CNP-induced cGMP signalling in neural differentiation and functional integration

由分泌的C型利钠肽(CNP)与其受体鸟苷酸环化酶B(GC-B)结合而触发的cGMP信号通路与多种生理功能有关。我们自己的研究表明，胚胎背根神经节神经元在脊髓背根进入区的轴突分支严重依赖于cGMP信号通路，其中包括CNP、GC-B和cGMP依赖的蛋白激酶Iα(cGKIα)。在第一个资助期，我们建立了用于表达图谱的GC-B-LacZ-Report小鼠系和GC-B-CreERT2小鼠系，结合适当的条件报告器，能够对单个GC-B表达的神经元进行可视化。我们可以证明，与DRG神经元类似，CNP/GC-B/cGKIα信号通路也调节胚胎后脑颅感觉神经节神经元的轴突分叉。利用各自的LacZ报告小鼠系对CNP和GC-B在中枢神经系统中的表达模式进行了分析，发现该配体及其受体在海马区有一个有趣的分布，海马区是一个参与记忆形成的大脑结构，对成人神经发生具有重要作用。我们确定了在海马区表达CNP和GC-B的神经元亚群以及这些表达模式的发育时间进程。因此，我们观察到海马结构中表达GC-B的齿状颗粒细胞(DGC)的数量随着年龄的增长而减少。将BrdU应用于GC-B-LacZ报告基因小鼠后的免疫细胞化学研究表明，GC-B阳性的DGCs在成年小鼠的大脑中得到补充。此外，我们还建立了一种用于GC-B阳性DGC的荧光标记的遗传学方法，目的是表征它们的电生理特性和连接。初步结果提示，GC-B在DGC兴奋性的控制中起作用。这些研究将继续进行，并辅之以行为测试。为此，我们建立了一个条件GC-B小鼠模型，允许特定组织的GC-B失活，这也将有助于与该网络中的其他小组合作，这些小组将研究GC-B在听觉系统和疼痛处理中的功能。将对GC-B杂合子与GC-B KO小鼠的基因标记神经元进行表达谱分析，以确定GC-B阳性神经元中cGMP信号的进一步分子成分和cGMP依赖的转录程序。这些研究将进一步阐明GC-B在神经元生长和突触整合过程中的作用，并将增进我们对cGMP信号受损的神经学后果的理解。

A cGMP signalling pathway triggered by binding of the secreted factor C-type natriuretic peptide (CNP) to its receptor guanylyl cyclase B (GC-B) has been linked to a remarkable variety of physiological functions. Our own investigations demonstrated that the bifurcation of axons from neurons of embryonic dorsaI root ganglia at the dorsal root entry zone of the spinal cord critically depends on a cGMP signalling pathway comprising CNP, GC-B and cGMP-dependent protein kinase I alpha (cGKI alpha). In the first funding period, we established a GC-B-LacZ-reporter mouse line for expression mapping and a GC-B-CreERT2 mouse line that in combination with an appropriate conditional reporter enables the visualization of individual GC-B-expressing neurons. We could demonstrate that, similar to DRG neurons, the CNP/GC-B/cGKI alpha signalling pathway also regulates axon bifurcation of neurons from cranial sensory ganglia at the embryonic hindbrain. The analysis of the expression patterns of CNP and GC-B in the central nervous system utilizing the respective LacZ-reporter mouse lines revealed an interesting distribution of the ligand and its receptor in the hippocampus, a brain structure involved in memory formation and prominent for adult neurogenesis. We determined the neuronal subpopulations that express CNP and GC-B in the hippocampus and the developmental time course of those expression patterns. Thereby, we observed an age-related decrease in the number of GC-B-expressing dentate granule cells (DGCs) in the hippocampal formation. Immunocytochemical studies following the application of BrdU to GC-B-LacZ-reporter mice demonstrated a replenishment of GC-B-positive DGCs in the adult brain. Moreover, we have established a genetic approach for fluorescent labelling of GC-B-positive DGCs with the aim to characterize their electrophysiological properties and connections. Preliminary results suggest a role of GC-B in the control of DGC excitability. These studies will be continued and complemented with behavioural tests. For this purpose, we have generated a conditional GC-B mouse model allowing tissue-specific inactivation of GC-B which will also be instrumental in collaborations with other groups within this network who will study GC-B function in the auditory system and in pain processing. Expression profiling of genetically-labelled neurons from GC-B heterozygous vs GC-B KO mice will be performed to identify further molecular components of cGMP signalling in GC-B-positive neurons and cGMP-dependent transcriptional programmes. These studies will further clarify the role of GC-B in the process of neuronal growth and synaptic integration and will improve our understanding of the neurological consequences of impaired cGMP signalling.