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 mitocho...

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&#8242;,7&#8242;-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>&#946;-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...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

5.9K Views.  Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, 45137-66731, Iran. 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.

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

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

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 ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

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Chapters in this video

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Analysis of β-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts