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

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.
<ol>
	<li>Preparation of buffers for mitochondrial isolation
	<ol>
		<li>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.</li>
		<li>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 &#176;C for at least 4 weeks.</li>
		<li>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 &#176;C for up to 3 days.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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&#8211;1.1.6 on the day of mitochondrial isolation and store them on ice until use.</li>
	</ol>
	</li>
	<li>Isolation of rat brain 
	NOTE: Decapitation and brain removal of male rats (150&#8211;200 g) were performed according to Sims and Anderson14.
	<ol>
		<li>Put a 30 mL beaker in an ice bucket and add 10 mL of cold IB to the beaker.</li>
		<li>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.</li>
		<li>Rapidly transfer the brain to the beaker containing cold IB.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Wash the tissue 2x with 30 mL of IB, transfer to a beaker containing cold IB, and finely mince the brain with scissors.</li>
		<li>Add 10 volumes of pre-cooled IB (about 10 mL of IB per rat brain).</li>
		<li>Transfer the tissue suspension to a 20 mL cold Dounce homogenizer.</li>
		<li>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.</li>
		<li>Transfer the homogenate to a pre-chilled 10 mL centrifuge tube and centrifuge at 1,300x&#160;g and 4 &#176;C for 3 min.</li>
		<li>Carefully decant the supernatant and transfer it to a pre-chilled 10 mL centrifuge tube and centrifuge at 21,000x g at 4 &#176;C for 10 min.</li>
		<li>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.</li>
		<li>Centrifuge in a fixed-angle rotor at 30,700x g at 4 &#176;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).</li>
		<li>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.</li>
		<li>Add 8 mL of IB to the mitochondrial fraction while gently stirring the mixture with a pipette.</li>
		<li>Centrifuge at 16,700x g at 4 &#176;C for 10 min and carefully remove the supernatant, leaving the bottom loose pellet undisturbed.</li>
		<li>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.</li>
		<li>Centrifuge at 6,900x g at 4 &#176;C for 10 min, which should produce a firm pellet (Figure 1A, right).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Protein concentration determination
NOTE: Protein concentration is measured using the method of Lowry et al.16.
<ol>
	<li>Preparation of solutions for Lowry assay
	<ol>
		<li>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.</li>
		<li>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 &#176;C for up to 6 months.</li>
		<li>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.</li>
		<li>Prepare BSA standards with final concentrations of 0, 20, 40, 60, 80, 100, and 120 &#181;g/mL by combining 0, 20, 40, 60, 80, 100, and 120 &#181;L of 1 mg/mL BSA, respectively, with enough DW to make 1000 &#181;L of solution.</li>
	</ol>
	</li>
	<li>Protein concentration measurement 
	NOTE: For greater accuracy, run this step in triplicate.
	<ol>
		<li>Add 50 &#181;L of standard solutions and mitochondrial homogenate to each well of a 96 well plate, followed by the addition of 45 &#181;L of solution A. Then, incubate the plate for 10 min in a warm water bath set at 50 &#176;C.</li>
		<li>Add 5 &#181;L of solution B and incubate the plate for 10 min in dark at room temperature (RT).</li>
		<li>Add 150 &#181;L of solution C and incubate the plate for 10 min in a warm water bath set at 50 &#176;C.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Dilute mitochondrial homogenate to 1 mg/mL with cold IB and placed in two 1.5 mL tubes (typically 195 &#956;L of mitochondria per tube).</li>
	<li>Add 5 &#181;L of 20% (v/v) Triton X-100 (diluted with DW) to one tube (as a positive control for maximum enzyme activity) and 5 &#181;L of DW to another tube (for control) followed by mixing with a stirrer.</li>
	<li>Incubate the tubes for 10 min in a warm water bath set at 30 &#176;C.</li>
	<li>Pellet mitochondria by centrifugation of tubes in a fixed-angle rotor at 16,000 x g and 4 &#176;C for 15 min.</li>
	<li>Carefully collect the resulting supernatants for assaying the activity of mitochondrial MDH using a standard spectrophotometric assay described in the following section.</li>
	<li>Calculate the integrity of mitochondrial membrane as follows: 
	<img alt="Equation 1" src="/files/ftp_upload/59883/59883equ01v4.jpg" /></li>
</ol>
4. MDH activity determination
NOTE: MDH activity was measured spectrophotometrically as described by Sottocasa et al.17.
<ol>
	<li>Preparation of solutions for MDH activity assay
	<ol>
		<li>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 &#176;C with 1.0 M NaOH.</li>
		<li>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.</li>
		<li>Prepare a 10 mM &#946;-NADH solution: prepare a 7.81 mg/mL solution using &#946;-NADH in Tris-HCl.</li>
	</ol>
	</li>
	<li>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 &#946;-NADH.
	<ol>
		<li>Set the spectrophotometer to 25 &#176;C and 340 nm. Pipette 890 &#181;L Tris-HCl buffer, 100 &#181;L of oxaloacetate solution, and 10 &#181;L of NADH solution to a blank cuvette and 880 &#181;L Tris-HCl buffer, 100 &#181;L of oxaloacetate solution, and 10 &#181;L of NADH solution into a sample cuvette.</li>
		<li>Incubate cuvettes in the spectrophotometer for 3&#8211;4 min&#160;and reference against blank.</li>
		<li>Then add 10 &#181;L 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.</li>
	</ol>
	</li>
</ol>
5. In vitro &#945;-synuclein, bovine insulin, and HEWL fibril formation
<ol>
	<li>Protein preparation
	<ol>
		<li>&#945;-synuclein: 
		NOTE: Expression and purification of recombinant &#945;-synuclein is performed as described by Hoyer et al.18 with some modifications, and the purity of &#945;-synuclein is confirmed by SDS-PAGE.
		<ol>
			<li>Dialyze purified &#945;-synuclein overnight against phosphate-buffered saline (PBS).</li>
			<li>Determine the protein concentration using an extinction coefficient of 5600 M-1&#160;cm-1 at 275 nm19.</li>
			<li>Aliquots protein in 1.5 mL tubes and store at -80 &#176;C until use.</li>
		</ol>
		</li>
		<li>Bovine insulin and HEWL:
		<ol>
			<li>Provide both bovine insulin and HEWL.</li>
			<li>Dissolve each protein in 50 mM glycine buffer (pH = 1.6; adjust the pH with HCl).</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Amyloid fibrillation induction
	<ol>
		<li>Preparation of solutions for amyloid fibrillation:
		<ol>
			<li>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.</li>
			<li>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 &#956;m filter paper, and keep away from light at 4 &#176;C until use. 
			NOTE: This solution can be store at 4 &#176;C for up to 4 weeks.</li>
			<li>Prepare ThT solution (1 mM): add 200 &#181;L of ThT stock solution (5 mM) to 800 &#181;L of ThT buffer. 
			NOTE: This solution can be stored at 4 &#176;C for up to 1 week.</li>
		</ol>
		</li>
		<li>In vitro &#945;-synuclein amyloid fibril formation:
		<ol>
			<li>Add aliquots (294 &#181;L) of protein solution (200 &#956;M) and 6 &#181;L of ThT solution (1 mM) to 1.5 mL tubes followed by stirring.</li>
			<li>Incubate the tubes in a thermomixer at 37 &#176;C under constant stirring at 800 rpm for 4 days.</li>
			<li>Take aliquots (10 &#181;L) of incubated samples after regular time intervals and add 490 &#181;L of ThT buffer, mixed thoroughly, and incubate for 5 min at RT.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>In vitro bovine insulin amyloid fibril formation:
		<ol>
			<li>Add 637 &#181;L of protein solution (250 &#181;M) to 1.5 mL tube. Then, add 13 &#181;L of ThT solution (1 mM) followed by stirring.</li>
			<li>Add aliquots (200 &#181;L) of protein solution (250 &#181;M) containing 20 &#181;M ThT to each well of a clear-bottomed 96 well plate and seal the plate with crystal clear sealing tape.</li>
			<li>Load the plate into a fluorescence plate reader and incubate at 57 &#176;C without agitation.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>In vitro HEWL amyloid fibril formation:
		<ol>
			<li>Add aliquots (200 &#181;L) of protein solution )1 mM) containing 20 &#181;M ThT to each well of a clear-bottomed 96 well plate and seal the plate with crystal clear sealing tape.</li>
			<li>Load the plate into a fluorescence plate reader and incubate at 57 &#176;C without agitation.</li>
			<li>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).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Treatment of mitochondria with amyloid fibrils, MDH release assay, and ROS measurement
<ol>
	<li>Incubation of isolated mitochondria with amyloid fibrils
	<ol>
		<li>Turn on centrifuge and warm water bath and set to 4 &#176;C and 30 &#176;C, respectively.</li>
		<li>Using IF, dilute mitochondrial homogenate to a final concentration of 1 mg/mL.</li>
		<li>Prepare two series of 1.5 mL tubes containing mitochondrial homogenates (one series for MDH release assay and another for mitochondrial ROS measurement).</li>
		<li>Add aliquots of fresh or amyloid fibrils of &#945;-synuclein, bovine insulin, or HEWL (at final concentrations of 5 &#181;M, 10 &#181;M, 20 &#181;M, and 25 &#181;M; use PBS or glycine buffer as a control) to mitochondrial homogenate (final volume = 200 &#181;L) (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.</li>
		<li>Incubate tubes containing mitochondrial suspensions for 30 min in warm water bath set to 30 &#176;C.</li>
		<li>Measure mitochondrial MDH release and ROS content as outlined in sections 6.2 and 6.3.</li>
	</ol>
	</li>
	<li>Mitochondrial MDH release assay
	<ol>
		<li>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.</li>
		<li>Calculate the release of MDH as a fraction of the maximum effect (Triton X-100) as follows:</li>
	</ol>
	</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/59883/59883equ02v4.jpg" />
<ol start="3">
	<li>Mitochondrial ROS measurement 
	NOTE: Mitochondrial ROS content is determined with the oxidation sensitive fluorogenic precursor dihydrodichlorocarboxyfluorescein diacetate (DCFDA)22.
	<ol>
		<li>Prepare solutions for mitochondrial ROS measurement. Prepare a 50 &#181;M DCFDA solution by dissolving in methanol (prepare fresh) and a 200 mM succinate solution by dissolving in DW.</li>
		<li>Pipette 191 &#181;L of incubated mitochondrial homogenate to each well of a 96 well plate and add 4 &#181;L of 50 &#181;M DCFDA (1 &#181;M final concentration) and 5 &#181;L of 200 mM succinate (5 mM final concentration).</li>
		<li>Incubate the plate for 30 min in a warm water bath set at 30 &#176;C while gently stirring.</li>
		<li>Load the plate into a fluorescence plate reader and measure fluorescence intensity, with excitation at 485 nm and emission at 530 nm.</li>
	</ol>
	</li>
</ol>
7. Statistical analysis
<ol>
	<li>Perform all experiments at least 2x or 3x with triplicate assays and conduct appropriate statistical tests. Here, the results are presented as mean &#177; SD, and a Student&#39;s paired t-test was utilized to calculate statistical significance. P-values less than 0.01 and 0.05 were considered statistically significant (*p &#60; 0.05; **p &#60; 0.01).</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+production+by+the+mitochondrial+respiratory+chain+in+isolated+rat+hepatocytes+and+liver+mitochondria:+studies+using+myxothiazol.">Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol.</a> Archives of Biochemistry and Biophysics. 405, 65-72 (2002).</li><li>Meratan, A. A., Ghasemi, A., Nemat-Gorgani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+integrity+and+amyloid+cytotoxicity:+a+model+study+involving+mitochondria+and+lysozyme+fibrillation+products.">Membrane integrity and amyloid cytotoxicity: a model study involving mitochondria and lysozyme fibrillation products.</a> Journal of Molecular Biology. 409, 826-838 (2011).</li><li>Katebi, B., Mahdavimehr, M., Meratan, A. A., Ghasemi, A., Nemat-Gorgani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+silibinin+on+insulin+amyloid+fibrillation,+cytotoxicity+and+mitochondrial+membrane+damage.">Protective effects of silibinin on insulin amyloid fibrillation, cytotoxicity and mitochondrial membrane damage.</a> Archives of Biochemistry and Biophysics. 659, 22-32 (2018).</li><li>Fink, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aggregation+and+fibrillation+of+alpha-synuclein.">The aggregation and fibrillation of alpha-synuclein.</a> Accounts of Chemical Research. 39, 628-634 (2006).</li><li>Diraviyam, K., Stahelin, R. V., Cho, W., Murray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+modeling+of+the+membrane+interaction+of+FYVE+domains.">Computer modeling of the membrane interaction of FYVE domains.</a> Journal of Molecular Biology. 328, 721-736 (2003).</li><li>Van Rooijen, B. D., Claessens, M., Subramaniam, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+bilayer+disruption+by+oligomeric+&#945;-synuclein+depends+on+bilayer+charge+and+accessibility+of+the+hydrophobic+core.">Lipid bilayer disruption by oligomeric &#945;-synuclein depends on bilayer charge and accessibility of the hydrophobic core.</a> Biochimica et Biophysica Acta. 1788, 1271-1278 (2009).</li><li>Kourie, J. I., Henry, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channel+formation+and+membrane-linked+pathologies+of+misfolded+hydrophobic+proteins:+the+role+of+dangerous+unchaperoned+molecules.">Ion channel formation and membrane-linked pathologies of misfolded hydrophobic proteins: the role of dangerous unchaperoned molecules.</a> Clinical and Experimental Pharmacology &amp; Physiology. 29, 741-753 (2002).</li><li>Bucciantini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherent+toxicity+of+aggregates+implies+a+common+mechanism+for+protein+misfolding+diseases.">Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.</a> Nature. 416, 507-511 (2002).</li><li>Bolognesi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANS+binding+reveals+common+features+of+cytotoxic+amyloid+species.">ANS binding reveals common features of cytotoxic amyloid species.</a> ACS Chemical Biology. 5, 735-740 (2010).</li><li>Posse, E., De Arcuri, B. F., Morero, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysozyme+interactions+with+phospholipid+vesicles:+relationships+with+fusion+and+release+of+aqueous+content.">Lysozyme interactions with phospholipid vesicles: relationships with fusion and release of aqueous content.</a> Biochimica et Biophysica Acta. 1193, 101-106 (1994).</li><li>Roqanian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphenols+protect+mitochondrial+membrane+against+permeabilization+induced+by+HEWL+oligomers:+possible+mechanism+of+action.">Polyphenols protect mitochondrial membrane against permeabilization induced by HEWL oligomers: possible mechanism of action.</a> International Journal of Biological Macromolecules. 103, 709-720 (2017).</li><li>Ulmer, T. S., Bax, A., Cole, N. B., Nussbaum, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+dynamics+of+micelle-bound+human+alphasynuclein.">Structure and dynamics of micelle-bound human alphasynuclein.</a> The Journal of Biological Chemistry. 280, 9595-9603 (2005).</li><li>Stockl, M., Fischer, P., Wanker, E., Herrmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+selectively+binds+to+anionic+phospholipids+embedded+in+liquid-disordered+domains.">Alpha-synuclein selectively binds to anionic phospholipids embedded in liquid-disordered domains.</a> Journal of Molecular Biology. 375, 1394-1404 (2008).</li><li>Devi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+import+and+accumulation+of+&#945;-synuclein+impair+complex+I+in+human+dopaminergic+neuronal+cultures+and+Parkinson+disease+brain.">Mitochondrial import and accumulation of &#945;-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain.</a> The Journal of Biological Chemistry. 283, 9089-9100 (2008).</li><li>Ghio, S., Kamp, F., Cauchi, R., Giese, A., Vassallo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+&#945;-synuclein+with+biomembranes+in+Parkinson's+disease-role+of+cardiolipin.">Interaction of &#945;-synuclein with biomembranes in Parkinson's disease-role of cardiolipin.</a> Progress in Lipid Research. 61, 73-82 (2016).</li><li>Petersen, C. A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+&#946;-peptide+is+imported+into+mitochondria+via+the+TOM+import+machinery+and+localized+to+mitochondrial+cristae.">The amyloid &#946;-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae.</a> Proceedings of National Academy of Sciences of the United States of America. 105, 13145-13150 (2008).</li><li>Costa, V., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+role+of+mitochondria+in+the+pathogenesis+of+Huntington's+disease.">Shaping the role of mitochondria in the pathogenesis of Huntington's disease.</a> EMBO Journal. 31, 1853-1864 (2012).</li><li>Vande Velde, C., Miller, T. M., Cashman, N. R., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+association+of+misfolded+ALS-linked+mutant+SOD1+with+the+cytoplasmic+face+of+mitochondria.">Selective association of misfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria.</a> Proceedings of National Academy of Sciences of the United States of America. 105, 4022-4027 (2008).</li><li>Oladzad Abbasabadi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+mitochondrial+membrane+integrity+induced+by+amyloid+aggregates+arising+from+variants+of+SOD1.">Disruption of mitochondrial membrane integrity induced by amyloid aggregates arising from variants of SOD1.</a> International Journal of Biological Macromolecules. 61, 212-217 (2013).</li></ol>

The authors have nothing to disclose.

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&#160;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,&#160;damage, and permeabilization of mitochondrial membranes upon the addition of &#945;-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&#160;and&#160;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&#160;bears a net positive charge over a broad range of pH levels and&#160;has a high affinity for anionic and neutral phospholipids31 (e.g., the mitochondrial membrane23), no mitochondrial membrane&#160;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 &#181;M 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 &#945;-synuclein fibrils (Figure 3 and&#160;Figure 4).
This observation may be attributed to the high capacity of &#945;-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 &#945;-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 &#945;-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 &#946;-peptide, &#945;-synuclein, Huntingtin, and ALS-linked mutant SOD1 to mitochondria35,37,38,39. Moreover, previous studies have demonstrated that amyloidogenic assemblies of amyloid &#946; (25&#8211;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.

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&#8217;s and Parkinson&#8217;s diseases6,7. In accordance with this issue, numerous reports have indicated binding and accumulation of amyloid &#946;-peptide, &#945;-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&#160;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 &#945;-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.

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

interactions with and membrane permeabilization of brain mitochondria by amyloid fibrils

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 &#945;-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.

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&#160;and enzyme release&#160;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 &#945;-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 &#945;-synuclein, bovine insulin, or HEWL amyloid fibrils to mitochondrial suspensions and following the procedures outlined. The&#160;preliminary experiments showed that the presence of ThT (up to 3 &#181;M) 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 &#945;-synuclein amyloid fibrils to mitochondria in a concentration-dependent manner. Although&#160;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 &#945;-synuclein fibrils led to a considerable increase in ROS content of brain mitochondria in a concentration-dependent manner (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59883/59883fig01.jpg" /> 
Figure 1: Mitochondrial isolation and membrane integrity determination.&#160;(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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59883/59883fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59883/59883fig02.jpg" /> 
Figure 2: Amyloid fibrillation of &#945;-synuclein, bovine insulin, and HEWL.&#160;(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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59883/59883fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59883/59883fig03.jpg" /> 
Figure 3: Mitochondrial MDH release upon interaction with monomer and amyloid fibril of &#945;-synuclein, bovine insulin, and HEWL.&#160;Release is expressed as the percentage of the maximum observed upon treatment with 0.5% (v/v) Triton X-100. Each value represents mean &#177; SD (n = 3; *p &#60; 0.05, **p &#60; 0.01). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59883/59883fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59883/59883fig04.jpg" /> 
Figure 4: Mitochondrial ROS content upon interaction with monomer and amyloid fibril of &#945;-synuclein, bovine insulin, and HEWL.&#160;The data are expressed as the percentage of values in control mitochondria. Each value represents the mean &#177; SD (n = 3; *p &#60; 0.05; **p &#60; 0.01). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59883/59883fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Mitochondrial homogenate (1 mg/mL)</td>
			<td>&#945;-synuclein monomer (200 &#181;M)</td>
			<td>&#945;-synuclein fibril (200 &#181;M)</td>
			<td>Isolation buffer</td>
			<td>PBS</td>
			<td>Triton x-100 20% (v/v)</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>25 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>25 &#181;L</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Monomer 25 &#181;M</td>
			<td>175 &#181;L</td>
			<td>25 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 5 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>5 &#181;L</td>
			<td>20 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 10 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>10 &#181;L</td>
			<td>15 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 20 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>20 &#181;L</td>
			<td>5 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 25 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>25 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Positive control</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>20 &#181;L</td>
			<td>0</td>
			<td>5 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Treatment of mitochondria with various concentrations of &#945;-synuclein amyloid fibrils.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;</td>
			<td>Mitochondrial homogenate (1 mg/mL)</td>
			<td>Bovine insulin monomer (250 &#181;M)</td>
			<td>Bovine insulin fibril (250 &#181;M)</td>
			<td>Isolation buffer</td>
			<td>Glycine buffer</td>
			<td>Triton x-100 20% (v/v)</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>25 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Glycine buffer</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>25 &#181;L</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Monomer 25 &#181;M</td>
			<td>175 &#181;L</td>
			<td>20 &#181;L</td>
			<td>0</td>
			<td>5 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 5 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>4 &#181;L</td>
			<td>21 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 10 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>8 &#181;L</td>
			<td>17 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 20 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>16 &#181;L</td>
			<td>9 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 25 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>20 &#181;L</td>
			<td>5 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Positive control</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>20 &#181;L</td>
			<td>0</td>
			<td>5 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 2: Treatment of mitochondria with various concentrations of bovine insulin amyloid fibrils.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;</td>
			<td>Mitochondrial homogenate (1 mg/mL)</td>
			<td>HEWL monomer (1 mM)</td>
			<td>HEWL fibril (1 mM)</td>
			<td>Isolation buffer</td>
			<td>Glycine buffer</td>
			<td>Triton x-100 20% (v/v)</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>25 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Glycine buffer</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>25 &#181;L</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Monomer 25 &#181;M</td>
			<td>175 &#181;L</td>
			<td>5 &#181;L</td>
			<td>0</td>
			<td>20 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 5 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>1 &#181;L</td>
			<td>24 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 10 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>2 &#181;L</td>
			<td>23 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 20 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>4 &#181;L</td>
			<td>21 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Fibril 25 &#181;M</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>5 &#181;L</td>
			<td>20 &#181;L</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Positive control</td>
			<td>175 &#181;L</td>
			<td>0</td>
			<td>0</td>
			<td>20 &#181;L</td>
			<td>0</td>
			<td>5 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 3: Treatment of mitochondria with various concentrations of HEWL amyloid fibrils.

Watch this Scientific Journal Video about Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils at JoVE.com

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

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.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

interactions-with-membrane-permeabilization-brain-mitochondria

Nanyang Technological University

Research

JoVE Journal

Biochemistry

5.9K Views.  Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, 45137-66731, Iran. In deze video beschrijven we een modelstudie voor het mechanisme van amyloïde toxiciteit op membraanniveau met behulp van rattenhersenmitochondriën als een in vitro biologisch model. Ondanks het accumuleren van rapport beschrijft mechanisme van membraan verstoringen door amyloïde aggregaat in fosfolipid model systemen onderzocht rechtstreeks gericht op de gebeurtenissen die beginnen bij de ontwikkeling van biologisch membraan. In de huidige video beschrijven we amyloïde fibrils van alfa-s...

Video: Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Neuroscience

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Medicine

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

Biology

Unveiling Mitochondrial Ultrastructure in Neurons Using STED Microscopy

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

Author Spotlight: Decoding Mitochondrial Aging 

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

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