Electrophysiological and genetic targeting of pruriceptors

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Several dermatoses are associated with chronic itch resulting in a high personal and societal burden. In contrast to model organisms, our understanding how the human nervous system detects and encodes itch signals under these and even under naïve conditions is still limited. In mice, specific, transcriptionally defined classes of sensory neurons, such as the Natriuretic polypeptide b (Nppb)-expressing cells have been linked to pruriceptive signaling in health and disease, but the lack of transcriptomic information has hampered such an approach in larger animals and humans. Instead, a function-based neuronal classification system has been used, creating an apparent disconnect and translational gap between the two classification strategies.To bridge this gap, we propose to detect functional and transcriptional characteristics of sensory neurons in the pig simultaneously, thereby defining pruriceptive neuronal classes in large animals. To achieve this, we will virally express optogenetic actuators in transcriptionally defined classes of porcine sensory neurons and record from transduced nerve fibers in vivo. Natural and optogenetic responses will be integrated in a harmonized classification system and pruritic stimuli can be mapped onto this new comprehensive taxonomy. A key prerequisite for this novel approach will be the availability of suitable promoter constructs used for AAV-based viral targeting. First, we will capture transcriptional heterogeneity in pig sensory neurons using single-cell transcriptomics and epigenomics and harness this information for unbiased viral targeting. Second, we will specifically target NPPB-expressing neurons, building on our detection of NPPB and several pruritogen receptors, including the interleukin 31 receptor (IL-31RA), in a group of human sensory neurons, which mimics expression patterns of these pruriceptive markers in mice.The strict co-expression of NPPB and IL-31RA opens an excellent route to study neuronal effects of IL-31, a key player driving skin inflammation and chronic itch in mouse models and several human dermatoses. We will therefore investigate responses to acute and chronic IL-31 stimulation in murine NPPB-neurons, both in vitro and in vivo, as a proxy for dermatitis-induced plasticity of sensory neurons. We will focus on the determination of cell-autonomous changes in neuronal excitability and identify potential mechanisms underlying these changes by measuring alterations in gene expression on the single-cell level. Subsequently, we will explore their causal relations with pharmacological and genetic approaches.In summary, we will define structure-function-relationships of pruriceptive nerve fibers in large animals and investigate the contribution of these fibers to chronic itch in mice. We envision that this knowledge will provide avenues for translation to humans, improving our understanding of itch encoding in health and disease.