A FOXO–Pak1 transcriptional pathway controls neuronal polarity

Abstract

Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. Here, we report that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo-dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. We also identify the gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development.[Keywords: FOXO; neuronal polarity; Pak1; transcription; axons; dendrites] Supplemental material is available at http://www.genesdev.org.

FoxO6 regulates memory consolidation and synaptic function

“…In all transfections, the anti-apoptotic protein Bcl-xl was coexpressed to rule out potential effects of cell survival on neuronal morphology. The expression of Bcl-xl had no effect on the morphology of neurons (Gaudilliere et al 2004;de la Torre-Ubieta et al 2010). Individual images of GFP-labeled DIV5 primary hippocampal neurons prepared from FoxO6 mutant or wild-type mice were captured in a blinded manner on a Nikon eclipse TE2000 epifluorescence microscope using a digital CCD camera (Diagnostic Instruments).…”

“…The next day and every third day, one-half of the volume was replaced with Neurobasal/B27 medium without serum. For neuronal morphology analyses, neurons were transfected at 1 d in vitro (DIV1) with a modified calcium phosphate protocol (Konishi et al 2002;de la Torre-Ubieta et al 2010), fixed at DIV5, and subjected to immunocytochemistry with the GFP antibody together with an antibody to the dendritic marker MAP2 (Sigma) and the axonal marker Tau-1 (Chemicon). In all transfections, the anti-apoptotic protein Bcl-xl was coexpressed to rule out potential effects of cell survival on neuronal morphology.…”

“…Cerebellar granule neurons (CGNs) were cultured and transfected 2 d after plating using a modified calcium phosphate method as described (Shalizi et al 2006;de la Torre-Ubieta et al 2010), with 500 ng of the p3xFoxO6 construct or the empty pGL3-basic luciferase construct and 50 ng of a renilla luciferase reporter construct (pRL-TK). To test the FoxO6 dependency of the p3xFoxO6 construct, 500 ng of wild-type FoxO6-GFP (FoxO6 WT-GFP) in the pEGFP-N1 vector or the pEGFP-N1 vector alone was also transfected.…”

“…During development, the FoxO family is required to establish neuronal polarity, and the ectopic expression of FoxO6 can rescue the neuronal polarity defects due to deficiency of the FoxO family (de la Torre-Ubieta et al 2010). In adults, FoxO family members play an important role in neural stem cell self-renewal and fate Renault et al 2009) and in neuronal apoptosis following epileptic seizures (Shinoda et al 2004).…”

FoxO6 affects Plxna4-mediated neuronal migration during mouse cortical development

“…Furthermore, it was shown, both in vivo and in vitro, that the single-FoxO3-deficient animal results in a similar decline in NSC number (10). FoxO6 was shown to be required for the regulation, in the hippocampus, of a set of genes involved in synaptic function (11) and is essential in Pak1-mediated cellular polarity in the cerebellum (12), demonstrating the importance of this forkhead member in the mammalian brain.…”