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Keywords:

  • Neural stem cell;
  • Neural differentiation;
  • Proliferation;
  • Progenitor cells;
  • Transcription factors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Forkhead Box Family of Transcription Factors
  5. Fox Factors and Adult Neurogenesis
  6. Conclusion
  7. Author Contributions
  8. Disclosure of Potential Conflicts of Interest
  9. References

New cells are continuously generated from immature proliferating cells in the adult brain in two neurogenic niches known as the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus and the sub-ventricular zone (SVZ) of the lateral ventricles. However, the molecular mechanisms regulating their proliferation, differentiation, migration and functional integration of newborn neurons in pre-existing neural network remain largely unknown. Forkhead box (Fox) proteins belong to a large family of transcription factors implicated in a wide variety of biological processes. Recently, there has been accumulating evidence that several members of this family of proteins play important roles in adult neurogenesis. Here, we describe recent advances in our understanding of regulation provided by Fox factors in adult neurogenesis, and evaluate the potential role of Fox proteins as targets for therapeutic intervention in neurodegenerative diseases. Stem Cells 2014;32:1398–1407


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Forkhead Box Family of Transcription Factors
  5. Fox Factors and Adult Neurogenesis
  6. Conclusion
  7. Author Contributions
  8. Disclosure of Potential Conflicts of Interest
  9. References

For a century, the generation of functionally integrated neurons from stem/progenitor cells was only considered to occur in the embryonic brain of mammals. The absence of neurogenesis in the adult brain was challenged in the late 1960s by Altman and Das who reported the production of new cells in the adult dentate gyrus (DG) of the hippocampus using the 3H-thymidine incorporation technique [1]. The original dogma was definitely overturned 30 years later with the development of a bromodeoxyuridine incorporation labeling method coupled with immunohistochemistry demonstrating the occurrence of neurogenesis in the adult brain [2]. Adult neurogenesis is observed in all mammals examined so far, including humans [3]. In the healthy adult brain, neurogenesis takes place in three specific areas: the DG of the hippocampus, the subventricular zone of the lateral ventricle (SVZ), and the hypothalamus [4, 5]. This process encompasses the proliferation of resident neural stem/progenitor cells and their subsequent differentiation, migration, and functional integration into the pre-existing synaptic network. This review aims at discussing recent advances in the molecular control of adult neurogenesis, focussing more particularly on the role of Fox proteins.

Adult Neurogenesis in the DG

The hippocampus is composed of two main parts: the Ammon's horn (subfielded into cornu ammonis 1—CA1, CA2, and CA3) and the DG (Fig. 1A). The DG is subdivided into three areas: the hilus, the granular cell layer (GCL), and the subgranular cell zone (SGZ). The formation of new neurons in this adult neurogenic zone is now well defined. Indeed, in the SGZ, at the interface of the hilus and the DG, new neurons originate from neural stem cells (NSCs) also named type-1 progenitors or radial glia-like stem cells. These radial glia-like cells divide very slowly or are quiescent. They send a long apical extension into the molecular layer that contacts blood vessels through vascular end feet. These cells are characterized by the expression of glial fibrillary acidic protein (GFAP), an intermediate filament protein. Recently, a second class of type-1 cells, characterized by short and horizontal processes, has been identified [6]. These cells appear to divide more quickly than radial glial-like cells. The lineage relationship between radial and horizontal type-1 cells remains unclear. Type-1 cells divide asymmetrically and give rise to type-2 precursor cells, subdivided in type-2a cells expressing Achaete-scute homolog 1 protein (Ascl1, also named mammalian achaete scute homolog-1 [Mash1]), and more mature type-2b cells expressing T-box brain protein 2 (Tbr2) [7]. Type-2b cells actively divide, expand the lineage, and finally generate type-3 cells, which are the most neuronally committed precursors and possess restricted proliferative capacity as compared to type-2b cells [7]. Type-3 cells exit the cell cycle and migrate on a short distance into the GCL, where they give rise to immature postmitotic neurons expressing doublecortin. Around 50% of immature neurons produced will die, and only few newborn granule cells will be stably integrated into the synaptic network of the DG (Fig. 1B).

image

Figure 1. Schematic representation of adult rodent neurogenesis and the expression of Fox family genes involved in neurogenesis. (A): Illustration of a sagittal section through the mouse brain showing location of neurogenic zones: the SGZ of the DG in the hippocampus and the subventricular zone of the LV where neurons are produced and migrate to the olfactory bulb via the rostral migratory stream (RMS). (B): Schematic summary of the neuronal differentiation cascade in the adult DG. Each cell type is characterized by the expression of specific markers. (C): Schematic representation of “pinwheel structure” and progenitor cell types and neurons in the SVZ/RMS/olfactory system based on the expression of localization and expression of specific markers. Abbreviations: BLBP, brain lipid-binding protein; DCX, doublecortin; DG, dentate gyrus; Dlx2, distal-less homeobox2; GFAP, glial fibrillary acid protein; LV, lateral ventricles; Mash1, mammalian achaete scute homolog 1; NeuN, neuronal nuclei; Prox1, prospero homeobox 1; PSA-NCAM, polysialated neural cell adhesion molecule; SGZ, subgranular zone; Sox2, SRY-related HMG-box gene 2; Tbr2, T-box brain protein 2.

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Adult Neurogenesis in the SVZ

The SVZ is a region that lies immediately beneath the ependymal layer on the lateral wall of the lateral ventricles. In the SVZ, the formation of new neurons is well defined. Indeed, neurogenesis originates from NSCs and generates large numbers of olfactory bulb (OB) interneurons [8]. The SVZ is composed of four main cell types: NSCs (type-B1 and -B2 cells), TAPs (transit amplifying progenitors, type-C cells), neuroblasts (type-A cells), and a layer of ciliated ependymal cells (type-E cells) [9] (Fig. 1C).

Type-B1 and -B2 have ultrastructural characteristics of radial glia, with multiple processes, mainly composed of intermediate Filaments rich in GFAP that intercalate extensively between other cells. Contrarily to type-B1 cells, which are localized at the interface between type-C cells and the ependymal layer, type-B2 cells lie between the type-C cells and the striatum. Only type-B1 cells extend an apical ending that contact the ventricle and a long basal process ending on blood vessels [10]. Type-B1 cells divide slowly to generate by successive asymmetric or symmetric division type-C cells or TAPs. TAPs represent the most important pool of proliferating progenitors. These cells are larger and more spherical than type-B cells [11]. Type-C cells further differentiate into neuroblasts also named type-A cells. This ultimate precursor cell type forms chain and migrates over long distances from the SVZ via the rostral migratory stream (RMS) toward the OB through a glial tube formed by astrocytes. In the OB, neuroblasts migrate radially toward glomeruli where they differentiate into granule cells or periglomerular interneurons [12].

At the level of the ventricular lumen, the apical endings of type-B1 cells are surrounded by two types of ependymal cells (type-E1 and -E2) forming a specific planar organization: the “pinwheel structure” (Fig. 1C). Type-E cells have several motile cilia at their apical surface and have been subdivided recently into two categories: type-E1 cells with multiple long cilia, and type-E2 cells comprised less than 5% of cells on the ventricular surface with long cilia, and type-E2 cells comprised less than 5% of cells on the ventricular surface with only two cilia [13]. It is known that cilia of type-E1 cells control cerebrospinal flow (CSF) and generate gradients of chemorepellents that guide anterior neuroblasts migration from the adult SVZ toward the OB [10, 14]. The role of cilia on type-E2 cells remains more elusive. They may serve as mechanical and chemical sensors for CSF [13].

Forkhead Box Family of Transcription Factors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Forkhead Box Family of Transcription Factors
  5. Fox Factors and Adult Neurogenesis
  6. Conclusion
  7. Author Contributions
  8. Disclosure of Potential Conflicts of Interest
  9. References

Forkhead box (Fox) proteins belong to a large family of functionally diverse transcription factors implicated in a wide variety of biological processes including cell cycle regulation, DNA repair, apoptosis, aging, cancer, diabetes, infertility, and neurogenesis [15-17]. In 1989, the founding member of Fox gene family was discovered in Drosophila, which, when mutated, gives the drosophila a two spiked-head appearance and so-called forkhead (fkh) [18]. One year later, the identification of the rat gene HNF-3A (hepatocyte nuclear factor-3A) [19] enabled the detection of a conserved 110 amino acid DNA-binding domain between Drosophila fkh and rat HNF-3A. This discovery permits the characterization of a previously unknown family of transcription factors carrying the fkh domain (also referred to as the winged-helix domain because of butterfly-like winged structure adopted by the DNA-bound Fox proteins) [20].

In 2000, the International Nomenclature Committee defined the Fox family as proteins having sequence homology to the canonical winged-helix/fkh DNA-binding domain. To date, the nomenclature of Fox family is based on a phylogenetic classification of protein sequences from more than 10 species. This enables to delineate 19 subclasses of Fox proteins (A to S). The Fox family proteins are termed using the following convention: all capital letters for human (e.g., FOXO1); only the F is capitalized for mouse (e.g., Foxo1), and the first letter and subclass letter capitalized for other chordates (e.g., FoxO1) [21].

Structure of Fox Transcription Factor Family

The canonical fkh domain consists of three α-helices, three β-sheets, a hydrophobic core, and two loops, which interact with the phosphate backbone of the target DNA molecule [22]. Fox transcription factors must bind to DNA to either activate or repress target gene expression. They preferentially bind to DNA at the Fox-recognized element, with the seven-nucleotide core consensus sequence 5′-(G/A)(T/C)(A/C)AA(C/T)A-3′ [23]. This core sequence is necessary but not sufficient for high affinity binding, which also depends on flanking sequences on both side of the core. In contrast with the highly conserved fkh domain, other functional regions of Fox proteins, such as trans-activation and trans-repression domains, are largely nonconserved. Still, several conserved domains and motifs have been identified within specific subclasses (Fig. 2).

image

Figure 2. Schematic representation of post-translational modifications of Fox family genes involved in the adult neurogenesis. The principal functional domains of Fox factors are shown together with the sites of modification and their potential modifying enzymes. P = sites of phosphorylation and A = sites of acetylation; green circles indicate positive regulation whereas red circles indicate negative regulation (inhibition). Gray circles indicate that the mechanism behind the modification remains unknown. Abbreviations: AMPK, AMP-activated protein kinase; CBP, CREB-binding protein; CDK, cyclin-dependent kinase; CHK2, checkpoint kinase 2; CK, casein kinase; DYRK1A, dual specificity tyrosine-phosphorylation-regulated kinase 1A; ERK, extracellular signal-regulated kinase; FKH DBD, forkhead DNA binding domain; IkKβ, Ikappaβ kinase; MAPK, mitogen-activated protein kinase; MST, mammalian Ste20-like kinase; NES, nuclear export system; NLS, nuclear localization system; NRD, negative regulatory domain; PKA, protein kinase A; PKB, protein kinase B; PLK, polo-like kinase; TAD, transactivation domain.

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Regulation of Fox Family

The mechanisms underlying the binding of Fox proteins to forkhead response elements in promoter regions have not been completely defined [24, 25]. Fox transcription factors may interact with a variety of cofactors, in particular members of nuclear receptor families, homeodomain proteins, which could induce changes in their DNA-binding ability and therefore their capability to promote or repress target gene expression [26]. Several Fox proteins have a unique ability to interact with highly compacted chromatin, thereby facilitating DNA remodeling and permit the binding of other transcription factors. This is followed by the recruitment of transcriptional coactivators such as p300/CREB-binding protein (p300/CBP), cyclin-dependent kinases 1 or 2 (CDK1 or CDK2), or steroid receptor coactivator-1, which are required for the assembly of a transcriptionally active complex [27].

The subcellular localization of Fox proteins, their stability, and DNA binding activity are mainly regulated by phosphorylation, acetylation, and ubiquitylation. Nuclear Fox proteins act as transcriptional regulators whereas cytoplasmic Fox proteins are inactive and often subject to proteosomal degradation. We will further describe the regulation of Fox classes that play key roles in adult neurogenesis (Fig. 2).

Foxos factors can be directly activated or inhibited by several kinases. More specifically, the activation of the phosphoinositide-3-kinase (PI3K)-protein kinase B/Akt pathway leads to inhibitory phosphorylation of Foxos. Indeed, these phosphorylations result in impairment of DNA binding ability [28], export of Foxos factors from the nucleus to the cytoplasm, where they are ubiquitinated and degradated [28-31]. Besides PI3K/Akt, numerous other kinases, including CDKs [32], extracellular signal-regulated kinases [33], or IkB kinases [34], inhibit transcriptional activity of FoxOs by phosphorylating multiple residues. In contrast, other kinases such as the stress-activated c-Jun N-terminal kinase, the energy sensing AMP-activated protein kinase, or mammalian Ste20-like kinases can increase the nuclear transport of Foxos and activate their transcriptional function upon oxidative and nutrient stress stimuli [35]. Like Foxos, Foxg1 is phosphorylated at conserved serine and threonine residues by AKT, leading to its subsequent inactivation by export to the cytoplasm. On the contrary, casein kinase I phosphorylation of Ser19 promotes Foxg1 nuclear import [36].

In contrast to the Foxo family, Foxm1 transcriptional activity correlates with its increased phosphorylation. Foxm1 expression and transcriptional activity are cell cycle-dependent. They are initiated at the onset of S-phase (synthesis phase) and continue throughout G2-phase and mitosis. Phosphorylation of Foxm1 is initiated by Cyclin-CDK complexes in early G1 and continued during G2- and M-phases (Mitosis phase) of the cell cycle [27]. More particularly, the C-terminal transcriptional activation domain of Foxm1 contains a cyclin-binding motif (LXL motif), and both CDK1/cyclin B and CDK2/cyclin E associate with Foxm1 during G1 and G2 phases, respectively. CDK-dependent phosphorylation stimulates the Foxm1 transcriptional activity, which correlates with binding to p300/CBP transcriptional coactivator. The effects of CDK phosphorylation on Foxm1 can be counteracted by the B55α regulatory subunit of protein phosphatase 2A (PP2A/B55α) [37]. In addition to association with Cyclin-CDK complexes, Foxm1 also binds to the cell cycle-inhibitory pocket protein pRb (retinoblastoma protein) and to the CDK-activating phosphatase Cdc25B in G1 and in G1/S, respectively [38, 39]. These proteins are known to be important cell cycle regulators and to regulate FoxM1 transcriptional activity via their effect on CDK's activity. To facilitate G2/M transition, Foxm1 is also phosphorylated by the mitotic kinase Polo-like kinase 1 (PLK1), which in turn feedback positively to upregulate PLK1 [40]. Foxm1 transcriptional activity also requires the presence of appropriate mitogenic signals involving the Raf/MEK/MAPK signaling pathway [41]. MAPK signaling-mediated Foxm1 phosphorylation has been shown to stimulate Foxm1 nuclear translocation and therefore transcriptional activation. The Foxj1 protein has two consensus tyrosine kinase sites in the forkhead domain but the mechanism by which Foxj1 is phosphorylated remains largely unknown.

Similar to phosphorylation, a control of activity through the balance of acetylation and/or deacetylation of lysines has also been observed for several members of the Fox family [42]. Acetylation of Fox factors by p300/CBP promotes cytoplasmic translocation and degradation [43]. Conversely, deacetylation of Fox factors by sirtuins enhances the targeting and activation of specific genes [35]. Importantly, the interaction between Fox factors and p300/CBP allows the recruitment of a coactivator complex to the promoters of specific genes and initiates transactivation of these genes. In addition, a recent study has shown that Fox transcription factors can undergo arginine methylation, which inhibits their phosphorylation [44, 45]. The degradation of Fox proteins is controlled by polyubiquitination and monoubiquitination on lysine residues [35, 46].

Fox Factors and Adult Neurogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Forkhead Box Family of Transcription Factors
  5. Fox Factors and Adult Neurogenesis
  6. Conclusion
  7. Author Contributions
  8. Disclosure of Potential Conflicts of Interest
  9. References

Studies of Fox protein function in vivo using mouse models have revealed both specific and redundant roles of Fox factors in development and tissue homeostasis, including the adult brain (Table 1).

Table 1. Fox factors involved in adult neurogenesis
GeneMicePhenotypeReferences
  1. Summary of mouse lines carrying fox gene mutations and the corresponding impact on adult neurogenesis.

  2. Abbreviations: DG, dentate gyrus; NSC, neural stem cell; OB, olfactory bulb.

Foxg1Foxg1-/-Die around birth, with a severe reduction in the size of the cerebral hemispheres.[47]
 Foxg1+/- and Frizzled9-Foxg1f1/f1Increased neuronal death and reduced postnatal DG neurogenesis.[48-50]
  NSCs prematurely exit the cell cycle and differentiate into neurons. 
 Overexpression of Foxgl by lentiviral infectionEnhances neurogenesis and proliferation of NSCs.[51]
Foxj1Foxj1-/-Majority die before weaning.[52-55]
  Left/right asymmetry with loss of motile cilia and flagella. Ependymal cells fail to differentiate. 
  Defects of proliferation and differentiation, which induced severe OB dysgenesis. 
Foxo1Foxo1-/-Die around E11.[56]
 GFAPcreNo appreciable changes in apoptosis are observed both in NSCs and neurons.[57]
 Foxo1f1/f1  
Foxo3Foxo3-/-Number of NSCs is reduced. NSCs have decreased self-renewal and impaired ability to generate different neural lineages.[58, 59]
 CaMKII-tTA;Loss of neural progenitors and increased apoptosis.[60]
 Foxo3-CA  
Foxo6Foxo6-/-No role in adult neurogenesis[61]
Foxo1/3/4GFAPcre;Enlarged brain.[57]
 Foxo1/3/4f1/f1Deregulation of NSC proliferation followed by a decline in NSC pool and neurogenesis. 
Foxm1Foxm1-/-Die perinatally between E13.5 and E17.5 due to multiple abnormalities (mainly cardiovascular and hepatocytes defects). Impairment of self-renewal of embryonic NSCs.[62-66]
 Math1Cre or NestinCre; Foxm1 f1/f1Delay in brain development.[67]

Foxg1

Foxg1 (formerly BF-1) is the only member of the forkhead G family. It is expressed in rapidly proliferating cell populations including the developing telencephalon where it regulates the rate of neurogenesis by keeping cells in a proliferative state [68]. Indeed, the loss of Foxg1 results in premature cell cycle exit and in differentiation of neocortical neural progenitors. Contrary to its role in regulating progenitor cell differentiation, the action of Foxg1 on cell cycle exit is independent of its DNA-binding properties [48].

Foxg1 is strongly expressed in the postnatal and adult telencephalon within the two neurogenic areas, the SVZ of the lateral ventricle and the DG of the hippocampus [48]. Haploinsufficiency for Foxg1 (Foxg1+/− mice) or deletion of Foxg1 during postnatal DG development by crossing the Foxg1fl/fl mice with a Frizzled9-CreER line (i.e., Foxg1-cKO mice) is associated with behavioral deficits (such as abnormal locomotion and contextual fear conditioning) consistent with hippocampal dysfunctions [47-49]. Indeed, Foxg1+/− and Foxg1-cKO mouse lines showed a marked reduction in size and an important malformation of the DG. This is associated with a failure of postnatal DG neurogenesis, characterized by a reduction in the number of NSCs [48, 50]. On the contrary, an overexpression of Foxg1 enhances neurogenesis. This effect is correlated with an increase of neurite outgrowth and an inhibition of gliogenesis [51]. Foxg1 cooperates with Bmi-1 (polycomb complex protein) to maintain forebrain NSC self-renewal and multipotency [69] mainly by repressing p21 expression (Table 2) [70].

Table 2. Target genes of Fox transcription factor involved in adult neurogenesis
MembersDNA binding consensus sequencesTarget genesRegulation (up [UPWARDS ARROW] or down [DOWNWARDS ARROW])Functional consequencesReferences
  1. Abbreviation: NSC, neural stem cell.

  2. The sequence binding sites are sourced from Jolma et al. [80] and Pazar, a public database of transcription factor and regulatory sequence annotation. http://www.pazar.info/.

Foxg1ATAAACAANWGTAAACAp21Cip1[DOWNWARDS ARROW]Maintains forebrain NSC selfrenewal, multipotency and increases proliferation.[70]
Foxj1TTTTGTTTGTTTGAnk-3, DNAI1, DNALI1, SPAG6, TEKT1/2[UPWARDS ARROW]Maintain an intact ependymal niche organization. Enhance the expression of cilia genes during multiciliated cell differentiation process.[71]
Foxo3GTAAACATGTTTACp27Kip1,[UPWARDS ARROW]Induce cell cycle arrest and cell fate decision[72-75]
  p21Cip1   
  Cyclin D[DOWNWARDS ARROW]  
  ASCL1/MASH1[DOWNWARDS ARROW]Blocks neuronal differentiation[59, 76]
Foxo1GTAAACATGTTTACBim, Bax[UPWARDS ARROW]Induce apoptosis[77]
Foxm1CCCAGCTGACCCACyclin A/B1, ATF2, cdc25a/b, Sox2[UPWARDS ARROW]Promote cell cycle progression[66, 78, 79]
  p21Cip1,[DOWNWARDS ARROW]Promote cell cycle progression[78]
  p27Kip1   

Foxg1 is also required for the survival of adult postmitotic neurons. In Foxg1+/− mice, the reduced size of the DG is caused by a cell survival decrease rather than reduced proliferation or granule cell differentiation [48]. Foxg1-cKO mice show an increased cell death exclusively in postmitotic neurons [50]. Ectopic expression of FoxG1 blocks neuronal death, whereas suppression of its expression induces death in otherwise healthy neurons [81]. To promote neuronal survival, Foxg1 cooperates with transducin-like enhancer of split 1 (TLE1) in a casein kinase 2- (CK2) and PI3K-Akt-dependent manner [82]. Phosphorylation of TLE1 at serine 239 by CK2 and phosphorylation of Foxg1 by Akt at Thr-271 are necessary for their neuronal survival-promoting activity. The exact stoichiometry between TLE1 and Foxg1 remains to be elucidated, but Foxg1 needs to physically interact with TLE1 to promote neuronal survival [82].

Foxj1

Three Foxj members have been identified: Foxj1, Foxj2, and Foxj3. Foxj2 and Foxj3 are closely related and are widely expressed during embryonic development including the brain but nothing is known about their role during central nervous system (CNS) development [83, 84]. Foxj1 is specifically expressed in epithelial cells containing classical motile cilia, including ependymal cells in the adult SVZ [52, 53]. Cilia are microtubule-based filamentous organelles classified in two types: nonmotile or primary cilia, which occur as single entity per cell, and motile cilia [85]. Primary cilia have widespread distribution in many cell types and play important roles in signal transduction. Motile cilia are usually present in large numbers exclusively in areas where a fluid movement is required. Notably, they contribute to ependymal cell differentiation and to migration of adult type-A progenitor cells from the SVZ toward the RMS [86, 87]. The targeted disruption of the mouse Foxj1 gene results mainly in left/right asymmetry with loss of motile cilia and flagella [52, 54].

In the postnatal/adult SVZ, the absence of Foxj1 is associated with defects in proliferation and differentiation resulting in severe OB dysgenesis [53]. Foxj1 is necessary for the differentiation of ependymal cells. Indeed, Foxj1−/− mice that do not exhibit aberrant left/right asymmetry survive into early adulthood, but their ependymal cells fail to differentiate and the surface of the cerebral ventricles is devoid of motile cilia. As a consequence, these knockout mice exhibit hydrocephalus. The core of a cilium, the so-called axoneme consists of a stereotypically arranged set of microtubules that originates at the basal body, which is anchored in the cortical actin cytoskeleton. Defective ciliogenesis in the absence of Foxj1 may result from the inability of basal bodies to migrate and anchor to the apical cytoskeleton leading to subsequent failure of axonemal formation [53]. Interestingly, primary cilia are normally present in Foxj1−/− mouse brains [53]. Thus, Foxj1 appears to be necessary for the biogenesis of motile cilia but not primary cilia.

The massive disruption of postnatal neurogenesis observed in the Foxj1−/− OB could not exclusively be linked to the absence of ependymal cells differentiation. Indeed, Foxj1 induction leads to differentiation of a subset of Foxj1+ radial glia into Foxj1+ astrocytes. Foxj1+ cells exhibit neurogenic potential in vivo and in vitro, suggesting that—at least a subset of them—function as postnatal and adult NSCs [53]. Furthermore, Foxj1-dependent maturation of the NSCs nonautonomously regulates progenitor proliferation in the SVZ. Altogether, these data support the hypothesis that Foxj1+ cells represent a subset of SVZ NSCs [53, 55].

The molecular mechanisms triggered downstream of Foxj1 in the adult brain remain largely unsolved. Foxj1 is a Shh-regulated gene in the neural tube [88] while it has been suggested to regulate expression and intracellular localization of ezrin [89, 90], a membrane-cytoskeleton linking factor implicated in establishing cell polarity in epithelial cells [91]. Whether this is the case in the adult brain remains to be demonstrated. Recently, it has been shown that ankyrin-3 (Ank-3) expression is regulated by Foxj1 in the adult SVZ (Table 2) [71]. Ank-3 is a large adaptor molecule that organizes lateral membrane domains in numerous different cell types [92]. In the adult SVZ, Ank-3 is specifically colocalized with Foxj1 cell population, that is, the ependymal cell lineage including some radial-glial cells and ependymal cells. In addition, disruption of Foxj1/Ank-3 pathway in vivo results in a dramatic reduction of neuroblasts production and brain ventricular wall organization [71]. Therefore, Foxj1/Ank-3 pathway is essential to maintain an intact ependymal niche organization and thus to allow a continuous production of newly generated neurons in the postnatal and adult brain.

Other Foxj1 regulatory factors have been identified. Indeed, it has been suggested that CP110 (centrosomal protein of 110 kDa), a centriolar-associated protein that blocks the ciliogenesis, may be important to allow Foxj1 to induce motile cilia biogenesis. When CP110 is overexpressed in Xenopus embryos, it inhibits ciliogenesis through ectopic expression Foxj1 [93]. RFX3, a member of the regulatory factor X (RFX) family of transcription factors, is also one such regulator of Foxj1. RFX3 is a key player in the formation of motile cilia in mouse brain ependymal cell cultures and acts, at least by regulating Foxj1 expression, by binding to its promoter [94]. In addition, RFX3 and Foxj1 share some common direct target genes, suggesting that both types of transcription factors co-operate to govern a specific motile ciliogenic program [93-95]. Indeed, a recent publication [96] has shown that RFX3, in association with Foxj1, induces the expression of the cilia-associated genes dynein axonemal intermediate chain 1, dynein axonemal light intermediate chain 1, sperm-associated antigen 6, Tektin 1, and Tektin 2 during multiciliated cell differentiation (Table 2).

Foxos

In mammals, there are four Foxos transcription factors: Foxo1, Foxo3, Foxo4, and Foxo6, which are encoded by distinct genes. Foxo6 is structurally and functionally distinct from the other isoforms, which are closely related to each other. Among their many biological functions, Foxo proteins are implicated in regulation of apoptosis, cell cycle progression, and oxidative stress resistance. They are also implied in glucose metabolism and suppression of tumorigenesis, properties mainly regulated by Foxo1. In the brain, the Foxos family is implicated in the regulation of NSCs self-renewal, proliferation, and differentiation during embryonic and adult neurogenesis.

Foxo1, Foxo3, and Foxo6 are expressed in the adult murine brain, while Foxo4 has not been found [97]. More specifically, Foxo1 and Foxo3 are broadly expressed in multiple cell types including adult multipotent progenitors in the two neurogenic areas [57, 58]. On the contrary, Foxo6 is particularly highly expressed in CA1 and CA3 areas of the hippocampus but not in the DG, and therefore has no role in adult neurogenesis [61].

The role of Foxo1 and Foxo3 has been documented by mouse models lacking one or both genes [56-58]. In Foxo3−/− or brain-specific conditional Foxo1/3/4 triple knockout mice, the brain is enlarged in perinatal stages with a premature burst in Neural Progenitor Cells (NPCs) proliferation, leading to their exhaustion and precocious significant decline of their pool, and finally to a lack of neurogenesis [57, 58]. The deletion of upstream regulators of Foxo3, in Igf1 (insulin-like growth factor 1) or Akt knockout mice, also results in the formation of smaller brains [98, 99]. On the contrary, enhanced activity of Foxo3, which may arise after insulin receptor/IGF signaling deficiency, leads to a loss of neural progenitors and increased apoptosis [60].

Foxo3 is responsible for cell cycle arrest by upregulating the CDK inhibitor p27Kip1 which blocks S phase progression through a direct inhibition of cyclinE/CDK2 and cyclinA/CDK2 complexes (Table 2) [72]. PI3K/Akt pathway induces inhibitory phosphorylation of Foxo3 in NPCs, leading to a reduction in p27Kip1 expression. It has also been reported that Foxo3 induces p21Cip1 expression and represses cyclins D expression (G1/S arrest) or it activated cyclin G2 (G0/G1 arrest), leading to cell cycle arrest (Table 2) [73-75]. Interestingly, mice deficient for p21 also showed an increase in early proliferation of NSCs followed by an accelerated age-dependent loss of NSCs self-renewal [100], suggesting that a pool of NSCs is generated at birth and is not indefinitely produced.

Among the Foxo3-regulated genes in NPCs are enzymes in carbon metabolism that act to combat reactive oxygen species by directing the flow of glucose and glutamine carbon into defined metabolic pathways [101]. In Foxo3 adult NPCs, an unexpected downregulation of glutamine and glucose metabolism occurred, that in turn leads to decreased pyruvate kinase and pentose phosphate pathway, respectively. These metabolic alterations contribute to a more oxidative cellular environment leading to accumulation of oxidative damage in Foxo3 KO NPCs.

More recently, it has been shown that Foxo3 maintains stemness and the NSCs pool in the adult niches by restraining master regulators of differentiated CNS cell fates [59]. Indeed, FoxO3 inhibits ASCL1/Mash1-dependent neurogenesis. ASCL1/Mash1 is a basic helix-loop-helix transcription factor that has been shown to be essential for neural differentiation during adult neurogenesis (Table 2) [59, 76]. Interestingly, ASCL1/Mash1 protein stabilization has been shown to mediate some of the cellular action of the PI3K-AKT signaling on neurogenesis [102]. Thus, it is conceivable that the PI3K-AKT signaling pathway coordinates ASCL1 stabilization with ASCL1 activation (via inhibition of the repressive effect of Foxo3 on ASCL1) to achieve a more potent induction of neurogenesis. How Foxo3 and ASCL1/Mash1 interact in vivo, and which ASCL1 transcriptional targets are inhibited remain to be established.

In the adult brain, Foxo1 has been identified as a key regulator of cell death. Activation of Foxo1 induces apoptosis by upregulating a number of cell death genes such as Bim or Bax (Table 2) [77]. In neurons, CDK1 phosphorylates Foxo1, and thereby disrupts its interaction with 14-3-3 proteins, resulting in its nuclear localization and increasing its transcriptional activity [103]. However, in Foxo1 knockout mice, no appreciable changes in apoptosis are observed both in NSCs and neurons [57].

Foxm1

Foxm1, also named Trident (mouse) or HFH-11B (human), binds promoter regions on a consensus “TAAACA” site similar to other forkhead proteins but with a unique affinity for tandem repeats. This may explain its relative low affinity for single copies of its forkhead consensus sequence [104]. Alternative splicing of two exons gives rise to three isoforms of Foxm1, the transcriptionally active Foxm1b and Foxm1c, and the transcriptionally inactive Foxm1a splice variants [105]. Foxm1 is a key regulator of the cell cycle, critical for G1/S phase transition and G2/M progression. Its expression and transcriptional activity depend on the progression of cell cycle and correlates with its phosphorylation level. Foxm1 protein and phosphorylation levels are diminished in quiescent cells, but both are upregulated during late G1-phase of the cell cycle and they persist throughout the G2- and M-phases [62, 106]. Foxm1 controls G1/S-phase transition by upregulating expression of several critical genes such as Cyclin A, activating transcription factor 2, and Cdc25A phosphatase, while downregulating protein levels of p21Cip1 and p27Kip1 through the activation of Skp2 and Cks1 genes (encoding subunits of the Skp1-Cullin1-F-box ubiquitin ligase complex) [78]. During G2 phase, Foxm1 also increases the expression of Cyclin B1 and cdc25b genes, which are required for mitotic entry and completion (Table 2) [79].

Foxm1 is ubiquitously expressed in embryonic tissues, particularly in proliferating cells of epithelial or mesenchymal origin [107]. Foxm1−/− mice die in utero between E13.5 and E17.5 due to multiple abnormalities in several organs including liver, lung, brain, and heart [63-65]. In adult tissues, FoxM1 expression is restricted to actively dividing cells and is eliminated in resting or terminally differentiating cells [106]. Therefore, Foxm1 is a typical proliferation-associated transcription factor. In situ hybridization revealed that Foxm1 mRNAs are highly expressed in NSCs and neural precursor cells during embryonic development [67, 108] and in both neurogenic areas in adult CNS (personal observations).

Deletion of Foxm1 impairs the self-renewal of embryonic NSCs and loss of Foxm1 is correlated with a lack of Sox2 expression, which seems to be directly activated by Foxm1 [66]. A conditional deletion of Foxm1 from precursors of cerebellar granule neurons (Math1-Cre and Nestin-Cre transgenes) caused delayed brain development [67]. Indeed, Foxm1-deficient cerebellar granule neuron precursors showed a significant delay in the G2/M transition probably through decreased expression of the target genes cyclin B1 and Cdc25b (Table 2). Furthermore, Foxm1 is a component of the Wnt signaling pathway and Foxm1-beta-catenin interaction is required for the activation of the canonical Wnt signaling pathway in NSCs [109]. Blockade of Wnt signaling reduces adult hippocampal neurogenesis both in vivo and in vitro. It is therefore tempting to speculate that Foxm1 is required for adult neurogenesis.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Forkhead Box Family of Transcription Factors
  5. Fox Factors and Adult Neurogenesis
  6. Conclusion
  7. Author Contributions
  8. Disclosure of Potential Conflicts of Interest
  9. References

Through transcriptional regulation of their target genes, Fox factors expressed in the adult brain and more specifically in neurogenic areas play crucial roles. Accumulating data demonstrate that a number of Fox transcription factors are present in vertebrate adult neurogenic areas. In addition, the animal model phenotypes exhibit various roles for Fox proteins during adult neurogenesis. They collectively demonstrate that several Fox are important determinants of adult neural cell fate. The data and results presented here underscore the enormous potential that Fox factors holds for our understanding of the development and homeostasis of NSCs. The key role of Fox factors in brain development is illustrated by the consequences of their mutation in humans. In particular, intellectual disability, autism, and speech disorders are observed as consequences of FOXG1, FOXP1, or FOXP2 mutations [110-112]. Moreover, a recent study showed that CNTNAP2 gene, which encodes Caspr2—a member of the neurexin superfamily of cell adhesion molecules—is a direct target of FOXP2 repression and is itself directly implicated in speech defects [113]. Future work aimed at delineating the role of specific Fox proteins, especially Foxm1 and Foxp2, in adult NSCs will enhance our understanding of the dynamic process of adult neurogenesis and is the first step toward strategies for cell replacement therapy after injury or neurodegenerative diseases. However, we have to keep in mind that deregulation of Fox protein signaling can significantly contribute to tumorigenesis and cancer progression [24, 114].

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Forkhead Box Family of Transcription Factors
  5. Fox Factors and Adult Neurogenesis
  6. Conclusion
  7. Author Contributions
  8. Disclosure of Potential Conflicts of Interest
  9. References

E.C.G and N.C.: conception and design and manuscript writing; R.V. and L.N.: manuscript editing and critical discussions; B.M.: conception and design, financial support, manuscript writing, and final approval of manuscript. E.C.G. and N.C. contributed equally to this article.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Forkhead Box Family of Transcription Factors
  5. Fox Factors and Adult Neurogenesis
  6. Conclusion
  7. Author Contributions
  8. Disclosure of Potential Conflicts of Interest
  9. References