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Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum
(2013)
This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.
The actin cytoskeleton is essential for many cellular functions, such as the regulation of cell morphology, cell migration and vesicle transport processes. The functional diversity of actin structures is reflected in a variety of distinct molecular mechanisms regulating the polymerization of actin filaments. The spontaneous polymerization of actin however is inhibited, by both the instability of small actin oligomers and by actin monomer binding proteins, which prevent the formation of such oligomers. Actin nucleation factors help to overcome this kinetic barrier of filament initiation and are essential for the generation of novel actin filaments at specified subcellular compartments. Spir proteins are the founding members of the novel class of WH2 domain containing actin nucleation factors. They initiate actin polymerization by binding of actin monomers to four WH2 domains in the central part of the protein. Despite their ability to nucleate actin polymerization in vitro by themselves, Spir proteins form a regulatory complex with the distinct actin nucleators of the formin subgroup of formins. Spir functions in the regulation of vesicular originated filamentous actin structures, vesicle transport processes and the assembly of the cleavage furrow during asymmetric meiotic cell divisions. The mammalian genome encodes two spir genes, spir-1 and spir-2. The corresponding proteins have an identical structural array and share a high degree of homology. In order to elucidate the Spir function in developing and adult mouse tissues, the yet unknown expression of the mouse spir-2 gene was addressed. Real-time PCR analysis revealed highest expression of spir-2 in oocytes, the brain, throughout the gastrointestinal tract, testis and kidney of adult mice. In situ hybridizations were performed to substantiate the cellular nature of spir gene expression. During embryogenesis in situ hybridizations show spir-2 to be expressed in the developing nervous system and intestine. In adult mouse tissues highest expression of spir-2 was detected in the epithelial cells of the digestive tract, in neuronal cells of the nervous system and in spermatocytes. In contrast to the more restricted expression of the mouse spir-1 gene, which is mainly found in the nervous system, oocytes and testis, the data presented here show a distinct and broader expression pattern of the spir-2 gene and by this support a more general cell biological function of the novel actin nucleators. In order to address the function of Spir proteins in the developing and adult nervous system, Spir-1 deficient mice were generated by a gene trap method. Spir-1 deficient mice are viable and provide a perfect tool to address the neurobiological function of the Spir-1 protein. Analyses of primary cortical neurons from Spir-1 deficient mice revealed a specific reduction of dendritic branchpoints and are the first description of a neuronal Spir-1 function. Further, a transgenic mouse line (thy1-GFP-M) was employed that expresses the green fluorescent protein (GFP) under the control of neuron specific elements from the thy1 promoter. GFP is thereby expressed in only a subset of neurons and labels the neurons in their entirety. Spir-1 deficient mice carrying the GFP transgene were generated and analyzed. It was found that Spir-1 deficient mice exhibit a reduced number of dendritic spines in the entorhinal cortex compared to wildtype littermates. All together this study gives novel information about the cell biological function of Spir and provides insights how cytoskeletal functions structure the mammalian neuronal network.