Yan Li

Cholesterol-rich lipid rafts are organizing platforms for many excitatory ion channels and receptors in neurons and for inflammatory receptors like toll-like receptor 4 (TLR4) in immune cells. Human dorsal root ganglion (hDRG) neurons that express nociceptor markers also express high levels of TLR4. Apolipoprotein A-I binding protein (AIBP) binds to TLR4 and promotes cholesterol depletion and lipid raft disruption in cells where TLR4 is expressed. The current study conducted with male and female hDRG demonstrates that TLR4-lipid rafts are expressed with higher density in pain- versus non-pain-associated tissues and localized to neurons expressing transient receptor potential vanilloid 1 (TRPV1) typically expressed by nociceptors. This increased density of TLR4-lipid rafts results in increased excitability of hDRG TRPV1 receptors. Additionally, whole-cell recordings show that disruption of TLR4-rafts with AIBP inhibited pain-associated ectopic action potential generation and evoked neuronal excitation in hDRG neurons. AIBP treatment impacted several action potential dynamics specifically in cells with ongoing ectopic action potentials rather than nonspontaneously active neurons. Finally, AIBP is shown to be localized with satellite cells in human DRG and its expression increases along with glutamine synthetase in pain-associated hDRG. Given that TLR4-raft expression in DRG neurons is largely confined to nociceptors, a TLR4-raft-specific therapeutic such as AIBP may have potential as a novel pain therapeutic with a low-side effect profile.

Spontaneous activity in dorsal root ganglion (DRG) neurons is a key driver of neuropathic pain in patients suffering from this largely untreated disease. While many intracellular signalling mechanisms have been examined in preclinical models that drive spontaneous activity, none have been tested directly on spontaneously active human nociceptors. Using cultured DRG neurons recovered during thoracic vertebrectomy surgeries, we showed that inhibition of mitogen-activated protein kinase interacting kinase (MNK) with tomivosertib (eFT508, 25 nM) reversibly suppresses spontaneous activity in human sensory neurons that are likely nociceptors based on size and action potential characteristics associated with painful dermatomes within minutes of treatment. Tomivosertib treatment also decreased action potential amplitude and produced alterations in the magnitude of after hyperpolarizing currents, suggesting modification of Na⁺ and K⁺ channel activity as a consequence of drug treatment. Parallel to the effects on electrophysiology, eFT508 treatment led to a profound loss of eIF4E serine 209 phosphorylation in primary sensory neurons, a specific substrate of MNK, within 2 min of drug treatment. Our results create a compelling case for the future testing of MNK inhibitors in clinical trials for neuropathic pain.

Solid tumors sculpt their microenvironments to maximize their growth and metastatic potential. Previously, we showed that the loss of p53 in head and neck squamous cell carcinoma (HNSCC) cells reprograms tumor-associated neurons (TANs) towards an adrenergic phenotype and promotes tumor progression. The impact of this neuronal reprogramming on the tumor immune microenvironment remains undefined. To understand how neuronal transdifferentiation regulates the antitumor immune response, we tested our hypothesis that shuttling cancer-derived microRNAs to TANs via exosomes induces an immunosuppressive neuronal phenotype in the tumor microenvironment (TME) that undermines the anti-tumor immune response. We first checked the stability of HNSCC-derived exosomes and their cargo (i.e., microRNA [miR]). Then, we assessed the miR transfer to TANs both in vitro and in vivo using HNSCC syngeneic models, and we measured miR enrichment in the trigeminal ganglia neurons after exosome injection. We used RNA sequencing to assess the trigeminal ganglia’s transcriptome and a single-cell multiplex enzyme-linked immunosorbent assay to evaluate the functional status of CD8⁺ T cells cocultured with cancer cells with or without TANs. Cancer cell–derived exosomes and their cargo were stable for up to 14 days in the TME and were taken up by TANs both in vitro and in vivo. The injection of exosomes into the peripheral nerves led to miR324a and miR21 upregulation in the associated trigeminal ganglionic soma, indicating that cancer-derived exosomes mediated crosstalk between cancer and neuronal cells. The RNA sequencing of human and mouse neuronal cells transfected with miR21 and miR324a revealed many differentially regulated immune pathways, including the neuroinflammation, T-cell receptor signaling, toll-like receptor, macrophage differentiation and growth inhibition, antigen-processing, interleukin (IL)-6, programmed cell death protein 1/programmed death-ligand 1, acute-phase response signaling, IL-8, wound-healing, IL-9, IL-17A, IL-23, IL-2, IL-10, IL-7, IL-4, IL-33, IL-36, CD28, CD40, signal transducer and activator of transcription 3, and nuclear factor kappa B pathways. Furthermore, the single-cell proteomic assay indicated that the CD8+ T cells cultured with neuronal cells were functionally distinct from the activated T cells co-cultured with neuronal and cancer cells. The co-culture with cancer cells and neuronal cells significantly downregulated the effector phenotype markers (e.g., granzyme B, interferon gamma, tumor necrosis factor alpha) in activated T cells, indicating that the interaction of cancer cells with neurons regulates the immunosuppressive TME. Our study showed the ability of cancer-derived exosomes to reprogram neurons and downstream to that to regulate the tumor immune environment in HNSCC. Our data suggest that transdifferentiating of TANs induce immunosuppression by direct inhibitory signal and immune checkpoint expression in the TME of HNSCC. Future studies are needed to investigate TANs as a potential target in immune oncology. Citation Format: Shamima Akhter, Tongxin Xie, Simone Anfossi, Adewale Adebayo, Frederico O. Gleber-Netto, Melanie Winkle, Masayoshi Shimitzu, Weilu Zhao, Megan L. Uhelski, Yan Li, George A. Calin, Moran Amit. Harmony between transdifferentiated neuron and immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-068.

Neuropathic pain is a leading cause of high-impact pain, is often disabling and is poorly managed by current therapeutics. Here we focused on a unique group of neuropathic pain patients undergoing thoracic vertebrectomy where the dorsal root ganglia is removed as part of the surgery allowing for molecular characterization and identification of mechanistic drivers of neuropathic pain independently of preclinical models. Our goal was to quantify whole transcriptome RNA abundances using RNA-seq in pain-associated human dorsal root ganglia from these patients, allowing comprehensive identification of molecular changes in these samples by contrasting them with non-pain-associated dorsal root ganglia. We sequenced 70 human dorsal root ganglia, and among these 50 met inclusion criteria for sufficient neuronal mRNA signal for downstream analysis. Our expression analysis revealed profound sex differences in differentially expressed genes including increase of IL1B, TNF, CXCL14 and OSM in male and CCL1, CCL21, PENK and TLR3 in female dorsal root ganglia associated with neuropathic pain. Coexpression modules revealed enrichment in members of JUN-FOS signalling in males and centromere protein coding genes in females. Neuro-immune signalling pathways revealed distinct cytokine signalling pathways associated with neuropathic pain in males (OSM, LIF, SOCS1) and females (CCL1, CCL19, CCL21). We validated cellular expression profiles of a subset of these findings using RNAscope in situ hybridization. Our findings give direct support for sex differences in underlying mechanisms of neuropathic pain in patient populations.

Chronic pain affects one in five of the general population and is the third most important cause of disability-adjusted life-years globally. Unfortunately, treatment remains inadequate due to poor efficacy and tolerability. There has been a failure in translating promising preclinical drug targets into clinic use. This reflects challenges across the whole drug development pathway, from preclinical models to trial design. Nociceptors remain an attractive therapeutic target: their sensitization makes an important contribution to many chronic pain states, they are located outside the blood–brain barrier, and they are relatively specific. The past decade has seen significant advances in the techniques available to study human nociceptors, including: the use of corneal confocal microscopy and biopsy samples to observe nociceptor morphology, the culture of human nociceptors (either from surgical or post-mortem tissue or using human induced pluripotent stem cell derived nociceptors), the application of high throughput technologies such as transcriptomics, the in vitro and in vivo electrophysiological characterization through microneurography, and the correlation with pain percepts provided by quantitative sensory testing. Genome editing in human induced pluripotent stem cell-derived nociceptors enables the interrogation of the causal role of genes in the regulation of nociceptor function. Both human and rodent nociceptors are more heterogeneous at a molecular level than previously appreciated, and while we find that there are broad similarities between human and rodent nociceptors there are also important differences involving ion channel function, expression, and cellular excitability. These technological advances have emphasized the maladaptive plastic changes occurring in human nociceptors following injury that contribute to chronic pain. Studying human nociceptors has revealed new therapeutic targets for the suppression of chronic pain and enhanced repair. Cellular models of human nociceptors have enabled the screening of small molecule and gene therapy approaches on nociceptor function, and in some cases have enabled correlation with clinical outcomes. Undoubtedly, challenges remain. Many of these techniques are difficult to implement at scale, current induced pluripotent stem cell differentiation protocols do not generate the full diversity of nociceptor populations, and we still have a relatively poor understanding of inter-individual variation in nociceptors due to factors such as age, sex, or ethnicity. We hope our ability to directly investigate human nociceptors will not only aid our understanding of the fundamental neurobiology underlying acute and chronic pain but also help bridge the translational gap.

Radiation therapy for brain tumors commonly induces cognitive dysfunction. The prefrontal cortex (PFC) is crucial for a diverse array of cognitive processes, however its role in radiation-induced cognitive dysfunction is unknown. We previously found that cranial irradiation impairs neuroplasticity along the hippocampal-PFC pathway. Herein, we hypothesized that brain irradiation directly affects the firing properties of PFC neurons, contributing to deficits in neuronal functions. In vivo recordings were used to monitor the firing activities of PFC neurons and local field potentials in both PFC and hippocampal CA1/subicular regions after cranial irradiation of Sprague Dawley rats. We further assessed the impacts of irradiation on axon initial segments (AIS) with immunofluorescence assays of PFC slices. We found that PFC neurons exhibited increased excitation three days after radiation and the timing of increased excitation coincided with elongation of the AIS. At two weeks, excitation levels returned to nearly normal levels however the population of spontaneously firing neurons decreased. While the number of NeuN-positive neurons in the PFC was not different, persistent neuronal injury, manifested as ATF-3 staining, was present at two weeks. Radiation also disrupted communication along the hippocampal-PFC pathway, with elongation of the phase lag between regions. Analysis of paired pulse ratios suggested that this was secondary to presynaptic dysfunction. Cranial irradiation excited and injured surviving PFC neurons and was associated with partial block of PFC’s functional coupling to the hippocampus. These deficits in the PFC may contribute to radiation-induced cognitive dysfunction.

Neuropathic pain encompasses a diverse array of clinical entities affecting 7–10% of the population, which is challenging to adequately treat. Several promising therapeutics derived from molecular discoveries in animal models of neuropathic pain have failed to translate following unsuccessful clinical trials suggesting the possibility of important cellular-level and molecular differences between animals and humans. Establishing the extent of potential differences between laboratory animals and humans, through direct study of human tissues and/or cells, is likely important in facilitating translation of preclinical discoveries to meaningful treatments. Patch-clamp electrophysiology and RNA-sequencing was performed on dorsal root ganglia taken from patients with variable presence of radicular/neuropathic pain. Findings establish that spontaneous action potential generation in dorsal root ganglion neurons is associated with radicular/neuropathic pain and radiographic nerve root compression. Transcriptome analysis suggests presence of sex-specific differences and reveals gene modules and signalling pathways in immune response and neuronal plasticity related to radicular/neuropathic pain that may suggest therapeutic avenues and that has the potential to predict neuropathic pain in future cohorts.

Nociceptors, sensory neurons in the DRG that detect damaging or potentially damaging stimuli, are key drivers of neuropathic pain. Injury to these neurons causes activation of translation regulation signaling, including the mechanistic target of rapamycin complex 1 (mTORC1) and mitogen-activated protein kinase interacting kinase (MNK) eukaryotic initiation factor (eIF) 4E pathways. This is a mechanism driving changes in excitability of nociceptors that is critical for the generation of chronic pain states; however, the mRNAs that are translated to lead to this plasticity have not been elucidated. To address this gap in knowledge, we used translating ribosome affinity purification in male and female mice to comprehensively characterize mRNA translation inScn10a-positive nociceptors in chemotherapy-induced neuropathic pain (CIPN) caused by paclitaxel treatment. This unbiased method creates a new resource for the field, confirms many findings in the CIPN literature and also find extensive evidence for new target mechanisms that may cause CIPN. We provide evidence that an underlying mechanism of CIPN is sustained mTORC1 activation driven by MNK1-eIF4E signaling. RagA, a GTPase controlling mTORC1 activity, is identified as a novel target of MNK1-eIF4E signaling. This demonstrates a novel translation regulation signaling circuit wherein MNK1-eIF4E activity drives mTORC1 via control of RagA translation. CIPN and RagA translation are strongly attenuated by genetic ablation of eIF4E phosphorylation, MNK1 elimination or treatment with the MNK inhibitor eFT508. We identify a novel translational circuit for the genesis of neuropathic pain caused by chemotherapy with important implications for therapeutics.SIGNIFICANCE STATEMENTNeuropathic pain affects up to 10% of the population, but its underlying mechanisms are incompletely understood, leading to poor treatment outcomes. We used translating ribosome affinity purification technology to create a comprehensive translational profile of DRG nociceptors in naive mice and at the peak of neuropathic pain induced by paclitaxel treatment. We reveal new insight into how mechanistic target of rapamycin complex 1 is activated in neuropathic pain pointing to a key role of MNK1-eIF4E-mediated translation of a complex of mRNAs that control mechanistic target of rapamycin complex 1 signaling at the surface of the lysosome. We validate this finding using genetic and pharmacological techniques. Our work strongly suggests that MNK1-eIF4E signaling drives CIPN and that a drug in human clinical trials, eFT508, may be a new therapeutic for neuropathic pain.

Mice with transgenic insertion of code for enhanced green fluorescent protein (EGFP) at the locus for glutamic acid decarboxylase 67 (GAD67), one of two key enzymes for the synthesis of γ-aminobutyric acid (GABA) were used to test whether the morphological properties of these neurons show plasticity with nerve injury. Physiological properties and the delivery of intracellular label to EGFP-expressing lamina II neurons was done using whole-cell patch-clamp in spinal cord slices from sham and chronic constriction injury (CCI) mice. As well, whole cell recordings were made of non-EGFP labeled cells to ascertain changes in overall inhibitory signaling following CCI. The EGFP labeled neurons in both sham and CCI mice exhibited islet, central and vertical cell morphological profiles but no radial cell profiles were observed. The length of cell dendrites was found to be significantly shorter in CCI mice for all cell profile types. The longest neurites averaged 155.96 ± 18.29 μm in CCI mice compared to 334.93 ± 29.48 μm in sham control mice. No change was observed in either passive or evoked membrane properties of EGFP-expressing neurons in CCI versus sham mice. Meanwhile, the frequency of miniature inhibitory post-synaptic currents of non-EGFP expressing spinal lamina II neurons was significantly reduced. These results suggest that reduced inhibitory output from GABA neurons occurs with nerve injury in part due to altered cell morphology.