Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD)

We are trying to quantitatively measure the stability of mitochondria associated ER membranes of various thicknesses or gap widths, which is important in regulating the pathophysiology of several neurodegenerative diseases, including AD. Recent studies on mitochondria-associated ER membranes, often called MAMs, have described them as one of the key factors in initiating amyloid pathology in the early stage of AD. The findings led to the development of a new hypothesis called the MAM hypothesis, which suggests that AD is exclusively a MAM-related disease.

Our protocol is the first to provide a quantitative tool to measure MAM stabilization in live cells, which has remained challenging so far. The biggest advantage of our protocol is that it uses the intrinsic dynamic property of mitochondria to quantitatively measure the changes in MAM stabilization upon tightening or loosening MAM gap widths in live cells.

Our focus in the immediate future is to improve and employ our state-of-the-art live cell imaging and kymographic technique to screen a series of FDA-approved synthetic or natural small molecule modulators of MAMs or MAM bound sigma one receptor, using a recently developed AD drug screening platform and understand their mechanism of action.

[Narrator] To transfect the cells with MAM stabilizers, replace the bone marrow mononuclear mixture with two milliliters of room temperature DMEM-F12 medium in each well. Combine nucleofector reagent with supplement one in a 4.5 to one ratio. Add 100 microliters of the nucleofector reagent to the mixture. Then vortex the DNA solution. Add one to five micrograms of DNA per treatment into separate one milliliter micro centrifuge tubes for each transfection. After aspirating the existing media from the cells, wash the cells once with 10 milliliters of PBS. Aspirate the PBS and add one milliliter of accutase directly to the cells. Incubate the cells for five to 10 minutes at 37 degrees Celsius. Then gently tap the side of the dish to dislodge the cells. Under a microscope, verify that the cells are loosened and flowing freely. Neutralize the acutants with 10 milliliters of DMEM-F12 medium and transfer the cell suspension to a clean 15 milliliter tube. Next, take 10 microliters of the suspended cells and add them to side A of a cell counting chamber. Then insert side A of the filled chamber into the main slot on the front of the cell counter. Press measure and record the number of cells per milliliter. Centrifuge the required number of cells at 300 G for five minutes. Then aspirate the supernatant carefully and resuspend the pellet in 100 microliters of nucleofector mix for each electroporation. Next, add 100 microliters of the resuspended cell suspension to one of the tubes containing DNA. Mix by pipetting the solution several times. Then transfer the DNA cell mixture into an electroporation cuvette and seal it with the provided lid. Select the appropriate nucleofector program on the device. Immediately add approximately 500 microliters of DMEM from a prefilled six-well plate into the cuvette using sterile pipettes. Mix gently once and transfer the electroporated cells and the medium to the corresponding well. To begin, place the six-well plate containing cells in the microscope chamber and adjust the microscope's focus until the cells become visible. To capture the red fluorescent protein signal, excite the fluorophore using a 594 nanometer laser and set the emission to 570 to 640 nanometers. For green fluorescent protein, use a 488 nanometer laser for excitation with 510 to 540 nanometer emission. Crop the scanning area to fit around the axon. A smaller scanning area will reduce processing time and simplify kymography generation. Set the interval to one frame per second and the total duration to 181 seconds. Click run to start the process. The axonal speed of MAM 1X labeled ER bound mitochondria was reduced by 50% compared to free and MAM 9X bound mitochondria. 26.6% of MAM 1X mitochondria were mobile versus 53.82% of free mitochondria and 44.79% of MAM 9X mitochondria. Retrograde movement was lower for MAM 1X mitochondria than Mito-RFP mitochondria and MAM 9X mitochondria. Anterograde movement of MAM 1X mitochondria was also reduced versus Mito-RFP. The stationary percentage of MAM 1X mitochondria was significantly higher than MAM 9X mitochondria, reflecting a reduction in motility, while the overall speed of MAM 1X mitochondria was significantly lower than MAM 9X mitochondria.