Mitochondrial dysfunction underlies many neurodegenerative diseases. Our protocol provides an important tool for examining mitochondrial dynamics in the axons, facilitating the study of neurological diseases involving axonal degeneration. By combining mitochondrial labeling, live cell imaging and induced pluripotent stem cell technology our protocol can be used to characterize mitochondrial trafficking along human axons and to analyze their morphologies.
Impaired mitochondrial transport and morphology can be observed in stem cell cultures and animal models of neurodegenerative diseases providing potential therapeutic targets for treating these diseases. After day thirty five of culture, dissociate the neurospheres differentiated from human induced pluripotent stem cells into small clusters with 1 mg/ml of cell detachment solution for two minutes at 37 degrees Celsius. At the end of the incubation collect the cell clusters by centrifugation and resuspend the pellet in one milliliter of NDM.
Plate about five clusters of cells in one hundred microliters of medium per polyornithine and lactose dehydrogenase elevating virus-free reduced growth factor basement membrane matrix coated 35 millimeter glass bottom dishes. Then add one milliliter of NDM supplemented with B27, cyclic adenosine monophosphate, insulin-like growth factor, human brain-derived neurotrophic factor and glial cell-derived neurotrophic factor to each culture. To visualize mitochondria along the axons of forebrain neurons first stain the neurons with 50 nanomolar red fluorescent dye in NDM for three minutes at 37 degree Celsius followed by two washes in warm NDM.
Next, place the culture on the stage of a fluorescence microscope and select the 40X objective. Under the phase-field identify the axons based on their morphological characteristics. To distinguish the direction of the mitochondrial movement along the axons clearly identify the cell bodies of neurons.
After distinguishing the cell body and axon adjust the exposure time and focus of the mitochondria in the axons and capture the mitochondrial transport within the axons every five seconds for a total of five minutes. To analyze mitochondrial transport in ImageJ open Fiji and select the Bio-Formats plugin. Use the Bio-Formats importer to import the time-lapse images of the tiff series and select standard ImageJ and Open all series.
Check autoscale split channels and click OK taking care to note the frame number and size of the images in pixels. After adjusting the brightness and contrast for all 60 frames use the segmented line to draw a line from the cell body to the terminal axon. To generate the Kymograph select the multiple Kymograph plugin and select the line width.
To measure the distance, time values and velocity for the moving mitochondria open tsp050607. txt in the macros plugin. And draw a segmented line over the trace of mitochondrial movement on the kymograph from the superior to the inferior region.
After drawing the line open read velocities from tsp in the macros plugin to read the segmented velocities corresponding to the line. Next open the original image and use the line tool to draw a line along the scale bar. Select analyze to measure the length of the line and convert the dx now and the distance units from pixels to micrometers.
Then change the time from pixel to seconds. To analyze the mitochondrial length and area within axons in ImageJ install the straighten underscore dot JAR plugin and open the axon image of interest. Convert the 32 bit image to eight bit and use the segmented line tool to trace the axon.
Select the Straighten underscore JAR plugin and set the width of filament wide line to 50 pixels. Trace the axon again to generate a straightened axon and adjust the image threshold. Select analyze set measurements perimeter fit ellipse and shape descriptors to set the measurement and use the line function to measure the scale bar in the original image as demonstrated.
Under analyze and to set scale enter the distance in pixels known distance and the unit of length to set the scale. Then select global to set the scale setting to all of the images. Finally select analyze and analyze particles to determine the area and select display results to view the resulting measurement.
After differentiation into telencephalic glutamatergic neurons the cells can be characterized by their T-B-R one and beta three tubulin marker expression. Staining with red fluorescent dye and time-lapse imaging allows evaluation of the axonal transport of mitochondria. A single mitochondrion can remain static or move in an anterograde or retrograde direction within an axon.
The velocity of the mitochondrial movement along the axon can be quantified as illustrated. For example using commercially available analysis software the percentage of motile mitochondria is significantly reduced in S-P-G-3-A neurons compared to wild type neurons. To analyze the mitochondrial area length and aspect ratio axons can straightened in the image analysis software and selected by adjusting the threshold.
The mitochondrial area perimeter length width and aspect ratio can then be obtained from the straightened axon. For example both the mitochondrial length and the aspect ratio are significantly reduced in S-P-G 15 neuron axons compared to control wild type axons. It is important to warm the medium and the microscope incubator to wash the cells gently and to allow the cells to stabilize for twenty minutes before imaging.
After live cell imaging the cultures can be fixed and subjected to immunostaining to examine the expression of other proteins of interest. It is challenging to examine mitochondrial dynamics in live human nerves. This technique provides the unique tool for the study of mitochondrial dynamics and nerve degeneration in neurological disease.
