MicroRNAs (miRNAs) function as master switches for post-transcriptional gene expression. Their genes are either located in the extragenic space or within host genes, but these intragenic miRNA::host gene interactions are largely enigmatic. The aim of this study was to investigate the location and co-regulation of all to date available miRNA sequences and their host genes in an unbiased computational approach. The majority of miRNAs were located within intronic regions of protein-coding and non-coding genes. These intragenic miRNAs exhibited both increased target probability as well as higher target prediction scores as compared to a model of randomly permutated genes. This was associated with a higher number of miRNA recognition elements for the hosted miRNAs within their host genes. In addition, strong indirect autoregulation of host genes through modulation of functionally connected gene clusters by intragenic miRNAs was demonstrated. In addition to direct miRNA-to-host gene targeting, intragenic miRNAs also appeared to interact with functionally related genes, thus affecting their host gene function through an indirect autoregulatory mechanism. This strongly argues for the biological relevance of autoregulation not only for the host genes themselves but, more importantly, for the entire gene cluster interacting with the host gene.

Preclinical research using different rodent model systems has largely contributed to the scientific progress in the pain field, however, it suffers from interspecies differences, limited access to human models, and ethical concerns. Human induced pluripotent stem cells (iPSCs) offer major advantages over animal models, i.e., they retain the genome of the donor (patient), and thus allow donor-specific and cell-type specific research. Consequently, human iPSC-derived nociceptors (iDNs) offer intriguingly new possibilities for patient-specific, animal-free research. In the present study, we characterized iDNs based on the expression of well described nociceptive markers and ion channels, and we conducted a side-by-side comparison of iDNs with mouse sensory neurons. Specifically, immunofluorescence (IF) analyses with selected markers including early somatosensory transcription factors (BRN3A/ISL1/RUNX1), the low-affinity nerve growth factor receptor (p75), hyperpolarization-activated cyclic nucleotide-gated channels (HCN), as well as high voltage-gated calcium channels (VGCC) of the Ca_V2 type, calcium permeable TRPV1 channels, and ionotropic GABA_A receptors, were used to address the characteristics of the iDN phenotype. We further combined IF analyses with microfluorimetric Ca²⁺ measurements to address the functionality of these ion channels in iDNs. Thus, we provide a detailed morphological and functional characterization of iDNs, thereby, underpinning their enormous potential as an animal-free alternative for human specific research in the pain field for unveiling pathophysiological mechanisms and for unbiased, disease-specific personalized drug development.

Introduction The medial prefrontal cortex (mPFC) is well known for its prominent role in in decision-making, reward-guided learning and memory formation. It receives dense input from cholinergic neurons originating in the basal forebrain and laterodorsal tegmental nucleus and mPFC neurons express nicotinic as well as muscarinic acetylcholine receptors. In addition, serotonergic neurons emanating from the medial and dorsal raphe nuclei project to various subregions of the PFC and both metabotropic and ionotropic serotonin (5-HT) receptors are expressed in cortical neurons. The actions of both neuromodulators on the mPFC are diverse and insufficiently well understood. Although single cell patch clamp is routinely used to study the role of cholinergic and serotonergic neurons in cortical networks, we provide here first insights in the potential of multielectrode array (MEA) recordings in slices as an efficient and fast screening tool to investigate the neurotransmitters and synaptic modulators on the entire mPFC network. Methods We recorded spontaneous and chemically induced network activity in acute mPFC brain slices with a thickness of 300µm obtained from 10-12 weeks old C57BL/6J mice. After cutting, slices were stored for 12 min in protective-N-methyl-D-glucamine artificial cerebrospinal fluid solution, followed by recovery in artificial cerebrospinal fluid (aCSF) for 45 min. In vitro MEA recordings were performed to assess baseline discharge activity as well as dose specific responses to carbachol (CCh 1µM, 5µM, 25µM, 50µM, 100µM) or 5-HT (doses: 1µM, 50µM, 100µM, 250µM, 500µM) administration to the bath solution. Both, mean spike frequency and the number of activated electrodes were analyzed and dose response curves, time course of activation as well as the net modulatory effect on network activity in the mPFC were compared. In addition, RT-qPCR data were obtained to identify expression levels of cholinergic receptors in the mPFC. Results Recordings revealed a generally low baseline discharge rate with a minority of electrodes showing spontaneous discharge activity. Exposure to CCh induced a significantly enhanced network activity at doses > 5 µM, indicated by a dose-dependent increase in mean spike frequency and a significantly higher number of active electrodes compared to baseline activity. By contrast, 5-HT modulated the activity in the mPFC in a U-shaped dose dependent manner with the highest mean spike frequency after administration of 10µM and 500µM. Although administration of 10µM, 250µM and 500µM of 5-HT showed excitatory effects the mean spike frequency and also the number of activated electrodes was significantly lower compared to CCh stimulation. Conclusion MEA recordings revealed excitatory effects of both CCh and 5-HT in the mPFC. The impact of cholinergic stimulation exceeded serotonergic excitatory effects. Our results support the potential of MEA recordings as an efficient and fast screening technology to assess neuromodulator effects on network activity in acute cortical brain slices.

Non-coding RNAs (ncRNAs) play a critical role in regulating gene expression during development and in the pathogenesis of diseases. In particular, microRNAs have been extensively studied in the context of neurogenesis, the differentiation of pain sensing nociceptive neurons and the pathogenesis of pain disorder, however, little is known about the developmental signatures of other ncRNA species throughout sensory neuron differentiation. Moreover, there is currently no information available about the general expression signatures of ncRNAs in human dorsal root ganglia (DRGs) harboring the cell bodies of primary afferent nociceptors. To bridge this knowledge gap, we developed a comprehensive atlas of small ncRNA species signatures during the differentiation of human induced pluripotent stem cell (iPSC)-derived nociceptive neurons. By employing a combination of iPSC-derived sensory neuron and human DRG long and short RNA co-sequencing, we identified specific signatures that describe the developmental processes and the signatures of all currently known small ncRNA species in detail. Our analysis revealed that different ncRNA species, including tRNAs, snoRNAs, lncRNAs, and piRNAs, are associated with different stages of sensory neuron differentiation and maturation. We retrieved pronounced similarities in ncRNA expression between human DRG and late-stage iPSC-derived sensory neurons, which further supports the use of iPSC-derived sensory neurons to uncover functional and regulatory changes in ncRNAs and their suitability as a as a human model system to bridge the translational gap between preclinical findings mostly from rodent models and our understanding of human disorders for the development of mechanism-based treatments. In summary, our findings provide important insights into the role of ncRNA species other than microRNAs in human nociceptors. The updated NOCICEPTRA2.0 Tool will be the first fully comprehensive searchable ncRNA database for human sensory neurons enabling researchers to investigate important hub ncRNA regulators in nociceptors in full detail.