Physiological role of sensory axon bifurcation in DRG neurons mediated by Npr2-dependent cGMP signaling

Field: Neuroscience (Life Sciences)

Studies on afferent axon projections from sensory dorsal root ganglia (DRG) neurons into the spinal cord have demonstrated that the growth cone splitting (bifurcation) of these axons at the dorsal root entry zone into two daughter branches, which elongate either in caudal or rostral direction, is controlled by a Npr2-mediated cGMP signaling cascade. The aim of the thesis was twofold with respect to this bifurcation-inducing cGMP signaling cascade: (1) the generation of a floxed Npr2 mouse model for behavioral studies in order to gain first in vivo insights into the physiological function of axon bifurcation at the dorsal root entry zone, and (2) the investigation of mechanisms that regulate Npr2 activity as well as the search for potential protein interactions of components of the cGMP signaling cascade. (1) The generation of a conditional Npr2 knockout mouse was necessary due to the fact that Npr2-mediated cGMP signaling also contributes to the development and function of other tissues such as bone by endochondral ossification and the meiotic arrest of oocytes. Therefore, the loss of Npr2 function had to be confined to DRG neurons in order to allow interpretable behavioral studies and to clarify whether the transmission of sensory stimuli by afferent axons is hampered by impaired axonal branching in the spinal cord. The Cre-loxP recombination system was used as a genetic tool and a transgenic mouse model was generated containing two loxP target sites flanking exon 17 and exon 18 of the Npr2 gene. Whereas a first attempt to generate a floxed Npr2 mouse resulted in a hypomorphic phenotype due to a splice site mutation, the second attempt succeeded with the creation of the intended conditional Npr2 mouse mutant. Using the Wnt1-Cre mouse as a driver for Cre expression, experimental animals were generated which displayed undisturbed bone development in combination with impaired axon bifurcation. First behavioral studies (Hargreaves test) have revealed that the acute pain perception is reduced in these animals due to the lack of axon bifurcation. In general, the floxed Npr2 mouse provides a valuable tool for the investigation of Npr2-related phenotypes and therefore should be instrumental in studies of bone development, oogenesis or other Npr2-mediated physiological processes. (2) In order to analyze the regulation of Npr2 activity, site-directed mutagenesis of Npr2 followed by subsequent determination of the receptor activity revealed that cGMP production by the guanylyl cyclase domain of the receptor depends on the phosphorylation of five phosphorylation sites (Ser-513, Thr-516, Ser-518, Ser-523 and Ser-526) within the kinase homology domain of Npr2. Furthermore, to ascertain whether the cGMP signaling pathway is engaged in cross-talk with other proteins or signaling pathways, the proximity-dependent biotin identification (BioID) labeling assay based on the promiscuous biotin ligase BirA* was applied to screen for potential interaction partners of Npr2 and cGKIα in cell lines. Thus, a number of Npr2-specifically and cGKIα- specifically purified proteins were identified by mass spectrometry. The in vitro data of the biotin labeling assay provided first information about new potential interactors of the cGMP signaling cascade. However, it remains for future studies to experimentally confirm the actual link to cGMP signaling and the participation in axon bifurcation.