In this video, we describe a model study for the mechanism of amyloid toxicity at the membrane level using rat brain mitochondria as an in vitro biological model. Despite accumulating report describing mechanism of membrane perturbations by amyloid aggregate in phospholipid model systems studied directly targeting the events beginning at developing biological membrane. In the present video, we describe amyloid fibrils of alpha-synuclein analyzing the rat brain mitochondria membrane as an in vitro biological model.
We believe that mitochondria as an organelle with well-characterized membranes may provide an extremely useful biological model system for molecular studies related to the mechanism of amyloid cytotoxicity at the membrane level relating to neurodegenerative diseases. Decapitate the rat with a small animal guillotine and remove the brain from the scale within one minute of the decapitation of the rat to limit possible deterioration of mitochondrial properties. It is important to work quickly and keep everything on ice throughout the procedure.
Wash the tissue twice with 30 milliliter of isolation buffer. Transfer to a beaker containing cold isolation buffer and finely mince the brain with scissors. Transfer the tissue suspension to a 20 milliliter cold dounce homogenizer.
Homogenize the tissue pieces using nine up and down strokes with a motorized pestle. Leave the mixture on ice for approximately 30 seconds after each set of three homogenization strokes to ensure the homogenate remains cold. Transfer the homogenate to a pre-chilled centrifuge tube and centrifuge at 1, 300 G at four Centigrade for three minutes.
Carefully decant the supernatant and transfer it to a pre-chilled centrifuge tube. Centrifuge at 21, 000 G at four Centigrade for 10 minutes. Discard the supernatant and resuspend the pellet in a density gradient medium by gently stirring the mixture with the pipette.
Centrifuge in a fixed angle rotor at 30, 700 G at four Centigrade for five minutes. Using a Pasteur pipette, remove the sharp end of material accumulated at the top, which mostly contain myelin. Add eight milliliter isolation buffer to the mitochondrial fraction while gently stirring the mixture with the pipette.
Centrifuge at 16, 700 G at four Centigrade for 10 minutes. Carefully remove the supernatant, leaving the bottom loose pellet undisturbed. Add one milliliter 10 milligram per milliliter fatty acid-free BSA to centrifuge tube while gently stirring the mixture with the tip of the pipette.
Make up the volume to five milliliter by adding isolation buffer. Centrifuge at 6, 900 G at four Centigrade for 10 minutes. This should produce a firm pellet.
Decant the supernatant and gently resuspend the mitochondrial pellet in isolation buffer. Dilute mitochondrial homogenate to one milligram per milliliter with cold isolation buffer and place in two 1.5 milliliter tubes, typically 195 microliter of mitochondria per tube. Add five microliter 20%volume per volume Triton X-100 to one tube as positive control for maximum enzyme activity and five microliter deionized water to another tube for control, followed by mixing with a stirrer.
Incubate the tubes for 10 minutes in warm water bath set to 30 Centigrade. Pellet mitochondria by centrifugation of tubes in a fixed angle rotor at 16, 000 G four Centigrade for 15 minutes. Carefully collect resulting supernatants for assessing the activity of mitochondrial malate dehydrogenase using a standard spectrophotometric assay described on the next part.
Calculate the integrity of mitochondrial membrane as follows. For malate dehydrogenase activity determination, pipette 890 and 880 microliter 50 millimolar Tris-HCL buffer to blank and sample test cuvettes respectively followed by 100 microliter 50 millimolar oxaloacetate and 10 microliter of 10 millimolar beta-NADH. Put the cuvettes in a spectrophotometer and reference against blank.
Add 10 microliter mitochondrial homogenate one milligram per milliliter to sample cuvette. Immediately mix by inversion. Record decrease in absorbance due to NADH oxidation at 340 nanometer for one minute.
For alpha-synuclein amyloid fibrillation, add 294 microliter of protein solution, 200 micromolar to each 1.5 milliliter tube. Then add six microliter ThT solution one millimolar. Mix the solutions and incubate the tubes in a thermomixer at 37 Centigrade under constant stirring at 800 RPM.
For bovine insulin amyloid fibrillation, add 637 microliter of protein solution, 250 micromolar to 1.5 milliliter tube. Then add 13 microliter ThT solution one millimolar followed by stirring. Add aliquots 200 microliter of protein solutions to each well of a clear bottom 96-well plate.
Seal the plate with crystal clear sealing tape. Load the plate into Cytation 5 fluorescence plate reader. Measure ThT fluorescence at 30-minute intervals for 12 hours.
Use excitation at 440 nanometer and emission at 485 nanometer. Select the wells. Shake the plate for five seconds before each measurement.
Set the temperature at 57 Centigrade without agitation. Prepare two series of 1.5 milliliter tubes containing mitochondrial homogenates, one series for MDH release assay and other for mitochondrial ROS measurement. Add aliquots of fresh or amyloid fibrils of alpha-synuclein, bovine insulin or hen egg white lysozyme to mitochondrial homogenate followed by gently pipetting.
Incubate tubes containing mitochondrial suspensions for 30 minutes in warm water bath set to 30 Centigrade. For malate dehydrogenase release assay, centrifuge incubated mitochondrial homogenates at 16, 000 G for 15 minutes. Carefully collect resulting supernatants for assaying the activity of mitochondrial MDH as described on protocol section part four.
Calculate the release of MDH as follows. For mitochondrial ROS measurement, pipette 191 microliter of incubated mitochondrial homogenate to each well of a 96-well plate. Add four microliter of 50 micromolar DCFDA.
Then add five microliter of 200 millimolar succinate. Incubate the plate for 30 minutes in warm water bath set to 30 Centigrade while gently stirring. Load the plate into a Cytation 5 fluorescence plate reader and measure fluorescence intensity according to the protocol section.
Mitochondrial membrane integrity was assessed by measuring MDH activity in the isolated mitochondria before and after membrane disruption and enzyme release by Triton X-100. As you see, we typically found that mitochondrial preparations are about 93%intact. ThT fluorescence assay was carried out to monitor growth of amyloid fibrils.
The curve for amyloid fibrillation of three proteins showed a typical sigmoidal pattern, reaching to a plateau at about 96, 12, and 96 hours for alpha-synuclein, bovine insulin, and hen egg white lysozyme respectively, suggesting formation of amyloid fibrils that was more confirmed by AFM measurement. Possible permeabilization and damage of mitochondrial membrane by amyloid fibrils was investigated by the release of mitochondrial MDH and mitochondrial ROS measurement. As you see in the left graph, substantial release of MDH was observed upon addition of the alpha-synuclein amyloid fibrils to mitochondria.
While a slight release was detected by insulin fibrils, hen egg white lysozyme fibrils were found to be ineffective. Right graph shows the effect of amyloid fibrils on mitochondrial ROS content. While no significant enhancement in mitochondrial ROS was observed upon addition of hen egg white lysozyme or insulin amyloid fibrils, treatment with alpha-synuclein fibrils led to a considerable increase in ROS content of brain mitochondria.
The present video describes a model study for the mechanism of fibula aggregate cytotoxicity at the membrane level, demonstrating how amyloid fibrils arising from various proteins may cause different degrees of membrane damage and permeabilization. While some topics of amyloid fibrils and their specific binding membrane appear to provide with the capacity to interact with and cause destabilization and subsequent perturbations of membranes. After this video, you will be able to investigate the interaction of native form, pre-fibrillar, and mature amyloid fibrils of different peptides and with mitochondria isolated from different tissues or various areas of the brain.
