The Multi-pronged Regulation of Adult Neurogenesis by Forkhead Box O Family Members
Authors: Timal S. Kannangara, Diane C. Lagace
Journal: Neuron (2018)

In this issue of Neuron, Schäffner et al., 2018Schäffner I. Minakaki G. Khan M.A. Balta E.-A. Schlotzer-Schredardt U. Schwarz T.J. Beckervordersandforth R. Winner B. Webb A.E. DePinho R. et al.FoxO function is essential for maintenance of autophagic flux and neuronal morphogenesis in adult neurogenesis.Neuron. 2018; 99 (this issue): 1188-1203Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar discover multiple effects of the Forkhead Box O (FoxO) transcription factor family on the different stages of adult neurogenesis, including the genesis of dendrites and spines regulated by FoxO-dependent autophagic activity. In this issue of Neuron, Schäffner et al., 2018Schäffner I. Minakaki G. Khan M.A. Balta E.-A. Schlotzer-Schredardt U. Schwarz T.J. Beckervordersandforth R. Winner B. Webb A.E. DePinho R. et al.FoxO function is essential for maintenance of autophagic flux and neuronal morphogenesis in adult neurogenesis.Neuron. 2018; 99 (this issue): 1188-1203Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar discover multiple effects of the Forkhead Box O (FoxO) transcription factor family on the different stages of adult neurogenesis, including the genesis of dendrites and spines regulated by FoxO-dependent autophagic activity. The mechanisms that control the birth, maturation, survival, and integration of neurons within the landscape of the mature brain remain an uncompleted puzzle. Since the discovery of adult-generated neurons, a wealth of data has characterized their properties at different stages in development (Gonçalves et al., 2016Gonçalves J.T. Schafer S.T. Gage F.H. Adult neurogenesis in the hippocampus: from stem cells to behavior.Cell. 2016; 167: 897-914Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). However, how these cells advance between the different developmental stages remains an active area of research. The dynamic process of adult neurogenesis begins with the radial glial-like neural stem cells (NSCs) that have the fundamental ability in vivo to self-renew and give rise to differentiated progeny. Within the dentate gyrus subfield of the hippocampus, populations of quiescent and activated NSCs reside in the subgranular zone and generate rapidly proliferating progenitor cells that develop into immature neurons. The majority of immature neurons do not survive to become neurons; however, within a few months, surviving neurons become indistinguishable from dentate granule neurons that were formed during embryonic development. Numerous intrinsic and extrinsic signals from the surrounding niche, along with inputs onto the cells at different stages, clearly regulate the distinct aspects of development (Gonçalves et al., 2016Gonçalves J.T. Schafer S.T. Gage F.H. Adult neurogenesis in the hippocampus: from stem cells to behavior.Cell. 2016; 167: 897-914Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). Many of these factors, such as morphogens, growth factors, cytokines, transcription factors, and metabolic components, are recognized as exerting a plethora of effects, including altering the NSC population, the rate of proliferation, cell survival, fate specification, and integration of the adult-generated cells. Thus, defining the orchestrated combination of spatial and temporal signals that regulate the dynamic process of adult neurogenesis is crucial for understanding how these cells contribute to brain function in health or disease states. The FoxO family of transcription factors is most often thought of as a central regulator of longevity. However, this family of four members (FoxO1, FoxO3, FoxO4, and FoxO5) is increasingly being recognized as governors of diverse gene expression programs and has a broad role in many cellular processes. Indeed, they are involved in cell-cycle regulation, apoptosis, and metabolism, in addition to regulating stem cells (Eijkelenboom and Burgering, 2013Eijkelenboom A. Burgering B.M. FOXOs: signalling integrators for homeostasis maintenance.Nat. Rev. Mol. Cell Biol. 2013; 14: 83-97Crossref PubMed Scopus (713) Google Scholar). Through these actions, FoxOs can respond to stress conditions, acting to maintain homeostasis over time and coordinate responses to environmental changes. Although most would not usually associate the genesis and maturation of an adult-born neuron as a stressful conditional per se, there is no doubt that developing cells need to coordinate multiple changes and are in a stage of constant flux. In this issue, work published by Schäffner et al., 2018Schäffner I. Minakaki G. Khan M.A. Balta E.-A. Schlotzer-Schredardt U. Schwarz T.J. Beckervordersandforth R. Winner B. Webb A.E. DePinho R. et al.FoxO function is essential for maintenance of autophagic flux and neuronal morphogenesis in adult neurogenesis.Neuron. 2018; 99 (this issue): 1188-1203Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar shows that adult-specific deletion of the FoxO family in mice has multiple short- and long-term effects on the process of adult neurogenesis as illustrated in Figure 1, in part through disruption of macroautophagy, a catabolic process that degrades macromolecules and organelles by delivering them to lysosomes. Schäffner et al. deleted FoxO1, FoxO2, and FoxO3 (referred to hereafter as FoxOs) using an inducible genetic knockout approach in adult mice to show that these transcription factors are necessary for the maintenance of hippocampal NSCs and progenitor cells in the adult brain. Within days of removal, there was a robust increase in dividing NSCs, followed by a slower depletion of neurogenesis that was not identified until 4 months. These findings are consistent with the ability of FoxOs to preserve a diverse number of stem cell pools (Genin et al., 2014Genin E.C. Caron N. Vandenbosch R. Nguyen L. Malgrange B. Concise review: forkhead pathway in the control of adult neurogenesis.Stem Cells. 2014; 32: 1398-1407Crossref PubMed Scopus (39) Google Scholar, Paik et al., 2009Paik J.H. Ding Z. Narurkar R. Ramkissoon S. Muller F. Kamoun W.S. Chae S.S. Zheng H. Ying H. Mahoney J. et al.FoxOs cooperatively regulate diverse pathways governing neural stem cell homeostasis.Cell Stem Cell. 2009; 5: 540-553Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, Renault et al., 2009Renault V.M. Rafalski V.A. Morgan A.A. Salih D.A. Brett J.O. Webb A.E. Villeda S.A. Thekkat P.U. Guillerey C. Denko N.C. et al.FoxO3 regulates neural stem cell homeostasis.Cell Stem Cell. 2009; 5: 527-539Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). The expression of FoxOs in mature granule cells next prompted them to use a retroviral approach to birthdate and ablate the FoxOs in the adult dividing progenitor cells in vivo. This resulted in a progressive decline in the survival of the FoxO null cells, suggesting that FoxOs are required for the survival of adult-generated neurons independent of their effects on stem cells or the proliferating progenitor cells. Notably, this effect was observed at a relatively long time (4 months) following ablation, after the normal synaptogenesis occured (Toni et al., 2007Toni N. Teng E.M. Bushong E.A. Aimone J.B. Zhao C. Consiglio A. van Praag H. Martone M.E. Ellisman M.H. Gage F.H. Synapse formation on neurons born in the adult hippocampus.Nat. Neurosci. 2007; 10: 727-734Crossref PubMed Scopus (468) Google Scholar). Further examination of dendrites and synapses revealed the surprising finding of abnormal maturation of FoxO null cells as early as 21 days following removal of FoxOs. The authors found distinct decreases in the dendritic length and complexity in FoxO null cells, as well as increased spine density in dendrites located within the molecular layer. Accompanying the morphology deficits of FoxO null cells were two additional unique properties, which altered the proximal dendrites located in the granule layer, close to the soma. The first property observed was the drastic shortening, or complete absence, of the single long apical dendrite characteristic to adult-generated neurons. In its place, FoxO null cells demonstrated either an early bifurcation or two primary processes originating at the soma, similar to embryonically generated dentate granule neurons found in the more superficial layers of the granule cell layer. While the effect of alterations to apical dendrites is unclear, one could reasonably assume that replacing a single apical dendrite with two primary processes could impact local dendritic processing simply by further compartmentalizing the integration of synaptic input (Branco and Häusser, 2010Branco T. Häusser M. The single dendritic branch as a fundamental functional unit in the nervous system.Curr. Opin. Neurobiol. 2010; 20: 494-502Crossref PubMed Scopus (223) Google Scholar). The second property observed was that the primary dendrites of the FoxO null cells contained a significant number of spines, a feature absent in normal, adult-generated neurons. Previous reports have shown that excitatory drive onto normal dentate granule cells is primarily distributed to dendrites in the molecular cell layer, which asks the question, what sort of function do granule cell layer spines have? As such, this feature may be indicative of an overall upregulation of non-specific spine formation throughout the dendritic tree, irrespective of presynaptic partner. To begin to isolate a possible mechanism and examine whether these neurogenic related effects were cell-autonomous, primary neurospheres were created from 8-week-old mice and retroviral approaches were used to remove FoxOs. In alignment with enhanced proliferation in FoxO cKO mice in vivo, this induced an increase in proliferation and self-renewal potential, as determined by single-cell neurosphere assay over several passages. In contrast to the in vivo models that had no differentiation phenotype, there was an increase in neurogenesis and a concurrent reduction in astrogenesis after in vitro differentiation. The authors suggest that this effect could be due to non-cell-autonomous effects that alter differentiation of the FoxO null cells in vivo. It is also possible that these differences may have arisen due to different cell populations analyzed since the in vivo experiments examined the cells in the subgranular zone of the dentate gyrus, and the in vitro experiments examined cells isolated from the subventricular zone (SVZ) of the lateral ventricles. To address the latter, future experiments examining the in vivo effects of FoxO depletion on the stem cells in the SVZ and the resulting neurogenesis in the olfactory bulb and/or cultured spheres from the hippocampus would be insightful. Given that FoxOs are implicated in the regulation of autophagy in a variety of cell types in vertebrates and invertebrates, the authors used multiple methods to measure autophagy. These methods included quantifying the markers of the autophagosome, as well as the autophagolysosomes that form from the fusion of autophagosomes with lysosomes. Additionally, they measured the genesis and turnover of autophagosomes to autophagolysosomes to assess autophagic flux. Their in vitro data consistently showed significantly more and larger autophagosomes and a lack of autophagolysosomes in the soma of both proliferating and differentiated FoxO null cells. In vivo retroviral-mediated removal of FoxO also produced a significant reduction in autophagic flux in the cell soma at 21 days, concurrent with the occurrence of morphological abnormalities in the developing neurons. These findings thus add to the growing appreciation for the regulatory role of autophagy in the spines of mature neurons (Nikoletopoulou and Tavernarakis, 2018Nikoletopoulou V. Tavernarakis N. Regulation and roles of autophagy at synapses.Trends Cell Biol. 2018; 28: 646-661Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and support the hypothesis that FoxO-dependent autophagy controls dendrite and spinogenesis in adult-generated neurons. These data set the stage for Schäffner et al. to directly test this hypothesis by examining if the enhancement of autophagic flux would be sufficient to reverse the morphological deficits in the developing neurons. Autophagy was activated using rapamycin, an inhibitor of the mTOR pathway, or trehalose, a natural disaccharide with mTOR-independent effects. Although both of these drugs have a wide variety of effects on multiple pathways including autophagy, treatment with either of these compounds in vitro produced strikingly similar abilities to induce autophagy in the single-cell neurosphere assay. Specifically, in control cells, treatment occurring either during proliferation or differentiation increased the number and size of autophagosomes, as well as the number of autophagolysosomes. In the FoxO null cells, treatment of the proliferating and differentiated cells also rescued the significant reduction in autophagic flux that was caused by strong absence of autophagolysosomes. The normalization of autophagic flux in the FoxO cells was also accompanied by functional effects that normalized levels of proliferation and differentiation to match that of the control cells. Therefore, these data support that the loss of FoxOs and reduction of autophagic flux, accompanied by enhanced proliferation and reduction in neuronal differentiation, can be prevented through enhancing autophagic flux cell autonomously in vitro. These findings also extend the growing number of connections between autophagy and adult neurogenesis that support that some, but not all, regulators of autophagy modify the proliferation and differentiation capacity of neural stem and progenitor cells (Dhaliwal et al., 2017Dhaliwal J. Trinkle-Mulcahy L. Lagace D.C. Autophagy and adult neurogenesis: discoveries made half a century ago yet in their infancy of being connected.Br. Plast. 2017; 3: 99-110Crossref PubMed Google Scholar). The authors then tested the in vivo effects of chronic treatment with rapamycin and trehalose immediately following retroviral removal of FoxOs for up to 42 days after the division and labeling of adult-generated neurons. Both of these treatments produced FoxO null neurons that had normalized similar apical dendrite lengths and spine location and density to control WT neurons. This also resulted in no effect on the survival of the adult-generated neurons, in alignment with their previous experiments showing that loss of FoxOs required 4 months to induce a survival effect in vivo. Future experiments using these treatments in the inducible FoxO mouse model would be required to determine if inducing autophagic activity could also reverse the significant alterations in the stem cells and proliferation progenitors, as well as the long-term effects on the deletion of the stem cell pool and survival. These findings are exciting and raise many novel questions about how FoxO-dependent autophagy controls dendrite and spine biogenesis. For example: What is the cargo that is within the autophagosomes? What is the distribution of the autophagosomes and autophagolysosomes within the dendrites of FoxO null mice compared to developing dendrites in control mice? In summary, the work by Schäffner et al. shows that the FoxO family is a major regulator of the development of adult-generated neurons through multiple independent effects as illustrated in Figure 1. There are robust increases in dividing stem and progenitor cells within days, significant alterations in dendritic morphology and spine location and density within a month, and a long-term effect on the depletion of neurogenesis that occurs through the exhaustion of the stem cells as well as a significant reduction in survival occurring independent of stem cell exhaustion within several months. The work also discovers that FoxO-dependent autophagic activity alters the genesis of dendrites and spines in adult-generated neurons, which has exciting implications for understanding spine dynamic in various brain diseases. FoxO Function Is Essential for Maintenance of Autophagic Flux and Neuronal Morphogenesis in Adult NeurogenesisSchäffner et al.NeuronSeptember 6, 2018In BriefSchäffner et al. identify FoxO transcription factors as critical regulators of autophagic flux in adult hippocampal neurogenesis and show that FoxO-dependent autophagic flux is necessary for morphological maturation and synaptic integration of adult-born hippocampal neurons. Full-Text PDF Open Archive