Generating Critical Knowledge on the NMP Peptide Signaling in the Peripheral Nervous System

About This Grant

PROJECT SUMMARY This new R21 application focuses on extending our work on the neuronal membrane proteasome derived peptides (NMP-peptides)1-7 and their link to peripheral neuron crosstalk and regulation. In this proposal, we present new data describing NMP-peptides presence and function on DRG neuron signaling in the PNS. In our previous studies using central nervous system (CNS) neurons, we developed new tools and protocols to purify, identify, and test specific NMP-derived peptides.2,4,5 In the peripheral nervous system (PNS), we made the important discovery that NMPs are expressed only on a subset (Mgpra3+ and Cystlr2+) of somatosensory DRG neurons.7 Using approaches developed for our CNS studies, we have found a preliminary set of NMP-peptide sequences that can lead to modulation of neuronal signaling in diverse populations of naïve DRG neuronal subtypes, including those that do not express NMPs. Taken together, we now hypothesize that specific NMP- peptides mediate ‘crosstalk’ between sensory DRG neuron subtypes (NMP(+) and NMP(-)) to induce downstream changes in neuronal signaling. Consistent with our hypothesis, we demonstrated that inhibition of NMPs modulate cell autonomous and cell non-autonomous responses to stimulation between DRG subtypes.7 In this proposal we aim to test our hypothesis in the PNS, using proteomics, calcium imaging, and single cell RNA sequencing studies with the following expected outcomes: 1) Identify sequences of PNS NMP-peptides following distinct stimuli; 2) Determine which NMP-peptides actively stimulate specific DRG subtypes; And 3) Show the NMP-peptide induced sensory neuron-specific transcriptional changes. Our goal with this application will be to reveal the actions of NMP-peptides in the PNS and provide a foundation for further studying this novel mechanism of crosstalk between sensory neurons that we have shown is critical for sensory behaviors such as touch and pain.7 NMP peptides offer a new opportunity in this area of DRG crosstalk and this R21 is foundational as a first of its kind investigation in NMP peptides and PNS function. To attain our goal, we will focus on the mouse DRG sensory neurons which has a variety of tools for evaluating the details of cell type specific biochemical, cellular, and calcium signaling changes. Specifically: Aim 1. To define and classify the stimulus induced NMP-derived peptides from DRG neurons, to test our working hypothesis that NMP expressing DRG neurons produce NMP-peptides following specific stimuli (depolarization or pruritogen stimulation). Aim 2. To study and identify the NMP-peptides that activate DRG neurons, to test our working hypothesis that active NMP-peptides have unique signature sequence and stimulate distinct subsets of DRG neurons. Aim 3. To determine the DRG-specific transcriptional programs stimulated by active NMP- peptides, to test our working hypothesis that NMP-peptides work to drive new downstream transcriptional changes in diverse populations of DRGs. This will begin to define a mechanistic link between specific NMP- peptides and the downstream autonomous and non-autonomous regulation of DRG subtypes.