Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.

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.

Differentiation Drives Widespread Rewiring of the Neural Stem Cell Chaperone Network.

Neural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Lifelong maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPC chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.

FOXO3 directly regulates an autophagy network to functionally regulate proteostasis in adult neural stem cells.

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.

FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice.

We and others have shown that embryonic and neonatal fibroblasts can be directly converted into induced neuronal (iN) cells with mature functional properties. Reprogramming of fibroblasts from adult and aged mice, however, has not yet been explored in detail. The ability to generate fully functional iN cells from aged organisms will be particularly important for in vitro modeling of diseases of old age. Here, we demonstrate production of functional iN cells from fibroblasts that were derived from mice close to the end of their lifespan. iN cells from aged mice had apparently normal active and passive neuronal membrane properties and formed abundant synaptic connections. The reprogramming efficiency gradually decreased with fibroblasts derived from embryonic and neonatal mice, but remained similar for fibroblasts from postnatal mice of all ages. Strikingly, overexpression of a transcription factor, forkhead box O3 (FoxO3), which is implicated in aging, blocked iN cell conversion of embryonic fibroblasts, whereas knockout or knockdown of FoxO3 increased the reprogramming efficiency of adult-derived but not of embryonic fibroblasts and also enhanced functional maturation of resulting iN cells. Hence, FoxO3 has a central role in the neuronal reprogramming susceptibility of cells, and the importance of FoxO3 appears to change during development.

FOXO transcription factors: key regulators of cellular quality control.

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.

Single-cell transcriptomics of peripheral blood in the aging mouse.

Compositional and transcriptional changes in the hematopoietic system have been used as biomarkers of immunosenescence and aging. Here, we use single-cell RNA-sequencing to study the aging peripheral blood in mice and characterize the changes in cell-type composition and transcriptional profiles associated with age. We identified 17 clusters from a total of 14,588 single cells. We detected a general upregulation of antigen processing and presentation and chemokine signaling pathways and a downregulation of genes involved in ribosome pathways with age. In old peripheral blood, we also observed an increased percentage of cells expressing senescence markers (Cdkn1a, and Cdkn2a). In addition, we detected a cluster of activated T cells exclusively found in old blood, with lower expression of Cd28 and higher expression of Bcl2 and Cdkn2a, suggesting that the cells are senescent and resistant to apoptosis.

CellBiAge: Improved single-cell age classification using data binarization.

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.

Understanding the aging hypothalamus, one cell at a time.

The hypothalamus is a brain region that integrates signals from the periphery and the environment to maintain organismal homeostasis. To do so, specialized hypothalamic neuropeptidergic neurons control a range of processes, such as sleep, feeding, the stress response, and hormone release. These processes are altered with age, which can affect longevity and contribute to disease status. Technological advances, such as single-cell RNA sequencing, are upending assumptions about the transcriptional identity of cell types in the hypothalamus and revealing how distinct cell types change with age. In this review, we summarize current knowledge about the contribution of hypothalamic functions to aging. We highlight recent single-cell studies interrogating distinct cell types of the mouse hypothalamus and suggest ways in which single-cell 'omics technologies can be used to further understand the aging hypothalamus and its role in longevity.

Single-cell analysis of the aging female mouse hypothalamus.

Alterations in metabolism, sleep patterns, body composition, and hormone status are all key features of aging. While the hypothalamus is a well-conserved brain region that controls these homeostatic and survival-related behaviors, little is known about the intrinsic features of hypothalamic aging. Here, we perform single nuclei RNA-sequencing of 40,064 hypothalamic nuclei from young and aged female mice. We identify cell type-specific signatures of aging in neuronal subtypes as well as astrocytes and microglia. We uncover changes in cell types critical for metabolic regulation and body composition, and in an area of the hypothalamus linked to cognition. Our analysis also reveals an unexpected female-specific feature of hypothalamic aging: the master regulator of X-inactivation, Xist, is elevated with age, particularly in hypothalamic neurons. Moreover, using machine learning, we show that levels of X-chromosome genes, and Xist itself, can accurately predict cellular age. This study identifies critical cell-specific changes of the aging hypothalamus in mammals, and uncovers a potential marker of neuronal aging in females.

Stem cell phenotype predicts therapeutic response in glioblastomas with MGMT promoter methylation.

A growing body of evidence supports the presence of a population of cells in glioblastoma (GBM) with a stem cell-like phenotype which shares certain biological markers with adult neural stem cells, including expression of SOX2, CD133 (PROM1), and NES (nestin). This study was designed to determine the relationship between the expression of these stem cell markers and the clinical outcome in GBM patients. We quantified the intensity of expression of the proteins CD133 and SOX2 by immunohistochemistry (IHC) in a cohort of 86 patients with IDH-wildtype GBM, and evaluated patient outcomes using Kaplan-Meier and Cox proportional hazards analysis. In our patients, MGMT promoter methylation status and age were predictors of overall survival and progression free survival. The levels of SOX2 and CD133 were not associated with outcome in univariate analysis; however, stratification of tumors based on low or high levels of CD133 or SOX2 expression revealed that MGMT methylation was a predictor of progression-free survival and overall survival only for tumors with high levels of expression of CD133 or SOX2. Tumors with low levels of expression of CD133 or SOX2 did not show any relationship between MGMT methylation and survival. This relationship between MGMT and stem cell markers was confirmed in a second patient cohort, the TCGA dataset. Our results show that stratification of GBM by the level of expression of CD133 and SOX2 improved the prognostic power of MGMT promoter methylation status, identifying a low-expressing group in which the clinical outcome is not associated with MGMT promoter methylation status, and a high-expressing group in which the outcome was strongly associated with MGMT promoter methylation status. These findings support the concept that the presence of a high stem cell phenotype in GBM, as marked by expression of SOX2 or CD133, may be associated with the clinical response to treatment.

Corticotropin Releasing Factor Binding Protein as a Novel Target to Restore Brain Homeostasis: Lessons Learned From Alcohol Use Disorder Research.

Stress is well-known to contribute to the development of many psychiatric illnesses including alcohol and substance use disorder (AUD and SUD). The deleterious effects of stress have also been implicated in the acceleration of biological age, and age-related neurodegenerative disease. The physio-pathology of stress is regulated by the corticotropin-releasing factor (CRF) system, the upstream component of the hypothalamic-pituitary-adrenal (HPA) axis. Extensive literature has shown that dysregulation of the CRF neuroendocrine system contributes to escalation of alcohol consumption and, similarly, chronic alcohol consumption contributes to disruption of the stress system. The CRF system also represents the central switchboard for regulating homeostasis, and more recent studies have found that stress and aberrations in the CRF pathway are implicated in accelerated aging and age-related neurodegenerative disease. Corticotropin releasing factor binding protein (CRFBP) is a secreted glycoprotein distributed in peripheral tissues and in specific brain regions. It neutralizes the effects of CRF by sequestering free CRF, but may also possess excitatory function by interacting with CRF receptors. CRFBP's dual role in influencing CRF bioavailability and CRF receptor signaling has been shown to have a major part in the HPA axis response. Therefore, CRFBP may represent a valuable target to treat stress-related illness, including: development of novel medications to treat AUD and restore homeostasis in the aging brain. This narrative review focuses on molecular mechanisms related to the role of CRFBP in the progression of addictive and psychiatric disorders, biological aging, and age-related neurodegenerative disease. We provide an overview of recent studies investigating modulation of this pathway as a potential therapeutic target for AUD and age-related neurodegenerative disease.

Adult Hippocampal Neurogenesis in Aging and Alzheimer's Disease.

Cognitive deficits associated with Alzheimer's disease (AD) severely impact daily life for the millions of affected individuals. Progressive memory impairment in AD patients is associated with degeneration of the hippocampus. The dentate gyrus of the hippocampus, a region critical for learning and memory functions, is a site of adult neurogenesis in mammals. Recent evidence in humans indicates that hippocampal neurogenesis likely persists throughout life, but declines with age and is strikingly impaired in AD. Our understanding of how neurogenesis supports learning and memory in healthy adults is only beginning to emerge. The extent to which decreased neurogenesis contributes to cognitive decline in aging and AD remains poorly understood. However, studies in rodent models of AD and other neurodegenerative diseases raise the possibility that targeting neurogenesis may ameliorate cognitive dysfunction in AD. Here, we review recent progress in understanding how adult neurogenesis is impacted in the context of aging and AD.

Changing and stable chromatin accessibility supports transcriptional overhaul during neural stem cell activation and is altered with age.

Neural stem cells (NSCs) in the adult and aged brain are largely quiescent, and require transcriptional reprogramming to re-enter the cell cycle. However, the mechanisms underlying these changes and how they are altered with age remain undefined. Here, we identify the chromatin accessibility differences between primary neural stem/progenitor cells in quiescent and activated states. These distinct cellular states exhibit shared and unique chromatin profiles, both associated with gene regulation. Accessible chromatin states specific to activation or quiescence are active enhancers bound by key pro-neurogenic and quiescence factors. In contrast, shared sites are enriched for core promoter elements associated with translation and metabolism. Unexpectedly, through integrated analysis, we find that many sites that become accessible during NSC activation are linked to gene repression and associated with pro-quiescence factors, revealing a novel mechanism that may preserve quiescence re-entry. Furthermore, we report that in aged NSCs, chromatin regions associated with metabolic and transcriptional functions bound by key pro-quiescence transcription factors lose accessibility, suggesting a novel mechanism of age-associated NSC dysfunction. Together, our findings reveal how accessible chromatin states regulate the transcriptional switch between NSC quiescence and activation, and how this switch is affected with age.

Altered Chromatin States Drive Cryptic Transcription in Aging Mammalian Stem Cells.

A repressive chromatin state featuring trimethylated lysine 36 on histone H3 (H3K36me3) and DNA methylation suppresses cryptic transcription in embryonic stem cells. Cryptic transcription is elevated with age in yeast and nematodes, and reducing it extends yeast lifespan, though whether this occurs in mammals is unknown. We show that cryptic transcription is elevated in aged mammalian stem cells, including murine hematopoietic stem cells (mHSCs) and neural stem cells (NSCs) and human mesenchymal stem cells (hMSCs). Precise mapping allowed quantification of age-associated cryptic transcription in hMSCs aged in vitro. Regions with significant age-associated cryptic transcription have a unique chromatin signature: decreased H3K36me3 and increased H3K4me1, H3K4me3, and H3K27ac with age. Genomic regions undergoing such changes resemble known promoter sequences and are bound by TBP even in young cells. Hence, the more permissive chromatin state at intragenic cryptic promoters likely underlies increased cryptic transcription in aged mammalian stem cells.

FOXO3 regulates a common genomic program in aging and glioblastoma stem cells.

Background: Glioblastoma (GBM) is an aggressive, age-associated malignant glioma that contains populations of cancer stem cells. These glioma stem cells (GSCs) evade therapeutic interventions and repopulate tumors due to their existence in a slowly cycling quiescent state. Although aging is well known to increase cancer initiation, the extent to which the mechanisms supporting GSC tumorigenicity are related to physiological aging remains unknown. Aims: Here, we investigate the transcriptional mechanisms by which Forkhead Box O3 (FOXO3), a transcriptional regulator that promotes healthy aging, affects GSC function and the extent to which FOXO3 transcriptional networks are dysregulated in aging and GBM. Methods and results: We performed transcriptome analysis of clinical GBM tumors and observed that high FOXO3 activity is associated with gene expression signatures of stem cell quiescence, reduced oxidative metabolism, and improved patient outcomes. Consistent with these findings, we show that elevated FOXO3 activity significantly reduces the proliferation of GBM-derived GSCs. Using RNA-seq, we find that functional ablation of FOXO3 in GSCs rewires the transcriptional circuitry associated with metabolism, epigenetic stability, quiescence, and differentiation. Since FOXO3 has been implicated in healthy aging, we then investigated the extent to which it regulates common transcriptional programs in aging neural stem cells (NSCs) and GSCs. We uncover a shared transcriptional program and, most strikingly, find that FOXO3-regulated pathways are associated with altered mitochondrial functions in both aging and GBM. Conclusions: This work identifies a FOXO-associated transcriptional program that correlates between GSCs and aging NSCs and is enriched for metabolic and stemness pathways connected with GBM and aging.

Taking Prisoners: Vimentin Cages Capture Proteasomes during NSC Activation.

Asymmetric partitioning of damaged proteins is thought to play a key role in preserving stem cell function with age. In this issue of Cell Stem Cell, Morrow et al. (2020) show that vimentin recruits proteasome machinery to aggresomes to control NSC proteostasis during quiescence exit.

Mechanisms of enhanced quiescence in neural stem cell aging.

The maintenance of neural stem cell function is vital to ensure neurogenesis throughout adulthood. During aging, there is a significant reduction in adult neurogenesis that correlates with a decline in cognitive function. Although recent studies have revealed novel extrinsic and intrinsic mechanisms that regulate the adult neural stem cell (NSC) pool and lineage progression, the precise molecular mechanisms that drive dysregulation of adult neurogenesis in the context of aging are only beginning to emerge. Recent studies have shed light on mechanisms that regulate the earliest step of adult neurogenesis, the activation of quiescent NSCs. Interestingly, the ability of NSCs to enter the cell cycle in the aged brain significantly declines suggesting a deepend state of quiescence. Given the likely contribution of adult neurogenesis to supporting cognitive function in humans, enhancing neurogenesis may be a strategy to combat age-related cognitive decline. This review highlights the mechanisms that regulate the NSC pool throughout adulthood and discusses how dysregulation of these processes may contribute to the decline in neurogenesis and cognitive function throughout aging.

Laminin alpha5 is essential for the formation of the zebrafish fins.

The vertebrate fin fold, the presumptive evolutionary antecedent of the paired fins, consists of two layers of epidermal cells extending dorsally and ventrally over the trunk and tail of the embryo, facilitating swimming during the embryonic and larval stages. Development of the fin fold requires dramatic changes in cell shape and adhesion during early development, but the proteins involved in this process are completely unknown. In a screen of mutants defective in fin fold morphogenesis, we identified a mutant with a severe fin fold defect, which also displays malformed pectoral fins. We find that the cause of the defect is a non-sense mutation in the zebrafish lama5 gene that truncates laminin alpha5 before the C-terminal laminin LG domains, thereby preventing laminin alpha5 from interacting with its cell surface receptors. Laminin is mislocalized in this mutant, as are the membrane-associated proteins, actin and beta-catenin, that normally form foci within the fin fold. Ultrastructural analysis revealed severe morphological abnormalities and defects in cell-cell adhesion within the epidermis of the developing fin fold at 36 hpf, resulting in an epidermal sheet that can not extend away from the body. Examining the pectoral fins, we find that the lama5 mutant is the first zebrafish mutant identified in which the pectoral fins fail to make the transition from an apical epidermal ridge to an apical fold, a transformation that is essential for pectoral fin morphogenesis. We propose that laminin alpha5, which is concentrated at the distal ends of the fins, organizes the distal cells of the fin fold and pectoral fins in order to promote the morphogenesis of the epidermis. The lama5 mutant provides novel insight into the role of laminins in the zebrafish epidermis, and the molecular mechanisms driving fin formation in vertebrates.

Enhancing Lysosomal Activation Restores Neural Stem Cell Function During Aging.

Adult neurogenesis supports cognitive and sensory functions in mammals and is significantly reduced with age. Quiescent neural stem cells are the source of new neurons in the adult brain and emerging evidence suggests that the failure of these cells to activate and re-enter the cell cycle is largely responsible for reduced neurogenesis in old animals. However, the molecular mechanisms supporting quiescence and activation in the adult and aged brain remain undefined. Recent work published by Leeman et al. in Science uncovers a novel role for lysosomes in supporting neural stem cell activation, and reveals that loss of lysosome function during aging contributes to reduced neural stem cell activity. Using a combination of transcriptomics and functional analysis, the authors show that quiescent and activated neural stem cells employ different branches of proteostasis networks, with quiescent stem cells particularly dependent on the lysosome-autophagy system. Excitingly, stimulation of lysosomal activity in the aged quiescent population significantly enhanced their ability to activate and increased the frequency of activated neural stem and progenitor cells within the neural stem cell niche. This work for the first time identifies lysosomal dysfunction as a cause of reduced neurogenesis during aging, and shows that enhancing lysosomal function is sufficient to restore healthy stem cell activity in the aged brain.

Regulation of FOXO Factors in Mammalian Cells.

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.