Nociceptor-immune interactomes reveal insult-specific immune signatures of pain.

Inflammatory pain results from the heightened sensitivity and reduced threshold of nociceptor sensory neurons due to exposure to inflammatory mediators. However, the cellular and transcriptional diversity of immune cell and sensory neuron types makes it challenging to decipher the immune mechanisms underlying pain. Here we used single-cell transcriptomics to determine the immune gene signatures associated with pain development in three skin inflammatory pain models in mice: zymosan injection, skin incision and ultraviolet burn. We found that macrophage and neutrophil recruitment closely mirrored the kinetics of pain development and identified cell-type-specific transcriptional programs associated with pain and its resolution. Using a comprehensive list of potential interactions mediated by receptors, ligands, ion channels and metabolites to generate injury-specific neuroimmune interactomes, we also uncovered that thrombospondin-1 upregulated by immune cells upon injury inhibited nociceptor sensitization. This study lays the groundwork for identifying the neuroimmune axes that modulate pain in diverse disease contexts.

Early-Life Gene Expression in Neurons Modulates Lasting Epigenetic States.

In mammals, the environment plays a critical role in promoting the final steps in neuronal development during the early postnatal period. While epigenetic factors are thought to contribute to this process, the underlying molecular mechanisms remain poorly understood. Here, we show that in the brain during early life, the DNA methyltransferase DNMT3A transiently binds across transcribed regions of lowly expressed genes, and its binding specifies the pattern of DNA methylation at CA sequences (mCA) within these genes. We find that DNMT3A occupancy and mCA deposition within the transcribed regions of genes is negatively regulated by gene transcription and may be modified by early-life experience. Once deposited, mCA is bound by the methyl-DNA-binding protein MECP2 and functions in a rheostat-like manner to fine-tune the cell-type-specific transcription of genes that are critical for brain function.

MeCP2 Represses the Rate of Transcriptional Initiation of Highly Methylated Long Genes.

Mutations in the methyl-DNA-binding repressor protein MeCP2 cause the devastating neurodevelopmental disorder Rett syndrome. It has been challenging to understand how MeCP2 regulates transcription because MeCP2 binds broadly across the genome and MeCP2 mutations are associated with widespread small-magnitude changes in neuronal gene expression. We demonstrate here that MeCP2 represses nascent RNA transcription of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex. By measuring the rates of transcriptional initiation and elongation directly in the brain, we find that MeCP2 has no measurable effect on transcriptional elongation, but instead represses the rate at which Pol II initiates transcription of highly methylated long genes. These findings suggest a new model of MeCP2 function in which MeCP2 binds broadly across highly methylated regions of DNA, but acts at transcription start sites to attenuate transcriptional initiation.

Activity-induced MeCP2 phosphorylation regulates retinogeniculate synapse refinement.

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MECP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here, we show that MeCP2 is phosphorylated at four residues in the mouse brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from the synapse refinement defect previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period.

Author Correction: Epigenetic regulation in psychiatric disorders.

In part c of Figure 1 in this article, the orientation of the tail of histone 3 with respect to the core region of this molecule was incorrect, and the amino acid residue K79 should not have been depicted in the tail. The corrected figure is shown below. The authors and editors thank T. Brown, R. Lober and C. Waker for bringing this error to our attention. Note that the present affiliation of the corresponding author and their current email address have been added to the correction notice.

Neuroma morphology: A macroscopic classification system.

INTRODUCTION/AIMS: Neuromas come in different shapes and sizes; yet the correlation between neuroma morphology and symptomatology is unknown. Therefore, we aim to investigate macroscopic traits of excised human neuromas and assess the validity of a morphological classification system and its potential clinical implications. METHODS: End-neuroma specimens were collected from prospectively enrolled patients undergoing symptomatic neuroma surgery. Protocolized images of the specimens were obtained intraoperatively. Pain data (Numeric rating scale, 0-10) were prospectively collected during preoperative interview, patient demographic and comorbidity factors were collected from chart review. A morphological classification is proposed, and the inter-rater reliability (IRR) was assessed. Distribution of neuroma morphology with patient factors, was described. RESULTS: Forty-five terminal neuroma specimens from 27 patients were included. Residual limb patients comprised 93% of the population, of which 2 were upper (8.0%) and 23 (92.0%) were lower extremity residual limb patients. The proposed morphological classification, consisting of three groups (bulbous, fusiform, atypical), demonstrated a strong IRR (Cohen's kappa = 0.8). Atypical neuromas demonstrated higher preoperative pain, compared with bulbous and fusiform. Atypical morphology was more prevalent in patients with diabetes and peripheral vascular disease. DISCUSSION: A validated morphological classification of neuroma is introduced. These findings may assist surgeons and researchers with better understanding of symptomatic neuroma development and their clinical implications. The potential relationship of neuroma morphology with the vascular and metabolic microenvironment requires further investigation.

Neuroma Analysis in Humans: Standardizing Sample Collection and Documentation.

INTRODUCTION: The biology of symptomatic neuromas is poorly understood, particularly the factors causing pain in human neuromas. Pain presence varies among and within individuals, with some having painful and nonpainful neuromas. To bridge these knowledge gaps, our group developed a protocol for assessing neuroma pain and collecting tissue for molecular analysis. This manuscript outlines our workflow and challenges and aims to inspire other centers to share their experiences with these tissues. METHODS: For every included patient and collected nerve or bone tissue specimens, we perform a detailed chart review and a multifaceted analysis of pain and pain perception immediately before surgery. We collect patient-reported outcome measures (PROMs) on pain, function, and mental well-being outcomes at preoperative assessment and at the 6-month follow-up postoperatively. Before surgery, the patient is assessed once again to obtain an immediate preoperative pain status and identify potential differences in pain intensity of different neuromas. Intraoperatively, specimens are obtained and their gross anatomical features are recorded, after which they are stored in paraformaldehyde or frozen for later sample analyses. Postoperatively, patients are contacted to obtain additional postoperative PROMs. RESULTS: A total of 220 specimens of nerve tissue have been successfully obtained from 83 limbs, comprising 95 specimens of neuromas and 125 specimens of nerves located proximal to the neuromas or from controls. CONCLUSIONS: Our approach outlines the methods combining specimen collection and examination, including both macroscopic and molecular biological features, with PROMs, encompassing physical and psychological aspects, along with clinical metadata obtained through clinical teams and chart review.

Comparative Gene Signature of Nociceptors Innervating Mouse Molar Teeth, Cranial Meninges, and Cornea.

BACKGROUND: The trigeminal ganglion (TG) collects afferent sensory information from various tissues. Recent large-scale RNA sequencing of neurons of the TG and dorsal root ganglion has revealed a variety of functionally distinct neuronal subpopulations, but organ-specific information is lacking. METHODS: To link transcriptomic and tissue-specific information, we labeled small-diameter neurons of 3 specific subpopulations of the TG by local application of lipophilic carbocyanine dyes to their innervation site in the dental pulp, cornea, and meninges (dura mater). We then collected mRNA-sequencing data from fluorescent neurons. Differentially expressed genes (DEGs) were analyzed and subjected to downstream gene set enrichment analysis (GSEA), and ion channel profiling was performed. RESULTS: A total of 10,903 genes were mapped to the mouse genome (>500 reads). DEG analysis revealed 18 and 81 genes with differential expression (log 2 fold change > 2, Padj < .05) in primary afferent neurons innervating the dental pulp (dental primary afferent neurons [DPAN]) compared to those innervating the meninges (meningeal primary afferent neurons [MPAN]) and the cornea (corneal primary afferent neurons [CPAN]). We found 250 and 292 genes differentially expressed in MPAN as compared to DPAN and to CPAN, and 21 and 12 in CPAN as compared to DPAN and MPAN. Scn2b had the highest log 2 fold change when comparing DPAN versus MPAN and Mmp12 was the most prominent DEG when comparing DPAN versus CPAN and, CPAN versus MPAN. GSEA revealed genes of the immune and mitochondrial oxidative phosphorylation system for the DPAN versus MPAN comparison, cilium- and ribosome-related genes for the CPAN versus DPAN comparison, and respirasome, immune cell- and ribosome-related gene sets for the CPAN versus MPAN comparison. DEG analysis for ion channels revealed no significant differences between the neurons set except for the sodium voltage-gated channel beta subunit 2, Scn2b . However, in each tissue a few ion channels turned up with robust number of reads. In DPAN, these were Cacna1b , Trpv2 , Cnga4 , Hcn1 , and Hcn3 , in CPAN Trpa1 , Trpv1 , Cacna1a , and Kcnk13 and in MPAN Trpv2 and Scn11a . CONCLUSIONS: Our study uncovers previously unknown differences in gene expression between sensory neuron subpopulations from the dental pulp, cornea, and dura mater and provides the basis for functional studies, including the investigation of ion channel function and their suitability as targets for tissue-specific analgesia.

Epigenomic profiling of mouse nucleus accumbens at single-cell resolution.

The nucleus accumbens (NAc) is a key brain region involved in reward processing and is linked to multiple neuropsychiatric conditions such as substance use disorder, depression, and chronic pain. Recent studies have begun to investigate NAc gene expression at a single-cell resolution, however, our understanding of the cellular heterogeneity of the NAc epigenomic landscape remains limited. In this study, we utilize single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) to map cell-type-specific differences in chromatin accessibility in the NAc. Our findings not only reveal the transcription factors and putative gene regulatory elements that may contribute to these cell-type-specific epigenomic differences but also provide a valuable resource for future studies investigating epigenomic changes that occur in neuropsychiatric disorders.

Migraine-associated gene expression in cell types of the central and peripheral nervous system.

BACKGROUND: Genome-wide association studies have implicated dozens of genes with migraine susceptibility, but it remains unclear in which nervous system cell types these genes are expressed. METHODS: Using single-cell RNA sequencing data from the central and peripheral nervous system, including the trigeminal ganglion, the expression of putative migraine-associated genes was compared across neuronal, glial and neurovascular cell types within these tissues. RESULTS: Fifty-four putative migraine-associated genes were expressed in the central nervous system, peripheral nervous system or neurovascular cell types analyzed. Six genes (11.1%) were selectively enriched in central nervous system cell types, three (5.5%) in neurovascular cell types, and two (3.7%) in peripheral nervous system cell types. The remaining genes were expressed in multiple cell types. CONCLUSIONS: Single-cell RNA sequencing of the brain and peripheral nervous system localizes each migraine-associated gene to its respective nervous system tissue and the cell types in which it is expressed. While the majority of migraine-associated genes are broadly expressed, we identified several cell-type-specific migraine-associated genes in the central nervous system, peripheral nervous system, and neurovasculature. Trial registration: not applicable.

Localization of migraine susceptibility genes in human brain by single-cell RNA sequencing.

BACKGROUND: Migraine is a debilitating disorder characterized by severe headaches and associated neurological symptoms. A key challenge to understanding migraine has been the cellular complexity of the human brain and the multiple cell types implicated in its pathophysiology. The present study leverages recent advances in single-cell transcriptomics to localize the specific human brain cell types in which putative migraine susceptibility genes are expressed. METHODS: The cell-type specific expression of both familial and common migraine-associated genes was determined bioinformatically using data from 2,039 individual human brain cells across two published single-cell RNA sequencing datasets. Enrichment of migraine-associated genes was determined for each brain cell type. RESULTS: Analysis of single-brain cell RNA sequencing data from five major subtypes of cells in the human cortex (neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells) indicates that over 40% of known migraine-associated genes are enriched in the expression profiles of a specific brain cell type. Further analysis of neuronal migraine-associated genes demonstrated that approximately 70% were significantly enriched in inhibitory neurons and 30% in excitatory neurons. CONCLUSIONS: This study takes the next step in understanding the human brain cell types in which putative migraine susceptibility genes are expressed. Both familial and common migraine may arise from dysfunction of discrete cell types within the neurovascular unit, and localization of the affected cell type(s) in an individual patient may provide insight into to their susceptibility to migraine.

Essential role of SIRT1 signaling in the nucleus accumbens in cocaine and morphine action.

Sirtuins (SIRTs), class III histone deacetylases, are well characterized for their control of cellular physiology in peripheral tissues, but their influence in brain under normal and pathological conditions remains poorly understood. Here, we establish an essential role for SIRT1 and SIRT2 in regulating behavioral responses to cocaine and morphine through actions in the nucleus accumbens (NAc), a key brain reward region. We show that chronic cocaine administration increases SIRT1 and SIRT2 expression in the mouse NAc, while chronic morphine administration induces SIRT1 expression alone, with no regulation of all other sirtuin family members observed. Drug induction of SIRT1 and SIRT2 is mediated in part at the transcriptional level via the drug-induced transcription factor ΔFosB and is associated with robust histone modifications at the Sirt1 and Sirt2 genes. Viral-mediated overexpression of SIRT1 or SIRT2 in the NAc enhances the rewarding effects of both cocaine and morphine. In contrast, the local knockdown of SIRT1 from the NAc of floxed Sirt1 mice decreases drug reward. Such behavioral effects of SIRT1 occur in concert with its regulation of numerous synaptic proteins in NAc as well as with SIRT1-mediated induction of dendritic spines on NAc medium spiny neurons. These studies establish sirtuins as key mediators of the molecular and cellular plasticity induced by drugs of abuse in NAc, and of the associated behavioral adaptations, and point toward novel signaling pathways involved in drug action.

Heterotopic Ossification Is Associated with Painful Neuromas in Transtibial Amputees Undergoing Surgical Treatment of Symptomatic Neuromas.

BACKGROUND: A relationship between nerve and osseous regeneration has been described. During the surgical treatment of symptomatic neuroma in transtibial amputees, we have noticed that heterotopic ossification (HO) depicted on preoperative radiographs appears to be associated with the location of symptomatic neuromas in both the peroneal and tibial nerve distributions. METHODS: Data were collected for transtibial amputees who underwent surgical management of symptomatic neuroma and were prospectively enrolled from 2018 through 2023. Preoperative radiographs were assessed for the presence of HO located at the distal fibula and tibia. The presence of a peroneal and/or tibial neuroma was based on findings contained within the operative reports. Pain levels were measured on a numeric rating scale (0-10). RESULTS: Sixty-five limbs of 62 amputees were include. Peroneal neuroma and presence of fibular HO (P=0.001), and tibial neuroma and presence of tibial HO (P=0.038) demonstrated an association. The odds of having a symptomatic peroneal neuroma with fibular HO present are greater than the odds of a symptomatic peroneal neuroma when fibular HO is absent (OR 9.3; 95%CI [1.9-45.6], P=0.006). Pre-operative pain scores were significantly higher for all patients with HO (P<0.001), those with fibular HO (P<0.001), and those with tibial HO (P<0.001), compared to patients without HO. CONCLUSIONS: In patients with symptomatic neuromas, preoperative pain was worse when HO was present in the transtibial amputee's residual limb. Further research on the neuroma-HO-complex in symptomatic amputees is required. LEVEL OF EVIDENCE: Therapeutic Level IV.

Biology and pathophysiology of symptomatic neuromas.

ABSTRACT: Neuromas are a substantial cause of morbidity and reduction in quality of life. This is not only caused by a disruption in motor and sensory function from the underlying nerve injury but also by the debilitating effects of neuropathic pain resulting from symptomatic neuromas. A wide range of surgical and therapeutic modalities have been introduced to mitigate this pain. Nevertheless, no single treatment option has been successful in completely resolving the associated constellation of symptoms. While certain novel surgical techniques have shown promising results in reducing neuroma-derived and phantom limb pain, their effectiveness and the exact mechanism behind their pain-relieving capacities have not yet been defined. Furthermore, surgery has inherent risks, may not be suitable for many patients, and may yet still fail to relieve pain. Therefore, there remains a great clinical need for additional therapeutic modalities to further improve treatment for patients with devastating injuries that lead to symptomatic neuromas. However, the molecular mechanisms and genetic contributions behind the regulatory programs that drive neuroma formation-as well as the resulting neuropathic pain-remain incompletely understood. Here, we review the histopathological features of symptomatic neuromas, our current understanding of the mechanisms that favor neuroma formation, and the putative contributory signals and regulatory programs that facilitate somatic pain, including neurotrophic factors, neuroinflammatory peptides, cytokines, along with transient receptor potential, and ionotropic channels that suggest possible approaches and innovations to identify novel clinical therapeutics.

Neuroma-to-Nerve Ratio: Does Size Matter?

BACKGROUND AND OBJECTIVES: Anatomic features of neuromas have been explored in imaging studies. However, there has been limited research into these features using resected, ex vivo human neuroma specimens. The aim of this study was to investigate the influence that time may have on neuroma growth and size, and the clinical significance of these parameters. METHODS: Patients who underwent neuroma excision between 2022 through 2023 were prospectively included in this study. Neuroma specimens were obtained after operative resection. Standardized neuroma size measurements, expressed as a neuroma-to-nerve ratio (NNR), were conducted with ImageJ software. Pain data (numeric rating scale, 0-10) were prospectively recorded during preoperative evaluation, and patient factors were collected from chart reviews. RESULTS: Fifty terminal neuroma specimens from 31 patients were included, with 94.0% of the neuromas obtained from individuals with amputations. Most neuromas were excised from the lower extremities (n = 44, 88.0%). The neuromas had a median NNR of 2.45, and the median injury to neuroma excision interval was 6.3 years. Larger NNRs were associated with a longer injury to neuroma excision interval and with a smaller native nerve diameter. In addition, sensory nerves were associated with a larger NNR compared with mixed nerves. NNR was not associated with preoperative pain or with anatomical nerve distribution. CONCLUSION: This study suggests that neuromas seem to continue to grow over time and that smaller nerves may form relatively larger neuromas. In addition, sensory nerves develop relatively larger neuromas compared with mixed nerves. Neuroma size does not appear to correlate with pain severity. These findings may stimulate future research efforts and contribute to a better understanding of symptomatic neuroma development.

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

Sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli to the central nervous system. Single-cell RNA sequencing has provided insights into the diversity of sensory ganglia cell types in rodents, nonhuman primates, and humans, but it remains difficult to compare cell types across studies and species. We thus constructed harmonized atlases of the DRG and TG that describe and facilitate comparison of 18 neuronal and 11 non-neuronal cell types across six species and 31 datasets. We then performed single-cell/nucleus RNA sequencing of DRG from both human and the highly regenerative axolotl and found that the harmonized atlas also improves cell type annotation, particularly of sparse neuronal subtypes. We observed that the transcriptomes of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The resources presented here can guide future studies in comparative transcriptomics, simplify cell-type nomenclature differences across studies, and help prioritize targets for future analgesic development.

Activity-Induced MeCP2 Phosphorylation Regulates Retinogeniculate Synapse Refinement.

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MeCP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here we show that MeCP2 is phosphorylated at four residues in the brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from that previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period. SIGNIFICANCE STATEMENT: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that predominantly affects girls. RTT is caused by loss of function mutations in a single gene MeCP2. Girls with RTT develop normally during their first year of life, but then experience neurological abnormalities including breathing and movement difficulties, loss of speech, and seizures. This study investigates the function of the MeCP2 protein in the brain, and how MeCP2 activity is modulated by sensory experience in early life. Evidence is presented that sensory experience affects MeCP2 function, and that this is required for synaptic pruning in the brain. These findings provide insight into MeCP2 function, and clues as to what goes awry in the brain when the function of MeCP2 is disrupted.

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.

Human OPRM1 and murine Oprm1 promoter driven viral constructs for genetic access to µ-opioidergic cell types.

With concurrent global epidemics of chronic pain and opioid use disorders, there is a critical need to identify, target and manipulate specific cell populations expressing the mu-opioid receptor (MOR). However, available tools and transgenic models for gaining long-term genetic access to MOR+ neural cell types and circuits involved in modulating pain, analgesia and addiction across species are limited. To address this, we developed a catalog of MOR promoter (MORp) based constructs packaged into adeno-associated viral vectors that drive transgene expression in MOR+ cells. MORp constructs designed from promoter regions upstream of the mouse Oprm1 gene (mMORp) were validated for transduction efficiency and selectivity in endogenous MOR+ neurons in the brain, spinal cord, and periphery of mice, with additional studies revealing robust expression in rats, shrews, and human induced pluripotent stem cell (iPSC)-derived nociceptors. The use of mMORp for in vivo fiber photometry, behavioral chemogenetics, and intersectional genetic strategies is also demonstrated. Lastly, a human designed MORp (hMORp) efficiently transduced macaque cortical OPRM1+ cells. Together, our MORp toolkit provides researchers cell type specific genetic access to target and functionally manipulate mu-opioidergic neurons across a range of vertebrate species and translational models for pain, addiction, and neuropsychiatric disorders.