ROLES OF GFL-RET SIGNALING IN BLADDER SENSATION

描述(申请人提供)：感觉活动异常在膀胱疾病中起主要作用，如间质性膀胱炎/痛性膀胱综合征(IC/PBS)，尿毒症的感觉神经病，以及神经退行性疾病中与年龄相关的膀胱功能障碍，如帕金森病。确定正常和疾病状态下调节尿路感觉的分子和遗传机制对于合理设计膀胱感觉障碍的治疗方案和风险分层至关重要。胶质细胞源性神经营养因子配体家族(GFL)是一类神经营养因子，可与辅受体GFRRET(1-4)结合，激活受体酪氨酸激酶α。GFL-RET信号促进感觉神经元存活和轴突生长，防止轴突变性。我们证明RET的激活导致关键的对接酪氨酸的磷酸化，结合细胞内的适配器，并激活特定的信号转导通路，如PLCγ和PI3K/MAPK，以调节自主和遗传神经元的增殖、存活和迁移，并收集系统祖细胞。我们发现Ret-docking酪氨酸在尿路和自主神经节中具有不同的组织特异性作用，并且Ret在躯体痛觉中很重要。我们的初步数据显示，RET在人感觉神经节(DRG)以及膀胱的尿路上皮、粘膜下和肌间神经中高表达，这表明GFL-RET信号在膀胱功能中的作用尚不清楚。我们观察到，Ret-Null小鼠严重减少了膀胱神经支配。使用我们独特的Ret突变小鼠，我们发现Ret感觉神经元支配膀胱，GDNF单倍体功能不全在急性膀胱炎模型中减轻疼痛。这些结果共同支持GFL-RET信号在膀胱功能和健康中的重要作用。我们假设GFL-RET信号在调节幼稚和受伤状态下的膀胱感觉中起关键作用，并且在人类中发现的RET突变影响感觉神经元的健康。提出了三个具体目标。在目标1中，我们将使用一组独特的RET突变小鼠来识别生理上相关的RET激活的信号通路，这些信号通路调节正常和损伤状态下的膀胱感觉。在目标2中，我们将利用钙成像和电生理学分析来确定特定的RET激活通路对膀胱背根神经节传入神经元选择性离子通道(TRPV1、TRPA1和电压门控钠钾通道)的调节。在目标3中，我们将破译在人类中发现的RET突变在初级感觉神经元存活、轴突生长和退化中的作用。这些结果将为GFL-RET信号如何被靶向治疗膀胱疾病的感觉障碍提供洞察力。GFLS目前正处于治疗神经退行性疾病和慢性疼痛的临床试验中。这将加速拟议研究的临床翻译。

DESCRIPTION (provided by applicant): Abnormal sensory activity plays a major role in bladder diseases such as interstitial cystitis/painful bladder syndrome (IC/PBS), sensory neuropathy in uremia, and age related bladder dysfunction in neurodegenerative diseases such as Parkinson's. Identifying molecular and genetic mechanisms regulating urinary tract sensation in normal and disease states is essential for rational design of therapies for bladder sensory disorders and risk stratification. The glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) are neurotrophic factors that bind to one of the coreceptors GFRα (1-4) and activate the receptor tyrosine kinase RET. GFL-RET signaling promotes sensory neuron survival and axonal growth and prevents axonal degeneration. We demonstrated that RET activation leads to phosphorylation of key docking tyrosines that bind intracellular adaptors and activate specific signal transduction pathways such as PLCγ and PI3K/MAPK to regulate proliferation, survival and migration of autonomic and eneteric neurons, and collecting system progenitors. We discovered distinct tissue-specific roles of Ret-docking tyrosines in the urinary tract and autonomic ganglia and that Ret is important in somatic pain sensation. Our preliminary data show high RET expression in human sensory ganglia (DRG) and in urothelial, submucosal and myenteric nerves in the bladder suggesting an unexplored role for GFL-RET signaling in bladder function. We observed that Ret-null mice have severely reduced bladder innervation. Using our unique Ret mutant mice we found that Ret+ sensory neurons innervate the bladder and that Gdnf haploinsufficiency reduces pain in an acute cystitis model. These results collectively support an important role for GFL-RET signaling in bladder function and health. We hypothesize that GFL-RET signaling has a critical role in regulating bladder sensation in naïve and injured states, and that RET mutations found in humans affect the health of sensory neurons. Three specific aims are proposed. In Aim 1 we will identify physiologically relevant RET-activated signaling pathways that regulate bladder sensation in normal and injured states using a battery of unique RET-mutant mice. In Aim 2 we will determine the regulation of select ion channels (TRPV1, TRPA1 and voltage-gated sodium and potassium channels) by specific RET- activated pathways in bladder afferent DRG neurons using calcium imaging and electrophysiology analysis. In Aim 3 we will decipher the roles of RET mutations found in humans in primary sensory neuron survival, axonal growth and degeneration. These results will provide insights into how GFL-RET signaling can be targeted for therapy in sensory dysfunction in bladder diseases. GFLs are currently in clinical trials for neurodegenerative disease and chronic pain. This will allow accelerated clinical translation of the proposed studies.