Secretin modulates the postnatal development of mouse cerebellar cortex via PKA- and ERK- dependent pathways

Abstract

The role of secretin (SCT) and secretin receptor (SCTR) in behavioral control has been extensively studied. Without an intact SCT/SCTR axis, multiple neurobehavioral functions including motor coordination, motor learning, social behavior and cognitive behavior are impaired in different mouse models. The cerebellum has been well-established as the control center for motor coordination and motor learning while there is growing acceptance for its importance in cognition. Notably, normal cerebellar function depends on well-orchestrated processes during morphogenesis and development. In particular, the cerebellum exhibits dramatic morphological change and considerable weight increase during the postnatal period due to Purkinje cell dendritogenesis and granular cell proliferation and migration. Of equal importance, apoptosis acts as part of a “quality-control” to help provide precise postnatal development. Nevertheless, it remains unclear whether the SCT/SCTR axis is involved in regulating postnatal morphogenesis and development of the cerebellum, which has been implicated in the pathogenesis of psychiatric disorders. In the present study, the potential role of SCT in cerebellar development was explored at multiple postnatal stages. Both SCT and SCTR were found to be constitutively expressed in the postnatal cerebellum with their expression levels significantly and consistently upregulated at the earliest postnatal stages. The expression of SCT was observed almost exclusively in Purkinje cells, whereas high expression levels of SCTR were shown in several cerebellar cell types, including Purkinje cells and granular cells. Using SCT knockout (Sct-/-) mice and their wild-type littermates (Sct+/+), it was demonstrated that SCT deprivation resulted in various abnormal developmental phenotypes, including reduced Purkinje cell density, underdeveloped Purkinje cell dendritic arbors, premature granular cell migration, and increased apoptotic activity. Furthermore, the signaling pathways utilized by SCT to regulate apoptosis were characterized. In Sct-/- mice, the phosphorylation levels of ERK1/2 and its downstream effector p90RSK were significantly decreased, whereas phosphorylated Akt remained unchanged. SCT deprivation further reduced the expression of CREB and its downstream survival proteins bcl-2 and bcl-xl. On ex vivo cultured para-sagittal cerebellar sections harvested from Sct-/- mice, SCT treatment significantly stimulated ERK1/2, p90RSK and CREB phosphorylation and bcl-2 and bcl-xl expression in a dose-dependent manner. In contrast, caspase-3 activation was negatively correlated with SCT dosage. The addition of PKA inhibitor H89, and/or ERK1/2 inhibitor U0126, or p90RSK inhibitor BI-D1870 all remarkably abolished the anti-apoptotic effect of SCT, which could only be fully abolished by co-treatment with H89 and U0126. Moreover, ERK1/2 and its downstream effector p90RSK were partially suppressed in the presence H89. In a similar manner, CREB phosphorylation and expressions of bcl-2 and bcl-xl were restored by SCT, and this recovery was partially suppressed by H89 or U0126 but was fully suppressed when both drugs were combined. Taken together, these results clearly indicate the convergence of PKA and ERK signaling pathways toward the CREB survival pathway and caspase-3 apoptotic pathway. In summary, the results of this study illustrate that SCT, as a previously unrecognized participant, is required for normal cerebellar postnatal development, emphasizing the necessary role of SCT in cerebellar-related functions.