Biology subjects

Lesnak, J. B.

Publications and source records attributed to Lesnak, J. B..

17 recordsLinked to original sources

bioRxiv2026

History of opioid use disorder correlates with altered neuroimmune signatures in the human dorsal root ganglia

Understanding mechanisms for the negative reinforcing effects of opioids are fundamental to improving patient care. Using human dorsal root ganglia (DRG) from donors with and without opioid use disorder (OUD), we set out to examine functional and molecular correlates of chronic opioid use. At a molecular level, we detected disrupted immune signatures throughout cells in the peripheral nervous system (PNS), spanning glial-neuroimmune interactions to changes in non-neuronal senescence. At the physiological level these changes are paired with sensitization of DRG sensory neurons. Our findings reveal a neuro-inflammatory state in the PNS of individuals with OUD. Targeting therapeutics towards these PNS-based mechanisms could be an approach to alleviating the negative reinforcing effects of opioids.

neuroscience↗

bioRxiv2026

Antibiotics modulate activity of mouse and human dorsal root ganglia neurons

Antibiotics are among the most prescribed medications worldwide, yet their direct effects on eukaryotic tissues remain largely unexplored. Here, we used in vitro calcium imaging to determine if clinically relevant concentrations of common antibiotics alter mouse and human dorsal root ganglion (DRG) neuron activity. We found that {beta}-lactams, macrolides, and tetracyclines induce calcium flux in sensory neurons within minutes of application. Regardless of antibiotic class, neuronal responses depend on extracellular calcium entry. Pharmacological manipulations further revealed that cephalexin responses depend on TRPA1 activity whereas doxycycline responses require mitochondrial reactive oxygen species (ROS) production. These findings demonstrate that clinically relevant concentrations of antibiotics directly modulate sensory neuron activity through divergent mechanisms, thus expanding our current understanding of antibiotic side effects.

neuroscience↗

bioRxiv2026

DRG meningeal tertiary lymphoid structures are regulated by B cells as a pronociceptive locus after peripheral nerve injury

B cell-derived IgG in the dorsal root ganglia (DRG) drives neuropathic pain after peripheral nerve injury (PNI), but the site of B cell organization is unclear. Here, PNI induced leukocyte clusters in the DRG meninges, enveloped by lymphatic endothelium and apposed to high endothelial venules. These clusters resemble tertiary lymphoid structures (TLSs) with germinal center-like features, including germinal center B cells and plasma cells, and follicular dendritic and follicular helper T cells. Single-cell RNA sequencing revealed enrichment of germinal center B cells in the DRG meninges after PNI. Germinal center B cells regulate TLS organization: TLSs were absent after deletion of Ezh2 from germinal center-experienced B cells. Intrathecal CD20 monoclonal antibody to locally deplete B cells also disrupted TLS organization. Conversely, intrathecal B cell transfer to B cell-deficient (muMT) mice was sufficient for TLS organization after PNI. Allodynia did not develop when TLS organization was disordered. Similar TLSs formed in pig DRG after tail docking and in human donors with chronic pain, where B cell receptor clonotype analysis confirmed functional maturity. Together, these data establish that germinal center B cells are required for TLS organization, and that disrupting this process abolishes the development of neuropathic pain after PNI.

neuroscience↗

bioRxiv2026

Leptin Drives Osteoarthritis Pain Through Sensory Neuron Reprogramming Independent of Cartilage Signaling

ObjectivePain in osteoarthritis (OA) is often discordant with structural joint damage, particularly in obesity-associated OA, where adipose-derived signals may drive nociception independently of cartilage pathology. Leptin has been demonstrated to be necessary, but not sufficient, to drive obesity-associated OA. Here, we tested the hypothesis that leptin mediates OA-associated pain through sensory neuron reprogramming rather than chondrocyte-intrinsic signaling, suggesting a fat-sensory nerve axis. DesignMale and female constitutive leptin-deficient (Ob/Ob), heterozygous (Ob/+), and wild-type (WT) mice, as well as chondrocyte-specific leptin receptor knockout mice (Aggrecan-CreERT2;LepRfl/fl), were challenged with destabilization of the medial meniscus (DMM) surgery to induce OA. Pain-related behaviors, joint pathology, serum cytokines, and lumbar dorsal root ganglia (DRG) transcriptomes were assessed. Human DRG cultures treated with leptin underwent transcriptomic profiling. Secondary analyses of human infrapatellar fat pad and synovium single-cell datasets evaluated leptin and leptin receptor expression patterns. ResultsChondrocyte-specific deletion of the leptin receptor did not mitigate OA pathology or pain. Global leptin-deficient (Ob/Ob) mice exhibited worse structural joint outcomes than WT and Ob/+ animals following DMM yet were robustly protected from OA-associated hyperalgesia - directly dissociating pain from structural pathology and demonstrating that leptin is involved in nociceptive sensitization. Serum cytokine profiles were sex-dependent and did not align with pain outcomes, separating systemic inflammation from nociceptive differences. Transcriptomic analysis of DRGs revealed that leptin drives enrichment of lipid metabolism, eicosanoid, and inflammatory programs, whereas leptin deficiency shifts sensory neurons toward a cytoskeletal remodeling state that does not sustain pain signaling. In human DRG cultures, leptin treatment produced a transcriptomic shift to enrich for neuronal excitability while vehicle treated cells were enriched for inflammatory signaling. Human infrapatellar fat pad and synovium transcriptomic data demonstrated adipocyte-enriched leptin expression and broad distribution of the leptin receptor across stromal, vascular, immune, and adipocyte populations. ConclusionsLeptin contributes to OA pain through neuro-immune crosstalk between adipose tissue and sensory neurons rather than through direct cartilage signaling. These findings identify leptin-associated neuronal programs linked to nociceptor sensitization and support targeting leptin-modulated neuro-immune pathways as a strategy to alleviate OA pain independently of structural disease progression.

physiology↗

bioRxiv2026

T-cell distribution in the dorsal root ganglion across species, sex, and age

T-cells infiltrate somatosensory ganglia in response to nerve damage, autoimmune disease, and infection, contributing to sensory abnormalities and pain. In naive states, T-cells are rare in the rodent dorsal root ganglion (DRG) but have been reported in human and non-human primates without known relevant exposures. It remains unclear whether there are inherent evolutionary or species differences in DRG T-cell residence. Using a comparative biology approach, we investigated the frequency and distribution of T-cells in the mammalian DRG across humans, non-human primates, pigs, and rodents, and in humans investigated the contributions of sex and age. Spatial transcriptomics and immunofluorescence independently verified the robust presence of DRG T-cells at similar levels in humans, non-human primates, and pigs, but were fewer in rats and largely absent in mice. In humans, premenopausal females were more likely to have elevated DRG endoneurial T-cells than post-menopausal females or adult males. T-cells were detected in human dorsal root ganglion at as early as two months of age but were less abundant within the perineuronal niche. Most human DRG T-cells expressed distinct markers consistent with a resident memory (Trm) phenotype. We discuss the importance of studying the functional roles of DRG-resident T-cells and raise broader considerations for modelling peripheral nervous system disease.

neuroscience↗

bioRxiv2026

Adipose-driven complement-lipid reprogramming controls nociceptive vulnerability in obesity-associated osteoarthritis

Obesity amplifies osteoarthritis (OA) pain disproportionately to joint damage, creating a major unmet clinical need for non-opioid interventions that act beyond the joint. Using OA as a translational model, we integrated serum multi-omics in obese mice with surgically induced OA and genetic and adipose-reconstitution models of complement factor D (FD). In humans, we analyzed longitudinal metabolomics data from the IDEA weight-loss trial and conducted functional studies in dorsal root ganglion (DRG) neurons. Adipose-derived FD emerged as a regulator of systemic immunometabolic state: FD deficiency in obese mice worsened pain sensitivity whereas restoring circulating FD normalized pain and inflammatory markers without altering joint structure. Cross-species lipid profiling identified conserved shifts in linoleic acid versus arachidonic acid-derived lipids that were associated with pain phenotypes in mice and with pain improvement in humans. Defined lipid cocktails modulated excitability and TRPV1 sensitivity in human DRG neurons, and transcriptomics of knee-innervating DRGs revealed diet and FD-dependent activation of complement and neuronal excitability pathways. Together, these findings define an adipose-complement-lipid axis that regulates nociceptive vulnerability independent of joint damage and identify extra-articular targets for translational, non-opioid OA pain therapies. One Sentence SummaryWe identify an adipose-complement-lipid axis that systemically regulates sensory neuron sensitization, providing a mechanistic basis for pain-structure discordance in obesity-associated osteoarthritis.

physiology↗

bioRxiv2026

Progressive neurodegeneration in human dorsal root ganglion from diabetes to painful neuropathy

Diabetic painful neuropathy (DPN) is characterized by neuropathic pain accompanied by loss of sensory function. We hypothesized that neurodegeneration in the dorsal root ganglion (DRG) could underlie DPN progression. To address this question, we performed multi-omic evaluation on DRGs from otherwise healthy organ donors, donors with diabetes but no neuropathy, and donors with clinically diagnosed DPN. We discovered that the first stages of neurodegeneration begin early in diabetes before the onset of DPN, with Nageotte nodule formation accompanied by apoptotic gene expression and decreased proportion of specific populations of A-fibers in DPN with remodeling of non-neuronal cells. Our findings define DPN as a neurodegenerative disorder of the DRG, identify molecular markers of disease stage, and highlight the need for early intervention to prevent irreversible neurodegeneration.

neuroscience↗

bioRxiv2025

A Reference Atlas of the Human Dorsal Root Ganglion

Somatosensory perception largely emerges from diverse peripheral sensory neurons whose cell bodies reside in dorsal root ganglia (DRG). Damage or dysfunction of DRG neurons is a major cause of chronic pain and sensory loss. In mice, deep single-cell transcriptomic profiling and genetically defined models have offered important clues into DRG function, but in humans, the cellular and molecular landscape of DRG neurons remains less understood. Here, we constructed a reference cell atlas of the human DRG by profiling transcriptomes of cells and nuclei from 126 donors sampled across cervical, thoracic, and lumbar DRGs. This atlas resolves 22 neuronal subtypes, including known and previously unrecognized subtypes linked to nociception, mechanosensation, thermosensation, and proprioception, as well as 10 types of non-neuronal cells. Cross-species integration, spatial transcriptomics, and microneurography enabled cell-type-specific comparisons of soma size and conduction velocity between species. Human DRG somata are larger across all cell types than their mouse counterparts, and the conduction velocities of human hair follicle innervating A-fibers are faster than in mice, suggesting a functional shift in rapid mechanical detection in humans. This integrated human DRG reference cell atlas provides a resource for exploring new molecular and physiological features of human DRG, which could help identify new strategies for treating chronic pain and other diseases of the peripheral nervous system.

neuroscience↗

bioRxiv2025

Peripheral nerve-targeting and pain-promoting transcriptomic signatures in early Guillain-Barr&eacute syndrome

Guillain-Barre syndrome (GBS) is an autoimmune disorder that causes weakness, sensory loss, autonomic dysfunction, and chronic neuropathic pain. The mediators responsible for driving early autoimmune injury in the most common GBS variant, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), remain incompletely understood. We performed single-cell and bulk RNA sequencing on peripheral blood mononuclear cells collected from early untreated AIDP-variant GBS patients and healthy controls to comprehensively deduce leukocyte transcriptome alterations and predict disease- and pain-driving interactions between pathogenic leukocytes and peripheral nervous system cells. We found that classical, intermediate, and non-classical monocytes were expanded and upregulated genes associated with type I and II interferons, JAK/STAT signaling, and NLRP3 inflammasome engagement. CD8+ T cells were highly proliferative and likewise upregulated JAK/STAT signaling. CD4+FOXP3+ regulatory T cells upregulated PRDM1 and CD74 in a signature that may indicate functional exhaustion. A subpopulation of highly activated intermediate monocytes upregulated genes related to angiogenesis and oncostatin M. Differential expression-based cell-cell interaction analysis between GBS leukocytes, Schwann cells, and sensory neurons predicted engagement of ligand-receptor pairs with nerve integrity and pain functions, including epiregulin, interferon-beta, adrenomedullin, clusterin, IL-6, IL-15, and CCL4. Functional validation demonstrated that CCL4 sensitizes human sensory neurons in vitro. These results unearth molecular interactions by which specific leukocyte populations in AIDP-variant GBS may participate in peripheral nerve injury and drive neuropathic pain. Many of these targets may be amenable to therapeutic modulation using available approved and investigational drugs, potentially providing drug repurposing opportunities.

neuroscience↗

bioRxiv2025

Enabling wider access to human molecular neuroscience research in pain: A simple preservation method for human dorsal root ganglion neurons in Hibernate A media

The use of human dorsal root ganglion (DRG) from organ donors opens the door for research into the molecular biology and physiology of human nociceptors; however, there are barriers to working with this tissue including logistical difficulties and limited access. We present an approach using Hibernate media to temporarily store either whole DRGs or dissociated DRG neurons prior to culturing and functional testing. Dissociation of DRGs following temporary storage (4-16hrs) in Hibernate media resulted in similar neuronal and immune cell yield as acutely dissociated DRGs. Neurons derived from DRGs stored in Hibernate media prior to dissociation exhibited similar electrophysiological properties and capsaicin responses as acutely dissociated DRG neurons. Similarly, neurons from acutely dissociated DRGs stored in Hibernate media (>24hrs) and shipped to geographically distant laboratories produced neuronal cultures displaying comparable electrophysiological properties as acutely cultured neurons. This approach overcomes insurmountable logistical burdens and increases access to freshly recovered human DRGs.

neuroscience↗

bioRxiv2025

Suzetrigine (VX-548) exhibits activity-dependent effects on human dorsal root ganglion neurons

Non-selective antagonists of voltage-gated sodium channels (VGSCs) provide effective local analgesia, but systemic administration is fraught with unwanted cardiovascular and central nervous system toxicity. The development of antagonists targeting VGSCs preferentially expressed in peripheral neurons, such as NaV1.7, 1.8, and 1.9, could mitigate these risks. One such new small molecule that selectively inhibits NaV1.8, is orally bioavailable, and has recent FDA approval for the treatment of acute pain is suzetrigine (VX-548). Here we tested the effects of this agent on human dorsal root ganglion (hDRG) neurons obtained from either patients undergoing surgical thoracic vertebrectomy or from organ donors in parallel electrophysiology studies across 2 laboratories. Bath application of 10 nM suzetrigine abolished spontaneous discharges previously shown to be associated with on-going neuropathic pain within minutes. In contrast, suppression of discharges evoked by intracellular current injection required more prolonged application times. These results suggest that suzetrigine will likely show efficacy versus spontaneous pain but may have less robust effects on evoked or breakthrough pain.

neuroscience↗

bioRxiv2025

Expansion of OSMR expression and signaling in the human dorsal root ganglion links OSM to neuropathic pain

RNA sequencing studies on human dorsal root ganglion (hDRG) from patients suffering from neuropathic pain show upregulation of OSM, linking this IL-6 family cytokine to pain disorders. In mice, however, OSM signaling causes itch behaviors through a direct effect on its cognate receptor expressed uniquely by pruriceptive sensory neurons. We hypothesized that an expansion in function of OSM-OSM receptor (OSMR) in sensory disorders in humans could be explained by species differences in receptor expression and signaling. Our in situ hybridization and immunohistochemical findings demonstrate broad expression of OSMR in DRG nociceptors and afferent fibers innervating the superficial and deep skin of humans. In patch-clamp electrophysiology, OSM directly activates human sensory neurons engaging MAPK signaling to promote action potential firing. Using CRISPR editing we show that OSM activation of MAPK signaling is dependent on OSMR and not LIFR in hDRG. Bulk, single-nuclei, and single-cell RNA-seq of OSM-treated hDRG cultures reveal expansive similarities in the transcriptomic signature observed in pain DRGs from neuropathic patients, indicating that OSM alone can orchestrate transcriptomic signatures associated with pain. We conclude that OSM-OSMR signaling via MAPKs is a critical signaling factor for DRG plasticity that may underlie neuropathic pain in patients.

neuroscience↗

bioRxiv2024

Multi-omic integration with human DRG proteomics highlights TNFα signalling as a relevant sexually dimorphic pathway

The peripheral nervous system (PNS) plays a critical role in pathological conditions, including chronic pain disorders, that manifest differently in men and women. To investigate this sexual dimorphism at the molecular level, we integrated quantitative proteomic profiling of human dorsal root ganglia (hDRG) and peripheral nerve tissue into the expanding omics framework of the PNS. Using data-independent acquisition (DIA) mass spectrometry, we characterized a comprehensive proteomic profile, validating tissue-specific differences between the hDRG and peripheral nerve. Through multi-omic analyses and in vitro functional assays, we identified sex-specific molecular differences, with TNF signalling emerging as a key sexually dimorphic pathway with higher prominence in males. Genetic evidence from genome-wide association studies (GWAS) further supports the functional relevance of TNF signalling in the periphery, while clinical trial data and meta-analyses indicate a sex-dependent response to TNF inhibitors. Collectively, these findings underscore a functionally sexual dimorphism in the PNS, with direct implications for sensory and pain-related clinical translation.

neuroscience↗

bioRxiv2024

Nageotte nodules in human DRG reveal neurodegeneration in painful diabetic neuropathy

Diabetic neuropathy is frequently accompanied by pain and loss of sensation attributed to axonal dieback. We recovered dorsal root ganglia (DRGs) from 90 organ donors, 19 of whom had medical indices for diabetic painful neuropathy (DPN). Nageotte nodules, dead sensory neurons engulfed by non-neuronal cells, were abundant in DPN DRGs and accounted for 25% of all neurons. Peripherin-and Nav1.7-positive dystrophic axons invaded Nageotte nodules, forming small neuroma-like structures. Using histology and spatial sequencing, we demonstrate that Nageotte nodules are mainly composed of satellite glia and non-myelinating Schwann cells that express SPP1 and are intertwined with sprouting sensory axons originating from neighboring neurons. Our findings solve a 100-year mystery of the nature of Nageotte nodules linking these pathological structures to pain and sensory loss in DPN.

neuroscience↗

bioRxiv2023

Brain-derived neurotrophic factor contributes to activity-induced muscle pain in male but not female mice

Activity-induced muscle pain increases release of interleukin-1{beta} (IL-1{beta}) in muscle macrophages and the development of pain is prevented by blockade of IL-1{beta}. Brain derived neurotrophic factor (BDNF) is released from sensory neurons in response to IL-1{beta} and mediates both inflammatory and neuropathic pain. Thus, we hypothesized that metabolites released during fatiguing muscle contractions activate macrophages to release IL-1{beta}, which subsequently activate sensory neurons to secrete BDNF. To test this hypothesis, we used an animal model of activity-induced pain induced by repeated intramuscular acidic saline injections combined with fatiguing muscle contractions. Intrathecal or intramuscular injection of inhibitors of BDNF-Tropomyosin receptor kinase B (TrkB) signaling, ANA-12 or TrkB-Fc, reduced the decrease in muscle withdrawal thresholds in male, but not in female, mice when given before or 24hr after, but not 1 week after induction of the model. BDNF messenger ribonucleic acid (mRNA) was significantly increased in L4-L6 dorsal root ganglion (DRG), but not the spinal dorsal horn or gastrocnemius muscle, 24hr after induction of the model in either male or female mice. No changes in TrkB mRNA or p75 neurotrophin receptor mRNA were observed. BDNF protein expression via immunohistochemistry was significantly increased in L4-L6 spinal dorsal horn and retrogradely labelled muscle afferent DRG neurons, at 24hr after induction of the model in both sexes. In cultured DRG, fatigue metabolites combined with IL-1{beta} significantly increased BDNF expression in both sexes. In summary, fatigue metabolites release, combined with IL-1{beta}, BDNF from primary DRG neurons and contribute to activity-induced muscle pain only in males, while there were no sex differences in the changes in expression observed in BDNF.

neuroscience↗

bioRxiv2022

The impact of sex and physical activity on the local immune response to muscle pain

Induction of muscle pain triggers a local immune response to produce pain and this mechanism may be sex and activity level dependent. The purpose of this study was to measure the immune system response in the muscle following induction of pain in sedentary and physically active mice. Muscle pain was produced via an activity-induced pain model using acidic saline combined with fatiguing muscle contractions. Prior to induction of muscle pain, mice (C57/BL6) were sedentary or physically active (24hr access to running wheel) for 8 weeks. The ipsilateral gastrocnemius was harvested 24hr after induction of muscle pain for RNA sequencing or flow cytometry. RNA sequencing revealed activation of several immune pathways in both sexes after induction of muscle pain, and these pathways were attenuated in physically active females. Uniquely in females, the antigen processing and presentation pathway with MHC II signaling was activated after induction of muscle pain; activation of this pathway was blocked by physical activity. Blockade of MHC II attenuated development of muscle hyperalgesia exclusively in females. Induction of muscle pain increased the number of macrophages and T-cells in the muscle in both sexes, measured by flow cytometry. In both sexes, the phenotype of macrophages shifted toward a pro-inflammatory state after induction of muscle pain in sedentary mice (M1+M1/2) but toward an anti-inflammatory state in physically active mice (M2+M0). Thus, induction of muscle pain activates the immune system with sex-specific differences in the transcriptome while physical activity attenuates immune response in females and alters macrophage phenotype in both sexes.

immunology↗

bioRxiv2022

Selective Androgen Receptor Modulator Microparticle Formulation Reverses Muscle Hyperalgesia in Mouse Model of Widespread Muscle Pain

Currently, there is a need for the generation of non-opioid analgesics for treating chronic pain. Preclinical and clinical studies demonstrate the analgesic effects of testosterone. However, treatment with testosterone is not feasible due to adverse effects. Selective androgen receptor modulators (SARMs) were developed to overcome these limitations by minimizing activation of androgenic side effects. First, we demonstrate SARM administration alleviates widespread muscle pain in male and female mice. We then developed a SARM-loaded PLGA microparticle formulation that reverses widespread muscle pain in two injections. In vitro and in vivo release kinetics demonstrate the microparticle formulation had sustained SARM release for 4 weeks. Antagonism of androgen receptors blocked the analgesic effects of the SARM microparticles. SARM treatment had no effect on cardiac or liver enzymes, cardiac histology, and did not produce rewarding behavior. These studies demonstrate SARM microparticles as a potential therapeutic for chronic muscle pain. One Sentence SummaryA selective androgen receptor modulator microparticle formulation alleviates widespread muscle pain in male and female mice while being non-toxic.

neuroscience↗