The Effect of Ultrasonication on the Fibrillar/ Oligomeric Structures of Aβ 1-42 at Different Concentrations

doi:10.21203/rs.3.rs-2107232/v1

Download PDF

Research Article

The Effect of Ultrasonication on the Fibrillar/ Oligomeric Structures of Aβ 1-42 at Different Concentrations

https://doi.org/10.21203/rs.3.rs-2107232/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 27 Aug, 2023

Read the published version in The Protein Journal →

You are reading this latest preprint version

The number of disease states linked the aberrant regular protein conformations to oligomers and amyloid fibrils. Amyloid beta 1–42 (Aβ₁₋₄₂) peptide is very hydrophobic and quickly forms the β-rich structure and fibrillar protein aggregates in some solutions and buffer conditions. Ultrasonication pulses can disrupt amyloid fibrils to smaller fragments and produce Aβ₁₋₄₂ peptides of different sizes and oligomers. Herein, we investigated the effects of buffer and ultrasonication on Aβ₁₋₄₂ structure at low and high concentrations. The electrophoresis and Western blot results showed that Aβ₁₋₄₂ fibrils were disrupted into different sizes after ultrasonication. The transmission electron microscopy results indicated Aβ₁₋₄₂ at low concentration (25 µM) in Ham’s/F12 phenol red-free culture medium formed short-size fragments and oligomers. In comparison, Aβ₁₋₄₂ at higher concentration (100 µM) formed fibrils that break down into smaller fragments after ultrasonication. However, after regrowth, it formed mature fibrils again. The cell cytotoxicity results indicated more toxicity of Aβ₁₋₄₂ oligomers formed at low concentration (25 µM) against PC12 cells than other forms. In conclusion, by applying ultrasonication pulses and controlling peptide concentration and buffer condition, we can rich Aβ₁₋₄₂ aggregates with a particular size and molecular structure.

Amyloid Fibrils

Protein Aggregation

Amyloid β Oligomers

Ultrasonic Pulses

Cell Toxicity

Protein misfolding and aggregation have been introduced as the molecular mechanism of some neurodegenerative diseases. Aggregated conformers are classified based on their size, structure, and solubility. Recently, highly toxic oligomers have been introduced as critical determinants in aggregate-induced toxicity [1–3]. Amyloid beta (Aβ) is one of the most critical aggregates responsible for Alzheimer’s disease (AD). Aβ peptides with different lengths, Aβ₁₋₄₀ or Aβ₁₋₄₂, have been used in different in vitro studies. They could exist as a monomer or larger soluble entities called Aβ oligomers with non-fibrillar structures and eventually insoluble fibrils. The soluble oligomers of Aβ₁₋₄₂ contain mixed intermolecular parallel and intramolecular antiparallel β-sheets. They have different types of assemblies: dimers, trimers, protofibrils, annular or pore-like oligomers, and spherical β-amyloid species (globulomers) [4–6]. Fibrils are unbranched, long supramolecular assemblies containing in-registered parallel β-sheets.

The cytotoxicity of Aβ₁₋₄₂ is related to the soluble oligomers that, due to their interaction with the lipid bilayer of the cell membrane and pore formation, cause an uncontrolled ion flux [5, 7, 8]. It is because oligomeric Aβ₁₋₄₂ can form the β-barrel in the cell membrane and permeabilize cells [9–11]. As mentioned above, different aggregated morphologies in neuronal cells exhibit different degrees of toxicity, and soluble oligomers are generally more toxic than amyloid fibrils [2, 12, 13]. Deposition of these aggregates is associated with various degenerative diseases, including AD, prion disease, and dialysis-related amyloidosis [14]. The in vitro protein aggregation mechanism is sensitive to subtle differences in environmental conditions such as buffer composition, agitation, and protein concentrations. Even small protein/ peptide concentration changes can lead to the formation of specific types of oligomers and intermediates [15–17]. In vitro studies indicated that a low concentration of Aβ₁₋₄₂ controls oligomers’ formation and favors a modest conformational conversion into fibrils. In contrast, its higher concentration would promote heterogeneous nucleation and aggregation to form fibrils of different sizes [18, 19].

Ultrasonication as an environmental factor has opposite effects on the formation and breakdown of fibrils [20]. Ultrasonic-dependent fragmentation is a fundamental approach to breaking Aβ₁₋₄₂ fibrils into smaller aggregates [20, 21]. The main reason is the repeated growth and collapse of bubbles, which are under the control of negative or positive pressures [22–24].

Herein, we decomposed Aβ₁₋₄₂ large fibrils in phosphate - NaCl buffer using ultrasonication. After recovering the monomers, oligomeric structures formed through ultrasonication. For this purpose, we modeled low and high concentrations of Aβ₁₋₄₂ peptide in different buffers using different techniques.

Materials

Aβ₁₋₄₂ was bought from Sigma-Aldrich (St. Louis, USA) and GL Biochem. (Beijing, China) as the lyophilized powder. Hexafluoroisopropanol (HFIP) was purchased from Merck Co. (Darmstadt, Germany). Thioflavin T (ThT), thiazolyl blue tetrazolium bromide (MTT), 8-Anilinonaphthalene-1-sulfonate (ANS), and Hoechst were purchased from Sigma-Aldrich Chem. Co. (St. Louis, USA). The PC12 rat pheochromocytoma cell line was purchased from Pasture Institute (Tehran, Iran). Cell culture plates were acquired from SPL (Beijing, China). Primary antibodies (ab201060 and ab224275) and the secondary antibody (ab6721) were bought from Abcam (Abcam Inc., Cambridge, MA). ECL Plus Kit was purchased from Bio-Rad (Bio-Rad Laboratories Inc., Hercules, CA, USA). Alexa Fluor 594 (clone Poly4064) was purchased from BioLegend (San Diego, CA, USA). PVDF was purchased from GE Healthcare (Biosciences, Stockholm, Sweden). All other materials were of analytical grade.

Methods

Preparation of Aβ Conformers. Aβ fibrils were formed according to the method described previously by us. Briefly, Aβ₁₋₄₂ (220 µM) was dissolved in 10 mM Na₂HPO₄, 100 mM NaCl, pH 7.4 and incubated at 37 ºC up to 5 h [25].

Other Aβ₁₋₄₂ peptide conformers were prepared by dissolving its powder in cold HFIP at a 2 mg/ml concentration and incubated at room temperature (RT) for 1 h. Then, the solution was divided into 25 µl aliquots, and HFIP was evaporated. The resulting Aβ₁₋₄₂ films were stored at − 20 °C until further experiments. Before any experiment, the peptide was dissolved in DMSO at a final concentration of 5 mM. Then resuspended in the suitable buffer as follows and treated with ultrasonication.

The high fibril concentration was formed by resuspension of this peptide in 10 mM HCl at a final concentration of 100 µM and incubating at 37 °C for 24 h.

The lower fibril concentration was prepared by diluting this solution to 25 µM in Ham’s/F12 phenol red-free culture medium.

Finally, preparation and propagation of oligomers were done by resuspension of Aβ₁₋₄₂ solution in DMSO into Ham’s/F12 (phenol red-free) at a final concentration of 100 µM incubated at 4 °C for 24 h [26].

Ultrasonication Treatments. Aβ₁₋₄₂ fibrils were placed on a water bath-type Ultrasonicator (Engotech, Zurich, Switzerland). Ultrasonication pulses were applied to both low (25 µM) and high (100 µM) concentrations of Aβ₁₋₄₂ solution for 120 min. The sonication output was set to 60 Hz and 280 watts, and the temperature was maintained at 4°C throughout the treatment. The Aβ₁₋₄₂ solution samples were named ultrasonicated high concentration (USH) and ultrasonicated low concentration (USL). In addition, a sample containing a high concentration of Aβ was also prepared and named as a non-ultrasonicated high concentration (NUSH).

Transmission Electron Microscopy (TEM). Amyloid conformers (NUSH, USH, and USL) were immediately placed on the 400-mesh carbon-coated copper grids. After 1 min incubation for adsorption of bio-macromolecules on the grid surface, the excess solution was removed with filter paper. Next, the grid’s surface was washed with deionized water. Then, the grids were negatively stained using 2% (w/v) uranyl acetate; after 15 min, the dried grids were used for TEM analysis. The electron micrographs were acquired using a transmission electron microscope (Model-EM208S) at 100 kV with a magnification of 200 K×. The images were prepared using a Digital camera. The electron micrographs were taken at Partow Rayan Rastak, Tehran, Iran.

Gel Electrophoresis and Western Blotting. Aβ₁₋₄₂ conformers were diluted to 5 µM into sample buffer (62.5 mM Tris, pH 6.8, 1% SDS, and 25% glycerol), separated on 12.5% polyacrylamide gels, and stained with Coomassie Brilliant Blue G-250. Gels were scanned using EPSON EU-88 image scanner (GE Healthcare, Chicago, USA). For semi-quantitative analysis, band intensities in the ranges of 10–60 and 60–180 kDa in SDS-PAGE (2–12 and more than 12 subunits, respectively) were measured using image processing software (Image J; National Institutes of Health, Bethesda, MD, USA).

For Western blot, Aβ₁₋₄₂ conformers were diluted to 2.5 mM in sample buffer. Then, the diluted samples were loaded onto the 12.5% SDS-PAGE. After electrophoretic separation, they were transferred to a polyvinylidene difluoride membrane (PVDF). The membranes were blocked with 3% BSA in Tris-buffered saline (TBS; 25 mM Tris–HCl, pH 7.4, 0.9% NaCl) containing 0.1% Tween 20 (TBS-T), washed for 10 min with TBS-T, and incubated with specific primary Aβ₁₋₄₂ antibody (Ab11132) overnight at 4°C. The day after, the membrane was washed three times, 10 min each, with TBS-T. Then, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG Goat antibody in TBS-T containing 0.5% BSA for 1 h at RT. Next, the membrane was rewashed with TBS-T three times, 10 min each, and then with TBS. Immune-reactive bands were visualized by the enhanced chemiluminescence (ECL) method on X-ray films. Subsequently, intensities of bands on the SAS-PAGE and WB membrane were quantified by using ImageJ software version 1.49t (NIH approved). So, the bands with less than 10 kDa, in the range of 10–50 kDa, and more than 50 kDa were considered the monomer, oligomer and fibril, respectively.

Dot Blot. Each Aβ₁₋₄₂ conformer was transferred and spotted (1 µg) on PVDF. Afterward, the membrane was blocked with 10% BSA in TBS-T for 1 h at 37°C. Next, it was incubated with rabbit polyclonal anti-Aβ₁₋₄₂ antibody for 2 h, at RT. Then the blots were washed in washing buffer, incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at RT for 1 h, and developed with ECL.

ThT Assay. Aβ₁₋₄₂ samples were diluted with ThT (0.4 µM ThT in 50 mM Na₂HPO₄ and 0.05% (w/v) NaN₃, pH 7.4). The fluorescence intensity (FI) was measured using a Cytation 3 microplate reader (BioTek Instruments, Inc. Winooski, VT, USA). The FI of ultrasonicated and non-ultrasonicated Aβ₁₋₄₂ at different time intervals of protein incubation at 37 °C was recorded at excitation and emission wavelengths of 450 and 482 nm, respectively.

ANS Fluorescence Assay. Diluted solutions (10 µM) of Aβ₁₋₄₂ in all forms of NUSH, USH, and USL were exposed to 20 µM of ANS at different incubation time points. The ANS fluorescence intensity at 500 nm was read after the excitation of the samples at 380 nm at different time intervals. The data was obtained using a Cytation 3 microplate reader.

Circular Dichroism (CD). Far-UV (190–260 nm) CD spectra explored changes in the secondary structure of the diluted conformers of oligomers, NUSH, USH, and USL solutions of Aβ₁₋₄₂ in 10 mM PBS, pH 7.4. These conformers were evaluated using a JASCO-J-810 Spectropolarimeter (JASCO Corporation, Tokyo, Japan), using a 5-mm path length cuvette at 25°C. Spectra were recorded with wavelength intervals of 1 nm, a response time of 4 s, and a scan rate of 100 nm/min. Each spectrum is the average of 4 scans of samples after subtraction of the related baseline. The noise component in data was smoothed using the JASCO J-810 software, including the fast Fourier-transform noise reduction method, which allows enhancement of most noisy spectra without distorting their peak shapes. The amounts of the secondary structures of the peptides were estimated using the Protein Secondary Structure Estimation Program (JWSSE-480), J-800 for windows. Yong plots were used as references for α-helix, β-sheet, β-turn, and random coil.

Cell Toxicity Assay. Aβ₁₋₄₂ toxicity was evaluated by cell viability assay using MTT. Therefore, we firstly cultured rat adrenal pheochromocytoma cells (PC12) in RPMI-1640 medium supplemented with 5% (v/v) fetal bovine serum (FBS), 10% (v/v) horse serum, and 1% (v/v) penicillin/ streptomycin. Then, PC12 cells were trypsinized and, after washing, were seeded in 96-well plates. After 24 h, different types of Aβ₁₋₄₂ peptides (NUSH, USH, and USL) were separately added to the PC12 cells. The cells were incubated for an additional 24 h at 37°C, and then the MTT solution (20 µl of 5 mg/ml) was added to the wells. After 4 h of incubation with MTT, the media were removed and replaced with 200 µl of DMSO. The color intensity of the formazan solution was quantified using an ELISA plate reader (Tecan, Zurich, Switzerland) at 570 nm. The control (untreated) cells were assumed 100% viable, and the viability of the Aβ₁₋₄₂ treated cells was calculated relative to the control and expressed as the percentages (%) of viability.

Immunofluorescence Assay of Aβ _1− 42 Cellular Uptake. PC12 cells were grown in RPMI-1640 medium supplemented with 5% fetal bovine serum, 10% horse serum, and 1% penicillin/ streptomycin. Then, cells were exposed to the IC₅₀ value of Aβ_1− 42 conformers. After 24 h, treated cells were washed with PBS and fixed in 70% chilled methanol for 10 m at RT. After PBS washing, nuclei were permeabilized in 0.3% Triton-X for 10 m at RT. Next, PC12 Cells were washed with PBS, and unspecific binding sites were blocked with 1% BSA for 30 m at RT. Then, cells were subjected to primary Aβ_1− 42 antibody at RT overnight. After that, cells were incubated with Alexa Fluor 594, as a fluorescent secondary antibody, for 1 h at RT, followed by counterstaining with Hochest 33258 solution. Finally, cells were scanned and imaged with a Cytation™ 3 microscope (BioTek Instruments, Inc. Winooski, VT, USA).

The TEM analyzes

TEM analysis was performed to obtain information about the shape and size of ultrasonicated Aβ₁₋₄₂ aggregates. Figure 1a shows long fibrillar aggregates of NUSH, while Fig. 1d shows Aβ₁₋₄₂ oligomers with spherical structures. Figures 1b and 1c show TEM images of USH and USL of Aβ₁₋₄₂. Comparing these two figures indicate a marked decrease in fibrillar size and the effective fragmentation of amyloid fibrils to smaller species and oligomers at low concentrations of Aβ₁₋₄₂ solution.

SDS-PAGE and Western blot profiles of Aβ₁₋₄₂ peptides at different conditions

Figure 2a shows the results of the SDS-PAGE study. From the left, the protein marker was in Lane 1. Lanes 2 to 4 show the oligomeric structure, 100 µM of Aβ1–42 fibrils without ultrasonication (NSH) and after ultrasonication (USH). It was highly degraded due to ultrasonication and converted to structures with less than 75 kDa. Lane 5 shows the Aβ₁₋₄₂ at a low concentration (25 µM) after ultrasonication. This treatment causes the conversion of the fibers to monomers and oligomers up to dodecamer structures. Figure 2b is the SDS-PAGE data's histogram and indicates each conformer's mean band intensity at different conditions.

Figure 2c shows the Western blot bands, and from left to right, it shows the oligomeric form of protein in lane 1, and the fibril with no ultrasonication in lane 2. Lanes 3 and 4 show the ultrasonicated low and high concentrations Aβ₁₋₄₂ peptides, respectively. As can be seen, ultrasonication breaks proteins and smears appear. However, two 45 and 25 kDa bands at lower concentrations (USL) indicate oligomeric structures.

Figure 2e shows the results of the Dot blot. It indicates the smaller species content at low and high concentrations of Aβ₁₋₄₂ peptides after ultrasonication. However, small oligomer content at a low concentration (25 µM) of Aβ₁₋₄₂ after ultrasonication was considerably higher than the others.

ThT fluorescence intensity and ANS binding assay

The interaction of Aβ₁₋₄₂ peptides with ThT, a dye reactive with β-sheet-rich conformers, and ANS, a hydrophobic dye, were studied. Figure 3a shows the enhancement of ThT fluorescence emission following its incubation with different forms of Aβ₁₋₄₂ peptides. NUSH showed higher fluorescence intensity after interaction with ThT, which was increased up to 3 h. However, the ultrasonicated forms showed lower fluorescence intensity changes. Figure 3a shows that the lowest ThT fluorescence intensity belongs to USL of Aβ₁₋₄₂, indicating the lowest fibrillar form.

Figure 3b shows the results of the ANS binding assay. The highest ANS fluorescence intensity was observed in the solutions containing oligomers and USL of Aβ after 150 min incubation. In contrast, the lowest fluorescence intensity was observed in the NUSH solution. It indicates the formation of Aβ₁₋₄₂ species with higher surface hydrophobicity, such as those observed in Aβ oligomers in the USL. These results suggest that ultrasonication shifts the equilibrium toward Aβ₁₋₄₂ fibrils depolymerization, monomer, and oligomer formation. Furthermore, these changes were more at lower Aβ₁₋₄₂ concentrations.

Secondary Structure of Different forms of Aβ_1-42

Figure 4a and Table 1 indicate oligomers’ higher percentage of β-turn and random coil. The NUSH solution showed a β-sheet-rich structure for fibrils (Fig. 4b and Table 1), while after ultrasonication, at higher concentration, the percentages of β-sheet decreased (from ~ 60% to ~ 37.5%) at the expense of β-turn (from 11–36%) and random coil (from 0 to 18%) conformation. At lower concentrations, the β-turn was also higher than in other structures (Table 1).

Table 1

The percentages of secondary structure for different forms of Aβ₁₋₄₂ peptides at different conditions was estimated from far-UV CD spectra using the mentioned software in the Methods.
	α-Helix	β-Sheet	Random Coil	β-Turn
Oligomer	11	-	48.8	40.2
NUSH	29.4	59.4	-	11.1
USL	18.4	12.8	22.1	46.7
USH	8.6	37.4	18	36

Cell toxicity assay of Aβ_1-42 at different conditions

The toxicity of different forms of Aβ peptide against PC12 cells was investigated using MTT assay and expressed as percentages of cell viability. The results in Fig. 5 indicated more toxicity of ultrasonicated Aβ_1-42 peptide (USL) than non-ultrasonicated one (NUSH) against these cells. The IC50 of oligomers was about 5 μM, and the IC50 of the USL was 10 μM, which is the highest cytotoxicity compared to other forms.

Uptake of Aβ_1-42 into the PC12 cells

The Aβ uptake by PC12 cells was studied by incubating the cells with a primary antibody against the Aβ_1–42peptide. Then, cells were stained with a secondary antibody against Aβ_1–42 and Hochest 33258 for the nucleus. Figs. 6a and 6b show the maximum and significant (p = 0.000) cellular uptake in cells exposed to the ultrasonicated-25 μM (USL)and AβOs.

In the present study, the stable fibrils of Aβ₁₋₄₂ (220 µM in phosphate buffer containing NaCl incubated at 37 °C) were subjected to destruction using ultrasonication. First, we tried to form the Aβ fibrils at high and low concentrations (100 and 25 µM) and oligomers by the standard methods of Stine et al. [25]. Then, we evaluated some properties of Aβ₁₋₄₂ before and after ultrasonication. Our findings show that ultrasonicated-25 µM of Aβ₁₋₄₂ was more toxic against PC12 cells than 100 µM before or after ultrasonication. The SDS-PAGE and Western blot data showed Aβ₁₋₄₂ fibrillation, mostly at 100 µM, before ultrasonication. Due to ultrasonication, the fibrils broke through into smaller species, resulting in oligomer formation. However, a low concentration of Aβ₁₋₄₂ adopts a predominantly smaller size aggregate and oligomeric structure. The changes in the fluorescence intensities of different forms of Aβ₁₋₄₂ in the presence of ThT and ANS were also other affirmative reasons for this finding. Dot blot analysis also indicated the presence of higher amounts of monomer and oligomer species after ultrasonication of 25 µM preparations than in the 100 µM preparations. We also showed the differences in the Aβ₁₋₄₂ structures at high and low concentrations using TEM images. Given the toxicity assay, we showed that USL was more toxic than other forms for PC12 cells due to their uptake by the cells, confirming their oligomeric structure.

The ThT-binding assay is a method of choice for detecting the presence of amyloid fibrils [27–29]. It showed a higher fluorescence intensity of NUSH, indicating more amyloid fibril formation. In contrast, the USL displays the lowest ThT fluorescence intensity, indicating the lowest fibrillar content. These data are consistent with the data obtained by TEM analysis. TEM examination of incubated NUSH also showed a tremendous amount of amyloid fibril content, while the lowest amount was observed at 25 µM solution after ultrasonication.

The highest ANS binding to the USL is another reason for Aβ oligomer formation. ANS is highly sensitive to the polarity of peptides and proteins. The fluorescence intensity increases upon interaction with the exposed hydrophobic regions in native or partially unfolded proteins [30]. Ultrasonic disruption of the fibrils increased to the extent of solvent exposure of hydrophobic Aβ₁₋₄₂ fragments and led to evaluate the hydrophobicity of Aβ using the ANS-binding assay. Aβ₁₋₄₂ oligomers are more hydrophobic [18] and, thus, bind more ANS. While hydrophobicity of both NUSH and USH were lower than USL.

Aβ₁₋₄₂ fibrils are composed of two in-register inter-strand parallel β-sheets connected by a bend between residues 25 and 30 [5, 31, 32]. Residues 10–16 are part of the first β-sheet in fibrils [11, 31, 32] and disordered in the pre-globulomer [33]. Two hydrophobic peptide segments within Aβ₁₋₄₂, residues 16–22 and 30–42, are solvent-exposed in the toxic Aβ₁₋₄₂ oligomers [18]. We also evaluated the secondary structure of the peptides by CD spectroscopy. The spectrum of NUSH would possess a sharp negative peak at about 220 nm, indicating that the peptide adopts mainly (~ 60%) the β-sheet conformation. In contrast, the spectra of other peptides show different characteristic peaks, indicating the decreased β-sheet percentage at the expense of β-Turn and random coil formation.

We also investigated the effect of ultrasonication on Aβ₁₋₄₂ fragmentation. According to the SDS-PAGE and Western blot analysis, ultrasonication of 25 µM of Aβ₁₋₄₂ induced a significant increase in the densities of the bands around 20–60 kDa. This pattern is compatible with the formation of Aβ₁₋₄₂ monomers (~ 10 kDa), oligomers (~ 25 kDa), and fibrils (~ 75 kDa) [34–36]. These results are in excellent agreement with those obtained from Dot blot in which the antibody recognized oligomers in USL more than those in USH solutions of Aβ₁₋₄₂. Furthermore, these data are compatible with the model that demonstrated two distinct pathways for the amyloid Aβ₁₋₄₂ soluble (oligomer) and insoluble (fibril) peptides [6, 37]. Similar studies have been performed on 25 µM [38] and 8 µM [39] of the Aβ₁₋₄₀ peptide, illustrating the role of environmental factors in the peptide structure.

A switch in mechanism with concentration has also been observed with Aβ₁₋₄₂. In a narrow concentration range (20 − 25 µM), spherical Aβ₁₋₄₂ oligomers formed that were positive for the oligomer-specific A11 antibody and showed a high capacity to disrupt lipid bilayers [18]. By contrast, at higher concentrations, a different type of oligomer was formed that did not react with the A11 antibody that did not disrupt lipid bilayers, despite having a similar size and secondary structure at low concentrations (< 20 µM) [18]. Studies such as these show the importance of recognizing the influence of concentration-dependent mechanistic changes and the need for accurate concentration measurements before experiments start[17].

The Aβ₁₋₄₂ amyloid fibrils in the membrane consist of two intermolecular β-sheets: β1, which is in the non-transmembrane (NTM) region (residues 17 − 28), and β2, is in the transmembrane (TM) region (residues 29 − 42) [5]. Under ultrasonication, a bubble was usually created around hydrophobic residues in the TM region after pressure becomes negative. The assembly of hydrophobic residues in the TM region acts as a nucleus for bubble formation, and the bubble breaks down the fibrils. The amyloid fibrils keep their β-sheet structure even in the bubble. When the pressure becomes positive, the bubbles shrink and collapse, water molecules crash against the hydrophilic residues in the NTM region; and then amyloids disrupt. The deformation and disruption of the β-sheet structure at the NTM region are possible because of the presence of Glu22 and Asp23 with negative electric charges. The repulsion between these residues is shielded in water but not in the bubble, which is the main reason for β-sheet structure destruction in the NTM region in the bubble. In the case of short amyloid fibrils like trimer, hexamer, and dodecamer, the Aβ peptides do not have enough hydrophobic residues to create a bubble. Thus, amyloid fibrils do not disrupt. Therefore, more time of negative pressure is required for bubble formation [22]. It appears that after ultrasonication, the possibility of antiparallel β-sheet formation at low concentrations of Aβ₁₋42 peptide is more than at high concentrations. It may be due to the repulsion between the negatively charged amino acids Glu22 and Asp23 and the critical concentration of micelle-like formation.. It has shown that after ultrasonication of both low and high concentrations, Aβ₁₋₄₂ fibrils break down to monomers and smaller species of aggregates. However, the kinetic, thermodynamic stability and energy landscape differences lead to possible conformations.

We examined the effect of the ultrasonic application on low and high concentrations of Aβ₁₋₄₂ peptide to study the simultaneous effects of the concentration and ultrasonication on Aβ₁₋₄₂ fibril disruption. The results indicated that due to ultrasonication of low concentration of Aβ₁₋₄₂, long amyloid fibrils disrupted and fragmented to smaller species of aggregates, including 2–12 mers. Furthermore, these small spherical Aβ₁₋₄₂ aggregates were more toxic against PC12 cells than the fibrillar aggregates at higher concentrations of Aβ₁₋₄₂. In addition, their characteristics were very similar to Aβ oligomers. The ThT and CD studies indicated the following order for β-sheet content in the Aβ1–42 peptides: NUSH > USH > USL > Oligomers.

Acknowledgment

The Ultrasonic experiments were done in the Prof. Mir F. Mousavi Lab., at Faculty of Science, TMU. The authors are also thankful of the Iranian National Science Foundation (INSF) project No. 95012277.

Funding

This study was financially supported by Tarbiat Modares University [Grant Number IG-39807] and the key projects of international scientific and technological innovation cooperation between governments [grant number 2017YFE0130100].

Conflict of Interest

No potential conflict of interest was reported by the authors.

Kirkitadze MD, Bitan G, Teplow DB (2002) Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies. J Neurosci Res 69(5):567–577. doi: 10.1002/jnr.10328
Ono K, Condron MM, Teplow DB (2009) Structure-neurotoxicity relationships of amyloid beta-protein oligomers. Proc Natl Acad Sci U S A 106(35):14745–14750. doi: 10.1073/pnas.0905127106
Cizas P, Budvytyte R, Morkuniene R, Moldovan R, Broccio M, Losche M, Niaura G, Valincius G, Borutaite V (2010) Size-dependent neurotoxicity of beta-amyloid oligomers. Arch Biochem Biophys 496(2):84–92. doi: 10.1016/j.abb.2010.02.001
Eisenberg D, Nelson R, Sawaya MR, Balbirnie M, Sambashivan S, Ivanova MI, Madsen AO, Riekel C (2006) The structural biology of protein aggregation diseases: Fundamental questions and some answers. Acc Chem Res 39(9):568–575. doi: 10.1021/ar0500618
Luhrs T, Ritter C, Adrian M, Riek-Loher D, Bohrmann B, Dobeli H, Schubert D, Riek R (2005) 3D structure of Alzheimer's amyloid-beta(1–42) fibrils. Proc Natl Acad Sci U S A 102(48):17342–17347. doi: 10.1073/pnas.0506723102
Barghorn S, Nimmrich V, Striebinger A, Krantz C, Keller P, Janson B, Bahr M, Schmidt M, Bitner RS, Harlan J, Barlow E, Ebert U, Hillen H (2005) Globular amyloid beta-peptide oligomer - a homogenous and stable neuropathological protein in Alzheimer's disease. J Neurochem 95(3):834–847. doi: 10.1111/j.1471-4159.2005.03407.x
Arispe N, Rojas E, Pollard HB (1993) Alzheimer disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum. Proc Natl Acad Sci U S A 90(2):567–571. doi: 10.1073/pnas.90.2.567
Ferguson N, Becker J, Tidow H, Tremmel S, Sharpe TD, Krause G, Flinders J, Petrovich M, Berriman J, Oschkinat H, Fersht AR (2006) General structural motifs of amyloid protofilaments. Proc Natl Acad Sci U S A 103(44):16248–16253. doi: 10.1073/pnas.0607815103
Kayed R, Sokolov Y, Edmonds B, McIntire TM, Milton SC, Hall JE, Glabe CG (2004) Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases. J Biol Chem 279(45):46363–46366. doi: 10.1074/jbc.C400260200
Kremer JJ, Pallitto MM, Sklansky DJ, Murphy RM (2000) Correlation of beta-amyloid aggregate size and hydrophobicity with decreased bilayer fluidity of model membranes. Biochemistry 39(33):10309–10318. doi: 10.1021/bi0001980
Cerf E, Sarroukh R, Tamamizu-Kato S, Breydo L, Derclaye S, Dufrene YF, Narayanaswami V, Goormaghtigh E, Ruysschaert JM, Raussens V (2009) Antiparallel beta-sheet: a signature structure of the oligomeric amyloid beta-peptide. Biochem J 421(3):415–423. doi: 10.1042/BJ20090379
Chromy BA, Nowak RJ, Lambert MP, Viola KL, Chang L, Velasco PT, Jones BW, Fernandez SJ, Lacor PN, Horowitz P, Finch CE, Krafft GA, Klein WL (2003) Self-assembly of Abeta(1–42) into globular neurotoxins. Biochemistry 42(44):12749–12760. doi: 10.1021/bi030029q
Ahmed M, Davis J, Aucoin D, Sato T, Ahuja S, Aimoto S, Elliott JI, Van Nostrand WE, Smith SO (2010) Structural conversion of neurotoxic amyloid-beta(1–42) oligomers to fibrils. Nat Struct Mol Biol 17(5):561–567. doi: 10.1038/nsmb.1799
Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem. 75(333 – 66. doi: 10.1146/annurev.biochem.75.101304.123901
Petkova AT, Leapman RD, Guo Z, Yau WM, Mattson MP, Tycko R (2005) Self-propagating, molecular-level polymorphism in Alzheimer's beta-amyloid fibrils. Science 307(5707):262–265. doi: 10.1126/science.1105850
Garvey M, Tepper K, Haupt C, Knupfer U, Klement K, Meinhardt J, Horn U, Balbach J, Fandrich M (2011) Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Abeta peptide. Biochem Biophys Res Commun 409(3):385–388. doi: 10.1016/j.bbrc.2011.04.141
Brender JR, Krishnamoorthy J, Sciacca MF, Vivekanandan S, D'Urso L, Chen J, La Rosa C, Ramamoorthy A (2015) Probing the sources of the apparent irreproducibility of amyloid formation: drastic changes in kinetics and a switch in mechanism due to micellelike oligomer formation at critical concentrations of IAPP. J Phys Chem B 119(7):2886–2896. doi: 10.1021/jp511758w
Ladiwala AR, Litt J, Kane RS, Aucoin DS, Smith SO, Ranjan S, Davis J, Van Nostrand WE, Tessier PM (2012) Conformational differences between two amyloid beta oligomers of similar size and dissimilar toxicity. J Biol Chem 287(29):24765–24773. doi: 10.1074/jbc.M111.329763
LeVine H 3rd (2004) Alzheimer's beta-peptide oligomer formation at physiologic concentrations. Anal Biochem 335(1):81–90. doi: 10.1016/j.ab.2004.08.014
Lee YH, Chatani E, Sasahara K, Naiki H, Goto Y (2009) A comprehensive model for packing and hydration for amyloid fibrils of beta2-microglobulin. J Biol Chem 284(4):2169–2175. doi: 10.1074/jbc.M806939200
Carulla N, Caddy GL, Hall DR, Zurdo J, Gairi M, Feliz M, Giralt E, Robinson CV, Dobson CM (2005) Molecular recycling within amyloid fibrils. Nature 436(7050):554–558. doi: 10.1038/nature03986
Okumura H, Itoh SG (2014) Amyloid fibril disruption by ultrasonic cavitation: nonequilibrium molecular dynamics simulations. J Am Chem Soc 136(30):10549–10552. doi: 10.1021/ja502749f
Barati AH, Mokhtari-Dizaji M, Mozdarani H, Bathaie Z, Hassan ZM (2007) Effect of exposure parameters on cavitation induced by low-level dual-frequency ultrasound. Ultrason Sonochem 14(6):783–789. doi: 10.1016/j.ultsonch.2006.12.016
Morch KA (2015) Cavitation inception from bubble nuclei. Interface Focus 5(5):20150006. doi: 10.1098/rsfs.2015.0006
Ghahghaei A, Bathaie SZ, Kheirkhah H, Bahraminejad E (2013) The protective effect of crocin on the amyloid fibril formation of Abeta42 peptide in vitro. Cell Mol Biol Lett 18(3):328–339. doi: 10.2478/s11658-013-0092-1
Stine WB, Jungbauer L, Yu C, LaDu MJ (2011) Preparing synthetic Abeta in different aggregation states. Methods Mol Biol 670:13–32. doi: 10.1007/978-1-60761-744-0_2
Chen ZA, Wang JL, Liu RT, Ren JP, Wen LQ, Chen XJ, Bian GX (2009) Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells. Cytotechnology 60(1–3):125–132. doi: 10.1007/s10616-009-9226-8
Gade Malmos K, Blancas-Mejia LM, Weber B, Buchner J, Ramirez-Alvarado M, Naiki H, Otzen D (2017) ThT 101: a primer on the use of thioflavin T to investigate amyloid formation. Amyloid 24(1):1–16. doi: 10.1080/13506129.2017.1304905
LeVine H 3 (1999) rd Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol. 309(274 – 84. doi: 10.1016/s0076-6879(99)09020-5
Bolognesi B, Kumita JR, Barros TP, Esbjorner EK, Luheshi LM, Crowther DC, Wilson MR, Dobson CM, Favrin G, Yerbury JJ (2010) ANS binding reveals common features of cytotoxic amyloid species. ACS Chem Biol 5(8):735–740. doi: 10.1021/cb1001203
Petkova AT, Yau WM, Tycko R (2006) Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils. Biochemistry 45(2):498–512. doi: 10.1021/bi051952q
Petkova AT, Ishii Y, Balbach JJ, Antzutkin ON, Leapman RD, Delaglio F, Tycko R (2002) A structural model for Alzheimer's beta -amyloid fibrils based on experimental constraints from solid state NMR. Proc Natl Acad Sci U S A 99(26):16742–16747. doi: 10.1073/pnas.262663499
Yu L, Edalji R, Harlan JE, Holzman TF, Lopez AP, Labkovsky B, Hillen H, Barghorn S, Ebert U, Richardson PL, Miesbauer L, Solomon L, Bartley D, Walter K, Johnson RW, Hajduk PJ, Olejniczak ET (2009) Structural characterization of a soluble amyloid beta-peptide oligomer. Biochemistry 48(9):1870–1877. doi: 10.1021/bi802046n
Xia W, Zhang J, Kholodenko D, Citron M, Podlisny MB, Teplow DB, Haass C, Seubert P, Koo EH, Selkoe DJ (1997) Enhanced production and oligomerization of the 42-residue amyloid beta-protein by Chinese hamster ovary cells stably expressing mutant presenilins. J Biol Chem 272(12):7977–7982. doi: 10.1074/jbc.272.12.7977
Walsh DM, Tseng BP, Rydel RE, Podlisny MB, Selkoe DJ (2000) The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain. Biochemistry 39(35):10831–10839. doi: 10.1021/bi001048s
Yanagisawa D, Taguchi H, Morikawa S, Kato T, Hirao K, Shirai N, Tooyama I (2015) Novel curcumin derivatives as potent inhibitors of amyloid beta aggregation. Biochem Biophys Rep. 4(357 – 68. doi: 10.1016/j.bbrep.2015.10.009
Gellermann GP, Byrnes H, Striebinger A, Ullrich K, Mueller R, Hillen H, Barghorn S (2008) Abeta-globulomers are formed independently of the fibril pathway. Neurobiol Dis 30(2):212–220. doi: 10.1016/j.nbd.2008.01.010
Soreghan B, Kosmoski J, Glabe C (1994) Surfactant properties of Alzheimer's A beta peptides and the mechanism of amyloid aggregation. J Biol Chem 269(46):28551–28554
Li L (2008) Mechanism of Early Stage Abeta Amyloid Formation. Case Western Reserve University

No competing interests reported.

Download PDF

Journal Publication

published 27 Aug, 2023

Read the published version in The Protein Journal →

Editorial decision: Major revision
23 Nov, 2022
Reviewers agreed at journal
10 Nov, 2022
Reviews received at journal
04 Oct, 2022
Reviewers agreed at journal
03 Oct, 2022
Reviewers invited by journal
01 Oct, 2022
Editor assigned by journal
27 Sep, 2022
Submission checks completed at journal
27 Sep, 2022
First submitted to journal
27 Sep, 2022

You are reading this latest preprint version

The Effect of Ultrasonication on the Fibrillar/ Oligomeric Structures of Aβ 1-42 at Different Concentrations

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials & Methods

Materials

Methods

Results

The TEM analyzes

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1