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

Summary

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.

Abstract

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⁺ and Cysltr2⁺ 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.

Introduction

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 function^7,8,9,10. Furthermore, proteasome-inhibitor-based chemotherapy induces painful neuropathies in 30-60% of patients, often characterized by chronic pain and altered sensation in the extremities^8,9,11,12,13. Interestingly, low-dose administration of these same inhibitors reduces sensitivity to painful stimuli, suggesting a complex role for the proteasome in sensory processing^6,14,15. 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 α, β, β, α subunits and mediates ubiquitin-independent protein degradation^17,18. Association with regulatory particles forms the 26S and 30S capped proteasome, which mediates ubiquitin-dependent protein degradation^4,17. 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^- 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⁺ and Cysltr2⁺ sensory neurons²⁰. A minimal number of the NMP⁺ cells clustered to a third cluster, in which no specific gene sets were identified, and this cluster was therefore left unassigned²⁰. MrgprA3⁺ and Cysltr2⁺ neurons are C-type nociceptors, sensitive to heat, mechanical, and pruritogenic stimuli, corresponding to CGRP-θ and SST neuronal subtypes, respectively^21,22. 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 αβ-methyleneadenosine 5'-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⁺ 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.

Protocol

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

1. Prepare Complete Saline Solution (CSS) containing 137 mM NaCl, 5.3 mM KCl, 1 mM MgCl₂·6H₂O, 25 mM sorbitol, 10 mM HEPES, and 3 mM CaCl₂·2H₂O. Adjust pH to 7.2 with NaOH and filter sterilize.
2. Prepare the tissue dissociation enzyme blend working solution (moderate trituration) by combining 190 µL of the stock (5 mg/mL in H₂O), 84 µL of 50 mM EDTA, and 6.5 mL of CSS. Prepare fresh and filter-sterilize before use.
3. Prepare tissue dissociation enzyme blend (low trituration)-papain solution containing 190 µL of enzyme blend stock solution, 150 µL of papain, 84 µL of 50 mM EDTA, and 6.5 mL of CSS. Prepare fresh and filter-sterilize before use.
4. 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.
5. 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.
6. Prepare FACS buffer by combining 1% FBS, 1x PBS, 25 mM HEPES, and 1% penicillin/streptomycin in autoclaved water. Prepare fresh and filter-sterilize.
7. 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 °C.

2. Preparation of single-cell DRG neuron suspension

1. Harvest all DRGs from 4-6 P20-P23 mice following previously described methods^20,23.
2. 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 °C for 20 min.
3. Spin down ganglia at 120 × 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 °C for 15 min.
4. Spin down at 120 × g for 2 min. Remove the supernatant and resuspend the ganglia in 500 µL of BSA/TI/DMEM solution.
5. Triturate the ganglia 12-16x with a 1 mL plugged fire-polished glass Pasteur pipette.
6. Filter out cellular debris by passing cells through a 40 µm 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.
7. Spin down at 120 × 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.

3. Antibody feeding to label NMP-positive neurons

1. Divide the DRG neuron suspension into two 1.5 mL tubes, each containing 500 µL, and label the two tubes: "primary" and "no primary".
2. Add 20 µL of goat anti-proteasome 20S subunit alpha 2 (PSMA2) antibody to the "primary" labeled tube (1:25 dilution).
   1. 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.
3. Incubate the cells under gentle agitation for 30 min at 37 °C.
4. Warm filtered, sterilized 1x PBS during primary antibody incubation.
5. Centrifuge the tubes at 200 × g for 2 min and remove 90% of the supernatant (discard).
6. Resuspend the neurons in 500 µL of warm 1x PBS. Incubate the tubes with gentle agitation for 3 min at 37 °C.
7. Repeat steps 3.5-3.6 for a total of three washes.
8. While the washing steps are in progress, prepare secondary antibody by diluting 4 µL of anti-goat IgG (555-nm fluorophore) in 1,000 µL of warm DRG media (1:250 dilution).
9. After the third wash, resuspend both the "primary" and "no primary" tubes in 500 µL of the secondary antibody solution.
   NOTE: From this point forward, protect the tubes from light by wrapping them with aluminum foil.
10. Incubate the tubes with gentle agitation for 40 min at 37 °C.
11. Wash the cells by repeating steps 3.5-3.6 for a total of three washes.
12. After the third wash, resuspend neurons in 300 µL 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.
13. 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.

4. Separation of NMP-positive (NMP⁺ ) and NMP-negative (NMP^- ) neurons via FACS sorting

1. Immediately after antibody feeding and labeling of DRG neurons, take the cells and coated tubes to a FACS sorter.
2. Add 7-aminoactinomycin D (7-AAD) (1:1,000) to both the "primary" and "no primary" tubes. Incubate for 10-30 min on ice.
   NOTE: 7-AAD is used to exclude dead cells from the sorting process.
3. 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.
4. Use the "no primary" labeled tube to set parameters and gating for unlabeled neurons.
5. Apply forward scatter (FSC) gating to isolate cells consistent with the size of sensory neurons. Sensory neuron diameters range from 10 to 50 µm.
6. 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).
7. Load the "primary" tube and set gating parameters as established with the "no primary" sample to detect unlabeled (NMP^-) and 555-positive (NMP⁺) neuronal populations.
8. Using a 100 µm nozzle and sorting pressure of 15-20 psi, sort the neurons into 555⁺ and 555^-.
9. Proceed to downstream analysis of sorted neurons.

5. Single-cell RNA sequencing of NMP⁺ and NMP^- neurons

1. Transfer the sorted cells to 1.5 mL tubes and place on ice. Transport the cells to a single-cell sequencing core.
2. Perform single-cell RNA sequencing on the NMP⁺ and NMP^- sorted neurons at a depth of 30,000 to 100,000 reads per cell.
3. 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 Villalón Landeros et al.²⁰.
4. Construct a list of specific gene markers to identify individual neuronal populations using available databases such as https://painseq.shinyapps.io/harmonized_painseq_v2/ or https://kleintools.hms.harvard.edu/tools/springViewer_1_6_dev.html?datasets/Sharma2019/all
5. 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).

6. Primary cultures of enriched NMP⁺ and NMP^- neurons

1. 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.
2. Once the coverslips are dry, coat them with 0.1 mg/mL poly-L-lysine hydrobromide (PLL) and place at 4 °C overnight.
3. 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.
4. Dilute 10 µL of 1 mg/mL laminin in 125 µL of DRG media and spot 20-30 µL on the center of each coverslip.
5. Coat the coverslips with laminin for at least 40 min before use.
6. Transfer the FACS-sorted cells to a 1.5 mL tube and centrifuge at 200 × g for 5 min to collect cells. Remove the supernatant and resuspend neurons in 50 µL of warm DRG media.
7. Aspirate laminin from the coverslip and allow the coverslip to dry.
8. Seed 15-20 µL 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.
9. 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.
10. Maintain cultures at 37 °C with 5% CO₂/95% humidity with 75% media changes every 2-3 days.

7. Cell-type-specific NMP expression analysis

1. Prepare a 140 mm culture plate or immunofluorescence staining tray by lining it with a large piece of parafilm.
2. 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.
3. Add 100 µL 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.
4. Dilute the goat anti-PSMA2 primary antibody in warm DRG media at a 1:20 dilution.
5. Using an aspirator fitted with a P10 unfiltered tip, remove the media from the coverslip slowly by aspirating from the edge. Add 100 µL of goat anti-PSMA2 antibody solution to the coverslips and incubate at room temperature for 40 min.
6. Carefully aspirate the primary antibody solution off the coverslips. Add 100 μL of PBS (at room temperature) and incubate for 3 min at room temperature to wash.
7. Aspirate PBS off the coverslip and repeat the addition and aspiration of PBS for a total of three washes.
8. Prepare anti-goat (e.g., 555-conjugated) secondary antibody in warm DRG media at a 1:250 dilution.
9. Following the third wash, add 100 µL 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.
10. Aspirate the secondary antibody solution off the coverslip and wash 3x as described in steps 7.6-7.7.
11. Remove the last PBS wash and fix the cells by adding 100 µL of fixative solution containing 4% paraformaldehyde (PFA), 4% sucrose in 1x PBS. Incubate at room temperature for 10 min.
12. Aspirate fixative and wash 3x by repeating steps 7.6-7.7.
13. Permeabilize the cells by adding 100 µL of 0.1% nonionic detergent (e.g., Triton X-100) in 1x PBS and incubate at room temperature for 5 min.
14. Aspirate permeabilizing solution and wash 3x by repeating steps 7.6-7.7.
15. Add 100 µL of blocking solution consisting of 5% FBS, 5% donkey serum in 1x PBS and incubate at room temperature for 40 min.
16. 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.
17. Remove blocking solution and add the primary antibody mix to the coverslip. Incubate at room temperature for 45 min.
18. Remove the antibody solution and wash 3x by repeating steps 7.6-7.7.
19. 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.
20. Remove the last PBS wash and add 100 µL of secondary antibody solution to the coverslips. Incubate at room temperature for 45 min.
21. Wash 3x by repeating steps 7.6-7.7.
22. Optional: Prepare nuclear stain at 1-10 µg/mL. Add 100 µL 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.
23. 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.
24. Place a 7 µL 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.
25. 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.
26. 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.

Results

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...

Discussion

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 central^25,26,27and 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...

Materials

List of materials used in this article

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