Electrophysiological and genetic targeting of pruriceptors

几种皮肤病与慢性瘙痒相关，导致高的个人和社会负担。与模式生物相比，我们对人类神经系统如何在这些条件下甚至在原始条件下检测和编码瘙痒信号的理解仍然有限。在小鼠中，特定的、转录定义的感觉神经元类别，如利钠肽B（Npp B）表达细胞，已与健康和疾病中的伤害性信号传导相关，但缺乏转录组信息阻碍了在较大动物和人类中的这种方法。相反，一个基于功能的神经元分类系统已被使用，创建一个明显的断开和两个分类策略之间的翻译gap.To弥合这一差距，我们建议同时检测在猪的感觉神经元的功能和转录特性，从而定义在大型动物的神经元类。为了实现这一点，我们将在转录定义的猪感觉神经元类别中病毒表达光遗传学致动器，并从体内转导的神经纤维记录。自然和光遗传学反应将被整合到一个统一的分类系统中，并且神经刺激可以映射到这个新的综合分类中。这种新方法的关键先决条件是用于基于AAV的病毒靶向的合适启动子构建体的可用性。首先，我们将使用单细胞转录组学和表观基因组学捕获猪感觉神经元中的转录异质性，并利用这些信息进行无偏见的病毒靶向。第二，我们将特异性靶向NPPB表达神经元，建立在我们在一组人类感觉神经元中检测NPPB和几种促神经元生成素受体，包括白细胞介素31受体（IL-31 RA）的基础上，其模拟小鼠中这些促神经元生成素标记物的表达模式。NPPB和IL-31 RA的严格共表达为研究IL-31的神经元效应开辟了一条极好的途径，在小鼠模型和几种人类皮肤病中，它是驱动皮肤炎症和慢性瘙痒的关键因素。因此，我们将研究急性和慢性IL-31刺激的小鼠NPPB-神经元，在体外和体内，作为一个代理皮炎诱导的可塑性的感觉神经元。我们将专注于确定神经元兴奋性的细胞自主变化，并通过测量单细胞水平上基因表达的改变来确定这些变化的潜在机制。随后，我们将探索它们的因果关系与药理学和遗传学的approaches.In总结，我们将定义的结构-功能-大型动物的痛觉神经纤维的关系，并调查这些纤维的贡献，在小鼠慢性瘙痒。我们预计这些知识将为人类提供翻译途径，提高我们对健康和疾病中瘙痒编码的理解。

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.