Journal: Biochemistry and Biophysics Reports

Article Title: Effect of methionine-35 oxidation on the aggregation of amyloid-β peptide

doi: 10.1016/j.bbrep.2015.07.017

Figure Lengend Snippet: Oxidation of Aβ40 peptide with H 2 0 2 in the presence of copper ions. MALDI MS spectra of Aβ42 incubated in the presence of 1% H 2 0 2 at pH 7.3 in 20 mM HEPES. Samples were taken at time intervals shown in the legend; A – native peptide; B – peptide was oxidized with H 2 0 2 for 40 min before adding copper ions. C-SDS Page of Aβ40: Control, untreated peptide, Aβox, oxidized with H 2 0 2 in the absence of copper ions and Aβ ox+Cu refers to oxidized peptide treated with H 2 0 2 in the presence of copper ions for 3 h.

Article Snippet: Lyophilized Aβ40 and Aβ42 peptides (ultra pure, recombinant) NaOH salts or HFIP forms were purchased from rPeptide (Athens, USA).

Techniques: Incubation, SDS Page, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Effect of methionine-35 oxidation on the aggregation of amyloid-β peptide

doi: 10.1016/j.bbrep.2015.07.017

Figure Lengend Snippet: Fibrillization of Aβ peptides with reduced and oxidized Met35 residues at pH 7.3, 20 mM HEPES, 100 mM NaCl, 5 μM ThT. A – Fibrillization of Aβ42: 4 μM Aβ42 37 °C: Curves correspond to k =(1.30±0.02) min −1 , t lag =11.8 min −1 for reduced and k =(3.83±0.05) min −1 , t lag =24.9 min −1 for Met35ox peptide. B-Fibrillization of Aβ40: 5 μM Aβ40; 50 °C; Curves correspond to k =(3.92±0.09) min −1 , t lag =22.6 min −1 for reduced peptide and k =(14.1±0.3) min −1 , t lag =14.0 for the oxidized peptide; C-TEM images of Aβ40 fibrils, left Aβ40ox; right – Aβ40 control.

Article Snippet: Lyophilized Aβ40 and Aβ42 peptides (ultra pure, recombinant) NaOH salts or HFIP forms were purchased from rPeptide (Athens, USA).

Techniques: Control

Journal: Alzheimer's Research & Therapy

Article Title: An integrated multi-study analysis of intra-subject variability in cerebrospinal fluid amyloid-β concentrations collected by lumbar puncture and indwelling lumbar catheter

doi: 10.1186/s13195-015-0136-z

Figure Lengend Snippet: Profile of percent change of the mean in Aβ40 for each subject studied by each sponsor with serial sampling of CSF for up to 40 hours post catheter placement. Percent change of the mean is on the y axis and time of day for each study sponsor is shown on the x axis. a AbbVie Inc., b Bristol-Myers Squibb (BMS), c Eli Lilly and Company, d Merck and Company, e Radboud University Medical Center (RUMC), f Washington University

Article Snippet: Mesor-to-peak amplitudes of diurnal fluctuation of Aβ40 were 4.61 % (AbbVie), –6.52 % (BMS), and –4.90 % (Lilly).

Techniques: Sampling

Journal: Alzheimer's Research & Therapy

Article Title: An integrated multi-study analysis of intra-subject variability in cerebrospinal fluid amyloid-β concentrations collected by lumbar puncture and indwelling lumbar catheter

doi: 10.1186/s13195-015-0136-z

Figure Lengend Snippet: Diurnal oscillation of Aβ peptides in participants from two different study cohorts. Data presented as mean-adjusted average Aβ levels of the group over time of day for all subjects. Data from Merck and Company for ( a ) Aβ40 and ( b ) Aβ42, and data from Washington University for ( c ) Aβ40 and ( d ) Aβ42. Mesor-to-peak amplitudes of diurnal fluctuation of Aβ40 were 5.48 % for Merck and 4.91 % for Washington University. Mesor-to-peak amplitudes of diurnal fluctuation of Aβ42 were 4.87 % for Merck and 4.26 % for Washington University. The cosine transformation for all datasets was significantly different from a straight line (all p <0.0001). CI confidence interval

Article Snippet: Mesor-to-peak amplitudes of diurnal fluctuation of Aβ40 were 4.61 % (AbbVie), –6.52 % (BMS), and –4.90 % (Lilly).

Techniques: Transformation Assay

Journal: Alzheimer's Research & Therapy

Article Title: An integrated multi-study analysis of intra-subject variability in cerebrospinal fluid amyloid-β concentrations collected by lumbar puncture and indwelling lumbar catheter

doi: 10.1186/s13195-015-0136-z

Figure Lengend Snippet: Diurnal oscillation of Aβ peptides in participants from three different study cohorts. Data presented as mean-adjusted average Aβ levels of the group over time of day for all subjects. Data from AbbVie Inc. for ( a ) Aβ40 and ( b ) Aβ42, from Bristol-Myers Squibb (BMS) for ( c ) Aβ40 and ( d ) Aβ42, and from Eli Lilly and Company for ( e ) Aβ40 and ( f ) Aβ42. Mesor-to-peak amplitudes of diurnal fluctuation of Aβ40 were 4.61 % (AbbVie), –6.52 % (BMS), and –4.90 % (Lilly). Mesor-to-peak amplitudes of diurnal fluctuation of Aβ42 were 4.29 % (AbbVie), –7.01 % (BMS), and –5.34 % (Lilly). The cosine transformation for all datasets were significantly different from a straight line except for Aβ42 from the AbbVie studies ( p = 0.06). CI confidence interval

Article Snippet: Mesor-to-peak amplitudes of diurnal fluctuation of Aβ40 were 4.61 % (AbbVie), –6.52 % (BMS), and –4.90 % (Lilly).

Techniques: Transformation Assay

Journal: Alzheimer's Research & Therapy

Article Title: An integrated multi-study analysis of intra-subject variability in cerebrospinal fluid amyloid-β concentrations collected by lumbar puncture and indwelling lumbar catheter

doi: 10.1186/s13195-015-0136-z

Figure Lengend Snippet: ANOVA of fixed effects for industry in-dwelling catheter studies for Aβ40 and Aβ42

Article Snippet: Mesor-to-peak amplitudes of diurnal fluctuation of Aβ40 were 4.61 % (AbbVie), –6.52 % (BMS), and –4.90 % (Lilly).

Techniques:

Journal: Alzheimer's Research & Therapy

Article Title: An integrated multi-study analysis of intra-subject variability in cerebrospinal fluid amyloid-β concentrations collected by lumbar puncture and indwelling lumbar catheter

doi: 10.1186/s13195-015-0136-z

Figure Lengend Snippet: Slope and significance for time × draw groups 1–3 interaction for Aβ40 and Aβ42

Article Snippet: Mesor-to-peak amplitudes of diurnal fluctuation of Aβ40 were 4.61 % (AbbVie), –6.52 % (BMS), and –4.90 % (Lilly).

Techniques:

Journal: Alzheimer's Research & Therapy

Article Title: An integrated multi-study analysis of intra-subject variability in cerebrospinal fluid amyloid-β concentrations collected by lumbar puncture and indwelling lumbar catheter

doi: 10.1186/s13195-015-0136-z

Figure Lengend Snippet: Profile plot of cerebrospinal fluid (CSF) ( a ) Aβ40 and ( b ) Aβ 42 from subjects in Lilly lumbar puncture studies given placebo for approximately 2 weeks with lumbar punctures obtained at baseline and endpoint

Article Snippet: Mesor-to-peak amplitudes of diurnal fluctuation of Aβ40 were 4.61 % (AbbVie), –6.52 % (BMS), and –4.90 % (Lilly).

Techniques:

Journal: Journal of the American Chemical Society

Article Title: Single Molecule Characterization of the Interactions between Amyloid-β Peptides and the Membranes of Hippocampal Cells

doi: 10.1021/ja3103567

Figure Lengend Snippet: Representative images of HpL cells incubated with (A) and without (B) 500 nM of fluorescently labeled Aβ42 that has undergone aggregation for 3–4 h. In each pair of images, the left is the phase contrast image, and the right is the fluorescent (TIRFM) image. Yellow species in the fluorescent image are oligomers (dually labeled), whereas green and red species are monomers (singly labeled). Scale bars are 3 μm. (C) Aβ40 and Aβ42 monomeric and oligomeric species were detectable over the limits of nonspecific interaction with the slide surface and over the background fluorescence of unlabeled cells 500 nM total peptide concentration, number of cells varied from 35 to 50 for each category, error bars are standard error of the mean, (SEM).

Article Snippet: Monomeric solutions of HiLyteFluor488 and HiLyteFluor647-labeled Aβ40 or Aβ42 (Anaspec, San Jose, CA) were prepared by dissolving the lyophilized peptide in 0.01 M NaOH followed by sonication over ice for 30 min (Bandelin Sonorex, Schalltec Morfelden-Walldorf, Germany) and subsequent flash freezing into aliquots.

Techniques: Incubation, Labeling, Fluorescence, Concentration Assay

Journal: Journal of the American Chemical Society

Article Title: Single Molecule Characterization of the Interactions between Amyloid-β Peptides and the Membranes of Hippocampal Cells

doi: 10.1021/ja3103567

Figure Lengend Snippet: (A) Representative TIRFM images with arrows depicting the increased fraction of Aβ oligomers on the surfaces of hippocampal cells. The oligomeric fraction of Aβ42 and Aβ40 (B) on cell membranes and in solution prior to incubation with cells (aggregation concentrations used were 1 μM Aβ42 and 2 μM Aβ40); Aβ species were analyzed at different times during the aggregation reaction. For characterization of species prior to incubation with cells, the data were derived from three separate incubations. The oligomeric fraction of Aβ interacting with cell membranes as a function of different incubation concentrations of Aβ42 (C) and Aβ40 (D) (ANOVA single factor p = 0.17 for Aβ40 and p = 0.65 for Aβ42); distribution of sizes of oligomers after 4 h of aggregation of Aβ42 (E) or Aβ40 (F) present in solution (prior to incubation with cells) and on cell membranes (aggregation concentrations used were 1 μM Aβ42 and 2 μM Aβ40, n = 30–50 cells). All error bars are SEM; * is p < 0.05; ** is p < 0.01; n.s. p > 0.05.

Article Snippet: Monomeric solutions of HiLyteFluor488 and HiLyteFluor647-labeled Aβ40 or Aβ42 (Anaspec, San Jose, CA) were prepared by dissolving the lyophilized peptide in 0.01 M NaOH followed by sonication over ice for 30 min (Bandelin Sonorex, Schalltec Morfelden-Walldorf, Germany) and subsequent flash freezing into aliquots.

Techniques: Incubation, Derivative Assay

Journal: Journal of the American Chemical Society

Article Title: Single Molecule Characterization of the Interactions between Amyloid-β Peptides and the Membranes of Hippocampal Cells

doi: 10.1021/ja3103567

Figure Lengend Snippet: (A,B) Representative frames taken from a live cell imaging experiment to monitor Aβ40 diffusion in the cell membrane; the Aβ40 molecules were labeled with HiLyte488 and HiLyte647 fluorophores. (A) Monomers and oligomers (488 channel); (B) species undergoing FRET (633 nm channel, Aβ oligomers). The trajectories obtained by linking the images of the Aβ species in (A) and (B) are shown in (C). From the ensemble plots, it is already apparent that, in contrast to the small species present in (A), the motion of the large oligomers (defined as species that undergo FRET) is highly restricted. (D) A representative plot of the MSDs as a function of time for a mobile Aβ40 species (yellow arrows and trajectories in (C); the diffusion coefficient obtained from the fit is D = 0.063 ± 0.020 μm 2 /s. (E) The diffusion coefficient as estimated by the MSD analysis as a function of species intensity (which correlates with size) for both Aβ42 and Aβ40. Each point represents an Aβ monomer or oligomer. (F) The estimated diffusion coefficient as a function of species intensity for only the oligomers of Aβ42 and Aβ40. Concentrations used are 1 μM Aβ42 and 2 μM Aβ40.

Article Snippet: Monomeric solutions of HiLyteFluor488 and HiLyteFluor647-labeled Aβ40 or Aβ42 (Anaspec, San Jose, CA) were prepared by dissolving the lyophilized peptide in 0.01 M NaOH followed by sonication over ice for 30 min (Bandelin Sonorex, Schalltec Morfelden-Walldorf, Germany) and subsequent flash freezing into aliquots.

Techniques: Live Cell Imaging, Diffusion-based Assay, Labeling

Journal: Journal of the American Chemical Society

Article Title: Single Molecule Characterization of the Interactions between Amyloid-β Peptides and the Membranes of Hippocampal Cells

doi: 10.1021/ja3103567

Figure Lengend Snippet: Distributions of particle displacements (JDs, blue histogram) per unit time (35 ms) of monomers and oligomers of Aβ42 (A) and Aβ40 (B) fit to the two-dimensional diffusion equation for three diffusing populations (eq , black line). The three components of the fit are shown in red, green, and cyan (A) and green, cyan, and purple (B), respectively. Concentrations used are 1 μM Aβ42 and 2 μM Aβ40.

Article Snippet: Monomeric solutions of HiLyteFluor488 and HiLyteFluor647-labeled Aβ40 or Aβ42 (Anaspec, San Jose, CA) were prepared by dissolving the lyophilized peptide in 0.01 M NaOH followed by sonication over ice for 30 min (Bandelin Sonorex, Schalltec Morfelden-Walldorf, Germany) and subsequent flash freezing into aliquots.

Techniques: Diffusion-based Assay

Journal: Journal of the American Chemical Society

Article Title: Single Molecule Characterization of the Interactions between Amyloid-β Peptides and the Membranes of Hippocampal Cells

doi: 10.1021/ja3103567

Figure Lengend Snippet: αB-Crystallin and clusterin inhibit the interaction of Aβ40 with the membranes of hippocampal cells. Representative TIRFM images of cells after incubation of the cells with fluorescently labeled Aβ40 monomers and oligomers in the absence of chaperones (A) or in the presence of either αB-crystallin (B) or clusterin (C). Phase contrast images are displayed (left) and fluorescence images (right). Single-color green or red species are HiLyteFluor488 and HiLyteFluor647-labeled Aβ40 monomers and dual-color species (which appears as yellow) are oligomers, the scale bar in each case is 5 μm. (D) The species density per 10 μm 2 cell area in the presence and absence of chaperones; 33–39 cells were analyzed for each sample. Significance testing was performed relative to “Aβ40 only”, *** p < 0.001; * p < 0.05; n.s. p > 0.05. The concentrations used are 2 μM for Aβ40 and both chaperones. Error bars are SEM.

Article Snippet: Monomeric solutions of HiLyteFluor488 and HiLyteFluor647-labeled Aβ40 or Aβ42 (Anaspec, San Jose, CA) were prepared by dissolving the lyophilized peptide in 0.01 M NaOH followed by sonication over ice for 30 min (Bandelin Sonorex, Schalltec Morfelden-Walldorf, Germany) and subsequent flash freezing into aliquots.

Techniques: Incubation, Labeling, Fluorescence

Journal: Journal of the American Chemical Society

Article Title: Single Molecule Characterization of the Interactions between Amyloid-β Peptides and the Membranes of Hippocampal Cells

doi: 10.1021/ja3103567

Figure Lengend Snippet: Diffusion Coefficients ( D ) and Relative Abundances ( A ) for All Experimental Conditions Studied in This Work

Article Snippet: Monomeric solutions of HiLyteFluor488 and HiLyteFluor647-labeled Aβ40 or Aβ42 (Anaspec, San Jose, CA) were prepared by dissolving the lyophilized peptide in 0.01 M NaOH followed by sonication over ice for 30 min (Bandelin Sonorex, Schalltec Morfelden-Walldorf, Germany) and subsequent flash freezing into aliquots.

Techniques: Diffusion-based Assay

Journal: eLife

Article Title: A novel monomeric amyloid β-activated signaling pathway regulates brain development via inhibition of microglia

doi: 10.7554/eLife.100446

Figure Lengend Snippet: ( a ) TNFα and IL-6 secretion (pg/ml) in wildtype microglia following lipopolysaccharide (LPS) stimulation in the absence or presence of Aβ40 (50 nM). *p < 0.05; **p < 0.01; n = 25 each group for TNFα and 11 each group for IL-6. ( b ) TNFα and MCP1 secretion (pg/ml) in wildtype microglia following LPS stimulation in the absence or presence of Aβ40 (500 nM) from Genscript. Effects on IL-1β secretion in were also performed with Genscript Aβ40. All other experiments in were performed with ApexBio Aβ40. *p < 0.05; **p < 0.01; n = 5–7 each group. ( c ) TNFα, IL-23, and IL-10 mRNA expression in wildtype microglia following LPS stimulation in the absence or presence of Aβ40 (400 nM). *p < 0.05; n = 6 each group. ( d ) IL-1β secretion (pg/ml) in control and App:Cx3cr1-Cre mutant microglia following LPS stimulation in the absence or presence of Aβ40. *p < 0.05; n = 8–12 each group. ( e ) TNFα (pg/ml) in control or Aplp2:Cx3cr1-Cre mutant microglia following LPS stimulation in the absence or presence of Aβ40 (400 nM). *p < 0.05; n = 9–13 each group. ( f ) IL-10 and IL-23 mRNA expression in control and App:Cx3cr1-Cre mutant microglia following LPS stimulation in the absence or presence of Aβ40 (400 nM). *p < 0.05; n = 6 each group. ( g ) IL-1β secretion (pg/ml) in fresh unelicited control and App:Cx3cr1-Cre mutant peritoneal macrophages following LPS stimulation in the absence or presence of Aβ40 (400 nM). *p < 0.05; n = 12 each group. ( h ) IL-1β secretion (pg/ml) in f control and Ric8a:Cx3cr1-Cre mutant microglia following LPS stimulation in the absence or presence of Aβ40 (500 nM). *p < 0.05; ***p < 0.001; n = 7–8 each group. ( i ) IL-6 mRNA expression in control and Ric8a:Cx3cr1-Cre mutant microglia following LPS stimulation in the absence or presence of Aβ40 (200 nM). *p < 0.05; n = 6 each group. ( j ) TNFα (pg/ml) in wildtype microglia in the absence or presence of Aβ40 oligomers aggregated (at 10 μM monomer equivalent). ***p < 0.001; n = 10 each group.

Article Snippet: For experiments other than assaying IL-1β secretion, microglia were treated with LPS at 20 ng/ml for 3 hr or at 5 ng/ml overnight and, if applicable, DMSO or Aβ40 (ApexBio and Genscript) was applied at the same time as LPS.

Techniques: Expressing, Control, Mutagenesis

Journal: eLife

Article Title: A novel monomeric amyloid β-activated signaling pathway regulates brain development via inhibition of microglia

doi: 10.7554/eLife.100446

Figure Lengend Snippet: ( a ) TNFα, IL-6, IL-1β, and MCP1 secretion (pg/ml) by wildtype microglia following lipopolysaccharide (LPS) stimulation in the absence or presence of Aβ40 (200 or 500 nM). *p < 0.05; ***p < 0.001; n = 8–14 each group. ( b ) TNFα and IL-1β secretion (pg/ml) by wildtype microglia following poly I:C stimulation in the absence or presence of Aβ40 (500 nM). *p < 0.05; **p < 0.01; n = 6–7 each group. ( c ) IL-6 and IL-1β mRNA induction in wildtype microglia following LPS stimulation in the absence or presence of Aβ40 (500 nM). *p < 0.05; n = 6 each group. ( d ) TNFα and IL-6 secretion (pg/ml) by control and App:Cx3cr1-Cre mutant microglia following LPS stimulation in the absence or presence of Aβ40 (200 nM). **p < 0.01; ***p < 0.001; n = 8 each group. ( e ) IL-6 and IL-1β mRNA induction in control and App:Cx3cr1-Cre mutant microglia following LPS stimulation in the absence or presence of Aβ40 (200 nM). *p < 0.05; **p < 0.01; n = 6 each group. ( f ) TNFα and IL-6 secretion (pg/ml) by control and App:Cx3cr1-Cre mutant peritoneal macrophages following LPS stimulation in the absence or presence of Aβ40 (500 nM). *p < 0.05; n = 6–7 each group. ( g ) TNFα and IL-6 secretion (pg/ml) by control and Ric8a:Cx3cr1-Cre mutant microglia following LPS stimulation in the absence or presence of Aβ40 (200 nM). ***p < 0.001; n = 12–14 each group. Figure 4—source data 1. Excel files for control and App and Ric8a mutant microglia/macrophage ELISA and qRT-PCR analysis undergoing Aβ40 stimulation.

Article Snippet: For experiments other than assaying IL-1β secretion, microglia were treated with LPS at 20 ng/ml for 3 hr or at 5 ng/ml overnight and, if applicable, DMSO or Aβ40 (ApexBio and Genscript) was applied at the same time as LPS.

Techniques: Control, Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Metabolites

Article Title: High Correlation among Brain-Derived Major Protein Levels in Cerebrospinal Fluid: Implication for Amyloid-Beta and Tau Protein Changes in Alzheimer’s Disease

doi: 10.3390/metabo12040355

Figure Lengend Snippet: Correlation coefficients among CSF major proteins and AD biomarkers in CN, MCI and AD.

Article Snippet: AD core markers were assayed by LSI Medience Corporation (Tokyo, Japan), using the following ELISA kits: Phinoscholar hTAU (10-992, Nipro Parma Corporation, Osaka, Japan) for (total) tau; Phinoscholar pTAU (10-994, Nipro) for p-tau (181); Human βAmyloid (1-40) ELISA Kit Wako II (298-64601, FUJIFILM Wako) for Aβ40; Human βAmyloid (42) ELISA Kit Wako High Sensitive (292-64501, FUJIFILM Wako) for Aβ42.

Techniques: