Biology subjects

Funk, G.

Publications and source records attributed to Funk, G..

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

Chronic lower limb pain is not associated with a loss of inhibitory neurons in the human lumbar spinal dorsal horn

The spinal dorsal horn is the primary processing site of nociceptive sensory input from the periphery. Excitatory spinal interneurons releasing glutamate can amplify this information before it is sent to the brain, whereas inhibitory neurons releasing GABA and/or glycine can suppress the outflow of nociceptive signals. An imbalance favoring excitation is thought to underlie certain aspects of chronic pain. Although rodent studies have identified spinal mechanisms underlying hyperalgesia and allodynia, little is known about the anatomical changes associated with chronic pain in the human spinal cord, a gap in knowledge we sought to address in this study. Using immunohistochemistry and in situ hybridization on lumbar spinal cord tissue recovered from organ donors, we characterized neuronal size and density across the human dorsal horn and confirmed the presence of the human equivalent of the lateral spinal nucleus in many individuals. Chronic lower limb pain was not associated with changes in neuronal density in the dorsal horn. Likewise, the ratio of excitatory (SLC17A6+) to inhibitory (PAX2+) neurons remained consistent across laminae for age, sex and chronic pain state, providing no evidence for selective loss of inhibitory neurons with chronic pain in humans. We found no differences in the size or density of the postsynaptic markers Homer1 and gephyrin between groups, suggesting glutamatergic and GABAergic postsynaptic sites remain structurally stable. These findings provide a thorough evaluation of cellular anatomy of the human dorsal horn and form a foundation for future studies investigating neuronal changes that may contribute to chronic pain in humans.

neuroscience↗

bioRxiv2026

Peripheral nerve-derived extracellular vesicles are dynamically regulated in chemotherapy-induced painful peripheral neuropathy

Communication between Schwann cells (SCs) and other cells in the peripheral nerve remains incompletely understood. Extracellular vesicles (EVs) are important mediators of cell-cell communication, however, understanding the function of EVs in vivo is challenging in part because of difficulty in determining the cell type from which EVs originate. To identify SC EVs in vivo, we created a novel P0-Cre-turbo-GFP/human-CD9-EV reporter mouse. EVs were isolated from sciatic nerves without disrupting cell integrity. SC-derived EVs were identified by high-resolution microscopy and fluorescence nanoparticle tracking analyses. To test whether sciatic nerve EV (snEV) populations are regulated under neuropathological conditions, we treated mice with the chemotherapy agent, paclitaxel, which induces neuropathic pain. Proteomes of healthy and neuropathic snEVs differed as determined by LC-MS/MS. Proteins essential for maintenance of axonal integrity and SC myelination were identified selectively in healthy snEVs, whereas neuropathic snEVs contained increased levels of metabolic enzymes and receptors associated with neuronal excitability. Neuropathic snEVs contained diminished levels of EVs derived from SCs. These EVs differed in size from normal snEVs and triggered altered cell-signaling responses in sensory neurons. The appearance of neuropathic EVs correlated with the development of pain-related behaviors. Our findings demonstrate that peripheral nerve EV physiology is dynamically regulated in peripheral neuropathy.

neuroscience↗

bioRxiv2026

Aquaporin-4 mislocalization from astrocyte endfeet prolongs survival in a prion-cerebral amyloid angiopathy model

Aquaporin 4 (AQP4) water channels are polarized to astrocytic endfeet at blood vessel interfaces, and lose polarity in vascular diseases, including stroke, chronic traumatic encephalopathy, and Alzheimers disease. AQP4 modulates water influx and efflux in the interstitial fluid, yet how AQP4 localization impacts cerebral amyloid angiopathy (CAA) remains poorly understood. Here we show that astrocytic end feet and AQP4 are displaced from amyloid-bearing vessels in a prion-CAA mouse model that expresses GPI-anchorless PrPC. Displacing AQP4 genetically through deleting alpha-syntrophin (Snta1-/-) led to a marked prolongation in survival, together with reduced microglial inflammation and C1q, in prion-CAA-affected mice. Additionally, synaptic structural proteins were better maintained. Finally, the level and distribution of prion aggregates were similar among the mice, indicating that prion conversion and spread was not affected. These results suggest that reducing AQP4 water channel function slows the decline in a vascular amyloid disease by reducing neuroinflammation.

neuroscience↗

bioRxiv2026

Cannabis Use by People with HIV is Associated with an Anti-Inflammatory Immunometabolic Phenotype in Monocyte-Derived Macrophages

Chronic neuroinflammation is associated with comorbidities in people with HIV (PWH) on antiretroviral therapy (ART). While cannabis use is associated with reduced neuroinflammation and neurocognitive impairment (NCI) in PWH, the underlying mechanisms are unknown. To address this gap in knowledge, we analyzed monocyte-derived macrophages (MDMs) from a cohort of 50 PWH and 33 people without HIV (mean age: 61.9 years), categorized by frequency of cannabis use (naive/low, moderate, daily). We performed immunocytochemistry, RNA sequencing, and qPCR on MDMs and quantified related biomarkers in donor plasma. In this cohort study, daily cannabis use in PWH was associated with less global neurocognitive deficits, and with an anti-inflammatory immunometabolic-phenotype in MDMs characterized by (1) a metabolic shift from glycolysis to oxidative phosphorylation, (2) higher mitochondrial numbers, (3) altered cytokine profiles (pro-inflammatory downregulation, anti-inflammatory upregulation), and (4) higher brain-derived neurotrophic factor (BDNF) expression. These cellular changes were corroborated by a plasma biomarker profile in PWH including (1) lower levels of growth differentiation factor 15 and soluble triggering receptor expressed on myeloid cells 2, and (2) higher mature BDNF/precursor BDNF ratios that correlated with better cognition. Thus, cannabis use may mitigate NCI in PWH by immunometabolically reprogramming MDM function towards an anti-inflammatory and neuroprotective state. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/709579v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@15fb1adorg.highwire.dtl.DTLVardef@189d79corg.highwire.dtl.DTLVardef@aa6a89org.highwire.dtl.DTLVardef@3852f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Nav1.8 mediates peripheral-to-central nociceptive transmission independently of central presynaptic mechanisms in human DRG-spinal cord circuits

Suzetrigine, a selective Nav1.8 blocker, despite its significant role as a non-opioid analgesic, exhibits modest efficacy. The potential enhancement of Nav1.8 blocker efficacy through engagement of Nav1.8 at central terminals remains uncertain. We established a donor-derived human organotypic nociceptive circuit to determine where and how clinically relevant analgesics suppress pain signaling. Using ELISA and immunohistochemistry, we quantified calcitonin gene-related peptide (CGRP) release from acute explants of adult human spinal cord, dorsal root ganglia (DRG), and an intact DRG-spinal cord preparation preserving primary afferent anatomy and directional signaling. In isolated spinal cords, capsaicin evoked concentration-dependent spinal CGRP release without compromising viability, and clinically used analgesics inhibited it, validating the assay. In the intact circuit, the application of capsaicin to the DRG cell bodies triggered CGRP release exclusively in the spinal cord, consistent with compartmentalized neuropeptide release at central terminals. Selective Nav1.8 inhibition with suzetrigine reduced spinal CGRP release only when applied to the DRG or nerve root, not the spinal cord, indicating that Nav1.8 regulates peripheral action potential propagation rather than presynaptic transmitter release. These findings establish the first intact human pain circuit-based assay to study analgesics and demonstrate that the analgesic efficacy of Nav1.8 inhibitors is unlikely to increase with improved CNS penetration.

neuroscience↗

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

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

Spatial transcriptomic profiling of human paravertebral sympathetic chain ganglia reveals diabetes-induced neuroplasticity

The paravertebral sympathetic chain ganglia (SCG) are autonomic ganglia critical for regulating the "fight-or-flight" response. Symptoms of sympathetic dysfunction are prevalent in diabetes, affecting up to 90% of patients. The molecular and cellular composition of the human SCG and its alteration in diabetes remains poorly defined. To address this gap, we performed spatial transcriptomic profiling of lumbar SCGs from diabetic and non-diabetic organ donors. We identified 3 three distinct neuronal populations, two noradrenergic (NA1 and NA2) and one cholinergic (CHO), based on tyrosine hydroxylase (TH) and SLC18A3 expression, respectively. We also characterized 9 non-neuronal populations consisting of Schwann cells, immune cells, fibroblasts, adipocytes, and endothelial cells. In diabetic SCGs, we observed a significant loss of myelinating Schwann cells and a phenotypic shift of cholinergic neurons toward a noradrenergic identity. Additionally, diabetes was associated with a significant reduction in the transcripts of vasodilatory neuropeptides, such as VIP and CALCA, suggesting a mechanism for impaired vascular control. Upstream regulator analysis highlighted altered neurotrophic signaling in diabetes, with enhanced NGF/TRKA and diminished BDNF/TRKB activity, potentially driven by target-derived cues. Comparison between SCG and dorsal root ganglia (DRG) neurons revealed ganglia-specific genes, like SCN3A and NPY (SCG) versus SCN10A and GPX1 (DRG), offering specific therapeutic targets for autonomic dysfunction or pain. Our findings provide a transcriptomic characterization of human SCG, revealing molecular signatures that underlie diabetic autonomic dysfunction. This work lays a foundation for the development of therapies to restore sympathetic function and avoid unintended autonomic effects in the development of analgesics. Significance StatementAutonomic dysfunction affects up to 90% of people with diabetes, yet the human sympathetic nervous system remains poorly molecularly defined. To address this gap, we present a spatial transcriptomic profile of the human sympathetic chain ganglia (SCG), revealing how diabetes affects the human autonomic nervous system. We show that diabetes shifts the cholinergic neuronal population to a noradrenergic phenotype and reduces vasodilation neuropeptide expression, potentially explaining impaired vascular control and thermoregulation. Comparative analysis of sympathetic and sensory ganglia reveals distinct gene profiles that may inform novel therapeutic strategies. These findings offer critical insight into the molecular drivers of diabetic autonomic neuropathy and lay the groundwork for safer, more precise treatments that selectively modulate autonomic or sensory function in chronic disease.

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

PrPC-induced signaling in human neurons activates phospholipase Cγ1 and an Arc/Arg3.1 response

Synaptic dysfunction and loss correlate with cognitive decline in neurodegenerative diseases, including Alzheimers disease (AD) and prion disease. Neuronal hyperexcitability occurs in the early stages of AD and experimental prion disease, prior to the onset of dementia, yet the underlying drivers are unclear. Here we identify an increase in the immediate early gene, Arc/Arg3.1, in the human prion disease-affected frontal cortex, suggestive of neuronal hyperactivity. To investigate early signaling events initiated by prion aggregates (PrPSc) in human neurons, we stimulated PrPC in human iPSC-derived excitatory neurons (iNs) with a known PrPSc-mimetic antibody (POM1), which recapitulated the Arc/Arg3.1 response within two hours. Proteomics, RNAseq, and a phosphokinase array in iNs revealed alterations in the EGF receptor and increased phosphorylated phospholipase C (PLC)-{gamma}1 (Y783), which was also observed in the cerebral cortex of prion-infected mice. Thus, PrPC ligands can induce a PLC-{gamma}1 intracellular signaling cascade together with an Arc response, suggestive of a neuronal activity response.

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↗

bioRxiv2025

Decreased KCC2 expression in the human spinal dorsal horn associated with chronic pain and long-term opioid use

Loss of GABAergic and glycinergic inhibitory efficacy in the spinal dorsal horn is associated with neuropathic pain and opioid-induced hyperalgesia in rodent models. Downregulation of the KCC2 chloride extrusion transporter is a key mechanism underlying this decreased inhibitory efficacy, but to-date there is no evidence supporting or opposing this hypothesis in humans. Here we demonstrate that KCC2 expression is decreased in superficial dorsal horn neurons of organ donors who died with a documented history of pain, or of long-term opioid use. We show profoundly decreased KCC2 dorsal horn membrane expression in a primary cohort associated with either chronic pain or opioid use, and in a replication cohort of mixed chronic pain and opioid use history. These results show that decreased dorsal horn inhibitory efficacy likely promotes chronic pain in humans and support the development of therapeutics augmenting KCC2 function as a treatment for chronic pain and opioid use disorders.

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↗

bioRxiv2024

Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 with functional confirmation

CRISPR-Cas9 editing is now the leading method for genome editing and is being advanced for the treatment of human disease. CRIPSR editing could have many applications for treatment of neurological diseases, including pain but traditional viral vector delivery approaches have neurotoxicity limiting their use. Overcoming these issues could open the door for genome editing treatments for diseases like intractable pain where the dorsal root ganglia (DRG) would be the desired target. To this end, we describe a simple method for viral-vector-independent transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. As proof of principle, we edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successful as demonstrated by the expression of the reporters as early as two days in vitro. CRISPR-Cas9 editing was confirmed at the genome level with insertion and deletion detection system T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca2+ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for intractable pain. TeaserWe describe a non-viral transfection method for CRISPR-Cas9 gene editing in human dorsal root ganglion neurons.

neuroscience↗

bioRxiv2023

NaV1.7 mRNA and protein expression in putative projection neurons of the human spinal dorsal horn

NaV1.7, a membrane-bound voltage-gated sodium channel, is preferentially expressed along primary sensory neurons, including their peripheral & central nerve endings, axons, and soma within the dorsal root ganglia and plays an integral role in amplifying membrane depolarization and pain neurotransmission. Loss- and gain-of-function mutations in the gene encoding NaV1.7, SCN9A, are associated with a complete loss of pain sensation or exacerbated pain in humans, respectively. As an enticing pain target supported by human genetic validation, many compounds have been developed to inhibit NaV1.7 but have disappointed in clinical trials. The underlying reasons are still unclear, but recent reports suggest that inhibiting NaV1.7 in central terminals of nociceptor afferents is critical for achieving pain relief by pharmacological inhibition of NaV1.7. We report for the first time that NaV1.7 mRNA is expressed in putative projection neurons (NK1R+) in the human spinal dorsal horn, predominantly in lamina 1 and 2, as well as in deep dorsal horn neurons and motor neurons in the ventral horn. NaV1.7 protein was found in the central axons of sensory neurons terminating in lamina 1-2, but also was detected in the axon initial segment of resident spinal dorsal horn neurons and in axons entering the anterior commissure. Given that projection neurons are critical for conveying nociceptive information from the dorsal horn to the brain, these data support that dorsal horn NaV1.7 expression may play an unappreciated role in pain phenotypes observed in humans with genetic SCN9A mutations, and in achieving analgesic efficacy in clinical trials.

neuroscience↗