Maintenance of a healthy proteome is essential for cellular homeostasis and loss of proteostasis is associated with tissue dysfunction and neurodegenerative disease. The mechanisms that support ...proteostasis in healthy cells and how they become defective during aging or in disease states are not fully understood. Here, we investigate the transcriptional programs that are essential for neural stem and progenitor cell (NSPC) function and uncover a program of autophagy genes under the control of the transcription factor FOXO3. Using genomic approaches, we observe that FOXO3 directly binds a network of target genes in adult NSPCs that are involved in autophagy, and find that FOXO3 functionally regulates induction of autophagy in these cells. Interestingly, in the absence of FOXO activity, aggregates accumulate in NSPCs, and this effect is reversed by TOR (target of rapamycin) inhibition. Surprisingly, enhancing FOXO3 causes nucleation of protein aggregates, but does not increase their degradation. The work presented here identifies a genomic network under the direct control of a key transcriptional regulator of aging that is critical for maintaining a healthy mammalian stem cell pool to support lifelong neurogenesis.
Forkhead box O (FOXO) transcription factors are central regulators of cellular homeostasis. FOXOs respond to a wide range of external stimuli, including growth factor signaling, oxidative stress, ...genotoxic stress, and nutrient deprivation. These signaling inputs regulate FOXOs through a number of posttranslational modifications, including phosphorylation, acetylation, ubiquitination, and methylation. Covalent modifications can affect localization, DNA binding, and interactions with other cofactors in the cell. FOXOs integrate the various modifications to regulate cell type-specific gene expression programs that are essential for metabolic homeostasis, redox balance, and the stress response. Together, these functions are critical for coordinating a response to environmental fluctuations in order to maintain cellular homeostasis during development and to support healthy aging.
Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct ...conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an “on-target” pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.
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•Ascl1 has pioneer activity, accessing closed chromatin to allow other factors to bind•Unlike other pioneer factors, Ascl1 binds its physiologic neural targets in fibroblasts•A trivalent chromatin domain predicts iN reprogramming ability in other cell types•Zfp238 is a direct Ascl1 target and critical mediator of iN cell reprogramming
Of three transcription factors that together convert different cell types into induced neuronal (iN) cells, Ascl1 leads the way as the “pioneer factor,” followed by Brn2 and Myt1l. A trivalent chromatin state at Ascl1 target genes predicts which types of cells will respond to reprogramming by these factors.
In the adult brain, the neural stem cell (NSC) pool comprises quiescent and activated populations with distinct roles. Transcriptomic analysis revealed that quiescent and activated NSCs exhibited ...differences in their protein homeostasis network. Whereas activated NSCs had active proteasomes, quiescent NSCs contained large lysosomes. Quiescent NSCs from young mice accumulated protein aggregates, and many of these aggregates were stored in large lysosomes. Perturbation of lysosomal activity in quiescent NSCs affected protein-aggregate accumulation and the ability of quiescent NSCs to activate. During aging, quiescent NSCs displayed defects in their lysosomes, increased accumulation of protein aggregates, and reduced ability to activate. Enhancement of the lysosome pathway in old quiescent NSCs cleared protein aggregates and ameliorated the ability of quiescent NSCs to activate, allowing them to regain a more youthful state.
Skeletal muscle stem cells, or “satellite cells” (SCs), are required for the regeneration of damaged muscle tissue. Although SCs self-renew during regeneration, the mechanisms that govern SC re-entry ...into quiescence remain elusive. We show that FOXO3, a member of the forkhead family of transcription factors, is expressed in quiescent SCs (QSCs). Conditional deletion of Foxo3 in QSCs impairs self-renewal and increases the propensity of SCs to adopt a differentiated fate. Transcriptional analysis of SCs lacking FOXO3 revealed a downregulation of Notch signaling, a key regulator of SC quiescence. Conversely, overexpression of Notch intracellular domain (NICD) rescued the self-renewal deficit of FOXO3-deficient SCs. We show that FOXO3 regulates NOTCH1 and NOTCH3 receptor expression and that decreasing expression of NOTCH1 and NOTCH3 receptors phenocopies the effect of FOXO3 deficiency in SCs. We demonstrate that FOXO3, perhaps by activating Notch signaling, promotes the quiescent state during SC self-renewal in adult muscle regeneration.
•FOXO3 is expressed in quiescent adult SCs•FOXO3 is required for self-renewal of SCs•FOXO3-deficient SCs display an increased propensity to differentiate•FOXO3 promotes Notch signaling, a key regulator of quiescence in adult SCs
Little is known about the molecular mechanisms that govern muscle stem cell self-renewal. Rando and colleagues show that FOXO3, a forkhead family transcription factor, activates Notch signaling, a key regulator of the quiescent state in muscle stem cells, and thereby promotes self-renewal during muscle regeneration.
Autophagy is a conserved catabolic pathway with emerging functions in mammalian neurodevelopment and human neurodevelopmental diseases. The mechanisms controlling autophagy in neuronal development ...are not fully understood. Here, we found that conditional deletion of the Forkhead Box O transcription factors FoxO1, FoxO3, and FoxO4 strongly impaired autophagic flux in developing neurons of the adult mouse hippocampus. Moreover, FoxO deficiency led to altered dendritic morphology, increased spine density, and aberrant spine positioning in adult-generated neurons. Strikingly, pharmacological induction of autophagy was sufficient to correct abnormal dendrite and spine development of FoxO-deficient neurons. Collectively, these findings reveal a novel link between FoxO transcription factors, autophagic flux, and maturation of developing neurons.
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•FoxOs control morphology and spine density of adult-born hippocampal neurons•Autophagic flux is severely impaired in FoxO-deficient, adult-born neurons•Induction of autophagy rescues dendrite and spine deficits of FoxO-ablated neurons
Schäffner 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.
•FOXO transcription factors are key regulators of autophagy and the ubiquitin–proteasome system.•FOXOs have been implicated in protection against proteotoxicity.•Reduced proteotoxicity is associated ...with protection against neurodegeneration.•FOXO and mTOR reciprocally regulate proteostasis, depending on nutrient availability.
FOXO transcription factors are conserved regulators of longevity downstream of insulin signaling. These transcription factors integrate signals emanating from nutrient deprivation and stress stimuli to coordinate programs of genes involved in cellular metabolism and resistance to oxidative stress. Here, we discuss emerging evidence for a pivotal role of FOXO factors in promoting the expression of genes involved in autophagy and the ubiquitin–proteasome system – two cell clearance processes that are essential for maintaining organelle and protein homeostasis (proteostasis). The ability of FOXO to maintain cellular quality control appears to be critical in processes and pathologies where damaged proteins and organelles accumulate, including aging and neurodegenerative diseases.
Summary
FOXO transcription factors (FOXOs) are central regulators of lifespan across species, yet they also have cell‐specific functions, including adult stem cell homeostasis and immune function. ...Direct targets of FOXOs have been identified genome‐wide in several species and cell types. However, whether FOXO targets are specific to cell types and species or conserved across cell types and throughout evolution remains uncharacterized. Here, we perform a meta‐analysis of direct FOXO targets across tissues and organisms, using data from mammals as well as Caenorhabditis elegans and Drosophila. We show that FOXOs bind cell type‐specific targets, which have functions related to that particular cell. Interestingly, FOXOs also share targets across different tissues in mammals, and the function and even the identity of these shared mammalian targets are conserved in invertebrates. Evolutionarily conserved targets show enrichment for growth factor signaling, metabolism, stress resistance, and proteostasis, suggesting an ancestral, conserved role in the regulation of these processes. We also identify candidate cofactors at conserved FOXO targets that change in expression with age, including CREB and ETS family factors. This meta‐analysis provides insight into the evolution of the FOXO network and highlights downstream genes and cofactors that may be particularly important for FOXO's conserved function in adult homeostasis and longevity.
FOXO transcription factors are central regulators of longevity from worms to humans. FOXO3, the FOXO isoform associated with exceptional human longevity, preserves adult neural stem cell pools. Here, ...we identify FOXO3 direct targets genome-wide in primary cultures of adult neural progenitor cells (NPCs). Interestingly, FOXO3-bound sites are enriched for motifs for bHLH transcription factors, and FOXO3 shares common targets with the proneuronal bHLH transcription factor ASCL1/MASH1 in NPCs. Analysis of the chromatin landscape reveals that FOXO3 and ASCL1 are particularly enriched at the enhancers of genes involved in neurogenic pathways. Intriguingly, FOXO3 inhibits ASCL1-dependent neurogenesis in NPCs and direct neuronal conversion in fibroblasts. FOXO3 also restrains neurogenesis in vivo. Our study identifies a genome-wide interaction between the prolongevity transcription factor FOXO3 and the cell-fate determinant ASCL1 and raises the possibility that FOXO3’s ability to restrain ASCL1-dependent neurogenesis may help preserve the neural stem cell pool.
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•FOXO3 directly binds over 2,000 targets in adult neural progenitor cells•FOXO3 targets overlap with those of ASCL1, a proneuronal bHLH factor•FOXO3 and ASCL1 are enriched at enhancers of genes involved in the Notch pathway•FOXO3 restrains ASCL1-dependent neurogenesis
The transcription factor FOXO3 functions as a prolongevity factor and acts to preserve stem cell pools in mammals. Now, Brunet and colleagues identify genome-wide binding sites for FOXO3 in adult neural stem/progenitor cells. FOXO3 shares common targets with the proneuronal factor ASCL1 in these cells and functions to restrain ASCL1-dependent neurogenesis. These results suggest that FOXO3 prevents depletion of the neural stem/progenitor reservoir in adults in part by inhibiting premature neurogenesis.
Aging is a major risk factor for many diseases. Accurate methods for predicting age in specific cell types are essential to understand the heterogeneity of aging and to assess rejuvenation ...strategies. However, classifying organismal age at single-cell resolution using transcriptomics is challenging due to sparsity and noise. Here, we developed CellBiAge, a robust and easy-to-implement machine learning pipeline, to classify the age of single cells in the mouse brain using single-cell transcriptomics. We show that binarization of gene expression values for the top highly variable genes significantly improved test performance across different models, techniques, sexes, and brain regions, with potential age-related genes identified for model prediction. Additionally, we demonstrate CellBiAge’s ability to capture exercise-induced rejuvenation in neural stem cells. This study provides a broadly applicable approach for robust classification of organismal age of single cells in the mouse brain, which may aid in understanding the aging process and evaluating rejuvenation methods.
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•CellBiAge classifies organismal age groups of single cells through binarization•CellBiAge model interpretation reveals cell-type-specific aging signatures•Generalizable to ML models, sc/snRNA-seq techniques, sexes, and mouse brain regions•The model captures exercise-induced rejuvenation in proliferating neural stem cells
Incorporating data binarization, Yu et al. develop the CellBiAge pipeline for accurate organismal age classification at the single-cell level in the mouse brain. CellBiAge demonstrates generalizability across techniques, sexes, and brain regions. Additionally, cell-type-specific models reveal distinct signatures and capture exercise-induced rejuvenation in proliferating neural stem cells.
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