Biology subjects

Sankaranarayanan, I.

Publications and source records attributed to Sankaranarayanan, I..

30 records · Page 2Linked to original sources

bioRxiv2024

Persistent changes in nociceptor translatomes govern hyperalgesic priming in mouse models

Hyperalgesic priming is a model system that has been widely used to understand plasticity in painful stimulus-detecting sensory neurons, called nociceptors. A key feature of this model system is that following priming, stimuli that do not normally cause hyperalgesia now readily provoke this state. We hypothesized that hyperalgesic priming occurs due to reorganization of translation of mRNA in nociceptors. To test this hypothesis, we used paclitaxel treatment as the priming stimulus and translating ribosome affinity purification (TRAP) to measure persistent changes in mRNA translation in Nav1.8+ nociceptors. TRAP sequencing revealed 161 genes with persistently altered mRNA translation in the primed state. We identified Gpr88 as upregulated and Metrn as downregulated. We confirmed a functional role for these genes, wherein a GPR88 agonist causes pain only in primed mice and established hyperalgesic priming is reversed by Meteorin. Our work demonstrates that altered nociceptor translatomes are causative in producing hyperalgesic priming.

neuroscience↗

bioRxiv2024

Deciphering the molecular landscape of human peripheral nerves: implications for diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is a prevalent complication of diabetes mellitus that is caused by metabolic toxicity to peripheral axons. We aimed to gain deep mechanistic insight into the disease process using bulk and spatial RNA sequencing on tibial and sural nerves recovered from lower leg amputations in a mostly diabetic population. First, our approach comparing mixed sensory and motor tibial and purely sensory sural nerves shows key pathway differences in affected nerves, with distinct immunological features observed in sural nerves. Second, spatial transcriptomics analysis of sural nerves reveals substantial shifts in endothelial and immune cell types associated with severe axonal loss. We also find clear evidence of neuronal gene transcript changes, like PRPH, in nerves with axonal loss suggesting perturbed RNA transport into distal sensory axons. This motivated further investigation into neuronal mRNA localization in peripheral nerve axons generating clear evidence of robust localization of mRNAs such as SCN9A and TRPV1 in human sensory axons. Our work gives new insight into the altered cellular and transcriptomic profiles in human nerves in DPN and highlights the importance of sensory axon mRNA transport as an unappreciated potential contributor to peripheral nerve degeneration.

neuroscience↗

bioRxiv2024

Mapping Somatosensory Afferent Circuitry to Bone Identifies Neurotrophic Signals Required for Fracture Healing

The profound pain accompanying bone fracture is mediated by somatosensory neurons, which also appear to be required to initiate bone regeneration following fracture. Surprisingly, the precise neuroanatomical circuitry mediating skeletal nociception and regeneration remains incompletely understood. Here, we characterized somatosensory dorsal root ganglia (DRG) afferent neurons innervating murine long bones before and after experimental long bone fracture in mice. Retrograde labeling of DRG neurons by an adeno-associated virus with peripheral nerve tropism showed AAV-tdT signal. Single cell transcriptomic profiling of 6,648 DRG neurons showed highest labeling across CGRP+ neuron clusters (6.9-17.2%) belonging to unmyelinated C fibers, thinly myelinated A{delta} fibers and A{beta}-Field LTMR (9.2%). Gene expression profiles of retrograde labeled DRG neurons over multiple timepoints following experimental stress fracture revealed dynamic changes in gene expression corresponding to the acute inflammatory (S100a8, S100a9) and mechanical force (Piezo2). Reparative phase after fracture included morphogens such as Tgfb1, Fgf9 and Fgf18. Two methods to surgically or genetically denervate fractured bones were used in combination with scRNA-seq to implicate defective mesenchymal cell proliferation and osteodifferentiation as underlying the poor bone repair capacity in the presence of attenuated innervation. Finally, multi-tissue scRNA-seq and interactome analyses implicated neuron-derived FGF9 as a potent regulator of fracture repair, a finding compatible with in vitro assessments of neuron-to-skeletal mesenchyme interactions.

cell biology↗

bioRxiv2024

VLK drives extracellular phosphorylation of EphB2 to govern the EphB2-NMDAR interaction and injury-induced pain

Phosphorylation of hundreds of protein extracellular domains is mediated by two kinase families, yet the significance of these kinases is underexplored. Here, we find that the presynaptic release of the tyrosine directed-ectokinase, Vertebrate Lonesome Kinase (VLK/Pkdcc), is necessary and sufficient for the direct extracellular interaction between EphB2 and GluN1 at synapses, for phosphorylation of the ectodomain of EphB2, and for injury-induced pain. Pkdcc is an essential gene in the nervous system, and VLK is found in synaptic vesicles, and is released from neurons in a SNARE-dependent fashion. VLK is expressed by nociceptive sensory neurons where presynaptic sensory neuron-specific knockout renders mice impervious to post-surgical pain, without changing proprioception. VLK defines an extracellular mechanism that regulates protein-protein interaction and non-opioid-dependent pain in response to injury. One-Sentence SummarySynaptic protein-protein interactions and pain are regulated by the presynaptic release of the extracellular kinase VLK in the spinal cord.

neuroscience↗

bioRxiv2023

Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia

Peripheral sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli including touch, temperature, and pain to the central nervous system. Recent advances in single-cell RNA-sequencing (scRNA-seq) have provided new insights into the diversity of sensory ganglia cell types in rodents, non-human primates, and humans, but it remains difficult to compare transcriptomically defined cell types across studies and species. Here, we built cross-species harmonized atlases of DRG and TG cell types that describe 18 neuronal and 11 non-neuronal cell types across 6 species and 19 studies. We then demonstrate the utility of this harmonized reference atlas by using it to annotate newly profiled DRG nuclei/cells from both human and the highly regenerative axolotl. We observe that the transcriptomic profiles of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The new resources and data presented here can guide future studies in comparative transcriptomics, simplify cell type nomenclature differences across studies, and help prioritize targets for future pain therapy development.

neuroscience↗

bioRxiv2022

Sex-differences in prostaglandin signaling: a semi-systematic review and characterization of PTGDS expression in human sensory neurons

There is increasing evidence of sex differences in underlying mechanisms causing pain in preclinical models, and in clinical populations. There are also important disconnects between clinical pain populations and the way preclinical pain studies are conducted. For instance, osteoarthritis pain more frequently affects women but most preclinical studies have been conducted using males in animal models. The most widely used painkillers, nonsteroidal anti-inflammatory drugs (NSAIDs), act on the prostaglandin pathway by inhibiting cyclooxygenase (COX) enzymes. The purpose of this study was to analyze the preclinical and clinical literature on the role of prostaglandins and COX in inflammation and pain. We aimed to specifically identify studies that used both sexes and investigate whether any sex-differences in the action of prostaglandins and COX inhibition had been reported, either in clinical or preclinical studies. We conducted a PubMed search and identified 369 preclinical studies and 100 clinical studies that matched our inclusion/exclusion criteria. Our analysis shows that only 17% of preclinical studies on prostaglandins used both sexes and, out of those, only 19% analyzed or reported data in a sex-aware fashion. In contrast, 79% of the clinical studies analyzed used both sexes. However, only 6% of those reported data in a sex-aware fashion. Interestingly, 14 out of 15 preclinical studies and 5 out of 6 clinical studies that analyzed data in a sex-aware fashion have identified sex-differences. This builds on the increasing evidence of sex-differences in prostaglandin signaling and the importance of sex-awareness in data analysis. The preclinical literature identifies a sex difference in prostaglandin D2 synthase (PTGDS) expression where it is higher in female than in male rodents in the nervous system. We experimentally validated that PTGDS expression is higher in female human dorsal root ganglia (DRG) neurons recovered from organ donors. Our semi-systematic literature review reveals a need for continued inclusivity of both male and female animals in prostaglandins studies and sex-aware analysis in data analysis in preclinical and clinical studies. Our finding of sex-differences in neuronal PTGDS expression in humans exemplifies the need for a more comprehensive understanding of how the prostaglandin system functions in the DRG in rodents and humans.

neuroscience↗

bioRxiv2022

Inducible co-stimulatory molecule (ICOS) alleviates Paclitaxel induced peripheral neuropathy via an IL-10-mediated mechanism in female mice

Chemotherapy-induced peripheral neuropathy (CIPN) is a primary dose-limiting side effect caused by antineoplastic agents, such as paclitaxel. This causes damage to peripheral nerves and the dorsal root ganglia (DRG). Currently, there are no effective treatments for CIPN, which can lead to long-term morbidity in cancer patients and survivors. Neuro-immune interactions occur in CIPN and have been implicated both in the development and progression of the disease and disease resolution. We investigated the potential role of Inducible co-stimulatory molecule (ICOS) in the resolution of CIPN pain-like behaviors in mice. ICOS is an immune checkpoint molecule that is expressed on the surface of activated T cells and promotes proliferation and differentiation of T cells. We found that intrathecal administration of ICOS agonist antibody (ICOSaa) alleviates mechanical hypersensitivity caused by paclitaxel and facilitates the resolution of mechanical sensitivity in female mice. Administration of ICOSaa reduced astrocyte-gliosis in the spinal cord and satellite cell gliosis in the DRG of mice previously treated with paclitaxel. Mechanistically, ICOSaa intrathecal treatment promoted pain resolution by increasing interleukin 10 (IL-10) expression in the dorsal root ganglion. In line with these observations, blocking IL-10 receptor (IL-10R) activity occluded the effects of ICOSaa treatment on CIPN behavior in female mice. Suggesting a broader activity in neuropathic pain, ICOSaa also partially resolved mechanical hypersensitivity in the spared nerve injury (SNI) model. Our findings support a model wherein ICOSaa administration induces IL-10 expression to facilitate neuropathic pain relief in female mice. ICOSaa treatment is in clinical development for solid tumors and given our observation of T cells in the human DRG, ICOSaa therapy could be developed for combination chemotherapy - CIPN clinical trials. HighlightsO_LIICOS agonist antibody (ICOSaa) promotes pain resolution in female mice C_LIO_LIDRG T cells appear to enter an anti-inflammatory phenotype by ICOSaa treatment C_LIO_LIICOSaa treatment increases DRG levels of IL-10 cytokine C_LIO_LIICOSaa effects in female mice are blocked by IL-10 sequestering treatment C_LI

neuroscience↗

bioRxiv2022

Meteorin alleviates Paclitaxel-induced peripheral neuropathy in mice

Chemotherapy-induced peripheral neuropathy (CIPN) is a challenging condition to treat, and arises due to severe, dose-limiting toxicity of chemotherapeutic drugs such as paclitaxel. This often results in debilitating sensory and motor deficits that are not effectively prevented or alleviated by existing therapeutic interventions. Recent studies have demonstrated the therapeutic effects of Meteorin, a neurotrophic factor, in reversing neuropathic pain in rodent models of peripheral nerve injury induced by physical trauma. Here, we sought to investigate the potential antinociceptive effects of recombinant mouse Meteorin (rmMeteorin) using a paclitaxel-induced peripheral neuropathy model in male and female mice. Paclitaxel treatment (4 x 4mg/kg, i.p.) induced hind paw mechanical hypersensitivity by day 8 after treatment. Thereafter, in a reversal dosing paradigm, five repeated injections of rmMeteorin (0.5 and 1.8mg/kg s.c. respectively) administered over 9 days produced a significant and long-lasting attenuation of mechanical hypersensitivity in both sexes. Additionally, administration of rmMeteorin (0.5 and 1.8mg/kg), initiated before and during paclitaxel treatment (prevention dosing paradigm), blocked the establishment of hind paw mechanical hypersensitivity. Repeated systemic administration of rmMeteorin in both dosing paradigms decreased histochemical signs of satellite glial cell reactivity as measured by glutamine synthetase and connexin43 protein expression in the DRG. Additionally, in the prevention administration paradigm rmMeteorin had a protective effect against paclitaxel-induced loss of intraepidermal nerve fibers. Our findings indicate that rmMeteorin has a robust and sustained antinociceptive effect in the paclitaxel-induced peripheral neuropathy model and the development of recombinant human Meteorin could be a novel and effective therapeutic for CIPN treatment. HighlightsO_LIMeteorin produces an antinociceptive effect in both male and female mice treated with paclitaxel. C_LIO_LISatellite glial cell reactivity induced by paclitaxel treatment is reversed by Meteorin. C_LIO_LIRetraction of intraepidermal nerve fibre (IENF) is blocked by Meteorin treatment in paclitaxel treated mice. C_LIO_LIFindings suggest a disease modifying effect of Meteorin in the mouse model of paclitaxel-induced peripheral neuropathy. C_LI

neuroscience↗

bioRxiv2021

RNA Profiling of Neuropathic Pain-Associated Human DRGs Reveal Sex-differences in Neuro-immune Interactions Promoting Pain

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 DRG 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 DRGs from these patients, allowing comprehensive identification of molecular changes in these samples by contrasting them with non-pain associated DRGs. We sequenced 70 human DRGs, including over 50 having mRNA libraries with neuronal mRNA. Our expression analysis revealed profound sex differences in differentially expressed genes including increase of IL1B, TNF, CXCL14, and OSM in male and including CCL1, CCL21, PENK and TLR3 in female DRGs associated with neuropathic pain. Co-expression modules revealed enrichment in members of JUN-FOS signaling in males, and centromere protein coding genes in females. Neuro-immune signaling pathways revealed distinct cytokine signaling 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.

neuroscience↗

bioRxiv2021

Spatial transcriptomics reveals unique molecular fingerprints of human nociceptors

Nociceptors are specialized sensory neurons that detect damaging or potentially damaging stimuli and are found in the dorsal root (DRG) and trigeminal ganglia. These neurons are critical for the generation of neuronal signals that ultimately create the perception of pain. These neurons are also primary targets for acute and chronic pain therapeutics. Single-cell transcriptomics on mouse nociceptors has transformed our understanding of pain mechanisms. We sought to generate equivalent information for human nociceptors with the goal of identifying transcriptomic signatures of nociceptors, identifying species differences and elucidating new drug targets. We used spatial transcriptomics to molecularly characterize transcriptomes of single dorsal root ganglion (DRG) neurons from 8 organ donors. We identified 12 clusters of human sensory neurons, 5 of which are C nociceptors; as well as 1 A{beta} nociceptor, 2 A{delta}, 2 A{beta} and 1 proprioceptor subtypes. By focusing on expression profiles for ion channels, G-protein coupled receptors (GPCRs) and other pharmacological targets, we provide a rich map of drug targets in the human DRG with direct comparison to mouse sensory neuron transcriptomes. We also compare human DRG neuronal subtypes to non-human primates showing conserved patterns of gene expression among many cell types, but divergence among specific nociceptor subsets. Finally, we identify sex differences in human DRG subpopulation transcriptomes, including a marked increase in CALCA expression in female pruritogen receptor enriched nociceptors. Our data open the door to development of drug discovery programs for new pain targets and unparalleled molecular characterization of clinical sensory disorders. One Sentence SummaryWe used spatial transcriptomics to molecularly characterize human sensory neurons, comparing them to mouse and non-human primate finding similarities but also divergence, in particular among drug targets.

neuroscience↗

bioRxiv2020

Convergence of peptidergic and non-peptidergic protein markers in the human dorsal root ganglion and spinal dorsal horn

Peripheral sensory neurons are characterized by their size, molecular profiles, and physiological responses to specific stimuli. In mouse, the peptidergic and non-peptidergic subsets of nociceptors are distinct and innervate different lamina of the spinal dorsal horn. The unique molecular signature and neuroanatomical organization of these neurons supports a labeled line theory for certain types of nociceptive stimuli. However, long standing evidence supports the polymodal nature of nociceptors in many species. We have recently shown that the peptidergic marker, CGRP, and the non-peptidergic marker, P2X3R, show largely overlapping expression at the mRNA level in human dorsal root ganglion (DRG). Herein, our aim was to assess the protein distribution of nociceptor markers, including their central projections, in the human DRG and spinal cord. Using DRGs obtained from organ donors, we observed that CGRP and P2X3R were co-expressed by approximately 33% of human DRG neurons and TrpV1 was expressed in ~60% of human DRG neurons. In the dorsal spinal cord, CGRP, P2X3R, TrpV1 and Nav1.7 protein stained the entirety of lamina II, with only P2XR3 showing a gradient of expression. This was confirmed by measuring the size of the substantia gelatinosa using Hematoxylin and Eosin staining of adjacent sections. Our findings are consistent with the known polymodal nature of most primate nociceptors and indicate that the central projection patterns of nociceptors are different between mice and humans. Elucidating how human nociceptors connect to subsets of dorsal horn neurons will be important for understanding the physiological consequences of these species differences.

neuroscience↗

bioRxiv2020

Sex differences in nociceptor translatomes contribute to divergent prostaglandin signaling in male and female mice

BackgroundThere are clinically relevant sex differences in acute and chronic pain mechanisms, but we are only beginning to understand their mechanistic basis. Transcriptome analyses of rodent whole dorsal root ganglion (DRG) have revealed sex differences, mostly in immune cells. We examined the transcriptome and translatome of the mouse DRG with the goal of identifying sex differences. MethodsWe used Translating Ribosome Affinity Purification (TRAP) sequencing and behavioral pharmacology to test the hypothesis that nociceptor (Nav1.8 expressing neurons) translatomes would differ by sex. ResultsWe found 66 genes whose mRNA were sex-differentially bound to nociceptor ribosomes. Many of these genes have known neuronal functions but have not been explored in sex differences in pain. We focused on Ptgds, which was increased in female mice. The mRNA encodes the prostaglandin D2 (PGD2) synthesizing enzyme. We observed increased Ptgds protein and PGD2 in female mouse DRG. The Ptgds inhibitor AT-56 caused intense pain behaviors in male mice but was only effective at high doses in females. Conversely, female mice responded more robustly to another major prostaglandin, PGE2, than did male mice. Ptgds protein expression was also higher in female cortical neurons, suggesting DRG findings may be generalizable to other nervous system structures. ConclusionsNociceptor TRAP sequencing (TRAP-seq) reveals unexpected sex differences in one of the oldest known nociceptive signaling molecule families, the prostaglandins. Our results demonstrate that translatome analysis reveals physiologically relevant sex differences important for fundamental protective behaviors driven by nociceptors.

neuroscience↗