Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Neurons rely on bidirectional transport of cargo along the axon to maintain functional synapsis and neuroconnectivity. Deficits in transport are thought to be critical contributors to the pathogenesis of several neurodegenerative diseases. We aim to identify mechanisms by which disease associated modifications to tau protein disrupt axonal transport.

We established that multiple pathological forms of tau protein disrupt normal axonal transport in mammalian neurons. This effect depends on changes to phosphorylation-based signaling pathways that regulate axonal transport. These disruptions represent a potential mechanism of tau induced neurotoxicity in disease.

Modified forms of specific proteins can disrupt normal fast axonal transport across several neurodegenerative diseases. However, we don't fully understand the underlying mechanisms of these effects. This provides a reproducible and simple protocol to identify how expression of disease associated proteins affect fast axonal transport in mammalian neurons.

This protocol provides a reproducible assay for axonal transport in mammalian primary neurons that is easily modifiable to include various cargo proteins along with the expression of proteins of interest with specific pathological modifications. It also allows for the targeted knockdown of other pathway components to test particular mechanistic details. To begin coat a four welled glass bottom chamber slide by adding 750 microliters of poly-d-lysine solution to each well.

Incubate the slide overnight at room temperature. After incubation, wash the slide four times with sterile deionized water, air dry, and store at four degrees Celsius if not used immediately. Next, perform a dissection of mouse pups.

Beginning at the foramen magnum, use micro dissection scissors to cut the skin and skull along the midline. Ensure to angle the lower tip of the scissors up against the skull to avoid damage to the brain tissue. Remove the overlying skin and carefully remove the skull To expose the brain.

Use forceps to hold the mouth or nose area and turn the skull, so the brain is facing downward over a Petri dish filled with 0.9%saline. Flip the brain out of the skull and cut the nerves and brainstem to release the brain into the saline. Place the Petri dish under a dissecting microscope.

To dissect the hippocampus, first, insert the scissors into the brain's midline, angle the scissors such that the top blade pushes one of the cerebral hemispheres outward and cut to separate the hemisphere away from the subcortical regions. Make a vertical cut through the cortex, such that the frontal cortex is anterior and the hippocampus is posterior to the cut site. Insert the lower tip of the scissors into the resulting hole and carefully cut along the apex of the cortex, stopping at the posterior end.

Using forceps, carefully swing out the cortex and finish the posterior cut. At this point, the hippocampus should resemble a crescent shape. Carefully trim the remaining cortex to maintain the hippocampus'crescent shape.

Next, remove the meninges from the hippocampus using forceps and scissors. Cut the hippocampus into four equal pieces and place them in a 15 milliliter conical tube with ice cold calcium and magnesium free buffer. Begin by dissecting the hippocampus from an embryonic mouse brain and place the dissected tissue in ice cold calcium and magnesium free buffer, or CMF.

Next, remove the CMF and rinse the tissue four times with fresh cold CMF, swirling the tube gently in between rinses. Then add filtered 0.125%tripsin solution and incubate for 15 minutes at 37 degrees Celsius, swirling every five minutes. After incubation, remove the trypsin solution and wash two times with ice cold CMF.

Then add three milliliters of trypsin inactivation solution. Next, to dissociate the cells, Triturate 30 times using two second draws with a 14 gauge needle attached to a three milliliter syringe. Continue trituration with two second draws 30 times using a 15 gauge needle, then 20 times using a 16 gauge needle, followed by 20 times with an 18 gauge needle.

Then perform three second draws using a 21 gauge needle 15 times to complete cell dissociation. Check for cell clumps by placing a droplet of the cell suspension on a microscope slide. Next, filter five milliliters of FBS using a 0.22 micron syringe filter.

Gently layer the cell suspension onto the FBS in a 15 milliliter conical tube. Centrifuge the cells at 200G at four degrees Celsius for five minutes. Remove FBS and resuspend the pellet in one milliliter of warm antibiotic-free neuro basal media.

Dilute the cell suspension in 0.4%tripin blue and obtain a cell count. Plate the cells in 750 microliters of antibiotic-free neuro basal media in a prewarmed poly-d-lysine coated four well chamber slide. Incubate the cells in a humidity controlled chamber at 37 degrees Celsius and 5%CO2.

Prepare for imaging using a confocal microscope two days after transfection of primary hippocampal neurons. Warm the live cell chamber attachment, 60x subjective, and lens oil to 37 degrees Celsius and equilibrate the chamber to 5%CO2. Using the eye pieces, identify transfected neurons in the channel containing the cargo protein.

Next, click on scan to image the neurons and identify the axon initial segment of the Psi five channel. Set the field of view to a rectangular box of 32 by 128 pixels and move it to image a region of the axon 50 to 150 microns distal to the axon initial segment. Record the directionality of the cargo transport and the orientation of the ROI, then press save as to save it as file.

Note that the dots on the box will appear at the top and right in subsequent images. Record the side of the image closest to the cell body and the side closest to the axon terminus to ensure correct orientation of the kymograph polyline. Set the 561 laser power to 1.40, the pinhole to 7.8, the size to 128 pixels squared, and the speed to 32 frames per second.

Adjust the gain to visualize the transport of individual cargo vesicles without being obscured by background signal. Select draw rectangular ROI from the ROI dropdown menu and draw the ROI so that it covers the entire field of view. Then right click and select use as stimulation ROI S1.Then, in the ND setup tab, click on acquire image to collect the pre stimulation reference image.

Then set stimulation to ROI S1, interval to no delay, duration of 1.64 seconds, and loops to seven. Then click on acquire image to collect post stimulation reference image. Select waiting, no acquisition, two minutes to provide a recovery period for fluorescent cargo to repopulate the bleached ROI.

Select acquisition, interval, no delay, duration five minutes, loops 8, 615. Click the apply stimulation settings button on the ND setup tab, followed by run now. Also collect transport videos from at least two neurons for analysis.

Reset the field of view to the entire image and then collect an image of the full neuron in the 561 and 647 channels, with the ROI box included. Record the XY coordinates where the image was taken for post fixation confirmation of protein expression. Live cell imaging of a transfected neuron expressing the cargo protein mA-Synaptophysin was performed using this protocol.

The external domain of neurofascin in the Axon initial segment was also labeled. Imaging of cargo transport was performed in the axonal region of interest. Further, post imaging immunofluorescence analysis confirmed the co-expression of tau protein and mA-Synaptophysin.