Cell Subtype-specific Analysis of Neuronal Membrane Proteasome in Somatosensory Neurons

We are investigating the mechanisms of how somatosensory neurons communicate through the recently discovered neuronal membrane proteasome or NNP to modulate how we sense touch, itch, and pain. We recently discovered the neuronal membrane proteasome, a specialized protein degradation complex found in some sensory neurons, which function to modulate sensitivity to touch, itch, and pain. With this protocol, we will be able to investigate the expression and modulation dynamics of the NMP in a cell type specific manner in health and disease conditions.

Our protocol enables the isolation of NMP expressing neurons from the non-NMP expressing neurons to be able to study their function with cell type specificity. Importantly, these isolated neuronal populations remain viable for downstream analyses. With these protocols, we will begin to investigate how different neuropathic conditions affect the expression and function of the NMP, and how this contributes to altered touch, itch, and pain sensation.

To begin, transfer the harvested dorsal root ganglia into a 15 milliliter conical tube. Using a pipette, add seven milliliters of tissue dissociation enzyme blend working solution to the tube and incubate at 37 degrees Celsius with gentle agitation for 20 minutes. Place the tube in a centrifuge and spin down the ganglia at 120 G for two minutes.

Carefully aspirate the supernatant without disturbing the pellet. Resuspend the ganglia in seven milliliters of low trituration tissue dissociation enzyme blend with papain. After centrifuging the tube again, aspirate and discard the supernatant.

Then re-suspend the ganglia in 500 microliters of BSA, TI, and DMEM solution. Using a one milliliter, plugged fire polished glass pasture pipette, triturate the ganglia 12 to 16 times. Pass the cell suspension through a 40 micrometer cell strainer to filter out cellular debris.

Wash the strainer with one milliliter of BSA, TI, and DMEM solution to collect the remaining dorsal root ganglia neurons. Transfer the filtered cell suspension into a 15 milliliter conical tube. Prepare an immunofluorescence staining tray by lining it with a large piece of parafilm.

Using sterile 5-45 forceps, transfer the cover slips from the tissue culture dish onto the parafilm, ensuring that the side containing dorsal root ganglia neurons faces upward. Slowly pipette 100 microliters of dorsal root ganglia media onto each cover slip to prevent the cells from drying out. Then dilute the go anti PSM alpha two primary antibody in warm dorsal root ganglia media to achieve a one to 20 dilution.

Using an aspirator fitted with a P10 unfiltered tip, slowly remove the media from the edge of each cover slip. Add 100 microliters of the go anti PSM alpha two antibody solution to each cover slip and incubate at room temperature for 40 minutes. After that, carefully aspirate the primary antibody solution from each cover slip.

Add 100 microliters of room temperature PBS to the cells and incubate at room temperature for three minutes to wash. Now, to prepare the secondary anti go antibody, dilute it in warm dorsal root ganglia media at a one to 250 dilution. After completing the third wash, add 100 microliters of the diluted secondary antibody solution to each cover slip.

Incubate the cover slips at room temperature for 40 minutes while protecting the cells from light. Then aspirate the secondary antibody solution from the cover slips and wash the cells three times, using PBS as described previously. After the final PBS wash, add 100 microliters of fixative solution containing 4%para formaldehyde, and 4%sucrose in PBS.

Once the cells are incubated at room temperature for 10 minutes, aspirate the fixative from each cover slip. Then wash the cells three times with PBS as previously described. To permeabolize the cells, add 100 microliters of 0.1%non ionic detergent in PBS.

Incubate the cover slips at room temperature for five minutes. Then aspirate the permeable solution from the cover slips and wash the cells three times, using PBS as previously described. Add 100 microliters of blocking solution composed of 5%fetal bovine serum, and 5%donkey serum in PBS to each cover slip.

Next, dilute rabbit anti CGRP, A selectin B four biotin conjugate, and chicken anti NFH in the blocking solution to prepare the dorsal root ganglion neuron cell type specific primary antibody mixture. Aspirate the blocking solution from the cover slips, then add 100 microliters of the prepared primary antibody mixture to each cover slip. Incubate the cover slips at room temperature for 45 minutes while protecting them from light.

Afterward, remove the primary antibody solution from the cover slips and wash the cells three times, using PBS as previously described. Now prepare the secondary antibody mixture by diluting streptavidin, donkey anti rabbit, and donkey anti chicken to one to 500 each in blocking solution. Aspirate the final PBS wash from each cover slip, then add 100 microliters of the prepared secondary antibody solution.

Incubate the cover slips at room temperature for 45 minutes in the dark. Aspirate the final wash from the cover slip. Using fine tip forceps, lift each cover slip from the parafilm and gently blot off excess liquid by touching the edge of the cover slip to a lint-free tissue.

Place a seven microliter droplet of mounting medium on a microscope slide. Carefully lower the cover slip onto the mounting medium with the cell side facing down, ensuring full contact with the medium. Place the prepared slides in a dark dry drawer or box to dry for at least one hour.

Seal the edge of the cover slip with fast dry nail polish and wait 10 to 15 minutes before imaging. Antibody feeding on live, dorsal root ganglion neurons revealed a subpopulation of neurons with surface accessible proteasomes, while control samples lacking primary antibody showed no surface labeling. Flow cytometry identified two distinct populations of dorsal root ganglion neurons, based on fluorescence intensity, separating NMP positive from NMP negative cells with clear gating regions established, using controls.

Quantification of flow sorted populations showed that approximately 4%of dorsal root ganglion neurons were NMP positive, while the remaining majority were NMP negative. Sorted NMP positive and NMP negative neurons retained viability and expressed cell type specific markers NFH, IB four, and CGRP, confirming the suitability of the workflow for downstream molecular analysis.