stained with DAPI (blue). Mitochondrial-bound HK-I-GFP (Aa, arrows) and detached, cytosolic HK-I-GFP (A, b and c, arrowheads), are indicated. Red arrows indicate fragmented nuclei presenting Aβ-induced apoptosis. Scale bar = 20 μm. B and C, Aβ-induced VDAC1 oligomerization, SH-SY5Y (B) and HeLa (C) cells were incubated without or with Aβ (5 μm or 10 μm). Forty-eight hours later the cells were analyzed for VDAC1 oligomerization as revealed by EGS-based cross-linking and immunoblotting using anti-VDAC1 antibodies. Quantitative analysis of the level of VDAC1 dimers relative to actin is presented in relative unit. D and E, SH-SY5Y cells were incubated with or without Aβ (5 or 10 μm) for 48 h and analyzed for cytochrome c release using anti-cytochrome c-specific antibodies. SH-SY5Y cells were treated with digitonin (0.0015%), and release of Cyto c from mitochondria (Mito) to the cytosolic fraction (Cyto) was analyzed by immunoblotting (D) as described under “Experimental Procedures.” Anti-ATP synthase 5a (ATPsyn5a) antibodies were used to demonstrate the absence of mitochondria in the cytosolic fraction. Cytochrome c release was also analyzed by immunocytochemistry using anti-cytochrome c-specific antibodies. Cells were also stained with DAPI (blue) and visualized by confocal microscopy (E). Cytochrome c staining is significantly reduced in the Aβ-treated cells (Eb and Ec) as compared with the control cells (Ea). Scale bars = 10 μm. RU, relative units.

Article Snippet: Celluspots peptide arrays were obtained from INTAVIS Bioanalytical Instruments (Koln, Germany). . Amyloid β (Aβ residues 1–42) and VDAC1 N-terminal ( 1 MAVPPTYADLGKSARDVFTKGYGFGL 26 ) peptides were synthesized by GL Biochem (Shanghai, China).

Techniques: Transfection, Incubation, Staining, Western Blot, Immunocytochemistry, Confocal Microscopy, Control

Journal: The Journal of Biological Chemistry

Article Title: The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy *

doi: 10.1074/jbc.M115.691493

Figure Lengend Snippet: Model describing how Aβ leads to mitochondria-mediated cell death. A, molecular dynamics simulation of the interaction between monomeric Aβ peptide (Gly-29–Ala-42) and the N-terminal domain of VDAC1 (Met-1–Gly-26) identifies two sites of interactions, one of which is the GXXXG motif. B, Aβ peptide (Leu-17–Ala-42 and VDAC1 possibly interact through electrostatic (negative charges in Aβ and positive charges in N-terminal domain of VDAC1 are presented by red and blue colors, respectively), hydrophobic, and/or via GXXXG motif interactions. The GXXXG motif in VDAC1 and the Aβ oligomers are colored green. C, proposed model for Aβ cell penetration and apoptosis induction. Monomeric Aβ can form protofibrils and soluble aggregates. Based on our results, we suggest that VDAC1 plays a key role in Aβ toxicity on several levels. In the plasma membrane, plVDAC1 interacts with Aβ oligomers via its N-terminal domain, leading to VDAC1 oligomerization and the formation of large pores composed of Aβ-VDAC1 heteromers. These large pores allow Aβ to enter the cell and interact with mitochondrial VDAC1, leading to detachment of VDAC1-bound HK-I and VDAC1 oligomerization, concomitant with the formation of heteromeric Aβ-VDAC1 large pores that allow release of Cyto c, and the subsequent induction of apoptosis. Non-membrane-penetrating VDAC1 N-Ter peptides inhibit Aβ cell entry, probably by interacting with Aβ, thus sequestering and preventing Aβ interacting with plVDAC1 and entering the cell to elicit a toxic effect. D, proposed model for VDAC1-based treatment in Alzheimer disease. VDAC1 is overexpressed in Alzheimer disease, leading to VDAC1 oligomerization and subsequent Cyto c release and apoptosis. This VDAC1-dependent apoptosis can be inhibited by preventing VDAC1 expression by VDAC1-siRNA or inhibiting VDAC1 oligomerization stimulated by Aβ by VDAC1-N-Ter peptide.

Article Snippet: Celluspots peptide arrays were obtained from INTAVIS Bioanalytical Instruments (Koln, Germany). . Amyloid β (Aβ residues 1–42) and VDAC1 N-terminal ( 1 MAVPPTYADLGKSARDVFTKGYGFGL 26 ) peptides were synthesized by GL Biochem (Shanghai, China).

Techniques: Clinical Proteomics, Membrane, Expressing