Translational profiling of bladder sensory nerves and their cell type identities using dissociation free single nucleus sequencing

摘要 外周轴突需要持续的维护和修复，以保持与目标的连通性。本地 轴突翻译在调节蛋白质和能量在整个生命周期内的动态平衡方面起着重要作用。曲目 在发育和成年期的活跃的信使核糖核酸的翻译，以及修复，再生和 翻译与环境线索相关的特定mRNAs子集存储在远端轴突中。在许多 这些活动会在目标站点迅速发生，以便在新的抄本之前立即做出反应 从细胞体定位。在疾病状态下，轴突平移改变可能会影响延长、退出、 修剪、分枝或存活。膀胱的传入(感觉)和传出(副交感)神经支配 和交感神经)对其功能至关重要。这些神经建立了错综复杂的联系 膀胱的区域和细胞类型，包括尿路上皮、常驻免疫细胞、平滑肌细胞和 血管，并可能使用局部平移来维持体内平衡并对损伤或生理做出反应 改变。在膀胱神经末梢中活跃翻译的基因的身份及其随后的 在膀胱中的作用还没有被研究过。在健康和疾病状态下定义主动翻译的mRNA 膀胱神经和胞体来源的身份将为深入了解 周围神经和泌尿系统之间的神经解剖和生理相互作用，其 在多种具有神经基础的膀胱疾病中的作用和帮助合理设计靶向治疗 旨在保护神经细胞的连接。这可能会影响人们对膀胱衰退的理解 衰老、衰弱的膀胱症状和功能障碍(如顽固性疼痛、大小便失禁、 尿频和尿急)在美国影响数百万人，神经退行性疾病和 影响膀胱功能的神经性疾病(糖尿病、艾滋病毒)。我们将建立方法和概念验证 健康雌、雄小鼠膀胱感觉、自主神经轴突翻译组的研究 在患有尿路感染的雌性小鼠中(Aim 1)，并通过开发无伪迹的Single来鉴定它们的同源细胞体 投射到膀胱的背根节感觉神经元的核RNA表达图谱(目标2)。这些方法 开发将克服膀胱、其神经和膀胱的组成、功能和解剖方面的独特挑战 与中枢或躯体感觉系统不同的相应体节，并作为基准 适用于其他下尿路器官。开发的方法、使用的遗传工具和新的数据 将公开分享，以便快速采用和加速研究，以更好地了解监管 神经末梢及其目标位置由包括基础研究人员和翻译研究人员在内的不同用户集合， 对尿路疾病和正常生理学感兴趣的计算生物学家和临床医生。

Abstract Peripheral axons need sustained maintenance and repair to maintain connectivity with their targets. Local axonal translation plays a role in regulation of protein and energy homeostasis across lifespan. The repertoire of active mRNA translation during development and adulthood, and the ability to repair, regenerate and translate specific subsets of mRNAs in relation to environmental cues is stored in distal axons. In many instances these activities take place rapidly for immediate response at target sites before new transcripts localize from the cell bodies. In disease states, altered axonal translation may affect elongation, withdrawal, pruning, branching or survival. In the bladder innervation by afferent (sensory) and efferent (parasympathetic and sympathetic) nerves is critical for its function. These nerves establish intricate connections with different regions of the bladder and cell types including the urothelium, resident immune cells, smooth muscle cells and blood vessels and likely employ local translation to maintain homeostasis and respond to injury or physiologic changes. The identity of genes that are actively translated in bladder nerve terminal and subsequently their role in bladder has not been studied. Defining actively translated mRNA in healthy and disease states in bladder nerves and the identity of the soma source will provide insights into the mechanisms of neuroanatomical and physiological interactions between the peripheral nervous and urinary systems, their roles in a number of bladder diseases with neuronal basis and aid in rationally designing targeted therapies aimed at preserving neuro-uro connections. This has potential to impact understanding of declining bladder function in ageing, debilitating bladder symptoms and dysfunction (such as intractable pain, incontinence, frequency and urgency to urinate) that affect millions of people in the US, neurodegenerative diseases and neuropathies (diabetes, HIV) affecting bladder function. We will establish methods and proof of concept studies to delineate the translatome in bladder sensory, autonomic axons in healthy male and female mice and in female mice with UTI (Aim 1) and identify their cognate cell bodies by developing artifact –free single nucleus RNA expression maps of DRG sensory neurons projecting to the bladder (Aim 2). The methods developed will overcome challenges unique to composition, function and anatomy of bladder, its nerves and corresponding soma that are different from the CNS or somatosensory system and serve as benchmark for application to other lower urinary tract organs. The methods developed, genetic tools used and novel data generated will be publicly shared for rapid adoption and accelerating studies to better understand regulation of nerve terminals with their target sites by diverse set of users including basic and translational researchers, computational biologists and clinicians interested in urinary tract disorders and normal physiology.