Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice.

Somatosensory neurons are highly heterogeneous with distinct types of neural cells responding to specific stimuli. However, the distribution and roles of cell-type-specific long intergenic noncoding RNAs (lincRNAs) in somatosensory neurons remain largely unexplored. Here, by utilizing droplet-based single-cell RNA-seq (scRNA-seq) and full-length Smart-seq2, we show that lincRNAs, but not coding mRNAs, are enriched in specific types of mouse somatosensory neurons. Profiling of lincRNAs from single neurons located in dorsal root ganglia (DRG) identifies 200 lincRNAs localized in specific types or subtypes of somatosensory neurons. Among them, the conserved cell-type-specific lincRNA CLAP associates with pruritus and is abundantly expressed in somatostatin (SST)-positive neurons. CLAP knockdown reduces histamine-induced Ca2+ influx in cultured SST-positive neurons and in vivo reduces histamine-induced scratching in mice. In vivo knockdown of CLAP also decreases the expression of neuron-type-specific and itch-related genes in somatosensory neurons, and this partially depends on the RNA binding protein MSI2. Our data reveal a cell-type-specific landscape of lincRNAs and a function for CLAP in somatosensory neurons in sensory transmission.

Zcchc12-Containing Nociceptors Are Required for Noxious Heat Sensation.

DRG neurons are classified into distinct types to mediate the somatosensation with different modalities. Recently, transcriptional profilings of DRG neurons by single-cell RNA-sequencing have provided new insights into the neuron typing and functional properties. Zinc-finger CCHC domain-containing 12 (Zcchc12) was reported to be the representative marker for a subtype of galanin-positive (Gal+) DRG neurons. However, the characteristics and functions of Zcchc12+ neurons are largely unknown. Here, we genetically labeled Zcchc12+ neurons in Zcchc12-CreERT2::Ai9 mice, and verified that Zcchc12 represented a new subpopulation of DRG neurons in both sexes. Zcchc12+ neurons centrally innervated the superficial laminae in spinal dorsal horn, and peripherally terminated as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. In addition, Zcchc12+ neurons also formed circumferential endings surrounding the hair follicles in hairy skin. Functionally, in vivo calcium imaging in DRGs revealed that Zcchc12+ neurons were polymodal nociceptors and could be activated by mechanical and noxious thermal stimuli. Behavioral tests showed that selective ablation of Zcchc12+ DRG neurons reduced the sensitivity to noxious heat in mice. Together, we identified a new subpopulation of Zcchc12+ nociceptors essential for noxious heat sensation.SIGNIFICANCE STATEMENTZcchc12 represents a new subpopulation of DRG neurons. The characteristics and functions of Zcchc12+ neurons are largely unknown. Here we genetically labeled Zcchc12+ neurons, and showed that the fibers of Zcchc12+ DRG neurons projected to superficial lamina at spinal dorsal horn, and innervated skin as free nerve endings in the epidermis and cluster-shaped fibers in the dermis of footpads and nearby. Functionally, Zcchc12+ DRG neurons responded to noxious mechanical and heat stimuli. Ablation of Zcchc12+ DRG neurons impaired the sensation of noxious heat in mice. Therefore, we identify a new subpopulation of DRG neurons required for noxious heat sensation.

Lanatoside C inhibits herpes simplex virus 1 replication by regulating NRF2 distribution within cells.

BACKGROUND: In the past decades, extensive research has been conducted to identify new drug targets for the treatment of Herpes simplex virus type 1 (HSV-1) infections. However, the emergence of drug-resistant HSV-1 strains remains a major challenge. This necessitates the identification of new drugs with novel mechanisms of action. Lanatoside C (LanC), a cardiac glycoside (CG) approved by the US Food and Drug Administration (FDA), has demonstrated anticancer and antiviral properties. Nevertheless, its potential as an agent against HSV-1 infections and the underlying mechanism of action are currently unknown. PURPOSE: This study aimed to investigate the antiviral activity of LanC against HSV-1 and elucidate its molecular mechanisms. METHODS: The in vitro antiviral activity of LanC was assessed by examining the levels of viral genes, proteins, and virus titers in HSV-1-infected ARPE-19 and Vero cells. Immunofluorescence (IF) analysis was performed to determine the intracellular distribution of NRF2. Additionally, an in vivo mouse model of HSV-1 infection was developed to evaluate the antiviral activity of LanC, using indicators such as intraepidermal nerve fibers (IENFs) loss and viral gene inhibition. RESULTS: Our findings demonstrate that LanC significantly inhibits HSV-1 replication both in vitro and in vivo. The antiviral effect of LanC is mediated by the perinuclear translocation of NRF2. CONCLUSIONS: LanC exhibits anti-HSV-1 effects in viral infections, which are associated with the intracellular translocation of NRF2. These findings suggest that LanC has the potential to serve as a novel NRF2 modulator in the treatment of viral diseases.

Senolytic Treatment Improve Small Intestine Regeneration in Aging.

Aging induces a series of alterations, specifically a decline in the stature and number of villi and crypts in the small intestine, thus compromising the absorbent capability of the villi. This investigation employed a senolytic combination of dasatinib and quercetin (D+Q) to examine its impact on the intestinal tract of elderly mice. Our findings demonstrate that D+Q treatment leads to a decrease in the expression of p21, p16, and Ki67, while concurrently triggering removal of apoptotic cells within the villi. Additionally, D+Q treatment exhibits the ability to promote growth in both the height and quantity of villi and crypts, along with stimulating nitric oxide (NO) production in aged mice. The study presented a model to assess strategies to alleviate age-related senescence in the intestinal tract of elderly mice. Importantly, D+Q showcases promising potential in enhancing intestinal functionality within the aging.

A direct spino-cortical circuit bypassing the thalamus modulates nociception.

Nociceptive signals are usually transmitted to layer 4 neurons in somatosensory cortex via the spinothalamic-thalamocortical pathway. The layer 5 corticospinal neurons in sensorimotor cortex are reported to receive the output of neurons in superficial layers; and their descending axons innervate the spinal cord to regulate basic sensorimotor functions. Here, we show that a subset of layer 5 neurons receives spinal inputs through a direct spino-cortical circuit bypassing the thalamus, and thus define these neurons as spino-cortical recipient neurons (SCRNs). Morphological studies revealed that the branches from spinal ascending axons formed a kind of disciform structure with the descending axons from SCRNs in the basilar pontine nucleus (BPN). Electron microscopy and calcium imaging further confirmed that the axon terminals from spinal ascending neurons and SCRNs made functional synaptic contacts in the BPN, linking the ascending sensory pathway to the descending motor control pathway. Furthermore, behavioral tests indicated that the spino-cortical connection in the BPN was involved in nociceptive responses. In vivo calcium imaging showed that SCRNs responded to peripheral noxious stimuli faster than neighboring layer 4 cortical neurons in awake mice. Manipulating activities of SCRNs could modulate nociceptive behaviors. Therefore, this direct spino-cortical circuit represents a noncanonical pathway, allowing a fast sensory-motor transition of the brain in response to noxious stimuli.

Circular RNAs Represent a Novel Class of Human Cytomegalovirus Transcripts.

Human cytomegalovirus (HCMV) infects a large portion of the human population globally. Several HCMV-derived noncoding RNAs are involved in the regulation of viral gene expression and the virus life cycle. Here, we reported that circRNAs are a new class of HCMV transcripts. We bioinformatically predict 704 candidate circRNAs encoded by the TB40/E strain and 230 encoded by the HAN strain. We also systematically compare circRNA features, including the breakpoint sequence consensus, strand preference, length distribution, and exon numbers between host genome-encoded circRNAs and viral circRNAs, and showed that the unique characteristics of viral circRNAs are correlated with their genome types. Furthermore, we experimentally confirmed 324 back-splice junctions (BSJs) from three HCMV strains, Towne, TB40/E, and Toledo, and identified 4 representative HCMV circRNAs by RNase R treatment. Interestingly, we also showed that HCMV contains alternative back-splicing circRNAs. We developed a new amplified FISH method that allowed us to visualize circRNAs and quantify the number of circRNA molecules in the infected cells. The competitive endogenous RNA network analysis suggests that HCMV circRNAs play important roles in viral DNA synthesis via circRNA-miRNA-mRNA networks. Our findings highlight that circRNAs are an important component of the HCMV transcriptome that may contribute to viral replication and pathogenesis. IMPORTANCE HCMV infects 40% to 100% of the human population globally and may be a life-threatening pathogen in immunocompromised individuals. CircRNA is a family of unique RNA that is the most newly found and remains unknown in many aspects. Our current studies computationally identified HCMV-encoded circRNAs and confirmed the existence of the HCMV circRNAs in the infected cells. We systematically compared the features between host and different viral circRNAs and found that the unique characteristics of circRNAs were correlated with their genome types. We also first reported that HCMV contained alternative back-splicing circRNAs. More importantly, we developed a new amplified FISH method which allowed us for the first time not only to visualize circRNAs but also to quantify the number of circRNA molecules in the infected cells. This work describes a novel component of HCMV transcriptome bringing a new understanding of HCMV biology and disease.

Single-cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic pain.

Peripheral nerve injury could lead to chronic neuropathic pain. Understanding transcriptional changes induced by nerve injury could provide fundamental insights into the complex pathogenesis of neuropathic pain. Gene expression profiles of dorsal root ganglia (DRG) in neuropathic pain condition have been studied. However, little is known about transcriptomic changes in individual DRG neurons after peripheral nerve injury. Here we performed single-cell RNA sequencing on dissociated mouse DRG cells after spared nerve injury (SNI). In addition to DRG neuron types that are found under physiological conditions, we identified three SNI-induced neuronal clusters (SNIICs) characterized by the expression of Atf3/Gfra3/Gal (SNIIC1), Atf3/Mrgprd (SNIIC2) and Atf3/S100b/Gal (SNIIC3). These SNIICs originated from Cldn9+/Gal+, Mrgprd+ and Trappc3l+ DRG neurons, respectively. Interestingly, SNIIC2 switched to SNIIC1 by increasing Gal and reducing Mrgprd expression 2 days after nerve injury. Inferring the gene regulatory networks after nerve injury, we revealed that activated transcription factors Atf3 and Egr1 in SNIICs could enhance Gal expression while activated Cpeb1 in SNIIC2 might suppress Mrgprd expression within 2 days after SNI. Furthermore, we mined the transcriptomic changes in the development of neuropathic pain to identify potential analgesic targets. We revealed that cardiotrophin-like cytokine factor 1, which activates astrocytes in the dorsal horn of spinal cord, was upregulated in SNIIC1 neurons and contributed to SNI-induced mechanical allodynia. Therefore, our results provide a new landscape to understand the dynamic course of neuron type changes and their underlying molecular mechanisms during the development of neuropathic pain.

Clinical Characteristics, Treatment Effectiveness, and Predictors of Response to Pharmacotherapeutic Interventions Among Patients with Herpetic-Related Neuralgia: A Retrospective Analysis.

BACKGROUND: The treatment for herpetic-related neuralgia focuses on symptom control by use of antiviral drugs, anticonvulsants, and tricyclic antidepressants. We aimed to explore the clinical characteristics associated with medication responsiveness, and to build a classifier for identification of patients who have risk of inadequate pain management. METHODS: We recruited herpetic-related neuralgia patients during a 3-year period. Patients were stratified into a medication-resistant pain (MRP) group when the pain decrease in the visual analogue scale (VAS) is < 3 points, and otherwise a medication-sensitive pain (MSP) group. Multivariate logistic regression was performed to determine the factors associated with MRP. We fitted four machine learning (ML) models, namely logistic regression, random forest, supporting vector machines (SVM), and naïve Bayes with clinical characteristics gathered at admission to identify patients with MRP. RESULTS: A total of 213 patients were recruited, and 132 (61.97%) patients were diagnosed with MRP. Subacute herpes zoster (HZ) (vs. acute, OR 8.95, 95% CI 3.15-29.48, p = 0.0001), severe lesion (vs. mild lesion, OR 3.84, 95% CI 1.44-10.81, p = 0.0084), depressed mood (unit increase OR 1.10, 95% CI 1.00-1.20, p = 0.0447), and hypertension (hypertension, vs. no hypertension, OR 0.36, 95% CI 0.14-0.87, p = 0.0266) were significantly associated with MRP. Among four ML models, SVM had the highest accuracy (0.917) and receiver operating characteristic-area under the curve (0.918) to discriminate MRP from MSP. Phase of disease is the most important feature when fitting ML models. CONCLUSIONS: Clinical characteristics collected before treatment could be adopted to identify patients with MRP.

Somatosensory Neuron Typing with High-Coverage Single-Cell RNA Sequencing and Functional Analysis.

Different physical and chemical stimuli are detected by the peripheral sensory receptors of dorsal root ganglion (DRG) neurons, and the generated inputs are transmitted via afferent fibers into the central nervous system. The gene expression profiles of DRG neurons contribute to the generation, transmission, and regulation of various somatosensory signals. Recently, the single-cell transcriptomes, cell types, and functional annotations of somatosensory neurons have been studied. In this review, we introduce our classification of DRG neurons based on single-cell RNA-sequencing and functional analyses, and discuss the technical approaches. Moreover, studies on the molecular and cellular mechanisms underlying somatic sensations are discussed.

Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity.

Sensory neurons are distinguished by distinct signaling networks and receptive characteristics. Thus, sensory neuron types can be defined by linking transcriptome-based neuron typing with the sensory phenotypes. Here we classify somatosensory neurons of the mouse dorsal root ganglion (DRG) by high-coverage single-cell RNA-sequencing (10 950 ± 1 218 genes per neuron) and neuron size-based hierarchical clustering. Moreover, single DRG neurons responding to cutaneous stimuli are recorded using an in vivo whole-cell patch clamp technique and classified by neuron-type genetic markers. Small diameter DRG neurons are classified into one type of low-threshold mechanoreceptor and five types of mechanoheat nociceptors (MHNs). Each of the MHN types is further categorized into two subtypes. Large DRG neurons are categorized into four types, including neurexophilin 1-expressing MHNs and mechanical nociceptors (MNs) expressing BAI1-associated protein 2-like 1 (Baiap2l1). Mechanoreceptors expressing trafficking protein particle complex 3-like and Baiap2l1-marked MNs are subdivided into two subtypes each. These results provide a new system for cataloging somatosensory neurons and their transcriptome databases.