Name: Cell Subtype-specific Analysis of Neuronal Membrane Proteasome in Somatosensory Neurons (Video) | JoVE Journal
Uploaded: 2025-10-10T13:20:00.000+00:00
Duration: 9 min 27 s
Description: Learn cell subtype-specific analysis of neuronal membrane proteasome in somatosensory neurons using cell sorting and immunofluorescence. This method enables isolation of viable NMP-expressing neurons for studying sensory modulation.

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5 minutes

E.V.L was supported by startup funds from Loyola University Chicago Stritch School of Medicine. We would like to thank Robert Ladd for his advice and assistance with FACS.

All methods described using experimental animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Loyola University of Chicago Stritch School of Medicine.
1. Preparation of digestion solutions, DRG media, and FACS media
<ol>
	<li>Prepare Complete Saline Solution (CSS) containing 137 mM NaCl, 5.3 mM KCl, 1 mM MgCl2&#183;6H2O, 25 mM sorbitol, 10 mM HEPES, and 3 mM CaCl2&#183;2H2O. Adjust pH to 7.2 with NaOH and filter sterilize.</li>
	<li>Prepare the tissue dissociation enzyme blend working solution (moderate trituration) by combining 190 &#181;L of the stock (5 mg/mL in H2O), 84 &#181;L of 50 mM EDTA, and 6.5 mL of CSS. Prepare fresh and filter-sterilize before use.</li>
	<li>Prepare tissue dissociation enzyme blend (low trituration)-papain solution containing 190 &#181;L of enzyme blend stock solution, 150 &#181;L of papain, 84 &#181;L of 50 mM EDTA, and 6.5 mL of CSS. Prepare fresh and filter-sterilize before use.</li>
	<li>Prepare BSA/TI/DMEM solution by combining 7.5 mg of trypsin inhibitor and 7.5 mg of BSA in 5 mL of DMEM/F12 medium. Prepare fresh and filter-sterilize.</li>
	<li>Prepare DRG neuron media by combining 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% glutamine, and 2% 50x B27 supplement in 430 mL of DMEM/F12.</li>
	<li>Prepare FACS buffer by combining 1% FBS, 1x PBS, 25 mM HEPES, and 1% penicillin/streptomycin in autoclaved water. Prepare fresh and filter-sterilize.</li>
	<li>Twenty-four hours before the start of the protocol, coat two 5 mL polystyrene round-bottom tubes by filling them with FACS buffer and placing them at 4 &#176;C.</li>
</ol>
2. Preparation of single-cell DRG neuron suspension
<ol>
	<li>Harvest all DRGs from 4-6 P20-P23 mice following previously described methods20,23.</li>
	<li>Transfer the DRGs to a 15 mL conical tube and add 7 mL of tissue dissociation enzyme blend (moderate trituration) working solution. Incubate with gentle agitation (by rotating) at 37 &#176;C for 20 min.</li>
	<li>Spin down ganglia at 120 &#215; g for 2 min. Carefully remove the supernatant without disturbing the pellet and resuspend the ganglia in 7 mL of tissue dissociation enzyme blend (low trituration)-papain working solution. Incubate with gentle agitation (by rotating) at 37 &#176;C for 15 min.</li>
	<li>Spin down at 120 &#215; g for 2 min. Remove the supernatant and resuspend the ganglia in 500 &#181;L of BSA/TI/DMEM solution.</li>
	<li>Triturate the ganglia 12-16x with a 1 mL plugged fire-polished glass Pasteur pipette.</li>
	<li>Filter out cellular debris by passing cells through a 40 &#181;m cell strainer. Wash the strainer with 1 mL of BSA/TI/DMEM solution to ensure collection of all DRG neurons. Transfer the cell suspension to a 15 mL conical tube.</li>
	<li>Spin down at 120 &#215; g for 2 min. Remove the supernatant and gently resuspend the pellet in 1 mL of DRG media. 
	NOTE: This is the single-cell DRG neuron suspension.</li>
</ol>
3. Antibody feeding to label NMP-positive neurons
<ol>
	<li>Divide the DRG neuron suspension into two 1.5 mL tubes, each containing 500 &#181;L, and label the two tubes: &#34;primary&#34; and &#34;no primary&#34;.</li>
	<li>Add 20 &#181;L of goat anti-proteasome 20S subunit alpha 2 (PSMA2) antibody to the &#34;primary&#34; labeled tube (1:25 dilution).
	<ol>
		<li>Optional: To test membrane integrity and specificity of the PSMA2 antibody, include an additional sample of primary DRGs incubated with chicken anti-neurofilament heavy chain (NF-H) (1:1,000) followed by anti-chicken (e.g., 488-conjugated) secondary (1:250) (step 3.10). 
		NOTE: The anti-NF-H labeling control should result in no cellular labeling if the neuronal membrane is intact.</li>
	</ol>
	</li>
	<li>Incubate the cells under gentle agitation for 30 min at 37 &#176;C.</li>
	<li>Warm filtered, sterilized 1x PBS during primary antibody incubation.</li>
	<li>Centrifuge the tubes at 200 &#215; g for 2 min and remove 90% of the supernatant (discard).</li>
	<li>Resuspend the neurons in 500 &#181;L of warm 1x PBS. Incubate the tubes with gentle agitation for 3 min at 37 &#176;C.</li>
	<li>Repeat steps 3.5-3.6 for a total of three washes.</li>
	<li>While the washing steps are in progress, prepare secondary antibody by diluting 4 &#181;L of anti-goat IgG (555-nm fluorophore) in 1,000 &#181;L of warm DRG media (1:250 dilution).</li>
	<li>After the third wash, resuspend both the &#34;primary&#34; and &#34;no primary&#34; tubes in 500 &#181;L of the secondary antibody solution. 
	NOTE: From this point forward, protect the tubes from light by wrapping them with aluminum foil.</li>
	<li>Incubate the tubes with gentle agitation for 40 min at 37 &#176;C.</li>
	<li>Wash the cells by repeating steps 3.5-3.6 for a total of three washes.</li>
	<li>After the third wash, resuspend neurons in 300 &#181;L of FACS buffer. Transfer the resuspended cells to separate 5 mL polystyrene round-bottom tubes and place on ice in an ice bucket with lid to protect from light.</li>
	<li>Place the pre-treated round-bottom tubes containing FACS buffer from the previous day in the ice bucket with the tubes containing the cells and proceed to the FACS sorting step.</li>
</ol>
4. Separation of NMP-positive (NMP+&#160;) and NMP-negative (NMP-&#160;) neurons via FACS sorting
<ol>
	<li>Immediately after antibody feeding and labeling of DRG neurons, take the cells and coated tubes to a FACS sorter.</li>
	<li>Add 7-aminoactinomycin D (7-AAD) (1:1,000) to both the &#34;primary&#34; and &#34;no primary&#34; tubes. Incubate for 10-30 min on ice. 
	NOTE: 7-AAD is used to exclude dead cells from the sorting process.</li>
	<li>Remove the FACS buffer from the tubes that were coated overnight and replace with 1 mL of fresh FACS buffer. These tubes serve as collection tubes for the sorted neurons.</li>
	<li>Use the &#34;no primary&#34; labeled tube to set parameters and gating for unlabeled neurons.</li>
	<li>Apply forward scatter (FSC) gating to isolate cells consistent with the size of sensory neurons. Sensory neuron diameters range from 10 to 50 &#181;m.</li>
	<li>Apply side scatter (SSC) gating to exclude dead cells and debris (lower side scatter) and to isolate events consistent with the granularity of DRG neurons (larger side scatter).</li>
	<li>Load the &#34;primary&#34; tube and set gating parameters as established with the &#34;no primary&#34; sample to detect unlabeled (NMP-) and 555-positive (NMP+) neuronal populations.</li>
	<li>Using a 100 &#181;m nozzle and sorting pressure of 15-20 psi, sort the neurons into 555+&#160;and 555-.</li>
	<li>Proceed to downstream analysis of sorted neurons.</li>
</ol>
5. Single-cell RNA sequencing of NMP+&#160;and NMP-&#160;neurons
<ol>
	<li>Transfer the sorted cells to 1.5 mL tubes and place on ice. Transport the cells to a single-cell sequencing core.</li>
	<li>Perform single-cell RNA sequencing on the NMP+&#160;and NMP-&#160;sorted neurons at a depth of 30,000 to 100,000 reads per cell.</li>
	<li>Analyze the scRNA-seq data using various freely available software programs to generate cell clusters according to specific gene expression profiles. For more details on the analysis steps and programs previously used, refer to Villal&#243;n Landeros et al.20.</li>
	<li>Construct a list of specific gene markers to identify individual neuronal populations using available databases such as&#160;https://painseq.shinyapps.io/harmonized_painseq_v2/ or https://kleintools.hms.harvard.edu/tools/springViewer_1_6_dev.html?datasets/Sharma2019/all</li>
	<li>Use the list of specific gene markers to identify the resulting cell clusters. 
	NOTE: The scRNA-seq analysis previously performed was conducted by an independent consulting company (www.cmcherryconsulting.com).</li>
</ol>
6. Primary cultures of enriched NMP+&#160;and NMP-&#160;neurons
<ol>
	<li>The day before the experiment, place 12 mm round coverslips in a 24-well culture plate (1 coverslip/well) and wash with 70% ethanol for 15 min. Then, rinse with sterile water 3x and air dry in a sterile tissue culture hood.</li>
	<li>Once the coverslips are dry, coat them with 0.1 mg/mL poly-L-lysine hydrobromide (PLL) and place at 4 &#176;C overnight.</li>
	<li>On the day of the experiment, remove the PLL, rinse the coverslips with sterile water 3x, and allow to dry in a tissue culture hood. 
	NOTE: The PLL can be saved and reused.</li>
	<li>Dilute 10 &#181;L of 1 mg/mL laminin in 125 &#181;L of DRG media and spot 20-30 &#181;L on the center of each coverslip.</li>
	<li>Coat the coverslips with laminin for at least 40 min before use.</li>
	<li>Transfer the FACS-sorted cells to a 1.5 mL tube and centrifuge at 200 &#215; g for 5 min to collect cells. Remove the supernatant and resuspend neurons in 50 &#181;L of warm DRG media.</li>
	<li>Aspirate laminin from the coverslip and allow the coverslip to dry.</li>
	<li>Seed 15-20 &#181;L of resuspended neurons on each coverslip. Carefully move the plate with seeded coverslips to a tissue culture incubator and incubate for at least 30 min (no more than 1 h) to allow the cells to adhere.</li>
	<li>Move the plate back to a tissue culture hood and add 1 mL of sterile warm DRG media to each well containing cells on coverslips. 
	NOTE: Lower the pipette tip to the bottom corner of the well and dispense slowly to avoid touching the coverslip and disrupting cells. Adding media too fast can create turbulence and dislodge cells.</li>
	<li>Maintain cultures at 37 &#176;C with 5% CO2/95% humidity with 75% media changes every 2-3 days.</li>
</ol>
7. Cell-type-specific NMP expression analysis
<ol>
	<li>Prepare a 140 mm culture plate or immunofluorescence staining tray by lining it with a large piece of parafilm.</li>
	<li>Using sterile 5/45 forceps, transfer the coverslips from the tissue culture dish to the parafilm on the staining tray, ensuring that the side containing the DRG neurons is facing up.</li>
	<li>Add 100 &#181;L of DRG media to each coverslip on the parafilm to prevent the cells from drying out. 
	NOTE: Add liquids by pipetting slowly just over the edge of the coverslip.</li>
	<li>Dilute the goat anti-PSMA2 primary antibody in warm DRG media at a 1:20 dilution.</li>
	<li>Using an aspirator fitted with a P10 unfiltered tip, remove the media from the coverslip slowly by aspirating from the edge. Add 100 &#181;L of goat anti-PSMA2 antibody solution to the coverslips and incubate at room temperature for 40 min.</li>
	<li>Carefully aspirate the primary antibody solution off the coverslips. Add 100 &#956;L of PBS (at room temperature) and incubate for 3 min at room temperature to wash.</li>
	<li>Aspirate PBS off the coverslip and repeat the addition and aspiration of PBS for a total of three washes.</li>
	<li>Prepare anti-goat (e.g., 555-conjugated) secondary antibody in warm DRG media at a 1:250 dilution.</li>
	<li>Following the third wash, add 100 &#181;L of the diluted secondary antibody to the coverslip and incubate at room temperature for 40 min. 
	NOTE: From this step forward, protect the cells from light.</li>
	<li>Aspirate the secondary antibody solution off the coverslip and wash 3x as described in steps 7.6-7.7.</li>
	<li>Remove the last PBS wash and fix the cells by adding 100 &#181;L of fixative solution containing 4% paraformaldehyde (PFA), 4% sucrose in 1x PBS. Incubate at room temperature for 10 min.</li>
	<li>Aspirate fixative and wash 3x by repeating steps 7.6-7.7.</li>
	<li>Permeabilize the cells by adding 100 &#181;L of 0.1% nonionic detergent (e.g., Triton X-100) in 1x PBS and incubate at room temperature for 5 min.</li>
	<li>Aspirate permeabilizing solution and wash 3x by repeating steps 7.6-7.7.</li>
	<li>Add 100 &#181;L of blocking solution consisting of 5% FBS, 5% donkey serum in 1x PBS and incubate at room temperature for 40 min.</li>
	<li>Prepare a DRG neuron cell-type-specific primary antibody mix according to the following dilutions in blocking solution: rabbit anti-calcitonin gene-related peptide (CGRP) (1:500); isolectin B4, biotin conjugate (IB4) (1:50); chicken anti-NF-H (1:1000). 
	NOTE: These antibodies do not interfere with each other and can be added together in the same solution.</li>
	<li>Remove blocking solution and add the primary antibody mix to the coverslip. Incubate at room temperature for 45 min.</li>
	<li>Remove the antibody solution and wash 3x by repeating steps 7.6-7.7.</li>
	<li>Prepare secondary antibody mix solution by diluting streptavidin (e.g., 647-conjugated), donkey anti-rabbit (e.g., 488-conjugated), and donkey anti-chicken (e.g., 405-conjugated) to 1:500 each.</li>
	<li>Remove the last PBS wash and add 100 &#181;L of secondary antibody solution to the coverslips. Incubate at room temperature for 45 min.</li>
	<li>Wash 3x by repeating steps 7.6-7.7.</li>
	<li>Optional: Prepare nuclear stain at 1-10 &#181;g/mL. Add 100 &#181;L of nuclear stain solution, incubate for 30 s at room temperature, and wash 2x with deionized water (diH2O) by repeating steps 7.6-7.7.</li>
	<li>Aspirate the last wash off the coverslip and using fine-tip forceps, lift the coverslip from the parafilm. Blot off excess liquid by gently touching the edge of the coverslip to a lint-free tissue.</li>
	<li>Place a 7 &#181;L droplet of mounting medium on a microscope slide. Gently place the coverslip over the mounting medium droplet, ensuring the cells are facing down and are in contact with the medium.</li>
	<li>Place the microscope slides with coverslips in a dark, dry drawer or box for at least 1 h to dry and seal the edge of the coverslip. Finish sealing the edge of the coverslips with fast-dry nail polish and wait 10-15 min before imaging.</li>
	<li>Image slides using a fluorescent or spinning-disk confocal microscope equipped with the appropriate light filters to visualize blue, green, red, and far-red fluorescence.</li>
</ol>

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<li>Paradise, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ApoE+isoforms+differentially+regulate+neuronal+membrane+proteasomes+to+shift+the+threshold+for+pathological+aggregation+of+endogenous+tau%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">ApoE isoforms differentially regulate neuronal membrane proteasomes to shift the threshold for pathological aggregation of endogenous tau.</a> bioRxiv. , (2022).</li>
<li>Banker, G., Goslin, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBanker%2C+G+%22Culturing+nerve+cells%22">Culturing nerve cells</a>. , 2nd edn, MIT Press. (1998).</li>
</ol>

The authors declare that they have no conflicts of interest.

Proteasomes are essential in all cells and can be found in all compartments throughout the cell19. The recent discovery of a specialized proteasome complex, the NMP, in neurons of the central25,26,27and peripheral20 nervous systems has changed our current understanding of the function of proteasomes. Previous work has shown that the NMP functions as a signaling proteasome that medi...

Proteasome function is essential for optimal development and maintenance of the peripheral nervous system (PNS)1,2,3,4,5,6. Both in vitro and in vivo studies using proteasome inhibitors demonstrate that proteasomal activity is crucial for neurodevelopment, myelination, excitability, and glial cell function7,8,9,10. Furthermore, proteasome-inhibitor-based chemotherapy induces painful neuropathies in 30-60% of patients, often characterized by chronic pain and altered sensation in the extremities8,9,11,12,13. Interestingly, low-dose administration of these same inhibitors reduces sensitivity to painful stimuli, suggesting a complex role for the proteasome in sensory processing6,14,15. However, despite these extensive studies, the specific function of proteasomes in the PNS has remained poorly understood.
Proteasomes are the primary protein degradation machinery in all domains of life and are essential for maintaining protein homeostasis16. The 20S core proteasome is composed of four heptameric rings of &#945;, &#946;, &#946;, &#945; subunits and mediates ubiquitin-independent protein degradation17,18. Association with regulatory particles forms the 26S and 30S capped proteasome, which mediates ubiquitin-dependent protein degradation4,17. In neurons, proteasomes have been identified in all cellular compartments, and their subcellular localization is known to influence function19. However, many commonly used proteasome inhibitors lack specificity to selectively target differentially localized proteasome populations.
Indeed, using membrane-impermeable proteasome inhibitors and antibody labeling in non-permeabilized cells, a neuron-specific proteasome complex was found to localize to the plasma membrane in a subset of somatosensory neurons20. scRNA-seq analysis of primary dorsal root ganglion (DRG) neurons, separated into NMP+ and NMP-&#160;populations via antibody feeding against a proteasome subunit and FACS sorting, resulted in 20 transcriptionally distinct cell clusters20. Further analysis revealed that the NMP+ cells clustered to only 3 of the 20 clusters. Using gene marker sets for specific neuronal subtypes, 13 different somatosensory neuron subtypes were identified20. The majority of NMP+ neurons clustered with MrgprA3+&#160;and Cysltr2+ sensory neurons20. A minimal number of the NMP+&#160;cells clustered to a third cluster, in which no specific gene sets were identified, and this cluster was therefore left unassigned20. MrgprA3+&#160;and Cysltr2+&#160;neurons are C-type nociceptors, sensitive to heat, mechanical, and pruritogenic stimuli, corresponding to CGRP-&#952; and SST neuronal subtypes, respectively21,22. Of the 20 identified cell clusters, 7 NMP- clusters expressed gene markers consistent with non-neuronal cells, including Schwann cells, satellite glia, and immune cells, and were also left unassigned20. These cell populations were expected, as it is not possible to separate non-neuronal from neuronal cells during DRG neuron culturing20.
Investigating its function, in vitro inhibition of the NMP altered neuronal excitability to KCl, histamine, and &#945;&#946;-methyleneadenosine 5&#39;-triphosphate stimulation20. In vivo, NMP inhibition reduced sensitivity to mechanical and pain stimuli with no effects on thermal sensation20. These findings reveal that the NMP is a key regulator of mechanical, pain, and itch sensation, and a potential therapeutic target for pain management20. However, the specific mechanisms linking NMP activity to pain development across different disease contexts remain poorly understood. Therefore, further investigation into NMP expression dynamics and function in pain- and neuropathy-associated conditions is warranted.
Here, this article describes a robust workflow (Figure 1) for identifying and isolating NMP+&#160;DRG neuronal populations. This approach enables cell-type-specific investigation of NMP expression dynamics using FACS sorting, scRNA-seq, and immunostaining. These approaches enable high-resolution analysis of NMP expression in a cell-type-specific manner, which can be used to investigate the role of the NMP in normal and various pain-associated conditions.

<table><tbody><tr><td>1 M HEPES buffer solution, pH 7.3</td><td>Quality Biological</td><td>118-089-721</td><td>FACS buffer</td></tr><tr><td>10x PBS</td><td>Thermo Fisher Scientific</td><td>70011-044</td><td>FACS buffer</td></tr><tr><td>16% Paraformaldehyde (PFA) stock solution; EM grade</td><td>EMS</td><td>15710</td><td>fixative solution</td></tr><tr><td>1x Phosphate-buffered saline (PBS)</td><td>Fisher Scientific</td><td>10-010-023</td><td>FACS washes</td></tr><tr><td>5 mL polystyrene round-bottom tube</td><td>Fisher Scientific</td><td>14-959-6</td><td>FACS tubes</td></tr><tr><td>7-Aminoactinomycin D (7-AAD)</td><td>Invitrogen</td><td>A1310</td><td>Labels dead cells</td></tr><tr><td>B27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td>culture medium</td></tr><tr><td>Bovine serum albumin</td><td>MilliporeSigma</td><td>A9647; CAS: 9048-46-8</td><td>BSA/TI/DMEM</td></tr><tr><td>Calcium chloride dihydrate (CaCl?-2H?O)</td><td>MilliporeSigma</td><td>C7902</td><td>complete saline solution</td></tr><tr><td>Chicken anti-neurofilament heavy chain (NF-H)</td><td>MilliporeSigma</td><td>AB5539</td><td>antibody</td></tr><tr><td>DMEM/F12 1:1</td><td>Thermo Fisher Scientific</td><td>11320033</td><td>culture medium; BSA/TI/DMEM</td></tr><tr><td>Donkey anti-goat IgG (H+L) Alexa Fluor Plus 555</td><td>Thermo Fisher Scientific</td><td>A32816</td><td>antibody</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor Plus 488</td><td>Thermo Fisher Scientific</td><td>A32790</td><td>antibody</td></tr><tr><td>DyLight 405 donkey anti-chicken</td><td>Jackson ImmunoResearch</td><td>703-475-155</td><td>antibody</td></tr><tr><td>EDTA</td><td>MilliporeSigma</td><td>E5134; CAS: 6381-92-6</td><td>Liberase TM/Liberase TL-papain working solutions</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>MilliporeSigma</td><td>A9647</td><td>culture medium/FACS buffer/blocking solution</td></tr><tr><td>Glass pasteur pipette, 9 inch</td><td>Fisher Scientific</td><td>1367820D</td><td>titruration</td></tr><tr><td>Goat anti-PSMA2</td><td>made in house</td><td>NA</td><td>antibody</td></tr><tr><td>HEPES powder</td><td>MilliporeSigma</td><td>H4034</td><td>complete saline solution</td></tr><tr><td>Hoechst</td><td>Thermo Fisher Scientific</td><td>H21492</td><td>nuclear stain</td></tr><tr><td>Horse serum</td><td>Thermo Fisher Scientific</td><td>26050070</td><td>blocking solution</td></tr><tr><td>IB4, biotin conjugate</td><td>MilliporeSigma</td><td>L2140</td><td>antibody</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020-1MG</td><td>culture dish</td></tr><tr><td>L-glutamine</td><td>Thermo Fisher Scientific</td><td>25030081</td><td>culture medium</td></tr><tr><td>Liberase trituration low (TL)</td><td>MilliporeSigma</td><td>54010200001</td><td>Liberase TL stock solution</td></tr><tr><td>Liberase trituration moderate (TM)</td><td>MilliporeSigma</td><td>5401119001</td><td>Liberase TM stock solution</td></tr><tr><td>Magnesium chloride hexahydrate (MgCl?-6H?O)</td><td>MilliporeSigma</td><td>M2393</td><td>complete saline solution</td></tr><tr><td>Mounting media</td><td>SouthernBiotech</td><td>0100-01</td><td>Fluoromount-G</td></tr><tr><td>Papain</td><td>Worthington</td><td>Cat#: 9001-73-4</td><td>Liberase TL-papain working solution</td></tr><tr><td>Penicillin/streptomycin</td><td>MilliporeSigma</td><td>15140122</td><td>culture medium/FACS buffer</td></tr><tr><td>Poly-L-lysine hydrobromide (PLL)</td><td>MilliporeSigma</td><td>P4707</td><td>culture dish</td></tr><tr><td>Potassium chloride (KCl)</td><td>MilliporeSigma</td><td>P5405</td><td>complete saline solution</td></tr><tr><td>Rabbit anti-CGRP</td><td>Immunostar</td><td>24112</td><td>antibody</td></tr><tr><td>sodium chloride (NaCl)</td><td>MilliporeSigma</td><td>S9888; CAS: 7647-14-5</td><td>complete saline solution</td></tr><tr><td>Sorbitol</td><td>MilliporeSigma</td><td>56755-M</td><td>complete saline solution</td></tr><tr><td>Streptavidin Alexa Fluor 647 conjugate</td><td>Thermo Fisher Scientific</td><td>S21374</td><td>antibody</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389-500G</td><td>fixative solution</td></tr><tr><td>Trypsin inhibitor</td><td>MilliporeSigma</td><td>T9253; CAS: 9035-91-8</td><td>BSA/TI/DMEM</td></tr></tbody></table>

cell subtype-specific analysis of neuronal membrane proteasome in somatosensory neurons

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

Sensory neuron signaling depends on functional proteasomes, and their subcellular localization influences function. A specialized proteasome complex, the neuronal membrane proteasome (NMP), was recently identified in a subpopulation of peripheral somatosensory neurons. The NMP localizes exclusively to the plasma membrane of the soma and both proximal and distal axons. Single-cell RNA sequencing (scRNA-seq) revealed that, under normal conditions, the NMP is predominantly expressed in MrgprA3+&#160;and Cysltr2+&#160;neurons, which mediate mechanical, itch, and pain sensation. Acute, selective NMP inhibition in the paw skin of mice reduced mechanical and pain sensitivity without affecting thermal sensation. Notably, unlike global proteasome inhibition, selective NMP inhibition does not cause neuropathy. In vitro studies suggest that the NMP facilitates neuron-to-neuron communication, potentially through the release of signaling peptides, and is essential for normal neuronal responses to stimulation. These findings identify the NMP as a key regulator of sensory neuron crosstalk required for normal touch, pain, and itch sensation. Thus, the NMP represents a potential target for pain management. This article presents methods for isolating both NMP-expressing and non-NMP-expressing neuronal populations using antibody feeding and fluorescence-activated cell sorting (FACS). These populations are suitable for cell-type-specific expression profiling via scRNA-seq or culture-based analyses of NMP expression dynamics. Together, these methods provide a robust workflow for investigating NMP expression dynamics and its contribution to pain-relevant disease conditions.

The ability to distinguish between the NMP and intracellular proteasomes relies on membrane-impermeable labeling approaches. Antibody feeding allows the use of proteasome subunit-specific antibodies on live neurons to label proteasomes accessible only from the outside of the cell. Due to their size and hydrophilicity, antibodies are unable to cross intact cell membranes24. Live neurons maintain membrane integrity, preventing antibodies from entering the cell. Using...

The neuronal membrane proteasome (NMP) was recently identified in a subset of somatosensory neurons as a key regulator of touch, pain, and itch perception. This article presents a robust workflow for analyzing cell-type-specific NMP expression and function using cell sorting, transcriptome profiling, and culture-based immunofluorescent analyses.

Sensory neuron signaling depends on functional proteasomes, and their subcellular localization influences function. A specialized proteasome complex, the ...

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.

cell-subtype-specific-analysis-neuronal-membrane-proteasome

Research

JoVE Journal

Neuroscience

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described.  The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Learn cell subtype-specific analysis of neuronal membrane proteasome in somatosensory neurons using cell sorting and immunofluorescence. This method enables isolation of viable NMP-expressing neurons for studying sensory modulation.

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

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