Investigating the role for Utrophin in age-related decline of the Merkel lineage

我们的触觉使人类存在的许多基本行为成为可能，允许我们吃东西， 沟通和生存。触觉反应性缺陷被认为是导致姿势下降的原因之一。 稳定性和抓地力，以及由此导致的跌倒频率的增加，这是决定 老年人的生活质量和独立生活能力。在哺乳动物中，不同的触觉质量 (曲率、纹理和振动)由驻留在皮肤中的触摸感受器编码，具有不同的生理 特性和形态末端器官；然而，其背后的细胞和分子机制 多样性在很大程度上是未知的。此外，对皮肤干细胞的调节负责 维持感知温柔触摸的细胞力感受器的周转，如默克尔细胞，是 未知。我们的实验室之前描述了一组位于表皮中的上皮干细胞。 皮肤中的触摸穹顶，被称为触摸穹顶干细胞(TDSCs)，负责维持 默克尔在动态平衡时期的血统。这项提案的长期目标是定义细胞和分子 维护默克尔家族血统的tDSC基础。我们的初步数据记录了一个戏剧性的与年龄相关的 人(40-90岁)和小鼠(2-32个月龄)皮肤中默克尔细胞数量下降。 然而，tDSC数字保持不变。我们证实，与年龄相关的默克尔细胞损失不是由于 成熟的Merkel细胞早熟退出或感觉传入神经支配缺陷，但由于tDSC减少 补充默克尔血统的祖辈能力。利用tDSC祖细胞中与年龄相关的缺陷 我们对年轻和老年小鼠皮肤和皮肤中的TDSCs进行了蛋白质组学和mRNA谱分析 发现粘着斑蛋白Utrn在老年人中显著下调 TDSCs。出生后第20天的Utrn空(Utrn-/-)小鼠的Merkel细胞数量与Wt 小鼠，不包括Utrn在默克尔细胞发育中的作用。然而，默克尔的细胞数量减少了 50%在2个月大的Utrn-/-小鼠中，为Utrn在年龄相关的功能中的作用提供了遗传学证据 TDSC对默克尔血统的维护。我们发现Nrg1-ERBB2信号轴是一种 Utrn在体外培养的小鼠和人上皮角质形成细胞及TDSCs中的表达。这些初步的 研究结果支持我们的中心假设：tDSC需要NRG1-ERBB2调节Utrn的表达 维持默克尔细胞的动态平衡。为了验证我们的假设，我们将使用小鼠模型对 干扰或挽救Utrn的表达，并评估其对默克尔细胞稳态的影响。使用体内血统 追踪工具和Nrg1条件等位基因，我们将研究tDSC的细胞机制 默克尔血统的维持和TD生态位中Nrg1表达的上游调控。最后，我们会 探讨TDSC中Utrn调控的分子机制和Utrn功能的下游效应。 我们的目标是首先揭示默克尔家族干细胞调控的遗传机制。

Our sense of touch enables numerous behaviors fundamental to human existence, allowing us to eat, communicate and survive. Deficits in tactile responsiveness are thought to contribute to the decline of postural stability and hand grip, and the resulting increase in falling frequency, which is a major factor determining quality of life and the ability to live independently for the elderly. In mammals, different tactile qualities (curvature, texture and vibration) are encoded by touch receptors residing in the skin with distinct physiological properties and morphological end-organs; however, the cellular and molecular mechanisms underlying this diversity are largely unknown. Furthermore, the regulation of skin stem cells that are responsible for maintaining the turnover of cellular mechanoreceptors that perceive gentle touch, such as Merkel cells, is unknown. Our laboratory previously characterized a population of epithelial stem cells that reside in epidermal touch domes in the skin, termed touch dome stem cells (TDSCs), that are responsible for maintaining the Merkel lineage during homeostasis. The long-term goal of this proposal is to define the cellular and molecular basis for TDSC maintenance of the Merkel lineage. Our preliminary data chronicles a dramatic age-related decline in Merkel cell numbers in human (40 – 90 years of age) and murine (2 – 32 months of age) skin. However, TDSC numbers remain unchanged. We confirmed that age-related loss of Merkel cells is not due to precocious exit of mature Merkel cells or defects in sensory afferent innervation but is due to diminished TDSC progenitor capacity to replenish the Merkel lineage. Leveraging this age-related deficit in TDSC progenitor capacity, we performed proteomics and mRNA profiling of TDSCs from young versus aged mouse skin and identified the focal adhesion-associated protein Utrophin (Utrn) to be dramatically downregulated in aged TDSCs. Utrn null (Utrn-/-) mice at postnatal day 20 displayed equal numbers of Merkel cells compared to Wt mice, excluding a role for Utrn in Merkel cell development. However, Merkel cell numbers were reduced by 50% in 2-month-old Utrn-/- mice providing genetic evidence for a functional role for Utrn in age-related maintenance of the Merkel lineage by TDSCs. We identified the Nrg1-Erbb2 signaling axis as a regulator of Utrn expression in mouse and human epithelial keratinocytes in vitro and TDSCs in vivo. These preliminary findings support our central hypothesis: Nrg1-Erbb2 regulation of Utrn expression is required for TDSC maintenance of Merkel cell homeostasis. To test our hypothesis, we will employ mouse models to genetically disrupt or rescue Utrn expression and assess its impact on Merkel cell homeostasis. Using in vivo lineage tracing tools and a Nrg1 conditional allele we will investigate the cellular mechanisms underlying TDSC maintenance of the Merkel lineage and upstream regulation of Nrg1 expression in the TD niche. Finally, we will interrogate the molecular mechanisms for Utrn regulation and downstream effectors of Utrn function in TDSCs. Our goal is to uncover first of its kind genetic mechanisms for stem cell regulation of the Merkel lineage.