Impaired neuronal maturation of hippocampal neural progenitor cells in mice lacking CRAF.

Verena Pfeiffer,Guadelupe Camarero,Rudolf Götz,Ulf Rüdiger Rapp,Robert Blum,Helmut Heinsen,Stephen D Ginsberg

doi:10.1371/journal.pone.0192067

Abstract

RAF kinases are major constituents of the mitogen activated signaling pathway, regulating cell proliferation, differentiation and cell survival of many cell types, including neurons. In mammals, the family of RAF proteins consists of three members, ARAF, BRAF, and CRAF. Ablation of CRAF kinase in inbred mouse strains causes major developmental defects during fetal growth and embryonic or perinatal lethality. Heterozygous germline mutations in CRAF result in Noonan syndrome, which is characterized by neurocognitive impairment that may involve hippocampal physiology. The role of CRAF signaling during hippocampal development and generation of new postnatal hippocampal granule neurons has not been examined and may provide novel insight into the cause of hippocampal dysfunction in Noonan syndrome. In this study, by crossing CRAF-deficiency to CD-1 outbred mice, a CRAF mouse model was established which enabled us to investigate the interplay of neural progenitor proliferation and postmitotic differentiation during adult neurogenesis in the hippocampus. Albeit the general morphology of the hippocampus was unchanged, CRAF-deficient mice displayed smaller granule cell layer (GCL) volume at postnatal day 30 (P30). In CRAF-deficient mice a substantial number of abnormal, chromophilic, fast dividing cells were found in the subgranular zone (SGZ) and hilus of the dentate gyrus (DG), indicating that CRAF signaling contributes to hippocampal neural progenitor proliferation. CRAF-deficient neural progenitor cells showed an increased cell death rate and reduced neuronal maturation. These results indicate that CRAF function affects postmitotic neural cell differentiation and points to a critical role of CRAF-dependent growth factor signaling pathway in the postmitotic development of adult-born neurons.

Highlights

Accelerated fibrosarcoma (RAF) kinases are a central part of the classical growth factor signaling cascade, the RAS-Rapidly accelerated fibrosarcoma (RAF)-MEK-ERK signaling pathway
We have previously reported that mice with a conditional elimination of BRAF showed a reduced ability of hippocampal progenitor cells to differentiate into granule cell neurons [28]
Western analysis using an antibody targeted against the N-terminus of CRAF confirmed expression of CRAF protein with a relative molecular weight of 74 kDa in the cerebellum, hippocampus, cortex and olfactory bulb of postnatal CRAF+/+ control (CRAF ct) mice (n>10, P10-postnatal day 30 (P30))

Summary

Introduction

Accelerated fibrosarcoma (RAF) kinases are a central part of the classical growth factor signaling cascade, the RAS-RAF-MEK-ERK signaling pathway This signaling cascade regulates proliferation, differentiation, survival and apoptosis of many cell types [1]. We show that postnatal mice lacking CRAF kinase exhibit strongly increased cell proliferation, cell-cycle abnormalities, failure of post-mitotic differentiation and enhanced apoptotic cell death in the dentate gyrus of the hippocampus. This shows that CRAF signaling has a specific impact on cell cycle regulation and differentiation in the early phase of postnatal hippocampal neurogenesis

Methods

Results

Conclusion