α synuclein apc  (Novus Biologicals)

Journal: Journal of the American Chemical Society

Article Title: Strain-Distinct α‑Synuclein and Tau Cross-Seeding Uncovered by Correlative Approach with Optical Photothermal Infrared Sub-Micron Imaging

doi: 10.1021/jacs.5c02811

Figure Lengend Snippet: Morphological characterization of αS/Tau PFFs using immuno-EM. (A) Immuno-EM showing amyloid fibril samples, costained for αSyn and Tau. Double signals only appear together in the hybrid PFFs (αS&Tau3R and αS&Tau4R). Small size gold (6 nm): αSyn; Big-size gold (12 nm): Tau. Scale bar = 200 nm. (B) Numbers of αSyn and Tau positive particles for each fibril are collected from 10 individual fields and shown in the dot-plots.

Article Snippet: For the immuno-TEM, fibrils were applied to carbon-coated copper grids, blocked with 1% BSA and 0.1% Tween-20 in PBS, and incubated with primary antibodies, mouse antihuman αSyn monoclonal (SMC-532, StressMarq Biosciences Inc.) and rabbit antihuman Tau polyclonal (SPC-802, StressMarq Biosciences Inc.).

Techniques:

Journal: Journal of the American Chemical Society

Article Title: Strain-Distinct α‑Synuclein and Tau Cross-Seeding Uncovered by Correlative Approach with Optical Photothermal Infrared Sub-Micron Imaging

doi: 10.1021/jacs.5c02811

Figure Lengend Snippet: Distinct amyloid structures are presented in different αSyn/Tau PFFs. (A) Schematic illustration of seeding activities for αS, Tau isoforms 0N3R, 2N4R, and their mixed and hybrid PFFs (B) Averaged and normalized O-PTIR spectra for heteromeric PFFs, respectively, formed with αS/Tau3R and αS/Tau4R, compared to solely αS PFF and αS monomer. (C) Averaged and normalized second derivatives of the O-PTIR spectra (normalized to the maximum value across all conditions, with αS&Tau3R set as 1.0), with indicated peak position specific to amyloid structures. (D) The intensity of various amyloid structures in αS/Tau and their heteromeric PFFs with the second derivatives method, is shown in the dot plot ( n = 5–8 samples/group). The significance of the multiple comparisons is provided in Data S3 ( Supporting Information ). (E) Acronym and secondary structures constitute different PFFs via peak fitting method. Amide I (1600–1700 cm –1 ) is resolved with a peak position specific to amyloid structures in (D). (F) Heatmaps of the original and average intensity of area under the curve in the O-PTIR spectra, corresponding to the amyloid band structures of each PFF sample. (G) The PCA of different PFFs are plotted with the first two principal components (PC1 and PC2).

Article Snippet: For the immuno-TEM, fibrils were applied to carbon-coated copper grids, blocked with 1% BSA and 0.1% Tween-20 in PBS, and incubated with primary antibodies, mouse antihuman αSyn monoclonal (SMC-532, StressMarq Biosciences Inc.) and rabbit antihuman Tau polyclonal (SPC-802, StressMarq Biosciences Inc.).

Techniques:

Journal: Journal of the American Chemical Society

Article Title: Strain-Distinct α‑Synuclein and Tau Cross-Seeding Uncovered by Correlative Approach with Optical Photothermal Infrared Sub-Micron Imaging

doi: 10.1021/jacs.5c02811

Figure Lengend Snippet: The seeding activity of αS/Tau PFFs on αSyn-A53T in GFP HEK cells. (A) The different seeding activities of various PFFs on the αSyn-A53T-GFP HEK cells at 24 and 48 h are shown in the fluorescent images (3 independent experiments/per group). Three characteristic inclusions per group are indicated with white arrows. Scale bars, 10 μm. (B–D) Quantifications of seeding effect as a function of time, the significance of the multiple comparisons is provided in Data S1 ( Supporting Information ) (E) Cell number per well recorded at each time point. (F) LDH assay of cells incubated with PFFs for 48 h, with a significance level set at P < 0.05.

Article Snippet: For the immuno-TEM, fibrils were applied to carbon-coated copper grids, blocked with 1% BSA and 0.1% Tween-20 in PBS, and incubated with primary antibodies, mouse antihuman αSyn monoclonal (SMC-532, StressMarq Biosciences Inc.) and rabbit antihuman Tau polyclonal (SPC-802, StressMarq Biosciences Inc.).

Techniques: Activity Assay, Lactate Dehydrogenase Assay, Incubation

Journal: Journal of the American Chemical Society

Article Title: Strain-Distinct α‑Synuclein and Tau Cross-Seeding Uncovered by Correlative Approach with Optical Photothermal Infrared Sub-Micron Imaging

doi: 10.1021/jacs.5c02811

Figure Lengend Snippet: Intracellular αSyn inclusions induced by different αSyn/Tau PFFs exhibit distinct morphology and phosphorylation levels. (A) Confocal images of αSyn-A53T-GFP HEK cells at 48 h post seeding show distinct morphology and phosphorylation levels of αSyn (p-129) and Tau (AT8) in αSyn inclusions formed with different PFFs. Cells are counterstained with DAPI. Scale bars are 10 μm, (B) colocalization p-129 and AT8 with αSyn-GFP inclusions after 48 h are evaluated with % colocalization and Pearson’s coefficient. Bar plots are shown as mean ± s.d. (30 fields ∼ 100 cells/per group), the significance of the multiple comparisons over time is provided in Data S2 ( Supporting Information ), (C) 3D reconstruction of confocal micrographs showing different patterns of p-129 and AT8 colocalization in the αSyn inclusions induced by αS + Tau3R and αS&Tau3R, (D) linear correlations analysis for p-129 and AT8 in cells exposed to αS + Tau3R and αS&Tau3R, (E) distinct solubility αSyn inclusions are examined with SDS-PAGE and subsequent blotting with antibodies αSyn 211 and αSyn-p129. Cell lysates are extracted with 1% TX-100, 1% Sarkosyl, (F) cell lysates are digested in proteinase K (PK, 2 μg/mL). After PK digestion, the high (H: ∼40–45 kDa) and low (L: ∼35 kDa) molecular weights of αSyn can be detected, indicated with arrows, (G) quantification of αSyn solubility in 1% TX-100 and 1% Sarkosyl are shown in the bar plot as mean ± s.d, (H) quantification of αSyn resistant to 2% PK is shown in the bar plot as mean ± s.d.

Article Snippet: For the immuno-TEM, fibrils were applied to carbon-coated copper grids, blocked with 1% BSA and 0.1% Tween-20 in PBS, and incubated with primary antibodies, mouse antihuman αSyn monoclonal (SMC-532, StressMarq Biosciences Inc.) and rabbit antihuman Tau polyclonal (SPC-802, StressMarq Biosciences Inc.).

Techniques: Phospho-proteomics, Solubility, SDS Page

Journal: Journal of the American Chemical Society

Article Title: Strain-Distinct α‑Synuclein and Tau Cross-Seeding Uncovered by Correlative Approach with Optical Photothermal Infrared Sub-Micron Imaging

doi: 10.1021/jacs.5c02811

Figure Lengend Snippet: Correlative analysis of fluorescence and O-PTIR microspectroscopy in cells seeded with different PFFs. (A) Bright-field overview of cells grown on borosilicate glass grids used to identify the same object for fluorescence and O-PTIR imaging modalities. White insert indicates the cells imaged with fluorescence (C) and O-PTIR microspectroscopy (B). Scale bars are 20 μm, (B) single energy O-PTIR images at the specific wavenumbers corresponding to 1658 cm –1 (α-helix/total proteins); 1628 cm –1 (β-sheet major parallel); 1640 cm –1 (random coils). Scale bars are 5 μm, (C) fluorescence image overlap with bright field for the selected cells. White inserts indicate cells collected for amyloid structure analysis: 1: cell with inclusions and 2: cell without inclusions. The same object is marked in the O-PTIR ratio maps in (D,E). Scale bars are 20 μm, (D) O-PTIR ratio maps show the distribution of major β-sheet structures (1628 cm –1 /1658 cm –1 ), overlay of the map with the masked visual image, (E) Overlays (yellow) of β-turns (1682 cm –1 , red), minor β-sheet (1695 cm –1 , red) with major β-sheet structures (green). Corresponding areas are indicated in (C,D) by white squares, (F) averaged and normalized second derivatives of O-PTIR spectra of cells with aggregates (red) and without aggregates (black) are compared the negative control (NC) groups (gray), (G) quantifications of major β-sheet structures intensity are shown in the bar plot as mean ± s.d. Spectra from five to seven cells in each group were pooled respectively for the statistical analysis, (H) averaged and normalized second derivatives of O-PTIR. A zoomed-in section of the β-sheet structure (1628 cm –1 ) is on the right of the main graph. No evident band for 1628 cm –1 was found for the cells seeded with Tau and αSyn Monomer, (I) heatmaps of the average intensity values of the second derivatives in the O-PTIR spectra, corresponding to the amyloid band structures of cells with inclusions in (H).

Article Snippet: For the immuno-TEM, fibrils were applied to carbon-coated copper grids, blocked with 1% BSA and 0.1% Tween-20 in PBS, and incubated with primary antibodies, mouse antihuman αSyn monoclonal (SMC-532, StressMarq Biosciences Inc.) and rabbit antihuman Tau polyclonal (SPC-802, StressMarq Biosciences Inc.).

Techniques: Fluorescence, Imaging, Negative Control

Journal: Journal of the American Chemical Society

Article Title: Strain-Distinct α‑Synuclein and Tau Cross-Seeding Uncovered by Correlative Approach with Optical Photothermal Infrared Sub-Micron Imaging

doi: 10.1021/jacs.5c02811

Figure Lengend Snippet: O-PTIR imaging guided by amyloid dyes depicting the subcellular distribution of amyloid structures in αSyn-GFP aggregate. (A) Confocal images for mature amyloids of the aggregates are labeled with Amytracker (red) and overlay with strain distinct αSyn-GFP aggregates (green) seeded from different PFFs at 60 h. Single intracellular aggregate is classified into core, high and low aggregate segments according to the overlapping of Amytracker and GFP. GFP-Core: (Amy+ and GFP+) in aggregates; GFP-High: (Amy– and GFP+) in aggregates; GFP-Low: No visible Amytracker and GFP labeling. White squares demonstrate the core regions of aggregates selected in each group. The compact aggregates seeded with αS&Tau4R can be classified into only core and low areas based on the above standards. Scale bars are 10 μm, (B) averaged and normalized second derivatives of O-PTIR spectra taken from the counterparts of the strain distinct αSyn-GFP aggregates. Shifted band of β-sheet structures from 1628 cm –1 to 1632 cm –1 is observed in the core of the αSyn seeded aggregates, and in the core and high of aggregates seeded with αS&Tau3R, (C) quantifications of the intensity of the major β-sheet structures are shown in the bar plot as mean ± s.d. Spectra from eight cells with typical aggregates in each group were included for statistical analysis, (D) averaged and normalized second derivatives of O-PTIR spectra of different subcellular parts of aggregates in strain distinct αSyn-GFP aggregates seeded with various PFFs, (E) heatmaps of the average intensity values of the second derivatives in the O-PTIR spectra of different subcellular parts of aggregates in strain distinct αSyn-GFP aggregates, corresponding to the amyloid band structures of cells with aggregates in (D). Intensity values are marked on the heatmap.

Article Snippet: For the immuno-TEM, fibrils were applied to carbon-coated copper grids, blocked with 1% BSA and 0.1% Tween-20 in PBS, and incubated with primary antibodies, mouse antihuman αSyn monoclonal (SMC-532, StressMarq Biosciences Inc.) and rabbit antihuman Tau polyclonal (SPC-802, StressMarq Biosciences Inc.).

Techniques: Imaging, Labeling

Journal: Journal of the American Chemical Society

Article Title: Strain-Distinct α‑Synuclein and Tau Cross-Seeding Uncovered by Correlative Approach with Optical Photothermal Infrared Sub-Micron Imaging

doi: 10.1021/jacs.5c02811

Figure Lengend Snippet: O-PTIR hyperspectral map distinguishes different patterns of β-sheet subcellular distributions in αSyn-GFP aggregates. (A) Overlayed confocal images of cells with aggregates seeded with different PFFs, Amytracker (red), GFP-αSyn aggregates (green). GFP-Core is indicated by a circle outlined with a black dashed line. White insert indicates the parts shown in the hyperspectral map. Scale bars are 5 μm, (B) hyperspectral map of β-sheets (1632 cm –1 ) normalized to total protein (1658 cm –1 ). The intensity of the rainbow represents level of β-sheets. The identical core region in A is marked on the hyperspectral map. The vicinity with high β-sheets (pericentral region) selected is indicated by the circle outlined with a red dashed line. Hyperspectral map of O-PTIR reveals different β-sheet subcellular distribution patterns of the aggregates. Scale bars are 2.5 μm, (C) 3D density plot with projection demonstrates the subcellular distribution of total β-sheets (1632 cm –1 ) in an individual cell with aggerate. The intensity of the rainbow represents levels of β-sheets, (D) averaged and normalized second derivatives of O-PTIR spectra from two different subcellular parts of aggregates in the circle (B) reveal different β-sheet subcellular distribution patterns of the aggregates. The crosses indicate the spot which corresponds to the spectra.

Article Snippet: For the immuno-TEM, fibrils were applied to carbon-coated copper grids, blocked with 1% BSA and 0.1% Tween-20 in PBS, and incubated with primary antibodies, mouse antihuman αSyn monoclonal (SMC-532, StressMarq Biosciences Inc.) and rabbit antihuman Tau polyclonal (SPC-802, StressMarq Biosciences Inc.).

Techniques:

Journal: Journal of the American Chemical Society

Article Title: Strain-Distinct α‑Synuclein and Tau Cross-Seeding Uncovered by Correlative Approach with Optical Photothermal Infrared Sub-Micron Imaging

doi: 10.1021/jacs.5c02811

Figure Lengend Snippet: Mechanisms of different types of αSyn/Tau PFFs cross-seeding αSyn aggregates presumed in the current study. (A) Pure αS seeds, but not solely Tau seeds, can serve as the template and initiate αSyn aggregates. (B) In mixed αS + Tau3R seeds, αS and Tau3R act in different roles in the formation of αSyn aggregates. αS seeds serve as the template and initiate αSyn aggregates. Only a small proportion of Tau3R seeds template or facilitate αSyn aggregates, while the others trigger a widespread intracellular Tau phosphorylation. The cross-seeding process leads to the lowest content of amyloid structures in the aggregates. (C) In hybrid αS&Tau3R seeds, αS&Tau3R copolymers act dependently to recruit αSyn and Tau monomers and form αSyn and Tau aggregates. The cross-seeding process leads to the highest content of amyloid structures in the aggregates.

Article Snippet: For the immuno-TEM, fibrils were applied to carbon-coated copper grids, blocked with 1% BSA and 0.1% Tween-20 in PBS, and incubated with primary antibodies, mouse antihuman αSyn monoclonal (SMC-532, StressMarq Biosciences Inc.) and rabbit antihuman Tau polyclonal (SPC-802, StressMarq Biosciences Inc.).

Techniques: Phospho-proteomics