Nociceptors are a type of sensory neuron that are integral to most forms of pain. Targeted disruption of nociceptor sensitization affords unique opportunities to prevent pain. An emerging model for nociceptors are sensory neurons derived from human stem cells. Here, we subjected five groups to high‐throughput sequencing: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC‐derived sensory neurons, mature co‐cultures containing hiPSC‐derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. Co‐culture of nociceptors and astrocytes promotes expression of transcripts enriched in DRG tissues. Comparisons of the hiPSC models to tissue samples reveal that many key transcripts linked to pain are present. Markers indicative of a range of neuronal subtypes present in the DRG were detected in mature hiPSCs. Intriguingly, translation factors were maintained at consistently high expression levels across species and culture systems. As a proof of concept for the utility of this resource, we validated expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissues and hiPSC samples. eIF5A is subject to a unique posttranslational hypusine modification required for its activity. Inhibition of hypusine biosynthesis prevented hyperalgesic priming by inflammatory mediators in vivo and diminished hiPSC activity in vitro. Collectively, our results illuminate the transcriptomes of hiPSC sensory neuron models. We provide a demonstration for this resource through our investigation of eIF5A. Our findings reveal hypusine as a potential target for inflammation associated pain in males.

Background and Purpose Translational controls pervade neurobiology. Nociceptors play an integral role in the detection and propagation of pain signals. Nociceptors can undergo persistent changes in their intrinsic excitability. Pharmacological disruption of nascent protein synthesis diminishes acute and chronic forms of pain‐associated behaviours. However, the targets of translational controls that facilitate plasticity in nociceptors are unclear. Experimental Approach We used ribosome profiling to probe the translational landscape in dorsal root ganglion (DRG) neurons from male Swiss‐Webster mice, after treatment with nerve growth factor and IL‐6. Expression dynamics of c‐Fos were followed with immunoblotting and immunohistochemistry. The involvement of ribosomal protein S6 kinase 1 (S6K1), a downstream component of mTOR signalling, in the control of c‐Fos levels was assessed with low MW inhibitors of S6K1 (DG2) or c‐Fos (T‐5224), studying their effects on nociceptor activity in vitro using multielectrode arrays (MEAs) and pain behaviour in vivo in Swiss‐Webster mice using the hyperalgesic priming model. Key Results c‐Fos was expressed in sensory neurons. Inflammatory mediators that promote pain in both humans and rodents promote c‐Fos translation. The mTOR effector S6K1 is essential for c‐Fos biosynthesis. Inhibition of S6K1 or c‐Fos with low MW compounds diminished mechanical and thermal hypersensitivity in response to inflammatory cues. Additionally, both inhibitors reduced evoked nociceptor activity. Conclusion and implications Our data show a novel role of S6K1 in modulating the rapid response to inflammatory mediators, with c‐Fos being one key downstream target. Targeting the S6 kinase pathway or c‐Fos is an exciting new avenue for pain‐modulating compounds.

The development of novel pain therapeutics hinges on the identification and rigorous validation of potential targets. Model organisms provide a means to test the involvement of specific genes and regulatory elements in pain. Here we provide a list of genes linked to pain‐associated behaviors. We capitalize on results spanning over three decades to identify a set of 242 genes. They support a remarkable diversity of functions spanning action potential propagation, immune response, GPCR signaling, enzymatic catalysis, nucleic acid regulation, and intercellular signaling. Making use of existing tissue and single‐cell high‐throughput RNA sequencing datasets, we examine their patterns of expression. For each gene class, we discuss archetypal members, with an emphasis on opportunities for additional experimentation. Finally, we discuss how powerful and increasingly ubiquitous forward genetic screening approaches could be used to improve our ability to identify pain genes. This article is categorized under: Neurological Diseases > Genetics/Genomics/Epigenetics Neurological Diseases > Molecular and Cellular Physiology

RNA stability is meticulously controlled. Here, we sought to determine if an essential post-transcriptional regulatory mechanism plays a role in pain.Nonsense-mediateddecay (NMD) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of roughly 10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target, UPF1, are expressed in murinedorsalrootganglion (DRG) sensory neurons. SMG1 protein is present in both the DRG and sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. We confirmed multiple NMD stability targets in sensory neurons including ATF4. ATF4 is preferentially translated during theintegratedstressresponse (ISR). This led us to ask if suspension of NMD induces the ISR. Inhibition of NMD increased eIF2-alpha phosphorylation and reduced the abundance of the eIF2-alpha phosphataseconstitutiverepressor ofeIF2-alphaphosphorylation (CReP). Finally, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 results in mechanical hypersensitivity in males and females that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through stimulation of the ISR.SIGNIFICANCE STATEMENT:Nociceptors undergo long-lived changes in their plasticity which may contribute to chronic pain. Translational regulation has emerged as a dominant mechanism in pain. Here, we investigate the role of a major pathway of RNA surveillance called NMD. Modulation of NMD is potentially beneficial for a broad array of diseases caused by frameshift or nonsense mutations. Our results suggest that inhibition of the rate-limiting step of NMD drives behaviors associated with pain through activation of the ISR. This work reveals complex interconnectivity between RNA stability and translational regulation and suggests an important consideration in harnessing the salubrious benefits of NMD disruption.

HuR is an RNA-binding protein implicated in RNA processing, stability, and translation. Previously, we examined protein synthesis in dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We found that the HuR consensus binding element was enriched in transcripts with elevated translation. HuR is expressed in the soma of nociceptors and their axons. Pharmacologic inhibition of HuR with the small molecule CMLD-2 reduced the activity of mouse and human sensory neurons. Peripheral administration of CMLD-2 in the paw or genetic elimination of HuR from sensory neurons diminished behavioral responses associated with NGF- and IL-6-induced allodynia in male and female mice. Genetic disruption of HuR altered the proximity of mRNA decay factors near a key neurotrophic factor (TrkA). Collectively, the data suggest that HuR is required for local control of mRNA stability and reveals a new biological function for a broadly conserved post-transcriptional regulatory factor.SIGNIFICANCE STATEMENTNociceptors undergo long-lived changes in excitability, which may contribute to chronic pain. Noxious cues that promote pain lead to rapid induction of protein synthesis. The underlying mechanisms that confer specificity to mRNA control in nociceptors are unclear. Here, we identify a conserved RNA-binding protein called HuR as a key regulatory factor in sensory neurons. Using a combination of genetics and pharmacology, we demonstrate that HuR is required for signaling in nociceptors. In doing so, we report an important mechanism of mRNA control in sensory neurons that ensures appropriate nociceptive responses to inflammatory mediators.