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

Meghan E. Imhoff; Heidi J. Morris; Eric Villalón Landeros

doi:10.3791/69061

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Method Article

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

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DOI:

10.3791/69061

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October 10th, 2025

Meghan E. Imhoff¹^,² , Heidi J. Morris¹^,² , Eric Villalón Landeros¹^,²

¹Department of Molecular Pharmacology and Neuroscience, Loyola University Chicago Stritch School of Medicine, ²Center for Translational Research and Education

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 function⁷^,⁸^,⁹^,¹⁰. Furthermore, ....