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Therapeutic Targets for Neurological Diseases 2015; 2: e500. doi: 10.14800/ttnd.500; © 2015 by Tomoka Wachi, et al. http://www.smartscitech.com/index.php/ttnd

RESEARCH HIGHLIGHT

Novel Functions of 14-3-3 Proteins in Neurogenesis and Neuronal Differentiation In Vivo Tomoka Wachi1, 2, Kazuhito Toyo-oka1, 2 1

Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco School of Medicine, San Francisco, California 94143, USA 2 Departments of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA Correspondence: Kazuhito Toyo-oka E-mail: [email protected] Received: December 30, 2014 Published online: February 1, 2015

During brain development, there are many essential steps for the proper formation of a functional brain, including neurogenesis and neuronal migration. Neuronal progenitor cells (NPCs) proliferate and symmetrically divide to expand their pools in the developing cerebral cortex. NPCs also asymmetrically divide to produce one neuronal progenitor cell and one neuron. These newly-born post-mitotic neurons radially migrate and reach the final destination in the cortical plate (CP) to finally form the six layers of the cortex. We previously found that the protein 14-3-3epsilon is important for neuronal migration and a responsible gene for the development of Miller-Dieker syndrome (MDS). Although fortunately we found the neuronal migration defects in 14-3-3epsilon knockout mice, there may be functional redundancies because there are seven isoforms in the family of 14-3-3 proteins, with high homology among them. Therefore, we produced the 14-3-3epsilon and 14-3-3zeta double knockout mice (14-3-3 dKO mice) and found that the dKO mice showed spontaneous epilepsy. We also found novel in vivo functions of the 14-3-3epsilon and 14-3-3zeta proteins in neurogenesis of radial glial cells (RGCs) as well as intermediate progenitor cells (IPCs) and in neuronal differentiation. In addition to the pathological defects seen in the dKO mice, we identified the molecular mechanisms involved in the neuronal differentiation defects and showed that the binding of 14-3-3 proteins to δ-catenin proteins regulated actin dynamics. Thus, 14-3-3 proteins are important for the key steps of brain development, including neurogenesis, neuronal migration and neuronal differentiation as well as their involvement in various brain morphological disorders, such as epilepsy. Keywords: Neurogenesis; Neuronal Differentiation; 14-3-3, Epilepsy; Cerebral Cortex; Neuronal Migration; Lissencephaly; Miller-Dieker Syndrome; Rho Family of GTPases; Actin To cite this article: Tomoka Wachi, et al. Novel Functions of 14-3-3 Proteins in Neurogenesis and Neuronal Differentiation In Vivo. Ther Targets Neurol Dis 2015; 1: e500. doi: 10.14800/ttnd.500.

Neuronal migration is one of the key developmental steps in brain development. If neuronal migration is disrupted, it results in severe brain morphological disorders such as lissencephaly and mental disorders such as schizophrenia [1]. Many genes responsible for neuronal migration have been identified, including, DCX, RELN, PAFAH1B1 and others. Doublecortin, DCX, was identified as a responsible gene for subcortical band heterotopia (SBH) in which an extra layer

of neurons is detected under the normal gray matter of the cortex [2, 3]. Gleeson et al revealed that DCX is a microtubule binding protein and important for neuronal migration in the cerebral cortex [4]. However, the Dcx knockout mice did not show neuronal migration defects because of the functional redundancy by Doublecortin-like kinase (Dclk). However, the Dcx and Dclk double knockout mice showed neuronal migration defects in the cortex and abnormal hippocampal

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Therapeutic Targets for Neurological Diseases 2015; 2: e500. doi: 10.14800/ttnd.500; © 2015 by Tomoka Wachi, et al. http://www.smartscitech.com/index.php/ttnd

development [5, 6]. The Reelin (Reln) gene was originally cloned as a gene responsible for the phenotypes seen in the reeler mice, including inverted layering in the cerebral cortex and morphological abnormalities in the hippocampus [7, 8]. Reelin binds to its receptor, very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) and activate the downstream signaling pathway including Dab1, Crk and CrkL [9-11]. The knockout mice of these Reelin-signaling proteins showed neuronal migration defects [10, 11]. PAFAH1B1, coding the protein Lis1, is a gene responsible for isolated lissencephaly sequence (ILS), and its point mutation or deletion result in ILS [12, 13]. The PAFAH1B1 knockout mice showed neuronal migration defects [14]. ILS is characterized by smooth brain surface (lissencephaly) without any other obvious defects when compared to Miller-Dieker syndrome (MDS) which is characterized by a more severe lissencephaly phenotype as well as additional defects, such as craniofacial abnormalities [15, 16] . Patients with ILS show a point mutation or deletion of the PAFAH1B1 gene. In contrast, patients with MDS have a longer chromosomal deletion at the chromosome 17p13.3. We proved that an approximately 1M base pair region in the 17p13.3 is critical for MDS [17]. 26 genes are localized in this critical region from the PAFAH1B1 gene to the Ywhae gene, coding the protein 14-3-3epsilon. 14-3-3 proteins are evolutionally conserved from plants to humans with very high homology [18, 19]. There are two isoforms in flies and worms, 13-15 in plants and 7 in mammals. There is high homology among isoforms, suggesting functional redundancy. By creating the Ywhae deficient mice, we previously found that 14-3-3 proteins are important for neuronal migration in the cerebral cortex as well as the hippocampus and proved the molecular mechanism that 14-3-3 binds to Cdk5-phosphorylated Ndel1 proteins, known Lis1-binding protein, and protect them from their dephosphorylation by Protein Phosphatase 2A (PP2A) [20] . In addition, the double heterozygotes of the PAFAH1B1 and the Ywhae genes showed more severe neuronal migration defects in the cortex and the hippocampus compared to each single knockout mouse [20]. Also, we found the novel functions of another 14-3-3 isoform, 14-3-3zeta protein, coded by the Ywhaz gene that is important for neuronal migration of pyramidal cells in the hippocampus by producing the Ywhag knockout mice [21]. Although the Ywhaz knockout mice did not show any defects in the cortex, there might be functional redundancy between the 14-3-3zeta and 14-3-3epsilon and/or other isoforms. To prove if there is functional redundancy between the 14-3-3zeta and 14-3-3epsilon and/or other isoforms, we

recently produced and analyzed the 14-3-3zeta and 14-3-epsilon double knockout mice (14-3-3 dKO mice) and identified novel functions in neurogenesis and neuronal differentiation [22]. More importantly, we found that the 14-3-3 dKO mice showed spontaneous seizures associated severe neuronal migration defects in the cerebral cortex. In the 14-3-3 dKO mice, neurogenesis was accelerated, and more radial glial cells (RGCs) and intermediate progenitor cells (IPCs) were produced throughout the cortex, suggesting an increased number of the post-mitotic neurons. In fact, in the 14-3-3 dKO mice RGCs asymmetrically divided more often to produce neurons, and IPCs more often symmetrically divided to produce two neurons instead of two IPCs. These findings were confirmed by in vitro experiments in which freshly-prepared RGCs and IPCs from the control mice and the 14-3-3 dKO mice were used. To confirm that the number of neurons is increased in the 14-3-3 dKO mice, we utilized in vitro neuronal differentiation assay using neuronal progenitor cells and proved that the ablation of the 14-3-3 proteins in neuronal progenitor cells resulted in increased neuronal differentiation in comparison to the control cells. These data indicate that the proteins14-3-3epsilon and 14-3-3zeta play important roles in neurogenesis and neuronal differentiation. Next, we explored the molecular mechanism of 14-3-3-mediated neuronal differentiation. We found an increased expression of δ-catenin and the decreased expression of β-catenin and αN-catenin in neuronal progenitor cells obtained from the 14-3-3 dKO mice. By using a variety of molecular techniques, we identified that 14-3-3 proteins bound to δ-catenin and its binding facilitated the degradation of δ-catenin proteins by the ubiquitin-mediated proteolysis system. We also showed that 14-3-3 proteins bound to the PKA-phosphorylated 1094 serine of δ-catenin proteins. Since the δ-catenin, β-catenin and αN-catenin proteins control actin dynamics through the Rho family of GTPases/Limk1/cofilin signaling cascade, we tested if the activity of these proteins were altered in the dKO mice. We found that the activity of the all of the Rho family of GTPases, RhoA, cdc42 and Rac1 was decreased in neuronal progenitor cells from the 14-3-3 dKO mice and the phosphorylation of Limk1 and cofilin was also decreased. Since unphosphorylated form of cofilin is active and can sever filamentous actin (F-actin), we confirmed the decreased staining of F-actin in the cortex of the 14-3-3 dKO mice. Finally, to test if δ-catenin, β-catenin and αN-catenin are involved in the neuronal differentiation defects seen in the 14-3-3 dKO mice, we performed several rescue experiments. As we expected, the knockdown of δ-catenin or overexpression of β-catenin and αN-catenin normalized the neuronal differentiation defects. Thus, we elucidated the detailed molecular mechanism of the stabilization of the

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Therapeutic Targets for Neurological Diseases 2015; 2: e500. doi: 10.14800/ttnd.500; © 2015 by Tomoka Wachi, et al. http://www.smartscitech.com/index.php/ttnd

δ-catenin proteins by 14-3-3 proteins and the new molecular mechanisms of neuronal differentiation by the signaling pathway involving 14-3-3, catenins, Rho family of GTPases, Limk1, cofilin and actin.

Research on Schizophrenia and Depression Young Investigator Award. We thank Brett Cornell for his suggestions and comments. References

Finally, we tested if the 14-3-3 dKO mice showed more severe neuronal migration defects than each single KO mice. As we expected, the 14-3-3 dKO mice showed more severe neuronal migration defects in the cortex. Interestingly, these neuronal migration defects were not able to be normalized by the knockdown of δ-catenin proteins. However, these defects were rescued by the active form of Ndel1 proteins in which three amino acids of the Cdk5 phosphorylation sites were replaced by glutamic acid to mimic the phosphorylation status. These data indicate the two different phenomena regulated by 14-3-3 proteins, neuronal differentiation and neuronal migration, are regulated by separate signaling pathways through the binding of the 14-3-3 proteins to the different targets. We clarified the novel functions of 14-3-3 proteins in neurogenesis and neuronal differentiation in addition to neuronal migration and new molecular mechanisms of neuronal differentiation in vivo. Taken together of the fact that the 14-3-3 dKO mice showed severe spontaneous seizure, these defects including neurogenesis, neuronal differentiation and neuronal migration are a part of etiology of seizure. Although to date there is no evidence that 14-3-3 is a responsible gene for epilepsy in humans, an association study should be done in the future as our research strongly suggests that the 14-3-3 genes are responsible for epilepsy. By doing this, the 14-3-3 genes may be used as biomarkers for screening for the development of epilepsy. Also, the 14-3-3 proteins are ubiquitously expressed in almost all tissues, suggesting that they are not potential pharmacological targets for lissencephaly, epilepsy and schizophrenia. However, they still may become a potential target. Although they are ubiquitously expressed, the administration of isoform-specific inhibitors of 14-3-3 proteins will be functional only in specific organs and cell types in a spatiotemporal manner because there are functional redundancies among the 14-3-3 isoforms. In fact, skin abnormalities were shown only in the knockout mice of Ywhas, coding the 14-3-3sigma [23, 24]. Also, although 14-3-3 isoforms share many binding partners, some proteins bind to the 14-3-3 proteins in an isoform specific manner [25]. Thus, it would be possible to use an isoform specific inhibitor as a potential medication of a variety of diseases, such as epilepsy, if the specific inhibitors are developed. Acknowledgements

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This study was supported by the National Alliance for

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