Because the FOXO proteins regulate diverse biological processes

Because the FOXO proteins regulate diverse biological processes

from cell survival to metabolism to longevity (Accili and Arden, 2004 and Salih and Brunet, 2008), our findings raise the possibility that SnoN1 may play a role in these fundamental biological processes. Characterization of DCX as a direct target gene of the SnoN1-FOXO1 transcriptional repressor complex highlights the importance of regulation of DCX gene expression in the control learn more of neuronal positioning in brain development and disease. In light of the dramatic consequence of DCX loss-of-function mutations in mental retardation and epilepsy it will be important to determine whether deregulation of SnoN1 and FOXO1 function might contribute to the pathogenesis of neurodevelopmental disorders of cognition and epilepsy. Interestingly, forced expression of DCX in the early postnatal period reduces subcortical band heterotopia and seizure threshold in an animal model of human double cortex syndrome ( Manent et al., 2009). Therefore, identification of a SnoN1-FOXO1 repressor complex as a regulator of DCX gene expression raises the prospect that manipulation of SnoN1 or FOXO1 function may provide a potential avenue of treatment for developmental disorders of cognition and epilepsy. shRNA Depsipeptide in vitro plasmids were produced by cloning the

following oligonucleotides into pBS/U6 or pBS/U6-cmvGFP (targeted sequence is underlined): SnoN1 RNAi: 5′-AACCAGTAGAGAATTATACAGTTGTTAACTATAACTGTATAATTCTCTACTGGTTCTTTTTTG-3′ and SnoN2 RNAi: 5′-AAGGCAGAGACAAATTCATCAATCCGTTAACAATTGATGAATTTGTCTCTG CCTTCTTTTTTG-3′. The pan-SnoN RNAi, FOXO RNAi, and FOXO1-RES expression and plasmids have been described (Bernard, 2004, Daitoku et al., 2004, Lehtinen et al., 2006, Sarker et al., 2005 and Yuan et al., 2008). The RNAi-resistant rescue construct (SnoN2-RES) was generated by using QuikChange Site-Directed Mutagenesis (Stratagene) and verified by sequencing. The cDNAs encoding the mutants SnoN1 1-539, SnoN1 1-477, SnoN1 1-366, and SnoN2 1-493 were

generated by PCR, subcloned into pcDNA3 or pEGFP-C2 (Clontech), and verified by sequencing. Granule neurons were prepared from postnatal day 6 (P6) Long-Evans rat pups and transfected either 8 hr, 2 days, or 4 days in vitro after plating by using a modified calcium phosphate method as described (Konishi et al., 2004) with indicated plasmids together with either GFP, DsRed, or β-galactosidase expression plasmid to visualize transfected neurons. To rule out the possibility that the effects of RNAi or protein expression on morphology were due to any effect of these manipulations on cell survival, the anti-apoptotic protein Bcl-xL was coexpressed in all neuronal transfections except those in which survival was assessed. The expression of Bcl-xL has little or no effect on axon or dendrite morphology (Gaudillière et al., 2004 and Konishi et al., 2004).

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