Dorsal Root Ganglion Sensory Neurons Research Articles

Computational modeling to study the impact of changes in Nav1.8 sodium channel on neuropathic pain

ObjectiveNav1.8 expression is restricted to sensory neurons; it was hypothesized that aberrant expression and function of this channel at the site of injury contributed to pathological pain. However, the specific contributions of Nav1.8 to neuropathic pain are not as clear as its role in inflammatory pain. The aim of this study is to understand how Nav1.8 present in peripheral sensory neurons regulate neuronal excitability and induce various electrophysiological features on neuropathic pain.MethodsTo study the effect of changes in sodium channel Nav1.8 kinetics, Hodgkin–Huxley type conductance-based models of spiking neurons were constructed using the NEURON v8.2 simulation software. We constructed a single-compartment model of neuronal soma that contained Nav1.8 channels with the ionic mechanisms adapted from some existing small DRG neuron models. We then validated and compared the model with our experimental data from in vivo recordings on soma of small dorsal root ganglion (DRG) sensory neurons in animal models of neuropathic pain (NEP).ResultsWe show that Nav1.8 is an important parameter for the generation and maintenance of abnormal neuronal electrogenesis and hyperexcitability. The typical increased excitability seen is dominated by a left shift in the steady state of activation of this channel and is further modulated by this channel’s maximum conductance and steady state of inactivation. Therefore, modified action potential shape, decreased threshold, and increased repetitive firing of sensory neurons in our neuropathic animal models may be orchestrated by these modulations on Nav1.8.ConclusionComputational modeling is a novel strategy to understand the generation of chronic pain. In this study, we highlight that changes to the channel functions of Nav1.8 within the small DRG neuron may contribute to neuropathic pain.

Electrophysiological properties of dorsal root ganglion neurons cultured on 3D silicon micro-pillar substrates

BackgroundSilicon-based micro-pillar substrates (MPS), as three-dimensional cell culture platforms with vertically aligned micro-patterned scaffolding structures, are known to facilitate high-quality growth and morphology of dorsal root ganglion (DRG) sensory neurons, promote neurite outgrowth and enhance neurite alignment. However, the electrophysiological aspects of DRG neurons cultured on silicon MPSs have not been thoroughly investigated, which is of greatest importance to ensure that such substrates do not disrupt neuronal homeostasis and function before their widespread adoption in diverse biomedical applications. New methodWe conducted whole-cell patch-clamp recordings to explore the electrophysiological properties of DRG neurons cultured on MPS arrays, utilizing a custom-made upright patch-clamp setup. ResultsOur findings revealed that DRG neurons exhibited similar electrophysiological responses on patterned MPS samples when compared to the control planar glass surfaces. Notably, there were no significant differences observed in the action potential parameters or firing patterns of action potentials between neurons grown on either substrate. Comparison with existing methodsIn the current study we for the first time confirmed that successful electrophysiological recordings can be obtained from the cells grown on MPS. ConclusionOur results imply that, despite the potential alterations caused by the cumulative trauma of tissue harvest and cell dissociation, essential functional cell properties of DRG neurons appear to be relatively maintained on MPS surfaces. Therefore, vertically aligned silicon MPSs could be considered as a potentially effective three-dimensional system for supporting a controlled cellular environment in culture.

ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke.

Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

Sensory neuron transient receptor potential vanilloid-1 channel regulates angiogenesis through CGRP in vivo.

Angiogenesis plays a key role in bone regeneration. The role of neurons of peripheral nerves involved in angiogenesis of bone defects needs to be explored. The transient receptor potential vanilloid 1 (TRPV1), a nociceptor of noxious stimuli, is expressed on sensory neurons. Apart from nociception, little is known about the role of sensory innervation in angiogenesis. Calcitonin gene-related peptide (CGRP), a neuropeptide secreted by sensory nerve terminals, has been associated with vascular regeneration. We characterized the reinnervation of vessels in bone repair and assessed the impact of TRPV1-CGRP signaling on early vascularization. We investigated the pro-angiogenic effect of neuronal TRPV1 in the mouse model of femur defect. Micro-CT analysis with Microfil® reagent perfusion demonstrated neuronal TRPV1 activation enhanced angiogenesis by increasing vessel volume, number, and thickness. Meanwhile, TRPV1 activation upregulated the mRNA and protein expression of vascular endothelial growth factor A (VEGF-A), cell adhesion molecule-1 (CD31), and CGRP. Immunostaining revealed the co-localization of TRPV1 and CGRP in dorsal root ganglia (DRG) sensory neurons. By affecting neuronal TRPV1 channels, the release of neuronal and local CGRP was controlled. We demonstrated that TRPV1 influenced on blood vessel development by promoting CGRP release from sensory nerve terminals. Our results showed that neuronal TRPV1 played a crucial role in regulating angiogenesis during bone repair and provided important clinical implications for the development of novel therapeutic approaches for angiogenesis.

Characterization of human iPSC-derived sensory neurons and their functional assessment using multi electrode array

Sensory neurons are afferent neurons in sensory systems that convert stimuli and transmit information to the central nervous system as electrical signals. Primary afferent neurons that are affected by non-noxious and noxious stimuli are present in the dorsal root ganglia (DRG), and the DRG sensory neurons are used as an in vitro model of the nociceptive response. However, DRG derived from mouse or rat give a low yield of neurons, and they are difficult to culture. To help alleviate this problem, we characterized human induced pluripotent stem cell (hiPSC) derived sensory neurons. They can solve the problems of interspecies differences and supply stability. We investigated expressions of sensory neuron related proteins and genes, and drug responses by Multi-Electrode Array (MEA) to analyze the properties and functions of sensory neurons. They expressed nociceptor, mechanoreceptor and proprioceptor related genes and proteins. They constitute a heterogeneous population of their subclasses. We confirmed that they could respond to both noxious and non-noxious stimuli. We showed that histamine inhibitors reduced histamine-induced neuronal excitability. Furthermore, incubation with a ProTx-II and Nav1.7 inhibitor reduced the spontaneous neural activity in hiPSC-derived sensory neurons. Their responsiveness was different from each drug. We have demonstrated that hiPSC-derived sensory neurons combined with MEA are good candidates for drug discovery studies where DRG in vitro modeling is necessary.

A mouse DRG genetic toolkit reveals morphological and physiological diversity of somatosensory neuron subtypes

Dorsal root ganglia (DRG) somatosensory neurons detect mechanical, thermal, and chemical stimuli acting on the body. Achieving a holistic view of how different DRG neuron subtypes relay neural signals from the periphery to the CNS has been challenging with existing tools. Here, we develop and curate a mouse genetic toolkit that allows for interrogating the properties and functions of distinct cutaneous targeting DRG neuron subtypes. These tools have enabled a broad morphological analysis, which revealed distinct cutaneous axon arborization areas and branching patterns of the transcriptionally distinct DRG neuron subtypes. Moreover, invivo physiological analysis revealed that each subtype has a distinct threshold and range of responses to mechanical and/or thermal stimuli. These findings support a model in which morphologically and physiologically distinct cutaneous DRG sensory neuron subtypes tile mechanical and thermal stimulus space to collectively encode a wide range of natural stimuli.

A199 ALKAL2/ALK AXIS PROMOTE PERSISTENT VISCERAL PAIN

Abstract Background Long-lasting changes in neural pain circuits precipitate the transition from acute to chronic pain in patients living with inflammatory bowel diseases (IBDs). While significant improvement in IBD therapy has been made to reduce inflammation, a large subset of patients continues to suffer throughout quiescent phases of the disease. Peripheral and central mechanisms contribute to the transition from acute to chronic pain during clinical remission. In the search of new treatments, we previously found that targeting the anaplastic lymphoma kinase (ALK) receptor could have therapeutic value for persistent pain conditions. Aims Here we investigated whether ALKAL2 (ALK ligand) /ALK signaling axis regulates chronic visceral pain. Methods We used the post-inflammatory dextran sodium sulfate (DSS) mouse model of colitis as a well-established rodent model of chronic visceral pain, in which animals display increased VHS 5 weeks after DSS administration in the absence of inflammation as described in IBD patients. To assess the role of ALKAL2/ALK signalling in chronic visceral pain, we pharmacologically blocked ALK receptor using clinically available compound Lorlatinib in the postcolitis model. Moreover we genetically depleted ALKAL2 in gut-innervating nociceptors that express the heat sensitive transient receptor potential vanilloid type 1 (TRPV1) channel, using shRNA vectorized in cell type specific Adeno-associated Viruses. Results We showed that ALKAL2 expression was significatively enhanced in lumbosacral (LS) dorsal root ganglia (DRG) sensory neurons in the post-colitis model. In naïve mice, ALKAL2 administration induced visceral hypersensitivity in an ALK dependant manner. In addition, depletion of ALKAL2 in TRPV1 nociceptors or blocking ALK with the clinically available compound Lorlatinib, reversed visceral hypersensitivity in the post-colitis model. Accordingly, phosphorylation of ALK receptor occurring at the LS of the spinal dorsal horn was reduced. Conclusions Overall, these findings provide novel insights into the mechanisms underlying the transition from acute to chronic visceral pain in IBD. Our work could generate a portfolio of clinically available anti-cancer drugs, including Lorlatinib, as novel pain therapeutics. Repurposed ALK inhibitor could facilitate pain management in in individuals with IBD. Funding Agencies CIHR

Acid-sensing ion channel 3 mediates pain hypersensitivity associated with high-fat diet consumption in mice.

Lipid-rich diet is the major cause of obesity, affecting 13% of the worldwide adult population. Obesity is a major risk factor for metabolic syndrome that includes hyperlipidemia and diabetes mellitus. The early phases of metabolic syndrome are often associated with hyperexcitability of peripheral small diameter sensory fibers and painful diabetic neuropathy. Here, we investigated the effect of high-fat diet-induced obesity on the activity of dorsal root ganglion (DRG) sensory neurons and pain perception. We deciphered the underlying cellular mechanisms involving the acid-sensing ion channel 3 (ASIC3). We show that mice made obese through consuming high-fat diet developed the metabolic syndrome and prediabetes that was associated with heat pain hypersensitivity, whereas mechanical sensitivity was not affected. Concurrently, the slow conducting C fibers in the skin of obese mice showed increased activity on heating, whereas their mechanosensitivity was not altered. Although ASIC3 knockout mice fed with high-fat diet became obese, and showed signs of metabolic syndrome and prediabetes, genetic deletion, and in vivo pharmacological inhibition of ASIC3, protected mice from obesity-induced thermal hypersensitivity. We then deciphered the mechanisms involved in the heat hypersensitivity of mice and found that serum from high-fat diet-fed mice was enriched in lysophosphatidylcholine (LPC16:0, LPC18:0, and LPC18:1). These enriched lipid species directly increased the activity of DRG neurons through activating the lipid sensitive ASIC3 channel. Our results identify ASIC3 channel in DRG neurons and circulating lipid species as a mechanism contributing to the hyperexcitability of nociceptive neurons that can cause pain associated with lipid-rich diet consumption and obesity.

P009 IL-17 mediates visceral hypersensitivity by sensitising colonic nociceptors

Abstract Background Comparison of cytokine expression in colitic tissue with respective cytokine receptor expression in pain sensing neurons highlights a likely contribution of IL-17 to the production of abdominal pain during flare. To explore this pro-nociceptive potential of IL-17 in colitis we examined the ability of IL-17 to stimulate sensory neurons and the colonic afferent response to noxious distension. Methods IL-17 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to IL-17 (10-300 ng/mL) alone and following pre-treatment with either TRPV1 or TRPA1 inhibitors. Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. In a separate set of experiments the effect of IL-17 was also examine on a neuron pure cell culture in which non-neuronal cells were removes by magnet assisted cell sorting. IL-17 mediated colonic nociceptor activation: electrophysiological recordings were made of lumbar splanchnic nerve activity and the response to noxious luminal distension of the isolated colorectum (ex-vivo), perfused with carbogenated Krebs solution, examined following pre-treatment with IL-17 (50 ng/mL) alone or in the presence of respective inhibitors. All experiments were performed using tissue from male C57BL/6J mice euthanaised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Consistent with the ability of IL-17 to stimulate sensory neurons including those classified as nociceptors by their co-sensitivity to capsaicin, pre-treatment with IL-17 also produced a significant sensitisation of the colonic afferent response to luminal distension at noxious distending pressures. The increase in [Ca2+]i produced by IL-17 in sensory neurons was blocked by pre-treament with a TRPV1 antagonist but not a TRPA1 antagonist. Furthermore, the proportion of neurons stimulated by IL-17 was comparable between standard DRG cell cultures and neuron pure cell cultures demonstrating a direct effect of IL-17 on sensory neurons. Finally, IL-17 mediated mechanosensitisation of colonic nociceptors was unaffected by TRPV1 inhibition. Conclusion Our findings demonstrate that IL-17 stimulates capsaicin-sensitive nociceptors and mediates visceral hypersensitivity in colonic nociceptors consistent with a causative role for IL-17 is the generation of abdominal pain in people with colitis.

P176 TYK2 inhibition attenuates the stimulation of sensory neurons with hyper-IL-6

Abstract Background Abdominal pain and diarrhoea are a cause of significant morbidity for people with colitis. Recent single cell RNA sequencing of sensory neurons innervating the gut highlights a likely role for sensory nerves in the pathophysiology of these symptoms based on the marked expression of cytokine receptors from the IL-6 receptor family in gut projecting sensory neurons. In keeping with the reported downstream signalling pathway for these receptors marked co-expression of TYK2 transcripts were also found along with IL-6 signal transducer (GP130) in colonic nociceptors characterised by expression of the capsaicin receptor TRPV1. These observations suggest that TYK2 inhibitors may ameliorate symptoms of pain and diarrhoea in people with colitis by preventing neurogenically mediated gut contractility and nociceptor activation. Methods TYK2 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to the hyper-IL-6 fusion protein of the soluble IL-6 receptor and IL-6, which transactivates the GP130 receptor subunit to mimic IL-6 family receptor signalling following pre-treatment with vehicle, the TYK2 inhibitor Deucravacitinib (100nM) or the pan-JAK inhibitor Pyridone-6 (2 μM). Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. All experiments were performed using tissue from male C57/B6 mice euthanised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Application of hyper-IL-6 produced a robust increase in [Ca2+]i levels in DRG sensory neurons which was significantly attenuated by pre-treatment with the TYK2 specific inhibitor Deucravacitinib (p<0.01) or the pan JAK inhibitor Pyridone-6 (p = <0.05). Conclusion Findings demonstrate that inhibition of TYK2 inhibitor prevents sensory neuron activation in response to IL-6 trans-signalling. This data suggests that TYK2 inhibitors may have utility for the treatment of abdominal pain in people with colitis.

P045 MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1

Abstract Background Abdominal pain is a cause of significant morbidity for people with colitis. We have previously shown that biopsy samples from people with Crohn’s disease (CD) are pro-nociceptive suggesting that peripheral nociceptor activation is a cause of pain in colitis. Furthermore, these effects were correlated with biopsy expression of macrophage metalloproteinase (MMP12), which was shown to stimulate mouse and human colonic nociceptors highlighting the potential utility of MMP12 inhibitors to act as visceral analgesics. Following on from this work we sought to determine the contribution of proteinase-activated receptor 1 (PAR1) to MMP12 mediated nociceptor activation. Methods MMP12 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to MMP12 or the PAR1 receptor agonists TRAP6 following pre-treatment with vehicle, the MMP12 inhibitor MMP408 or the PAR1 antagonist SCH79797. Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. MMP12 mediated colonic nociceptor activation: electrophysiological recordings were made of lumbar splanchnic nerve activity and of the isolated colorectum (ex-vivo) perfused with carbogenated Krebs solution, and responses determined following application with MMP12 alone or in the presence of respective MMP12 or PAR1 inhibitors. All experiments were performed using tissue from male C57/B6 mice euthanised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Application of MMP12 or TRAP6 produced a robust increase in [Ca2+]i levels in DRG sensory neurons classified as nociceptors by their co-sensitivity to capsaicin. These effects were significantly attenuated by pre-treatment with respective MMP12 or PAR1 inhibitors, with the response to MMP12 also being inhibited by PAR1 antagonist pre-treatment. Consistent with this pattern of nociceptor stimulation, the application of MMP12 also a robust activation of colonic afferents that was abolished by inhibition of MMP12 or PAR1 activity. Conclusion Findings demonstrate that MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1.

Mitochondrial impairment, decreased sirtuin activity and protein acetylation in dorsal root ganglia in Friedreich Ataxia models

Friedreich ataxia (FA) is a rare, recessive neuro-cardiodegenerative disease caused by deficiency of the mitochondrial protein frataxin. Mitochondrial dysfunction, a reduction in the activity of iron-sulfur enzymes, iron accumulation, and increased oxidative stress have been described. Dorsal root ganglion (DRG) sensory neurons are among the cellular types most affected in the early stages of this disease. However, its effect on mitochondrial function remains to be elucidated. In the present study, we found that in primary cultures of DRG neurons as well as in DRGs from the FXNI151F mouse model, frataxin deficiency resulted in lower activity and levels of the electron transport complexes, mainly complexes I and II. In addition, altered mitochondrial morphology, indicative of degeneration was observed in DRGs from FXNI151F mice. Moreover, the NAD+/NADH ratio was reduced and sirtuin activity was impaired. We identified alpha tubulin as the major acetylated protein from DRG homogenates whose levels were increased in FXNI151F mice compared to WT mice. In the mitochondria, superoxide dismutase (SOD2), a SirT3 substrate, displayed increased acetylation in frataxin-deficient DRG neurons. Since SOD2 acetylation inactivates the enzyme, and higher levels of mitochondrial superoxide anion were detected, oxidative stress markers were analyzed. Elevated levels of hydroxynonenal bound to proteins and mitochondrial Fe2+ accumulation was detected when frataxin decreased. Honokiol, a SirT3 activator, restores mitochondrial respiration, decreases SOD2 acetylation and reduces mitochondrial superoxide levels. Altogether, these results provide data at the molecular level of the consequences of electron transport chain dysfunction, which starts negative feedback, contributing to neuron lethality. This is especially important in sensory neurons which have greater susceptibility to frataxin deficiency compared to other tissues.

A phenotypic screening platform for chronic pain therapeutics using all-optical electrophysiology.

Chronic pain associated with osteoarthritis (OA) remains an intractable problem with few effective treatment options. New approaches are needed to model the disease biology and to drive discovery of therapeutics. We present an in vitro model of OA pain, where dorsal root ganglion (DRG) sensory neurons were sensitized by a defined mixture of disease-relevant inflammatory mediators, here called Sensitizing PAin Reagent Composition or SPARC. Osteoarthritis-SPARC components showed synergistic or additive effects when applied in combination and induced pain phenotypes in vivo. To measure the effect of OA-SPARC on neural firing in a scalable format, we used a custom system for high throughput all-optical electrophysiology. This system enabled light-based membrane voltage recordings from hundreds of neurons in parallel with single cell and single action potential resolution and a throughput of up to 500,000 neurons per day. A computational framework was developed to construct a multiparameter OA-SPARC neuronal phenotype and to quantitatively assess phenotype reversal by candidate pharmacology. We screened ∼3000 approved drugs and mechanistically focused compounds, yielding data from over 1.2 million individual neurons with detailed assessment of functional OA-SPARC phenotype rescue and orthogonal "off-target" effects. Analysis of confirmed hits revealed diverse potential analgesic mechanisms including ion channel modulators and other mechanisms including MEK inhibitors and tyrosine kinase modulators. Our results suggest that the Raf-MEK-ERK axis in DRG neurons may integrate the inputs from multiple upstream inflammatory mediators found in osteoarthritis patient joints, and MAPK pathway activation in DRG neurons may contribute to chronic pain in patients with osteoarthritis.

Molecular Mechanisms of Intratumoral Nerve Recruitment and Perineural Invasion Elucidated with Spatial Transcriptomics and CRISPR Activation

Perineural invasion (PNI) is an aggressive manifestation of tumor-nerve interactions associated with postoperative recurrence, metastasis, pain, and decreased survival. Hence, PNI is included in the staging criteria of several malignancies and often an indication for treatment intensification using adjuvant radiotherapy. However, the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown-hindering the development of new therapies targeting this key pathological process. Moreover, prior studies were limited by a lack of cell-type information, spatial context, and/or a fragmented focus on a small number of pathways. Using pancreatic ductal adenocarcinoma (PDAC) as an exemplar given the exceptionally high frequency of PNI in this malignancy, we performed the first comprehensive, cell-type specific, and spatially resolved whole-transcriptome analysis of human PDAC to identify molecular mediators of tumor-nerve crosstalk and PNI. We constructed 12 custom tissue microarrays (TMAs) derived from matched malignant regions with and without tumor-nerve proximity (n = 288 cores). We performed whole-transcriptome digital spatial profiling (DSP) to independently determine mRNA abundance from the malignant, fibroblast, and nerve compartments through optical sectioning. We mapped malignant subtypes we previously identified onto the spatial data and found strong (p<0.0001) positive nerve associations with the mesenchymal, basaloid, and neural-like progenitor subtypes and a negative nerve association with the classical subtype. Numerous genes expressed by malignant cells were enriched (e.g., MMP2, PLXND1, NRP1) or depleted (e.g., SEMA3B) in association with radial distance from nerves, including recapitulation of prior literature. To functionally explore these candidate mediators of tumor-nerve crosstalk, we derived genetically-engineered murine organoids (KrasLSL-G12D/+; Trp53FL/FL; Rosa26-dCas9-VPR) and transduced them with guide RNAs to overexpress subtype-specific transcription factors or candidate genes from the spatial analysis. We quantified (1) cancer cell invasion through extracellular matrix using cultured dorsal root ganglia (DRG) sensory neurons as the chemoattractant, and (2) the role of cancer-intrinsic signaling on nerve recruitment/outgrowth by applying conditioned media or exogenous proteins to cultured DRG sensory neurons and tracking their growth with live imaging. Our results suggest that the mechanisms enabling cancer cells to recruit nerves into the tumor microenvironment are distinct from those facilitating perineural invasion. This study has transformed our understanding of how cancer cells and the peripheral nervous system collaborate to promote tumor growth, survival, and dissemination, and is now guiding prioritization of therapeutic strategies that synergize with adjuvant radiotherapy in the burgeoning field of cancer neuroscience.

Transcriptome and secretome profiling of sensory neurons reveals sex differences in pathways relevant to insulin sensing and insulin secretion.

Sensory neurons in the dorsal root ganglia (DRG) convey somatosensory and metabolic cues to the central nervous system and release substances from stimulated terminal endings in peripheral organs. Sex-biased variations driven by the sex chromosome complement (XX and XY) have been implicated in the sensory-islet crosstalk. However, the molecular underpinnings of these male-female differences are not known. Here, we aim to characterize the molecular repertoire and the secretome profile of the lower thoracic spinal sensory neurons and to identify molecules with sex-biased insulin sensing- and/or insulin secretion-modulating activity that are encoded independently of circulating gonadal sex hormones. We used transcriptomics and proteomics to uncover differentially expressed genes and secreted molecules in lower thoracic T5-12 DRG sensory neurons derived from sexually immature 3-week-old male and female C57BL/6J mice. Comparative transcriptome and proteome analyses revealed differential gene expression and protein secretion in DRG neurons in males and females. The transcriptome analysis identified, among others, higher insulin signaling/sensing capabilities in female DRG neurons; secretome screening uncovered several sex-specific candidate molecules with potential regulatory functions in pancreatic β cells. Together, these data suggest a putative role of sensory interoception of insulin in the DRG-islet crosstalk with implications in sensory feedback loops in the regulation of β-cell activity in a sex-biased manner. Finally, we provide a valuable resource of molecular and secretory targets that can be leveraged for understanding insulin interoception and insulin secretion and inform the development of novel studies/approaches to fathom the role of the sensory-islet axis in the regulation of energy balance in males and females.

Pamoic acid-induced peripheral GPR35 activation improves pruritus and dermatitis.

Pruritic dermatitis is a disease with a considerable unmet need for treatment and appears to present with not only epidermal but also peripheral neuronal complications. Here, we propose a novel pharmacological modulation targeting both peripheral dorsal root ganglion (DRG) sensory neurons and skin keratinocytes. GPR35 is an orphan G-protein-coupled receptor expressed in DRG neurons and has been predicted to downregulate neuronal excitability when activated. Modulator information is currently increasing for GPR35, and pamoic acid (PA), a salt-forming agent for drugs, has been shown to be an activator solely specific for GPR35. Here, we investigated its effects on dermatitic pathology. We confirmed GPR35 expression in peripheral neurons and tissues. The effect of PA treatment was pharmacologically evaluated in cultured cells in vitro and in in vivo animal models for acute and chronic pruritus. Local PA application mitigated acute non-histaminergic itch and, consistently, obstructed DRG neuronal responses. Keratinocyte fragmentation under dermatitic simulation was also dampened following PA incubation. Chronic pruritus in 1-chloro-2,4-dinitrobenzene and psoriasis models were also moderately but significantly reversed by the repeated applications of PA. Dermatitic scores in the 1-chloro-2,4-dinitrobenzene and psoriatic models were also improved by its application, indicating that it is beneficial for mitigating disease pathology. Our findings suggest that pamoic acid activation of peripheral GPR35 can contribute to the improvement of pruritus and its associated diseases.

Members of the CUGBP Elav-like family of RNA-binding proteins are expressed in distinct populations of primary sensory neurons.

Primary sensory dorsal root ganglia (DRG) neurons are diverse, with distinct populations that respond to specific stimuli. Previously, we observed that functionally distinct populations of DRG neurons express mRNA transcript variants with different 3' untranslated regions (3'UTRs). 3'UTRs harbor binding sites for interaction with RNA-binding proteins (RBPs) for transporting mRNAs to subcellular domains, modulating transcript stability, and regulating the rate of translation. In the current study, analysis of publicly available single-cell RNA-sequencing data generated from adult mice revealed that 17 3'UTR-binding RBPs were enriched in specific populations of DRG neurons. This included four members of the CUG triplet repeat (CUGBP) Elav-like family (CELF): CELF2 and CELF4 were enriched in peptidergic, CELF6 in both peptidergic and nonpeptidergic, and CELF3 in tyrosine hydroxylase-expressing neurons. Immunofluorescence studies confirmed that 60% of CELF4+ neurons are small-diameter C fibers and 33% medium-diameter myelinated (likely Aδ) fibers and showed that CELF4 is distributed to peripheral termini. Coexpression analyses using transcriptomic data and immunofluorescence revealed that CELF4 is enriched in nociceptive neurons that express GFRA3, CGRP, and the capsaicin receptor TRPV1. Reanalysis of published transcriptomic data from macaque DRG revealed a highly similar distribution of CELF members, and reanalysis of single-nucleus RNA-sequencing data derived from mouse and rat DRG after sciatic injury revealed differential expression of CELFs in specific populations of sensory neurons. We propose that CELF RBPs may regulate the fate of mRNAs in populations of nociceptors, and may play a role in pain and/or neuronal regeneration following nerve injury.

Anti-inflammatory mechanism of electroacupuncture involves the modulation of multiple systems, levels and targets and is not limited to “driving the vagus-adrenal axis”

In October 2021, an international collaborative study on the use of electroacupuncture (EA) to treat inflammation was published in the journal Nature by Dr. Qiufu Ma’s team. Based on the results of EA on inflammation in the mouse model of lipopolysaccharide inflammatory storm, the study showed that the distal effect of acupuncture can be achieved by “driving the vagus-adrenal axis (through the adrenal medulla, by releasing catecholamines).” PROKR2Cre-marked sensory neurons, which innervate the deep hindlimb fascia but not the abdominal fascia, are crucial for driving this axis. The study suggests the existence of specificity distribution of acupoints, that different EA stimulation intensities or different needle penetration depths have different therapeutic effects, that photosensitive stimulation may be a substitute for needle acupuncture, and that massage, stretching and body movements may also activate PROKR2Cre-markable dorsal root ganglion sensory neurons and elicit anti-inflammatory effects. However, results of some other studies are contrary to the conclusions of Ma’s team. For examples: low-intensity EA at GB30 point significantly reduced the inflammation in the rat model of persistent inflammation, which is more relevant to the real daily acupuncture practice, and this effect was partly related to the adrenal cortex and associated with the stimulation of corticosterone and adrenocorticotropic hormone; manual acupuncture (similar to the low-intensity EA) at KI3, Zhichuan point (an extra point), etc. was effective in a severe COVID-19 patient with sepsis; stimulating ST25 with low-intensity EA or manual acupuncture was effective against gastrointestinal inflammations; the above mentioned points are not in an area enriched with PROKR2Cre-marked sensory nerve endings. Evidence shows that the mechanism of EA against inflammation includes modulating multi-systems, multi-levels and multi-targets, which does not limit to “driving the vagus-adrenal axis.”Please cite this article as: Fan AY. Anti-inflammatory mechanism of electroacupuncture involves the modulation of multiple systems, levels and targets and is not limited to “driving the vagus-adrenal axis.” J Integr Med. 2023; 21(4):320–323.

Integrin-Driven Axon Regeneration in the Spinal Cord Activates a Distinctive CNS Regeneration Program.

The peripheral branch of sensory dorsal root ganglion (DRG) neurons regenerates readily after injury unlike their central branch in the spinal cord. However, extensive regeneration and reconnection of sensory axons in the spinal cord can be driven by the expression of α9 integrin and its activator kindlin-1 (α9k1), which enable axons to interact with tenascin-C. To elucidate the mechanisms and downstream pathways affected by activated integrin expression and central regeneration, we conducted transcriptomic analyses of adult male rat DRG sensory neurons transduced with α9k1, and controls, with and without axotomy of the central branch. Expression of α9k1 without the central axotomy led to upregulation of a known PNS regeneration program, including many genes associated with peripheral nerve regeneration. Coupling α9k1 treatment with dorsal root axotomy led to extensive central axonal regeneration. In addition to the program upregulated by α9k1 expression, regeneration in the spinal cord led to expression of a distinctive CNS regeneration program, including genes associated with ubiquitination, autophagy, endoplasmic reticulum (ER), trafficking, and signaling. Pharmacological inhibition of these processes blocked the regeneration of axons from DRGs and human iPSC-derived sensory neurons, validating their causal contributions to sensory regeneration. This CNS regeneration-associated program showed little correlation with either embryonic development or PNS regeneration programs. Potential transcriptional drivers of this CNS program coupled to regeneration include Mef2a, Runx3, E2f4, and Yy1. Signaling from integrins primes sensory neurons for regeneration, but their axon growth in the CNS is associated with an additional distinctive program that differs from that involved in PNS regeneration.SIGNIFICANCE STATEMENT Restoration of neurologic function after spinal cord injury has yet to be achieved in human patients. To accomplish this, severed nerve fibers must be made to regenerate. Reconstruction of nerve pathways has not been possible, but recently, a method for stimulating long-distance axon regeneration of sensory fibers in rodents has been developed. This research uses profiling of messenger RNAs in the regenerating sensory neurons to discover which mechanisms are activated. This study shows that the regenerating neurons initiate a novel CNS regeneration program which includes molecular transport, autophagy, ubiquitination, and modulation of the endoplasmic reticulum (ER). The study identifies mechanisms that neurons need to activate to regenerate their nerve fibers.

Peripheral myeloid cells control resolution of painful Chemotherapy-Induced Peripheral Neuropathy (CIPN) (S23.001)

<h3>Objective:</h3> The objective of this study is to identify endogenous mediators controlling CIPN pain resolution. <h3>Background:</h3> Chemotherapies contribute to decreased cancer mortality but are associated with severe side effects including Chemotherapy-Induced Peripheral Neuropathy (CIPN). CIPN presents as a distal symmetric polyneuropathy causing numbness, tingling, and in a subset of patients, pain. Painful CIPN is a main cause for chemotherapy dose reduction, treatment cessation and reduced quality of life. A subset of painful CIPN patients will have pain resolution while others maintain pain persistency, however the mechanisms that mediate pain resolution are unknown and constitute a large gap of knowledge. <h3>Design/Methods:</h3> We developed painful CIPN models in mice using the chemotherapeutic paclitaxel, where mice treated with specific injection protocols develop resolving or persistent mechanical hypersensitivity measured via von Frey. Using these models, we performed bulk RNA sequencing on hind paw, whole dorsal root ganglion (DRG), DRG sensory neurons and spinal cord at initiation, pre-resolution, post-resolution, and persistency time points. DEGs were selected (P-adj &lt;0.05, FC &gt; 2, RPKM &gt; 1) and PANTHER analysis was performed to identify biological processes. To investigate the contribution of particular cell types in these tissues, we used the inducible diphtheria toxin receptor ablation model. <h3>Results:</h3> PANTHER analysis identified upregulation of immune system related genes in the hind paw during resolution of Model-1 not seen in Model-2. As myeloid cells are known to contribute to immune system related genes in the periphery, we ablated myeloid cells. We identified that CD11b+ peripheral, but not IBA1+ central, myeloid cells are critical for painful CIPN resolution. Further RNA sequencing analysis identified pro-inflammatory pathways correlated with painful CIPN resolution. <h3>Conclusions:</h3> These studies provide mechanistic insights for CIPN pain resolution and identify a unique therapeutic strategy for permanent treatment of painful CIPN. Future studies will delineate the myeloid cell sub-type and factors required for CIPN pain resolution. <b>Disclosure:</b> Mr. Naratadam has nothing to disclose. Prof. Akopian has nothing to disclose.