Muscarinic receptor signalling pathways regulating axonal regeneration and sprouting in adult neurons

以往对胚胎交感神经元的研究表明，乙酰胆碱（ACh）对毒蕈碱受体的激动作用可以调节神经元的兴奋性。此外，在多种胚胎神经细胞类型中，发育过程中的ACh信号传导可以调节生长锥运动，然而，被动员的细胞内通路仍未被探索。我们最近首次发现毒蕈碱乙酰胆碱1型受体（M1R）是成人感觉神经元神经突生长的调节因子。2017年，我们发现M1R拮抗剂是神经突生长的有效刺激剂，重要的是可以预防和逆转几种啮齿动物感觉神经病变模型中的神经损伤（Calcutt等）。临床投资，2017)。最近，通过在培养的成人感觉神经元中过度表达M1R或其无应答的DREAD突变体，我们证明了该受体是抑制神经突生长途径的抑制成分（Sabbir等）。神经科学前沿，2018)。活性M1R抑制轴突生长，部分是通过动员G蛋白G13，引起微管蛋白解聚。******这项工作的总体目标是确定乙酰胆碱信号通过M1R在体内发育感觉神经元中的作用，并表征连接M1R与轴突细胞骨架调节的关键信号转导成分。具体目的是：******目的1：确定诱导和/或条件敲除M1R或胆碱乙酰转移酶（ChAT；以消耗乙酰胆碱）对体内感觉神经元分化和成熟的影响。***目的2：在培养的神经元中，解剖由乙酰胆碱调节的M1R下游的信号转导通路，以抑制神经突起的生长。* * * * * * HQP培训。每个目标将由一名博士或硕士学生执行。在目标1中，学生将学习小鼠遗传学，神经元发育以及小鼠系的衍生和维持的基础知识。此外，在1-2年期间，学生还将在培养的感觉神经元上进行体外工作，同时建立菌落。在目标2中，另一名学生将学习原代神经元和克隆细胞系培养以及一系列用于解剖M1R信号和功能的药理学和生化方法。******影响。我们之前的工作表明，M1R可以靶向治疗几种神经性疾病。我们正在商业化，并有一家公司，WinSanTor Inc (www.winsantor.com)，正在美国和加拿大进行早期的二期试验。本文所述的工作计划将有助于这一过程，通过增强我们对这种导致发育期间和成人神经系统中感觉神经元表型改变的新型m1r介导途径的基础知识

Previous studies in embryonic sympathetic neurons revealed that acetylcholine (ACh) agonism at muscarinic receptors could modulate neuronal excitability. Furthermore, in a variety of embryonic neuronal cell types ACh signalling during development can modulate growth cone motility, however, the intracellular pathways mobilized remain unexplored. For the first time we recently identified the muscarinic acetylcholine type 1 receptor (M1R) as a regulator of neurite outgrowth in adult sensory neurons. In 2017 we revealed that antagonists of the M1R were potent stimulators of neurite outgrowth and importantly could prevent and reverse nerve damage in several rodent models of sensory neuropathy (Calcutt et al. J Clin Invest, 2017). Recently, using over-expression of M1R or its non-responding DREAD mutant in cultured adult sensory neurons we demonstrated that this receptor was an inhibitory component of a pathway constraining neurite outgrowth (Sabbir et al. Frontiers in Neuroscience, 2018). Active M1R suppressed axonal outgrowth, in part, via mobilization of the G protein, G13, which caused depolymerization of tubulin. ******The overall objective of this program of work is to determine the role of acetylcholine signaling via M1R in vivo in developing sensory neurons and to characterize the key signal transduction components linking M1R with regulation of the axonal cytoskeleton. The specific aims are:******Objective 1: Determine impact of inducible and/or conditional knockout of M1R or choline acetyltransferase (ChAT; to deplete acetylcholine) on sensory neuron differentiation and maturation in vivo.***Objective 2: In cultured neurons dissect signal transduction pathways downstream from M1R that are modulated by acetylcholine to constrain neurite outgrowth.******HQP training. Each Objective will be performed by one PhD or MSc student. In Objective 1 the student will learn the basics of mouse genetics, neuronal development and derivation and maintenance of mouse lines. In addition during years 1-2 the student will also perform in vitro work on cultured sensory neurons while setting up colonies. In Objective 2 the other student will learn primary neuron and clonal cell line culture and a range of pharmacological and biochemical approaches for dissecting M1R signaling and function.******Impact. Our previous work shows the M1R can be targeted for therapy in several neuropathic diseases. We are commercializing and have a company, WinSanTor Inc (www.winsantor.com), that is performing early phase 2 trials in the US and Canada. The work program described herein will contribute to this process by enhancing our fundamental knowledge of this novel M1R-mediated pathway leading to altered sensory neuron phenotype during development and in the adult nervous system.**