Traditionally, peripheral sensory neurons are assumed as the exclusive transducers of external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nerve fiber interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of afferents and adjacent connexin 43 contacts in native skin and have applied a pipeline based on expansion microscopy to quantify these parameter in skin sections of healthy participants versus patients with small fiber neuropathy. We further derived a fully human co-culture system, visualizing ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and potential interaction sites advances research at the neuro-cutaneous unit. These findings are crucial on the way to decipher the mechanisms of cutaneous nociception.

The honeybee (Apis mellifera) represents a model organism for social insects displaying behavioral plasticity. This is reflected by an age-dependent task allocation. The most protruding tasks are performed by young nurse bees and older forager bees that take care of the brood inside the hive and collect food from outside the hive, respectively. The molecular mechanism leading to the transition from nurse bees to foragers is currently under intense research. Circular RNAs, however, were not considered in this context so far. As of today, this group of non-coding RNAs was only known to exist in two other insects, Drosophila melanogaster and Bombyx mori. Here we complement the state of circular RNA research with the first characterization in a social insect. We identified numerous circular RNAs in the brain of A. mellifera nurse bees and forager bees using RNA-Seq with exonuclease enrichment. Presence and circularity were verified for the most abundant representatives. Back-splicing in honeybee occurs further towards the end of transcripts and in transcripts with a high number of exons. The occurrence of circularized exons is correlated with length and CpG-content of their flanking introns. The latter coincides with increased DNA-methylation in the respective loci. For two prominent circular RNAs the abundance in worker bee brains was quantified in TaqMan assays. In line with previous findings of circular RNAs in Drosophila, circAmrsmep2 accumulates with increasing age of the insect. In contrast, the levels of circAmrad appear age-independent and correlate with the bee’s task. Its parental gene is related to amnesia-resistant memory. We provide the first characterization of circRNAs in a social insect. Many of the RNAs identified here show homologies to circular RNAs found in Drosophila and Bombyx, indicating that circular RNAs are a common feature among insects. We find that exon circularization is correlated to DNA-methylation at the flanking introns. The levels of circAmrad suggest a task-dependent abundance that is decoupled from age. Moreover, a GO term analysis shows an enrichment of task-related functions. We conclude that circular RNAs could be relevant for task allocation in honeybee and should be investigated further in this context.

Fibromyalgia syndrome (FMS) is a heterogeneous chronic pain syndrome characterized by musculoskeletal pain and other key co-morbidities including fatigue and a depressed mood. FMS involves altered functioning of the central and peripheral nervous system (CNS, PNS) and immune system, but the specific molecular pathophysiology remains unclear. Anti-cholinergic treatment is effective in FMS patient subgroups, and cholinergic signaling is a strong modulator of CNS and PNS immune processes. Therefore, we used whole blood small RNA-sequencing of female FMS patients and healthy controls to profile microRNA regulators of cholinergic transcripts (CholinomiRs). We compared microRNA profiles with those from Parkinson’s disease (PD) patients with pain as disease controls. We validated the sequencing results with quantitative real-time PCR (qRT-PCR) and identified cholinergic targets. Further, we measured serum cholinesterase activity in FMS patients and healthy controls. Small RNA-sequencing revealed FMS-specific changes in 19 CholinomiRs compared to healthy controls and PD patients. qRT-PCR validated miR-182-5p upregulation, distinguishing FMS patients from healthy controls. mRNA targets of CholinomiRs bone morphogenic protein receptor 2 and interleukin 6 signal transducer were downregulated. Serum acetylcholinesterase levels and cholinesterase activity in FMS patients were unchanged. Our findings identified an FMS-specific CholinomiR signature in whole blood, modulating immune-related gene expression.

Fabry disease (FD) is caused by α-galactosidase A (AGAL) enzyme deficiency, leading to globotriaosylceramide accumulation (Gb3) in several cell types. Pain is one of the pathophysiologically incompletely understood symptoms in FD patients. Previous data suggest an involvement of hypoxia and mitochondriopathy in FD pain development at dorsal root ganglion (DRG) level. Using immunofluorescence and quantitative real-time polymerase chain reaction (qRT PCR), we investigated patient-derived endothelial cells (EC) and DRG tissue of the GLA knockout (KO) mouse model of FD. We address the question of whether hypoxia and mitochondriopathy contribute to FD pain pathophysiology. In EC of FD patients (P1 with pain and, P2 without pain), we found dysregulated protein expression of hypoxia-inducible factors (HIF) 1a and HIF2 compared to the control EC (p < 0.01). The protein expression of the HIF downstream target vascular endothelial growth factor A (VEGFA, p < 0.01) was reduced and tube formation was hampered in the P1 EC compared to the healthy EC (p < 0.05). Tube formation ability was rescued by applying transforming growth factor beta (TGFβ) inhibitor SB-431542. Additionally, we found dysregulated mitochondrial fusion/fission characteristics in the P1 and P2 EC (p < 0.01) and depolarized mitochondrial membrane potential in P2 compared to control EC (p < 0.05). Complementary to human data, we found upregulated hypoxia-associated genes in the DRG of old GLA KO mice compared to WT DRG (p < 0.01). At protein level, nuclear HIF1a was higher in the DRG neurons of old GLA KO mice compared to WT mice (p < 0.01). Further, the HIF1a downstream target CA9 was upregulated in the DRG of old GLA KO mice compared to WT DRG (p < 0.01). Similar to human EC, we found a reduction in the vascular characteristics in GLA KO DRG compared to WT (p < 0.05). We demonstrate increased hypoxia, impaired vascular properties, and mitochondrial dysfunction in human FD EC and complementarily at the GLA KO mouse DRG level. Our data support the hypothesis that hypoxia and mitochondriopathy in FD EC and GLA KO DRG may contribute to FD pain development.

Fibromyalgia syndrome (FMS) is a debilitating widespread chronic pain condition of unclear pathophysiology. We studied small noncoding RNAs as potential classifiers and mediators of FMS. Blood and keratinocyte microRNAs (miRs) and transfer RNA fragments (tRFs) were profiled by small RNA-sequencing within a comprehensively phenotyped female cohort of 53 patients with FMS vs 34 healthy controls (hCOs) and 15 patients with major depression and chronic physical pain (disease controls). Small RNAs were quantified via RNA-sequencing and candidates validated via qRT-PCR. MicroRNAs and tRFs were tested for association with FMS symptoms and their potential regulatory roles. miR and tRF profiles were altered in FMS compared to hCO in whole blood (n = 69; n = 22) and keratinocytes (n = 41; n = 55). Receiver operating characteristic analysis of blood miR candidates hsa-miR-148a-3p and hsa-miR-182-5p, and tRF candidate tRF-21-WB8647O5D levels separated FMS from hCO. In blood, hsa-miR-182-5p and hsa-miR-576-5p upregulation was validated via qRT-PCR, showing even higher expression in disease control, while TRF-20-40KK5Y93 was selectively increased in FMS. MicroRNAs in blood and keratinocytes were associated with how widespread pain manifested in patients. Keratinocyte tRFs correlated with loss of skin innervation. In blood, altered small RNAs were linked to immune and RNA processes, whereas in keratinocytes, adhesion and epithelial functions were targeted. Modulated tRFs shared sequence motifs in patients with FMS, which may promote concerted pathway regulation. Our findings show miRs/tRFs as key small RNAs dysregulation in FMS pathophysiology and open new perspectives for FMS diagnostics, symptom monitoring, and clinical management.

Neuropathic pain substantially affects the mental and physical well-being of patients and magnifies the socio-economic burden on the healthcare system. It is important to understand the molecular mechanisms underlying chronic pain to effectively target it. To investigate peripheral mechanisms relevant to pain signalling, we isolated nerve terminals from mouse footpads of wild type and Na _V 1.9-deficient mice, which exhibit impaired pain sensation and chronification. The isolated peripheral terminals are functionally active and capable of endocytosis. They contain both pre- and postsynaptic proteins in samples from mice and humans. Components of the protein translational machinery and mitochondria are enriched in nerve terminals, and these components are altered in Na _V 1.9 deficient terminals. An unbiased proteomic analysis of nerve terminals from footpads of Na _V 1.9 knockout mice shows dysregulation of the mitogen-activated protein kinase and extracellular regulated kinase 1/2 pathways. Preliminary proteomic analysis reveals a strong similarity between mouse and human skin samples, highlighting the broad and translational value of our approach. Our findings uncover peripheral signalling mechanisms involved in nociception, paving the way for unprecedented translational approaches using human biomaterials in pain research.