Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

Ramin Zadali; Ebrahim  Rostampour Ghareghozloo; Mohammad Ramezani; Vahid Hassani; Yasin Rafiei; Shiva  Mohammadi Chiyaneh; Ali Akbar Meratan

doi:10.3791/59883

JoVE Journal > Biochemistry

生物化学

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

Published: September 28, 2019

doi:

10.3791/59883

Ramin Zadali¹, Ebrahim Rostampour Ghareghozloo*¹, Mohammad Ramezani*¹, Vahid Hassani*¹, Yasin Rafiei*¹, Shiva Mohammadi Chiyaneh¹, Ali Akbar Meratan¹

¹Department of Biological Sciences,Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, 45137-66731, Iran

Summary

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

Abstract

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from α-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Introduction

Amyloid-related disorders, known as amyloidoses, constitute a large group of diseases defined by the appearance of insoluble protein deposits in different tissues and organs¹^,². Among them, neurodegenerative disorders are the most frequently forms in which protein aggregates appear in the central or peripheral nervous system². Although a number of mechanisms have been proposed to be involved in the toxicity of amyloid aggregates³, a growing body of evidence points to cell membrane disruption and permeabilization as the primary mechanism of amyloid pathology⁴^,⁵. In addition to plasma membrane, internal organelles (i.e., mitochondria) may also be affected.

Interestingly, emerging evidence suggests that mitochondrial dysfunction plays a critical role in the pathogenesis of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases⁶^,⁷. In accordance with this issue, numerous reports have indicated binding and accumulation of amyloid β-peptide, α-synuclein, Huntingtin, and ALS-linked mutant SOD1 proteins to mitochondria⁸^,⁹^,¹⁰^,¹¹. The mechanism of membrane permeabilization by amyloid aggregates is thought to occur either through formation of discrete channels (pores) and/or through a nonspecific detergent-like mechanism⁵^,¹²^,¹³. It is noteworthy that most of these conclusions have been based on reports involving phospholipid model systems, and studies directly targeting the events occurring in biological membranes are rare. Clearly, these artificial lipid bilayers do not necessarily reflect the intrinsic properties of biological membranes, including those of mitochondria, which are heterogeneous structures and composed of a wide variety of phospholipids and proteins.

In the present study, mitochondria isolated from rat brains are used as an in vitro biological model to examine the destructive effects of amyloid fibrils arising from α-synuclein (as an amyloidogenic protein), bovine insulin (as a model peptide showing significant structural homology with human insulin involved in injection-localized amyloidosis), and hen egg white lysozyme (HEWL; as a common model protein for study of amyloid aggregation). The interactions and possible damage of mitochondrial membranes induced by amyloid fibrils are then investigated by observing the release of mitochondrial malate dehydrogenase (MDH) (located in the mitochondrial matrix) and mitochondria reactive oxygen species (ROS) enhancement.

Protocol

All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of Medical Sciences of Tehran University. Maximal efforts were made to minimize suffering and detrimental effects to the rats by sharpening the guillotine blades and applying resolute and swift movements of the blade.

1. Brain homogenization and mitochondrial isolation

NOTE: All reagents for mitochondrial isolation were prepared according to Sims and Anderson¹⁴.

Preparation of buffers for mitochondrial isolation
1. Prepare a 100 mM Tris-HCl solution: weigh 0.605 g of Tris-HCl and dissolve it in approximately 40 mL of deionized water (DW) in a beaker. Transfer the solution to a 50 mL volumetric flask and increase the final volume to 50 mL by adding DW.
2. Prepare a 10 mM EDTA solution: weigh 0.202 g of EDTA and dissolve it in approximately 40 mL of DW in a beaker. Transfer the solution to a 50 mL volumetric flask and increase the final volume to 50 mL by adding DW.
  NOTE: Both solutions can be stored at 4 °C for at least 4 weeks.
3. Prepare a Tris-HCl/EDTA/sucrose stock solution: add 30 mL of 100 mM Tris-HCl and 30 mL of 10 mM EDTA in a beaker. Weigh 32.86 g of sucrose and add it to the beaker while stirring with a magnetic stirrer. When the sucrose is dissolved, transfer the solution to a 100 mL volumetric flask and increase the final volume to 100 mL by adding DW.
  NOTE: This solution can be store at 4 °C for up to 3 days.
4. Prepare an isolation buffer (IB): add 100 mL of Tris-HCl/EDTA/sucrose stock to approximately 150 mL of DW. Adjust the pH to 7.4 by adding 0.1 M HCl. Increase the final volume to 300 mL by adding DW.
5. Prepare 15% (v/v) density gradient medium with IB by adding 1.5 mL of density gradient medium to 8.5 mL of cold IB.
6. Prepare 10 mg/mL bovine serum albumin (BSA) solution: weigh 20 mg of fatty acid-free BSA and dissolve it in approximately 1.5 mL DW in a 2 mL vial. Increase the final volume to 2 mL by adding DW and store it on ice until use.
  NOTE: Freshly prepare the solutions from steps 1.1.4–1.1.6 on the day of mitochondrial isolation and store them on ice until use.
Isolation of rat brain
NOTE: Decapitation and brain removal of male rats (150–200 g) were performed according to Sims and Anderson¹⁴.
1. Put a 30 mL beaker in an ice bucket and add 10 mL of cold IB to the beaker.
2. Decapitate the rat with a small animal guillotine and remove the brain from the skull within 1 min of decapitation to limit deterioration of mitochondrial properties.
3. Rapidly transfer the brain to the beaker containing cold IB.
Brain homogenization and preparation of mitochondria
NOTE: Mitochondrial fractions were isolated according to the protocol described by Sims and Anderson¹⁴, with some modifications as described previously¹⁵. It is important to work quickly and keep everything on ice throughout the procedure.
1. Wash the tissue 2x with 30 mL of IB, transfer to a beaker containing cold IB, and finely mince the brain with scissors.
2. Add 10 volumes of pre-cooled IB (about 10 mL of IB per rat brain).
3. Transfer the tissue suspension to a 20 mL cold Dounce homogenizer.
4. Homogenize the tissue pieces using nine up-and-down strokes with a motorized pestle.
  NOTE: Leave the mixture on ice for approximately 30 s after each set of three homogenization strokes to ensure that the homogenate remains cold.
5. Transfer the homogenate to a pre-chilled 10 mL centrifuge tube and centrifuge at 1,300x g and 4 °C for 3 min.
6. Carefully decant the supernatant and transfer it to a pre-chilled 10 mL centrifuge tube and centrifuge at 21,000x g at 4 °C for 10 min.
7. Discard the supernatant and resuspend the pellet in a cold 15% density gradient medium solution (5 mL for each brain) by gently stirring the mixture with a pipette.
8. Centrifuge in a fixed-angle rotor at 30,700x g at 4 °C for 5 min using slow acceleration (45 s from 0 rpm to 500 rpm followed by normal acceleration) and deceleration (no brakes). This should produce two distinct bands of material (Figure 1A, left).
9. Using a Pasteur pipette, remove the sharp band of material accumulated at the top of the gradient, which mostly contains myelin. Then, remove the density gradient medium solution overlying the material (in band 2) as much as possible without losing any of the enriched mitochondrial fraction in band 2.
  NOTE: The band 2 contains both the synaptic and non-synaptic mitochondria.
10. Add 8 mL of IB to the mitochondrial fraction while gently stirring the mixture with a pipette.
11. Centrifuge at 16,700x g at 4 °C for 10 min and carefully remove the supernatant, leaving the bottom loose pellet undisturbed.
12. Add 1 mL of 10 mg/mL fatty acid-free BSA to the centrifuge tube while gently stirring the mixture with the tip of a pipette. Increase the final volume to 5 mL per brain by adding IB.
13. Centrifuge at 6,900x g at 4 °C for 10 min, which should produce a firm pellet (Figure 1A, right).
14. Decant the supernatant and gently resuspend the mitochondrial pellet in IB, aliquot them into 0.5 mL tubes, and store in liquid nitrogen until use.

2. Protein concentration determination

NOTE: Protein concentration is measured using the method of Lowry et al.¹⁶.

Preparation of solutions for Lowry assay
1. Prepare solution A: weigh 0.4 g of NaOH and 2 g of Na₂CO₃ and dissolve in 80 mL of DW, then transfer the solution to a 100 mL volumetric flask and increase the volume to 100 mL by adding DW.
2. Prepare solution B: weigh 0.1 g of potassium sodium tartrate and 0.05 g of CuSO₄ and dissolve in 8 mL of DW, then transfer the solution to a 10 mL volumetric flask and increase the volume to 10 mL by adding DW.
  NOTE: Both solutions A and B can remain stable at 4 °C for up to 6 months.
3. Prepare solution C: Add 0.5 mL of folin solution to 7.5 mL of DW.
  NOTE: Prepare solution C fresh and keep away from light until use.
4. Prepare BSA standards with final concentrations of 0, 20, 40, 60, 80, 100, and 120 µg/mL by combining 0, 20, 40, 60, 80, 100, and 120 µL of 1 mg/mL BSA, respectively, with enough DW to make 1000 µL of solution.
Protein concentration measurement
NOTE: For greater accuracy, run this step in triplicate.
1. Add 50 µL of standard solutions and mitochondrial homogenate to each well of a 96 well plate, followed by the addition of 45 µL of solution A. Then, incubate the plate for 10 min in a warm water bath set at 50 °C.
2. Add 5 µL of solution B and incubate the plate for 10 min in dark at room temperature (RT).
3. Add 150 µL of solution C and incubate the plate for 10 min in a warm water bath set at 50 °C.
4. Load the plate into a plate reader and record the absorbance values for standards and mitochondrial suspension at 650 nm. Then, using a calibration curve, calculate the protein content of the mitochondria.

3. Mitochondrial membrane integrity determination

NOTE: Mitochondrial membrane integrity is confirmed by measuring malate dehydrogenase (MDH) activity in isolated mitochondria before and after membrane disruption by Triton X-100.

Dilute mitochondrial homogenate to 1 mg/mL with cold IB and placed in two 1.5 mL tubes (typically 195 μL of mitochondria per tube).
Add 5 µL of 20% (v/v) Triton X-100 (diluted with DW) to one tube (as a positive control for maximum enzyme activity) and 5 µL of DW to another tube (for control) followed by mixing with a stirrer.
Incubate the tubes for 10 min in a warm water bath set at 30 °C.
Pellet mitochondria by centrifugation of tubes in a fixed-angle rotor at 16,000 x g and 4 °C for 15 min.
Carefully collect the resulting supernatants for assaying the activity of mitochondrial MDH using a standard spectrophotometric assay described in the following section.
Calculate the integrity of mitochondrial membrane as follows:

4. MDH activity determination

NOTE: MDH activity was measured spectrophotometrically as described by Sottocasa et al.¹⁷.

Preparation of solutions for MDH activity assay
1. Prepare a 50 mM Tris-HCl solution (pH = 7.5): prepare a 7.9 mg/mL solution in DW using Tris-HCl, and adjust the pH to 7.5 at 25 °C with 1.0 M NaOH.
2. Prepare a 50 mM oxaloacetate solution: prepare a 6.6 mg/mL solution using oxaloacetate in Tris-HCl.
  NOTE: This solution is not stable once in solution and should be prepared immediately prior to use.
3. Prepare a 10 mM β-NADH solution: prepare a 7.81 mg/mL solution using β-NADH in Tris-HCl.
MDH activity determination
NOTE: Final assay concentrations in a 1.0 mL reaction mixture are 50 mM Tris-HCl, 5 mM oxaloacetate, and 0.1 mM β-NADH.
1. Set the spectrophotometer to 25 °C and 340 nm. Pipette 890 µL Tris-HCl buffer, 100 µL of oxaloacetate solution, and 10 µL of NADH solution to a blank cuvette and 880 µL Tris-HCl buffer, 100 µL of oxaloacetate solution, and 10 µL of NADH solution into a sample cuvette.
2. Incubate cuvettes in the spectrophotometer for 3–4 min and reference against blank.
3. Then add 10 µL of mitochondrial homogenate (1 mg/mL) to the sample cuvette, immediately mix by inversion, and record decreases in absorbance due to NADH oxidation at 340 nm for 1 min.

5. In vitro α-synuclein, bovine insulin, and HEWL fibril formation

Protein preparation
1. α-synuclein:
  NOTE: Expression and purification of recombinant α-synuclein is performed as described by Hoyer et al.¹⁸ with some modifications, and the purity of α-synuclein is confirmed by SDS-PAGE.
  1. Dialyze purified α-synuclein overnight against phosphate-buffered saline (PBS).
  2. Determine the protein concentration using an extinction coefficient of 5600 M^-1cm^-1 at 275 nm¹⁹.
  3. Aliquots protein in 1.5 mL tubes and store at -80 °C until use.
2. Bovine insulin and HEWL:
  1. Provide both bovine insulin and HEWL.
  2. Dissolve each protein in 50 mM glycine buffer (pH = 1.6; adjust the pH with HCl).
  3. Determine bovine insulin and HEWL concentration using an extinction coefficient of 1.0 and 2.63 for 1.0 mg/mL at 276 nm and 280 nm, respectively²⁰^,²¹.
Amyloid fibrillation induction
1. Preparation of solutions for amyloid fibrillation:
  1. Prepare thioflavin T (ThT) buffer: weigh 0.3 g of NaH₂PO₄ and dissolve it in 80 mL of DW, adjust the pH to 6.5, and increase the volume to 100 mL by adding DW.
  2. Prepare ThT stock solution (5 mM): weigh 1.6 mg of ThT and dissolve it in 1 mL of ThT buffer, pass through a 0.22 μm filter paper, and keep away from light at 4 °C until use.
    NOTE: This solution can be store at 4 °C for up to 4 weeks.
  3. Prepare ThT solution (1 mM): add 200 µL of ThT stock solution (5 mM) to 800 µL of ThT buffer.
    NOTE: This solution can be stored at 4 °C for up to 1 week.
2. In vitro α-synuclein amyloid fibril formation:
  1. Add aliquots (294 µL) of protein solution (200 μM) and 6 µL of ThT solution (1 mM) to 1.5 mL tubes followed by stirring.
  2. Incubate the tubes in a thermomixer at 37 °C under constant stirring at 800 rpm for 4 days.
  3. Take aliquots (10 µL) of incubated samples after regular time intervals and add 490 µL of ThT buffer, mixed thoroughly, and incubate for 5 min at RT.
  4. Set excitation and emission slit widths as 5 nm and 10 nm, respectively, and measure ThT fluorescence by excitation at 440 nm and emission at 485 nm using a fluorescence spectrophotometer.
3. In vitro bovine insulin amyloid fibril formation:
  1. Add 637 µL of protein solution (250 µM) to 1.5 mL tube. Then, add 13 µL of ThT solution (1 mM) followed by stirring.
  2. Add aliquots (200 µL) of protein solution (250 µM) containing 20 µM ThT to each well of a clear-bottomed 96 well plate and seal the plate with crystal clear sealing tape.
  3. Load the plate into a fluorescence plate reader and incubate at 57 °C without agitation.
  4. Measure ThT fluorescence at 30 min intervals, with excitation at 440 nm and emission at 485 nm, for 12 h.
    NOTE: Shake the plate for 5 s before each measurement.
4. In vitro HEWL amyloid fibril formation:
  1. Add aliquots (200 µL) of protein solution )1 mM) containing 20 µM ThT to each well of a clear-bottomed 96 well plate and seal the plate with crystal clear sealing tape.
  2. Load the plate into a fluorescence plate reader and incubate at 57 °C without agitation.
  3. Measure ThT fluorescence at 2 h intervals, with excitation at 440 nm and emission at 485 nm, for 4 days.
    NOTE: For all three proteins, amyloid fibril formation is confirmed by atomic force microscopy (Figure 2B).

6. Treatment of mitochondria with amyloid fibrils, MDH release assay, and ROS measurement

Incubation of isolated mitochondria with amyloid fibrils
1. Turn on centrifuge and warm water bath and set to 4 °C and 30 °C, respectively.
2. Using IF, dilute mitochondrial homogenate to a final concentration of 1 mg/mL.
3. Prepare two series of 1.5 mL tubes containing mitochondrial homogenates (one series for MDH release assay and another for mitochondrial ROS measurement).
4. Add aliquots of fresh or amyloid fibrils of α-synuclein, bovine insulin, or HEWL (at final concentrations of 5 µM, 10 µM, 20 µM, and 25 µM; use PBS or glycine buffer as a control) to mitochondrial homogenate (final volume = 200 µL) (see Table 1, Table 2, and Table 3) followed by gently stirring the solution with a pipette.
  NOTE: For the MDH release assay, use Triton X-100 (at a final concentration of 0.5% [v/v]) as a positive control for maximum enzyme release.
5. Incubate tubes containing mitochondrial suspensions for 30 min in warm water bath set to 30 °C.
6. Measure mitochondrial MDH release and ROS content as outlined in sections 6.2 and 6.3.
Mitochondrial MDH release assay
1. Centrifuge the incubated mitochondrial homogenates at 16,000 x g for 15 min, then carefully collect the resulting supernatants for assaying the activity of mitochondrial MDH as described in section 4.
2. Calculate the release of MDH as a fraction of the maximum effect (Triton X-100) as follows:

Mitochondrial ROS measurement
NOTE: Mitochondrial ROS content is determined with the oxidation sensitive fluorogenic precursor dihydrodichlorocarboxyfluorescein diacetate (DCFDA)²².
1. Prepare solutions for mitochondrial ROS measurement. Prepare a 50 µM DCFDA solution by dissolving in methanol (prepare fresh) and a 200 mM succinate solution by dissolving in DW.
2. Pipette 191 µL of incubated mitochondrial homogenate to each well of a 96 well plate and add 4 µL of 50 µM DCFDA (1 µM final concentration) and 5 µL of 200 mM succinate (5 mM final concentration).
3. Incubate the plate for 30 min in a warm water bath set at 30 °C while gently stirring.
4. Load the plate into a fluorescence plate reader and measure fluorescence intensity, with excitation at 485 nm and emission at 530 nm.

7. Statistical analysis

Perform all experiments at least 2x or 3x with triplicate assays and conduct appropriate statistical tests. Here, the results are presented as mean ± SD, and a Student's paired t-test was utilized to calculate statistical significance. P-values less than 0.01 and 0.05 were considered statistically significant (*p < 0.05; **p < 0.01).

Representative Results

The protocol describes a model for studying the interactions of amyloid fibril with rat brain mitochondria as an in vitro biological model. For mitochondrial preparation, 15% (v/v) density gradient medium was used to remove myelin as major contamination of brain tissue¹⁴. As shown in Figure 1A, centrifugation at 30,700 x g produced two distinct bands of material, myelin (as the major component of band 1) and band 2, which contains enriched mitochondrial fraction.

By modifying the protocol described by Sims and Anderson¹⁴, a mitochondrial suspension containing both synaptic and non-synaptic mitochondria was prepared. For preparation of pure non-synaptic mitochondria, Sims and Anderson previously detailed the protocol¹⁴. At the final step of centrifugation,10 mg/mL fatty acid-free BSA was added to obtain a firm mitochondrial pellet (Figure 1A). Mitochondrial membrane integrity was assessed by measuring MDH activity in isolated mitochondria before and after membrane disruption and enzyme release by Triton X-100, as described in the protocol.

It was typically found that mitochondrial preparations were about 93% intact (Figure 1B). A ThT fluorescence assay was carried out to monitor the growth of amyloid fibrils. As shown in Figure 2A, the curve for amyloid fibrillation of three proteins showed a typical sigmoidal pattern, which is in line with nucleation-dependent polymerization models of these proteins as reported previously²³^,²⁴^,²⁵. The highest ThT fluorescence emission for α-synuclein, bovine insulin, and HEWL was observed after 96 h, 12 h, and 96 h, respectively, suggesting formation of amyloid fibrils (Figure 2A). This was further confirmed by AFM measurement.

As illustrated in Figure 2B, well-defined mature fibrils with typical amyloid morphology were observed for three proteins incubated under amyloidogenic conditions. Interactions and possible damage and permeabilization of mitochondrial membranes by amyloid fibrils were investigated. This was accomplished by monitoring the release of mitochondrial MDH and mitochondrial ROS measurement upon the addition of α-synuclein, bovine insulin, or HEWL amyloid fibrils to mitochondrial suspensions and following the procedures outlined. The preliminary experiments showed that the presence of ThT (up to 3 µM) had no effect on MDH release and mitochondrial ROS content (data not shown).

As shown in Figure 3, substantial release of MDH was observed upon the addition of α-synuclein amyloid fibrils to mitochondria in a concentration-dependent manner. Although slight release was detected upon the addition of bovine insulin amyloid fibrils, HEWL fibrils were found to be ineffective (Figure 3). While no significant enhancement in mitochondrial ROS content was observed upon the addition of bovine insulin or HEWL amyloid fibrils, treatment with α-synuclein fibrils led to a considerable increase in ROS content of brain mitochondria in a concentration-dependent manner (Figure 4).

Figure 1: Mitochondrial isolation and membrane integrity determination. (A) Left: typical appearance of a centrifuge tube after resuspending the pellet in cold 15% density gradient medium solution following by centrifugation. Myelin is the major component of band 1, which accumulates at the top. Band 2 contains the highly enriched mitochondrial fraction. Right: a firm pellet was produced after the addition of 10 mg/mL fatty-acid-free BSA and centrifugation at 6900 x g. (B) Mitochondrial membrane integrity was determined by measuring MDH activity in isolated mitochondria before and after membrane disruption by Triton X-100. Please click here to view a larger version of this figure.

Figure 2: Amyloid fibrillation of α-synuclein, bovine insulin, and HEWL. (A) Kinetics of amyloid fibril formation indicated by increasing fluorescence intensity of ThT at 485 nm. (B) AFM images of three proteins are shown, which were incubated under amyloidogenic conditions for regular times. Scale bars represent 500 nm. Please click here to view a larger version of this figure.

Figure 3: Mitochondrial MDH release upon interaction with monomer and amyloid fibril of α-synuclein, bovine insulin, and HEWL. Release is expressed as the percentage of the maximum observed upon treatment with 0.5% (v/v) Triton X-100. Each value represents mean ± SD (n = 3; *p < 0.05, **p < 0.01). Please click here to view a larger version of this figure.

Figure 4: Mitochondrial ROS content upon interaction with monomer and amyloid fibril of α-synuclein, bovine insulin, and HEWL. The data are expressed as the percentage of values in control mitochondria. Each value represents the mean ± SD (n = 3; *p < 0.05; **p < 0.01). Please click here to view a larger version of this figure.

	Mitochondrial homogenate (1 mg/mL)	α-synuclein monomer (200 µM)	α-synuclein fibril (200 µM)	Isolation buffer	PBS	Triton x-100 20% (v/v)
Control	175 µL	0	0	25 µL	0	0
PBS	175 µL	0	0	0	25 µL	0
Monomer 25 µM	175 µL	25 µL	0	0	0	0
Fibril 5 µM	175 µL	0	5 µL	20 µL	0	0
Fibril 10 µM	175 µL	0	10 µL	15 µL	0	0
Fibril 20 µM	175 µL	0	20 µL	5 µL	0	0
Fibril 25 µM	175 µL	0	25 µL	0	0	0
Positive control	175 µL	0	0	20 µL	0	5 µL

Table 1: Treatment of mitochondria with various concentrations of α-synuclein amyloid fibrils.

	Mitochondrial homogenate (1 mg/mL)	Bovine insulin monomer (250 µM)	Bovine insulin fibril (250 µM)	Isolation buffer	Glycine buffer	Triton x-100 20% (v/v)
Control	175 µL	0	0	25 µL	0	0
Glycine buffer	175 µL	0	0	0	25 µL	0
Monomer 25 µM	175 µL	20 µL	0	5 µL	0	0
Fibril 5 µM	175 µL	0	4 µL	21 µL	0	0
Fibril 10 µM	175 µL	0	8 µL	17 µL	0	0
Fibril 20 µM	175 µL	0	16 µL	9 µL	0	0
Fibril 25 µM	175 µL	0	20 µL	5 µL	0	0
Positive control	175 µL	0	0	20 µL	0	5 µL

Table 2: Treatment of mitochondria with various concentrations of bovine insulin amyloid fibrils.

	Mitochondrial homogenate (1 mg/mL)	HEWL monomer (1 mM)	HEWL fibril (1 mM)	Isolation buffer	Glycine buffer	Triton x-100 20% (v/v)
Control	175 µL	0	0	25 µL	0	0
Glycine buffer	175 µL	0	0	0	25 µL	0
Monomer 25 µM	175 µL	5 µL	0	20 µL	0	0
Fibril 5 µM	175 µL	0	1 µL	24 µL	0	0
Fibril 10 µM	175 µL	0	2 µL	23 µL	0	0
Fibril 20 µM	175 µL	0	4 µL	21 µL	0	0
Fibril 25 µM	175 µL	0	5 µL	20 µL	0	0
Positive control	175 µL	0	0	20 µL	0	5 µL

Table 3: Treatment of mitochondria with various concentrations of HEWL amyloid fibrils.

Discussion

A wealth of experimental results supports the hypothesis that the cytotoxicity of fibrillar aggregates is significantly associated with their ability to interact with and permeabilize biological membranes⁴^,⁵. However, most of the data are based on artificial lipid bilayers that do not necessarily reflect the intrinsic properties of biological membranes, which are heterogeneous structures with a wide variety of phospholipids and proteins. Here, using brain mitochondria as an in vitro biological membrane, a model for studying fibrillar aggregates cytotoxicity at the membrane level is described.

During the experiment, it is important to work quickly and keep everything on ice to prepare functionally active mitochondria with intact membranes. Moreover, mitochondrial concentration (based on total protein measurement) is an important factor affecting membrane-amyloid fibril interaction; thus, it must be keep constant throughout the protocol. In the present study, mitochondrial preparations contained both synaptic and non-synaptic mitochondria. For pure non-synaptic mitochondrial preparation, 40%, 23%, and 15% (v/v) density gradient medium should be used, as described by Sims and Anderson¹⁴. Because the mitochondria is an organelle with a well-characterized membrane, it may serve as an extremely useful biological model system in molecular studies related to the mechanisms of amyloid cytotoxicity at the membrane level (especially relating to neurodegenerative diseases). This protocol allows investigation of the interactions and extent of mitochondrial membranes permeabilization by looking into the release of various molecules and enzymes located in different compartments (i.e., those embedded in the outer and inner membranes and those located in the intermembrane space or mitochondrial matrix).

After mitochondrial membrane integrity confirmation (Figure 1B) and amyloid fibril formation (Figure 2), the interaction, damage, and permeabilization of mitochondrial membranes upon the addition of α-synuclein, bovine insulin, and HEWL amyloid fibrils was investigated. The results demonstrated clear differences in the extent of membrane permeabilization and mitochondrial ROS enhancement induced by amyloid fibrils (Figure 3 and Figure 4), suggesting variations in the capability of various amyloid fibrils to interact with and damage the mitochondrial membranes.

For membrane permeabilization initiation, amyloid fibrils must reach the mitochondrial surface. It has been shown that association, localization, and insertion of proteins to charged membranes is dependent on nonspecific electrostatic interactions and the hydrophobic nature of proteins²⁶^,²⁷^,²⁸. In regard to amyloid toxicity, a growing body of evidence strongly implicates a key role of protein aggregate surface hydrophobicity in their cytotoxicity²⁹^,³⁰. Although HEWL bears a net positive charge over a broad range of pH levels and has a high affinity for anionic and neutral phospholipids³¹ (e.g., the mitochondrial membrane²³), no mitochondrial membrane permeabilization and ROS enhancement were observed (Figure 3 and Figure 4). An explanation for this observation may be related to a reduction of surface hydrophobicity of HEWL fibrils due to lateral fibril-fibril interactions²³^,³², leading to loss of the ability to cause membrane damage.

For bovine insulin, despite a significant exposure of hydrophobic regions in the course of fibril formation²², a slight enzyme release was observed upon the addition of 25 µM amyloid fibrils (Figure 3) without significant effects on mitochondrial ROS content (Figure 4). As both brain mitochondria and bovine insulin fibrils bear a negative zeta potential at their surfaces²⁴, it is suggested that repulsive forces between mitochondrial membrane and bovine insulin amyloid fibrils may account for their inability to effectively interact with and damage mitochondrial membranes²⁴. The highest permeabilization and mitochondrial ROS increments were observed upon addition of α-synuclein fibrils (Figure 3 and Figure 4).

This observation may be attributed to the high capacity of α-synuclein for interacting with biological membranes that have negatively charged surfaces³³^,³⁴, such as mitochondria. To this regard, some studies have shown specific binding and importation of α-synuclein into mitochondria, where they become predominantly associated with the inner membrane³⁵. Interestingly, Ghio et al. reported that binding, mediated by cardiolipin (a phospholipid uniquely enriched in inner mitochondrial membrane), of α-synuclein may be an important mechanism for association and disruption of mitochondrial membranes³⁶.

Interestingly, there is clear evidence for binding and accumulation of various amyloidogenic peptides and proteins including amyloid β-peptide, α-synuclein, Huntingtin, and ALS-linked mutant SOD1 to mitochondria³⁵^,³⁷^,³⁸^,³⁹. Moreover, previous studies have demonstrated that amyloidogenic assemblies of amyloid β (25–35)¹⁵ and SOD 1⁴⁰ have the ability to interact with and cause mitochondrial membrane permeabilization. According to the literature, it is suggested that amyloid fibrils are too large to cross the membrane; therefore, average fibril size should not be a factor in the apparent lack of effects induced by bovine insulin and HEWL amyloid fibrils. Although further studies are needed to elucidate the detailed mechanisms involved, it is proposed that the biophysical features of amyloid fibrils (i.e., surface charge, hydrophobicity, and possessing a specific sequence-targeting mitochondrial membrane) may play an important role in their capacity to interact with and damage biological membranes.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by grants from the Research Council of the Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, Iran.

Materials

2′,7′-Dichlorodihydrofluorescein diacetate	Sigma	35845
Ammonium sulfate	Merck	1012171000
Black 96-well plate	Corning
Black Clear-bottomed 96-well plate	Corning
Bovine insulin	Sigma	I6634
Bovine Serum Albumin (BSA)	Sigma	A2153
BSA essentially fatty acid-free	Sigma	A6003
Centrifuge	Sigma
Crystal clear sealing tape	Corning
CuSO4	Sigma	451657
Dialysis bag (cut off 2 KDa)	Sigma	D2272
Dounce homogenizer	Potter Elvehjem
EDTA	Sigma	E9884
Fluorescence plate reader	BioTek
Fluorescence spectrophotometer	Cary Eclipse VARIAN
Folin	Merck	F9252
Glycine	Sigma	G7126
Guillotine	Made in Iran
HCl	Merck	H1758
Hen Egg White Lysozyme (HEWL)	Sigma	L6876
Na2CO3	Sigma	S7795
NaH2PO4	Sigma	S7907
NaOH	Merck	S8045
Oxaloacetate	Sigma	O4126
Percoll	GE Healthcare
Phosphate Buffer Saline (PBS)	Sigma	CS0030
PMSF	Sigma	P7626
Potassium sodium tartrate	Sigma	217255
Quartz cuvette	Sigma
Spectrophotometer	analytik jena	SPEKOL 2000 model
Succinate	Sigma	S2378
Sucrose	Merck	1076871000
Thermomixer	Eppendorph
Thioflavin T	Sigma	T3516
Tris-HCl	Merck	1082191000
Triton X-100	Sigma	T9284
Tryptone	QUELAB
Water bath	Memmert
Yeast Extract	QUELAB
β-NADH	Sigma	N8129

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Zadali, R., Ghareghozloo, E. R., Ramezani, M., Hassani, V., Rafiei, Y., Chiyaneh, S. M., Meratan, A. A. Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils. J. Vis. Exp. (151), e59883, doi:10.3791/59883 (2019).