Role of NGF signaling in the bladder after spinal cord injury

 描述(申请人提供)：我们最近发表了NGF受体p75是治疗脊髓损伤的潜在靶点：口服LM11A-31后脊髓损伤(SCI)后的运动功能得到改善，LM11A-31是一种针对p75的小分子。通过观察病理性proNGF与p75结合的阻断，证实了LM11A-31有效的体内靶向p75，这表明该药物可用于p75与proNGF或其他前神经营养因子相互作用而产生类似病理效应的其他系统。我们已经发表了在中枢神经系统损伤后ProNGF水平迅速升高，并被释放到脑脊液中。与同时结合TrkA和p75的成熟神经生长因子不同，proNGF选择性地结合p75，从而激活p75‘S下游的细胞凋亡级联反应。神经生长因子与脊髓损伤后的膀胱功能障碍以及膀胱过度活动和间质性膀胱炎/痛性膀胱综合征有关。在此条件下和脊髓损伤后，尿中NGF水平显著升高，提示NGF可作为一般膀胱功能障碍的生物标志物，并可能作为改善膀胱功能的靶点。然而，尽管长期以来人们对此感兴趣，旨在中和NGF作用的研究却导致了好坏参半的结果。我们认为，在我们将NGF及其受体作为缓解膀胱功能障碍的治疗靶点之前，有必要更详细地了解NGF及其受体在膀胱生物学中所起的作用。我们发现在脊髓损伤后不久，proNGF和proBDNF被释放到尿液中，而成熟的NGF在几天后被释放。这些前神经营养因子似乎诱导负责建立通透性屏障的伞状细胞的凋亡，因为LM11A-31的应用导致脊髓损伤后伞状细胞的滞留。因此，我们推测在脊髓损伤后伞状细胞中表达的p75在伞状细胞的凋亡中起作用。除了伞状细胞外，我们还发现在脊髓损伤后尿路上皮增生期，p75也表达于增殖的前体细胞。LM11A-31的应用减弱了这种增殖反应，提示前神经营养因子和p75在调节脊髓损伤诱导的增殖中起额外的作用。因此，我们推测p75在脊髓损伤后尿路上皮再生中起双重作用，(1)通过诱导脊髓损伤后不久伞状细胞的凋亡，以及(2)通过调节 祖细胞的增殖，从而补充丢失的伞状细胞。在这一假设下，我们提议确定p75是否诱导脊髓损伤后尿路上皮伞状细胞的死亡(目标1)，这将由有条件的p75小鼠来解决，其中p75在伞形细胞中被选择性地缺失；确定p75在脊髓损伤后尿路上皮细胞增殖中的作用(目标2)，这将通过分析脊髓损伤后小鼠的尿路上皮祖细胞中的p75缺失来解决；以及确定LM11A-31是否可以改善脊髓损伤后的膀胱功能(目标3)，将使用新的光学标测方法、单单位传入神经记录、通透性测量和膀胱测量(CMG)并同时进行尿括约肌肌电图(EMG)来解决。我们相信，这项研究的结果将改变我们目前对神经营养素在膀胱中作用的理解，并激励现有的针对神经营养素在各种泌尿系统疾病中改善膀胱功能的努力。

 DESCRIPTION (provided by applicant): We have recently published that a NGF receptor, p75, is a potential therapeutic target for the spinal cord injured: motor function after spinal cor injury (SCI) improved after oral delivery of LM11A-31, a small molecule that was designed to target p75. Effective in vivo targeting of p75 by LM11A-31 was confirmed by observation of a block in binding of a pathological proNGF to p75, suggesting that the drug can be used in other systems where p75 exerts similar pathological effects upon interaction with proNGF or other proneurotrophins. We have published that ProNGF levels increase rapidly after injury to the CNS, being released into the cerebrospinal fluid. Unlike mature NGF that binds both TrkA and p75, proNGF binds p75 selectively, thereby activating p75's downstream apoptotic cascade. NGF has been implicated in bladder dysfunction after SCI as well as in overactive bladder and interstitial cystitis/painful bladder syndromes. Under these conditions and after SCI, a significan increase in NGF levels is observed in the urine, suggesting that NGF can be a biomarker for general bladder dysfunction and may be targeted to improve bladder function. Despite the long-standing interest, however, studies that aimed at neutralizing NGF action has led to mixed results. We believe a more detailed understanding of the roles that NGF and its receptors play in bladder biology will be necessary before we focus on NGF and related neurotrophins as therapeutic target for alleviating bladder dysfunction. We have found that proNGF and proBDNF are released into the urine shortly after SCI, while mature NGF is released days later. These proneurotrophins appear to induce apoptosis of umbrella cells that are responsible for establishing the permeability barrier, as LM11A-31 administration led to retention of umbrella cells after SCI. We thus hypothesize that p75 that is expressed in umbrella cells play a role in apoptosis of umbrella cells after SCI. In addition to umbrella cells, we found that p75 is also expressed in proliferating progenitors of the urothelium during the period of urothelial hyperplasia after SCI. LM11A-31 administration attenuated this hyperplasic response, suggesting that proneurotrophins and p75 play an additional role in regulating SCI-induced hyperplasia. We thus hypothesize that p75 plays dual roles in the regeneration of the urothelium after SCI, (1) by inducing apoptosis of the umbrella cells shortly after SCI, and (2) by regulating proliferation of the progenitor cells, thereby replenishing the lost umbrella cells. Under this hypothesis, we propose to determine whether p75 induces death of umbrella cells in the urothelium after SCI (Aim 1), which will be addressed by conditional p75 mice, in which p75 is selectively deleted in umbrella cells; to determine the role of p75 in proliferation of the urothelium after SCI (Aim 2), which will be addressed by analyzing mice after SCI in which p75 is deleted in urothelial progenitors; and to determine whether LM11A-31 improves bladder function after SCI (Aim 3), which will be addressed using novel optical mapping approaches, single-unit afferent nerve recording, permeability measurements and cystometrograms (CMG) with simultaneous urethral sphincter electromyograms (EMG). We believe the outcome of this study will change our current understanding on neurotrophin action in the bladder and energize the existing effort to target neurotrophin in improving bladder function in various urological conditions.