cGMP signalling in pain processing and regeneration after peripheral nerve injury

疼痛刺激是由位于背根神经节和三叉神经节的感觉神经元感知的。我们和其他实验室先前的研究表明，感觉神经元的疼痛加工与cGMP的产生、cGMP依赖性蛋白激酶I （cGKI）的激活和富含半胱氨酸的蛋白4 （CRP4，以前称为CRP2）的磷酸化有关。最近，我们观察到感觉神经元中CRP4的磷酸化是由c型利钠肽（CNP）引起的，而不是由心房利钠肽（ANP）或NO引起的，这表明CNP/GC-B/cGMP/cGKI/CRP4信号通路参与了该神经元群体的疼痛加工。我们将使用具有GC-B、cGKI或CRP4感觉神经元特异性缺失的条件敲除小鼠，研究该信号通路在坐骨神经挤压损伤引起的慢性疼痛中的体内功能，该模型涉及初级传入神经元的退化和再生。特别是，我们将描述CNP-和gc - b阳性细胞群在伤害系统中的定位，CNP释放的动力学，疼痛刺激后背根神经节中cGMP产生的时空动力学，以及该信号通路的下游机制。在阐明了CNP表达细胞在伤害感知系统中的定位后，我们还计划在这种情况下表征组织特异性CNP敲除小鼠。与上述信号通路相反，NO-GC并不在感觉神经元中表达，而是在覆盖感觉神经元表面的卫星胶质细胞中表达。一些研究表明，周围神经损伤后，卫星胶质细胞增殖并参与疼痛加工。考虑到周围神经损伤后感觉神经元中NO合成酶的表达明显上调，很可能NO作为旁分泌信使增加卫星胶质细胞cGMP的产生。这种神经-胶质串扰可能有助于卫星细胞的增殖及其保护作用，防止周围神经损伤后感觉神经元的丧失。然而，卫星神经胶质细胞和其他伤害感觉系统细胞中的NO-GC亚型对周围神经损伤后疼痛加工和再生的相对贡献尚不清楚。因此，我们将利用缺乏NO-GC亚型的条件敲除小鼠来研究NO-GC信号在坐骨神经挤压损伤引起的慢性疼痛中的体内功能。总之，该项目将提供更多关于不同cGMP信号通路如何促进神经损伤后疼痛加工和再生的信息。长期目标是发现靶向cGMP信号是否可以作为治疗慢性疼痛的新方法。

Painful stimuli are detected by sensory neurons whose cell somata are located in dorsal root ganglia and trigeminal ganglia. Previous studies in our and other labs revealed that pain processing in sensory neurons is associated with cGMP production, activation of cGMP-dependent protein kinase I (cGKI) and phosphorylation of cysteine-rich protein 4 (CRP4; formerly named CRP2). Recently, we observed that CRP4 phosphorylation in sensory neurons is evoked by C-type natriuretic peptide (CNP), but not by atrial natriuretic peptide (ANP) or NO, suggesting that a CNP/GC-B/cGMP/cGKI/CRP4 signalling pathway contributes to pain processing in this neuronal population. Using conditional knockout mice with a sensory neuron-specific deletion of GC-B, cGKI or CRP4, we will investigate the in vivo function of this signalling pathway during chronic pain induced by sciatic nerve crush injury, a model that involves degeneration and regeneration of primary afferent neurons. In particular, we will characterize the localization of CNP- and GC-B-positive cell populations in the nociceptive system, the kinetics of CNP release, the spatiotemporal dynamics of cGMP production in dorsal root ganglia after painful stimulation, and downstream mechanisms of this signalling pathway. After elucidation of the localization of CNP-expressing cells in the nociceptive system, we also plan to characterize tissue-specific CNP knockout mice in this context. In contrast to the signalling pathway mentioned above, NO-GC is not expressed in sensory neurons but in satellite glial cells that cover the surface of sensory neurons. Several studies demonstrated that after peripheral nerve injury, satellite glial cells proliferate and contribute to pain processing. Considering that NO synthase expression is markedly upregulated in sensory neurons after peripheral nerve injury, it is very likely that NO acts as a paracrine messenger to increase cGMP production in satellite glial cells. This neuron-glia crosstalk might contribute to the proliferation of satellite cells and their protective role to prevent loss of sensory neurons after peripheral nerve injury. However, the relative contribution of NO-GC isoforms in satellite glial cells and other cells of the nociceptive system to pain processing and regeneration after peripheral nerve injury is poorly understood. We will therefore investigate the in vivo functions of NO-GC signalling during chronic pain induced by sciatic nerve crush injury using conditional knockout mice lacking NO-GC isoforms. Altogether, this project will provide more information on how different cGMP signalling pathways contribute to pain processing and regeneration after nerve injury. The long-term goal is to find out whether targeting cGMP signalling could serve as a new approach for treatment of chronic pain.