Figure 1.
Characterizing the morphological and tinctorial properties of the aggregates.
(A) Table describing the peptides and aggregation conditions used in this study. (B–D) Transmission Electron Microscopy (TEM) images showing the morphology of the aggregates used in this study: (B) α-synuclein fibrils (α-syn), (C) gelsolin fibrils, and (D) Aβ1–40 fibrils. (E) Congo Red (CR, red bars) and Thioflavin T (ThT, yellow bars) bound to the aggregates shown in B–D. All the samples were used at 65 µg/ml and CR and ThT were used at 10 µM and 20 µM, respectively. The buffer for the CR and ThT binding assays was 5 mM potassium phosphate and 150 mM NaCl at pH 7.4. For ThT: Ex = 450 nm and Em = 465–520 nm. For CR: absorbance at 540/447 nm.
Figure 2.
Amyloid fibrils maintained their amyloid architecture after proteolytic digestion and acetone extraction.
(A) Aβ1–40, α-syn or gelsolin peptides (65 µg/ml) in a fibrillar (upper gel) or soluble (lower gel) state were incubated in the absence or presence of 0.13 µg/ml (1∶500, w/w) proteinase K (PK) for 2 h at 42°C. The digestion was conducted in 50 mM sodium phosphate, pH 7.4, 150 mM NaCl buffer. The reaction was stopped by boiling the samples in Laemmli buffer with 2% SDS and the samples were resolved by 16% SDS-PAGE. Western blot using 6E10 (Aβ1–40), syn-1 (α-syn) or a gelsolin-specific antibody is presented. (B) The same reaction described in panel A was performed in the presence of 20 µM of thioflavin T (ThT) and the florescence was monitored every 10 min. Ex = 440 nm and Em = 485 nm. (C) Aβ1–40 amyloid fibrils at 65 µg/ml concentration were diluted in 1 volume (1 V) of PBS, hexane, acetone or chloroform and centrifuged (16,000 g) for 10 min at 4°C. The pellet was resuspended in phosphate buffer with 20 µM ThT and the fluorescence measured. An aliquot of undiluted/uncentrifuged fibrils was used as the load. Ex = 450 nm and Em = 465–520 nm.
Figure 3.
Effect of proteinase K digestion and acetone precipitation on the protein content of a complex biological extract.
(A and B) The complex biological extract was obtained by mechanical disruption of wild type C. elegans worms followed by a brief centrifugation (700 g for 3 min) to remove unlysed worms. Aβ1–40 fibrils (0.2% w/w protein concentration) were added to the worm post debris supernatant (PDS) and the samples were digested with PK (1∶500) for 2 h at 42°C followed by acetone precipitation. An aliquot before PK digestion (load), after PK digestion (+ PK) and after PK digestion and acetone precipitation (+ PK/acetone) were resolved by SDS-PAGE (A) or the protein was quantified by BCA assay (B). In the panel A, the upper gel is silver stained and the lower gel is a Western blot for Aβ using the 6E10 antibody. (C–F) TEM images of PDS. PDS was incubated in the absence (C) or in the presence of 0.2% Aβ1–40 fibrils (E) before the PK/acetone step. PDS incubated in the absence (D) or in the presence of 0.2% Aβ1–40 fibrils (F) was digested with PK and precipitated with acetone. Note that amyloid fibrils are present only in the samples to which Aβ1–40 fibrils were added (E and F).
Figure 4.
Dot blot using the LOC antibody that recognizes a generic amyloid epitope.
Aβ1–40, α-syn or gelsolin peptides (65 µg/ml) in a fibrillar or soluble state were incubated in the absence (A) or in the presence (B) of PK (1∶500) for 2 h at 42°C then spotted onto nitrocellulose membrane loaded with LOC antibody.
Figure 5.
Immunoprecipitation of amyloid fibrils using the LOC antibody.
(A) Schematic of the protocol used to isolate amyloid fibrils. (B) Aβ1–40, (C) gelsolin or (D) α-syn amyloid fibrils (0.2% w/w) were added to worm PDS, digested with PK for 2 h at 42°C and precipitated with 1 volume of cold acetone. The pellet was resuspended in buffer containing LOC antibody and the IP was performed as described in Materials and Methods. As a negative control, we performed the IP in the absence of LOC antibody (beads). The samples were resolved by SDS-PAGE (16% tris-tricine gels) and probed for Aβ1–40, gelsolin, or α-syn by western blotting.
Figure 6.
Immunoprecipitation of amyloid fibrils from tissue extracts of a C. elegans strain that overexpresses human Aβ1–42 peptide.
(A) Worm post debris supernatant (PDS) from N2 (wild type) or CL2006 (Aβ) worms were applied to SDS-PAGE (Load) or processed as described in Figure 5A (Eluate) before being applied to SDS-PAGE. N2 (wild type) worms were used at day 1 of adulthood, whereas CL2006 (Aβ) worms were used at days 1, 5 or 8 of adulthood. The gel was transferred to nitrocellulose membrane that was probed for Aβ (≈4 kDa) using the 6E10 antibody or for tubulin (≈55 kDa) as a loading control. The amount of sample applied to the gel was 10 fold higher for the eluate (10X) when compared with the load (1X). In lane 9, synthetic Aβ1–40 peptide (2 ng) was used as a standard for Aβ. Note that the peptide Aβ1–40 runs faster than the Aβ synthesized in the CL2006 worms. (B) Quantification of Aβ bands of panel (A). Since the eluate fractions do not contain tubulin, we normalized the eluate bands using the tubulin bands of the load samples. The quantification was made using Fiji software and the bars represent the standard deviation of two experiments.
Figure 7.
Use of the IP protocol to detect Aβ amyloid fibrils in cerebrospinal fluid (CSF).
(A) Different amounts of Aβ1–40 amyloid fibrils were added to human CSF and the samples were processed as described in the schematic of Figure 5A. As observed by western blot using the Aβ antibody 6E10, picograms of Aβ fibrils were detected in the eluted fraction of the immunoprecipitated sample. (B) The same experiment described in the panel A was conducted with CSF from patients diagnosed with Alzheimer’s disease and the respective age-matched control. A representative example of one of three CSF samples tested is shown. As positive control, we spiked 750 pg of Aβ1–40 amyloid fibrils into human CSF from healthy controls. The absence of detection of soluble Aβ in the CSF is due to its digestion by PK.