The project has accomplished with the tasks planned. We have identified nociceptor induced gene expression in purified nociceptive sensory neurons (SN), both in naïve and injured states in mice and rats. Some of the identified targets were evaluated in the in vivo models and in vitro assays. Proteome profiling was performed on samples obtained from naïve and vincristine-treated (VCR) animals, identifying potential biomarkers for disease prevention and diagnosis for neuropathic pain, and new targets for drug development. The two animal models of neuropathic pain used in the project were set up and pharmacological studies performed with reference compounds, tool compounds for new targets and combinations of both. Spinal microdialysis in awake rats was validated with reference compounds. Macrophage medium transfer experiments and co-culture experiments point to a complex, bi-directional crosstalk between glial cells and neurons. Our co-culture systems have been evaluated under different sensitization options, measuring the effects of various mediators on nociceptor sensitization. Additional experiments were performed on myeloid cells and were incorporated into a publication (Liang et al. Pain, in press). Neuron purification experiments in combination with MEA and calcium imaging experiments successfully identified a functional role for non-neuronal cells in establishing/promoting excitability of SN. Commercially available neonatal rDRGs and explanted primary rDRGs from naïve and VCR rats cultivated in miniaturized format showed a good morphology and the functional presence of pain relevant channels. Phenotypic analysis of neurons in 384-well format was proved and first results using a VCR were obtained. In relation to hiPSC work, a cell line was registered and used throughout the project. The protocols for the production, cryopreservation, transport and differentiation of SN, hiPSC-derived macrophage (iPSCdMΦ), and co-cultures have been developed. We established the lineage-specific TRPV1-mCherry reporter line, that proved the nociceptive sensory identity, and the iPSCdMΦ differentiation and definition of co-culture conditions, that maintains the full macrophage and SN functionality. SN differentiation was also implemented at Axxam, demonstrating robustness of the method and its applicability for larger screening purposes. We have demonstrated the feasibility to perform a FLIPR-based, HTS functional assay, using co-cultures or monoculture of hiPSC-derived SNs and supernatant from macrophages activated with LPS. This assay has been used for the study perturbations in the neuro-excitability induced by Veratridine. On the other hand, we developed a HCS phenotypic assay in 384-well plate format, to monitor modification in the neurite structure and outgrowth, using VCR as control of neurotoxicity. The assay can be used for in vitro functional and phenotypic testing and screening of compounds.
Exploitation: the SMEs, AXXAM and L&B, and the research institute NMI, have already included the assays and models used and developed in NGN-PET as new services and are being offered to the scientific and academic partners. At KCL, several cell-type specific RNA-seq datasets have been generated and published. A public database has been created to allow other biologists easy access. EST and GRT, have already analyzed two targets, but the list of genes and proteins identified is huge and the validation of new targets is a continuous process at both pharmaceutical companies. Those targets with sufficient efficacy, pharmacokinetic features, and safety profile, could become the starting point of a program to find a new modality for pharmacological intervention. GRT and EST have plans to incorporate the developed co-cultures to their HCS/HTS or MEA systems in the project workflows. The two neuropathic pain models have been already used for other internal projects at both companies. The hiPSC-derived cell types will be part of the drug discovery process from now on. Besides the work done with macrophages, several other glial cells have to be developed and studied, one by one or in combination. The exploitation plans will contribute to support European leadership in achieving a sounder knowledge on chronic pain pathways, crucial in guiding the discovery of effective analgesics and of clinically useful biomarkers and help improve the health care of patients.
Dissemination: the partners have actively worked to disseminate the project results by participating to scientific conferences, social media, publications of project proceedings and results, targeting both the general public and the research community.
