Protein homeostasis and degradation in quiescent neural stem cells.

Tissue stem cells are maintained in the adult body throughout life and are crucial for tissue homeostasis as they supply newly functional cells. Quiescence is a reversible arrest in the G0/G1 phase of the cell cycle and a strategy to maintain the quality of tissue stem cells. Quiescence maintains stem cells in a self-renewable and differentiable state for a prolonged period by suppressing energy consumption and cell damage and depletion. Most adult neural stem cells in the brain maintain the quiescent state and produce neurons and glial cells through differentiation after activating from the quiescent state to the proliferating state. In this process, proteostasis, including proteolysis, is essential to transition between the quiescent and proliferating states associated with proteome remodeling. Recent reports have demonstrated that quiescent and proliferating neural stem cells have different expression patterns and roles as proteostatic molecules and are affected by age, indicating differing processes for protein homeostasis in these two states in the brain. This review discusses the multiple regulatory stages from protein synthesis (protein birth) to proteolysis (protein death) in quiescent neural stem cells.

Fluctuation of lysosomal protein degradation in neural stem cells of the postnatal mouse brain.

Lysosomes are intracellular organelles responsible for degrading diverse macromolecules delivered from several pathways, including the endo-lysosomal and autophagic pathways. Recent reports have suggested that lysosomes are essential for regulating neural stem cells in developing, adult and aged brains. However, the activity of these lysosomes has yet to be monitored in these brain tissues. Here, we report the development of a new probe to measure lysosomal protein degradation in brain tissue by immunostaining. Our results indicate that lysosomal protein degradation fluctuates in neural stem cells of the hippocampal dentate gyrus, depending on age and brain disorders. Neural stem cells increase their lysosomal activity during hippocampal development in the dentate gyrus, but aging and aging-related disease reduce lysosomal activity. In addition, physical exercise increases lysosomal activity in neural stem cells and astrocytes in the dentate gyrus. We therefore propose that three different stages of lysosomal activity exist: the state of increase during development, the stable state during adulthood and the state of reduction due to damage caused by either age or disease.

Direct visualization of the transition status during neural differentiation by dual-fluorescent reporter human pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) can differentiate into neurons and glia via neural progenitor cells and are widely used for neurogenic studies. However, directly visualizing the transition status during the neural differentiation of live cells is difficult. Here, targeting NEUROG2 (NGN2) and TUBB3 as markers of neurogenic cells and neurons, respectively, we established TUBB3EGFP/NGN2TagRFP dual-reporter hiPSCs using CRISPR-Cas9 technology. We induced the differentiation of cortical neurons from dual-reporter hiPSCs, successfully visualizing cell-fate conversion in two-dimensional (2D) culture and 3D organoids. The reporter cells were used to monitor drug effects to enhance neural induction, responses to gene knockdown, transplantation to the embryonic mouse brain, and live imaging at single-cell resolution. Notably, the earliest REELIN-positive neurons showed a distinctive migration pattern, and their production was accelerated by HES1-function loss. Together, these results demonstrate the potential for dual-reporter hiPSCs in therapeutic neural regeneration strategies and studies on human cortical development.

Functional rejuvenation of aged neural stem cells by Plagl2 and anti-Dyrk1a activity.

The regenerative potential of neural stem cells (NSCs) declines during aging, leading to cognitive dysfunctions. This decline involves up-regulation of senescence-associated genes, but inactivation of such genes failed to reverse aging of hippocampal NSCs. Because many genes are up-regulated or down-regulated during aging, manipulation of single genes would be insufficient to reverse aging. Here we searched for a gene combination that can rejuvenate NSCs in the aged mouse brain from nuclear factors differentially expressed between embryonic and adult NSCs and their modulators. We found that a combination of inducing the zinc finger transcription factor gene Plagl2 and inhibiting Dyrk1a, a gene associated with Down syndrome (a genetic disorder known to accelerate aging), rejuvenated aged hippocampal NSCs, which already lost proliferative and neurogenic potential. Such rejuvenated NSCs proliferated and produced new neurons continuously at the level observed in juvenile hippocampi, leading to improved cognition. Epigenome, transcriptome, and live-imaging analyses indicated that this gene combination induces up-regulation of embryo-associated genes and down-regulation of age-associated genes by changing their chromatin accessibility, thereby rejuvenating aged dormant NSCs to function like juvenile active NSCs. Thus, aging of NSCs can be reversed to induce functional neurogenesis continuously, offering a way to treat age-related neurological disorders.

Novel Roles of Small Extracellular Vesicles in Regulating the Quiescence and Proliferation of Neural Stem Cells.

Neural stem cell (NSC) quiescence plays pivotal roles in avoiding exhaustion of NSCs and securing sustainable neurogenesis in the adult brain. The maintenance of quiescence and transition between proliferation and quiescence are complex processes associated with multiple niche signals and environmental stimuli. Exosomes are small extracellular vesicles (sEVs) containing functional cargos such as proteins, microRNAs, and mRNAs. The role of sEVs in NSC quiescence has not been fully investigated. Here, we applied proteomics to analyze the protein cargos of sEVs derived from proliferating, quiescent, and reactivating NSCs. Our findings revealed fluctuation of expression levels and functional clusters of gene ontology annotations of differentially expressed proteins especially in protein translation and vesicular transport among three sources of exosomes. Moreover, the use of exosome inhibitors revealed exosome contribution to entrance into as well as maintenance of quiescence. Exosome inhibition delayed entrance into quiescence, induced quiescent NSCs to exit from the G0 phase of the cell cycle, and significantly upregulated protein translation in quiescent NSCs. Our results suggest that NSC exosomes are involved in attenuating protein synthesis and thereby regulating the quiescence of NSCs.

Lysosomes and signaling pathways for maintenance of quiescence in adult neural stem cells.

Quiescence is a cellular strategy for maintaining somatic stem cells in a specific niche in a low metabolic state without senescence for a long period of time. During development, neural stem cells (NSCs) actively proliferate and self-renew, and their progeny differentiate into both neurons and glial cells to form mature brain tissues. On the other hand, most NSCs in the adult brain are quiescent and arrested in G0/G1 phase of the cell cycle. Quiescence is essential in order to avoid the precocious exhaustion of NSCs, ensuring a sustainable source of available stem cells in the brain throughout the lifespan. After receiving activation signals, quiescent NSCs reenter the cell cycle and generate new neurons. This switching between quiescence and proliferation is tightly regulated by diverse signaling pathways. Recent studies suggest significant involvement of cellular proteostasis (homeostasis of the proteome) in the quiescent state of NSCs. Proteostasis is the result of integrated regulation of protein synthesis, folding, and degradation. In this review, we discuss regulation of quiescence by multiple signaling pathways, especially bone morphogenetic protein and Notch signaling, and focus on the functional involvement of the lysosome, an organelle governing cellular degradation, in quiescence of adult NSCs.

Enhanced lysosomal degradation maintains the quiescent state of neural stem cells.

Quiescence is important for sustaining neural stem cells (NSCs) in the adult brain over the lifespan. Lysosomes are digestive organelles that degrade membrane receptors after they undergo endolysosomal membrane trafficking. Enlarged lysosomes are present in quiescent NSCs (qNSCs) in the subventricular zone of the mouse brain, but it remains largely unknown how lysosomal function is involved in the quiescence. Here we show that qNSCs exhibit higher lysosomal activity and degrade activated EGF receptor by endolysosomal degradation more rapidly than proliferating NSCs. Chemical inhibition of lysosomal degradation in qNSCs prevents degradation of signaling receptors resulting in exit from quiescence. Furthermore, conditional knockout of TFEB, a lysosomal master regulator, delays NSCs quiescence in vitro and increases NSC proliferation in the dentate gyrus of mice. Taken together, our results demonstrate that enhanced lysosomal degradation is an important regulator of qNSC maintenance.

Multilayered gene control drives timely exit from the stem cell state in uncommitted progenitors during Drosophila asymmetric neural stem cell division.

Self-renewal genes maintain stem cells in an undifferentiated state by preventing the commitment to differentiate. Robust inactivation of self-renewal gene activity following asymmetric stem cell division allows uncommitted stem cell progeny to exit from an undifferentiated state and initiate the commitment to differentiate. Nonetheless, how self-renewal gene activity at mRNA and protein levels becomes synchronously terminated in uncommitted stem cell progeny is unclear. We demonstrate that a multilayered gene regulation system terminates self-renewal gene activity at all levels in uncommitted stem cell progeny in the fly neural stem cell lineage. We found that the RNA-binding protein Brain tumor (Brat) targets the transcripts of a self-renewal gene, deadpan (dpn), for decay by recruiting the deadenylation machinery to the 3' untranslated region (UTR). Furthermore, we identified a nuclear protein, Insensible, that complements Cullin-mediated proteolysis to robustly inactivate Dpn activity by limiting the level of active Dpn through protein sequestration. The synergy between post-transcriptional and transcriptional control of self-renewal genes drives timely exit from the stem cell state in uncommitted progenitors. Our proposed multilayered gene regulation system could be broadly applicable to the control of exit from stemness in all stem cell lineages.

Deubiquitinating enzymes regulate Hes1 stability and neuronal differentiation.

Hairy and enhancer of split 1 (Hes1), a basic helix-loop-helix transcriptional repressor protein, regulates the maintenance of neural stem/progenitor cells by repressing proneural gene expression via Notch signaling. Previous studies showed that Hes1 expression oscillates in both mouse embryonic stem cells and neural stem cells, and that the oscillation contributes to their potency and differentiation fates. This oscillatory expression depends on the stability of Hes1, which is rapidly degraded by the ubiquitin/proteasome pathway. However, the detailed molecular mechanisms governing Hes1 stability remain unknown. We analyzed Hes1-interacting deubiquitinases purified from mouse embryonic stem cells using an Hes1-specific antibody, and identified the ubiquitin-specific protease 27x (Usp27x) as a new regulator of Hes1. We found that Hes1 was deubiquitinated and stabilized by Usp27x and its homologs ubiquitin-specific protease 22 (Usp22) and ubiquitin-specific protease 51 (Usp51). Knockdown of Usp22 shortened the half-life of Hes1, delayed its oscillation, and enhanced neuronal differentiation in mouse developing brain, whereas mis-expression of Usp27x reduced neuronal differentiation. These results suggest that these deubiquitinases modulate Hes1 protein dynamics by removing ubiquitin molecules, and thereby regulate neuronal differentiation of stem cells.

Expression dynamics and functions of Hes factors in development and diseases.

Hes genes, encoding basic helix-loop-helix (HLH) transcriptional repressors, are mammalian homologues of Drosophila hairy and Enhancer of split genes, both of which are required for normal neurogenesis in Drosophila. There are seven members in the human Hes family, Hes1-7, which are expressed in many tissues and play various roles mainly in development. All Hes proteins have three conserved domains: basic HLH (bHLH), Orange, and WRPW domains. The basic region binds to target DNA sequences, while the HLH region forms homo- and heterodimers with other bHLH proteins, the Orange domain is responsible for the selection of partners during heterodimer formation, and the WRPW domain recruits corepressors. Hes1, Hes5, and Hes7 are known as downstream effectors of canonical Notch signaling, which regulates cell differentiation via cell-cell interaction. Hes factors regulate many events in development by repressing the expression of target genes, many of which encode transcriptional activators that promote cell differentiation. For example, Hes1, Hes3, and Hes5 are highly expressed by neural stem cells, and inactivation of these genes results in insufficient maintenance of stem cell proliferation and prematurely promotes neuronal differentiation. Recently, it was shown that the expression dynamics of Hes1 plays crucial roles in proper developmental timings and fate-determination steps of embryonic stem cells and neural progenitor cells. Here, we discuss some key features of Hes factors in development and diseases.

The roles and mechanism of ultradian oscillatory expression of the mouse Hes genes.

Somites, metameric structures, give rise to the vertebral column, ribs, skeletal muscles and subcutaneous tissues. In mouse embryos, a pair of somites is formed every 2h by segmentation of the anterior parts of the presomitic mesoderm. This periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic expression of the basic helix-loop-helix gene Hes7. Hes7 oscillation is regulated by negative feedback with a delayed timing. This process has been mathematically simulated by differential-delay equations, which predict that negative feedback with shorter delays would abolish oscillations or produce dampened but more rapid oscillations. We found that reducing the number of introns within the Hes7 gene shortens the delay and abolishes Hes7 oscillation or results in a more rapid tempo of Hes7 oscillation, increasing the number of somites and vertebrae in the cervical and upper thoracic region. We also found that Hes1, a Hes7-related gene, is expressed in an oscillatory manner by many cell types, including fibroblasts and neural stem cells. In these cells, Hes1 expression oscillates with a period of about 2-3h, and this oscillation is important for cell cycle progression. Furthermore, in neural stem cells, Hes1 oscillation drives cyclic expression of the proneural genes Ascl1 and Neurogenin2 and regulates multipotency. Hes1 expression oscillates more slowly in embryonic stem cells, and Hes1 oscillation regulates their fate preferences. Taken together, these results suggest that oscillatory expression with short periods (ultradian oscillation) is important for many biological events.

Novel and robust transplantation reveals the acquisition of polarized processes by cortical cells derived from mouse and human pluripotent stem cells.

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apico-basal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.

The role of Hes genes in intestinal development, homeostasis and tumor formation.

Notch signaling regulates intestinal development, homeostasis and tumorigenesis, but its precise downstream mechanism remains largely unknown. Here we found that inactivation of the Notch effectors Hes1, Hes3 and Hes5, but not Hes1 alone, led to reduced cell proliferation, increased secretory cell formation and altered intestinal structures in adult mice. However, in Apc mutation-induced intestinal tumors, inactivation of Hes1 alone was sufficient for reducing tumor cell proliferation and inducing differentiation of tumor cells into all types of intestinal epithelial cells, but without affecting the homeostasis of normal crypts owing to genetic redundancy. These results indicated that Hes genes cooperatively regulate intestinal development and homeostasis and raised the possibility that Hes1 is a promising target to induce the differentiation of tumor cells.

Hes1 oscillations contribute to heterogeneous differentiation responses in embryonic stem cells.

Embryonic stem (ES) cells can differentiate into multiple types of cells belonging to all three germ layers. Although ES cells are clonally established, they display heterogeneous responses upon the induction of differentiation, resulting in a mixture of various types of differentiated cells. Our recent reports have shown that Hes1 regulates the fate choice of ES cells by repressing Notch signaling, and that the oscillatory expression of Hes1 contributes to various differentiation responses in ES cells. Here we discuss the mechanism regulating the intracellular dynamics in ES cells and how to trigger the lineage choice from pluripotent ES cells.

Ultradian oscillations in Notch signaling regulate dynamic biological events.

Notch signaling regulates many dynamic processes; accordingly, expression of genes in this pathway is also dynamic. In mouse embryos, one dynamic process regulated by Notch is somite segmentation, which occurs with a 2-h periodicity. This periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic expression of the Notch effector gene Hes7. Loss of Hes7 expression and sustained expression of Hes7 result in identical and severe somite defects, suggesting that Hes7 oscillation is required for proper somite segmentation. Mathematical models of this oscillator have been used to generate and test hypothesis, helping to uncover the role of negative feedback in regulating the oscillator. Oscillations of another Notch effector gene, Hes1, plays an important role in maintenance of neural stem cells. Hes1 expression oscillates with a period of about 2-3h in neural stem cells, whereas sustained Hes1 expression inhibits proliferation and differentiation of these cells, suggesting that Hes1 oscillations are important for their proper activities. Hes1 inhibits its own expression as well as the expression of the proneural gene Neurogenin2 and the Notch ligand Delta1, driving oscillations of these two genes. Delta1 oscillations in turn maintain neural stem cells by mutual activation of Notch signaling, which re-activates Hes1 to close the cycle. Hes1 expression also oscillates in embryonic stem (ES) cells. Cells expressing low and high levels of Hes1 tend to differentiate into neural and mesodermal cells, respectively. Furthermore, Hes1-null ES cells display early and uniform neural differentiation, indicating that Hes1 oscillations act to promote multipotency by generating heterogeneity in both the differentiation timing and the fate choice. Taken together, these results suggest that Notch signaling can drive short-period oscillatory expression of Hes7 and Hes1 (ultradian oscillation) and that ultradian oscillations are important for many biological events.

Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling.

Embryonic stem (ES) cells display heterogeneous responses upon induction of differentiation. Recent analysis has shown that Hes1 expression oscillates with a period of about 3-5 h in mouse ES cells and that this oscillating expression contributes to the heterogeneous responses: Hes1-high ES cells are prone to the mesodermal fate, while Hes1-low ES cells are prone to the neural fate. These outcomes of Hes1-high and Hes1-low ES cells are very similar to those of inactivation and activation of Notch signaling, respectively. These results suggest that Hes1 and Notch signaling lead to opposite outcomes in ES cell differentiation, although they work in the same direction in most other cell types. Here, we found that Hes1 acts as an inhibitor but not as an effector of Notch signaling in ES cell differentiation. Our results indicate that sustained Hes1 expression delays the differentiation of ES cells and promotes the preference for the mesodermal rather than the neural fate by suppression of Notch signaling.

The cyclic gene Hes1 contributes to diverse differentiation responses of embryonic stem cells.

Stem cells do not all respond the same way, but the mechanisms underlying this heterogeneity are not well understood. Here, we found that expression of Hes1 and its downstream genes oscillate in mouse embryonic stem (ES) cells. Those expressing low and high levels of Hes1 tended to differentiate into neural and mesodermal cells, respectively. Furthermore, inactivation of Hes1 facilitated neural differentiation more uniformly at earlier time. Thus, Hes1-null ES cells display less heterogeneity in both the differentiation timing and fate choice, suggesting that the cyclic gene Hes1 contributes to heterogeneous responses of ES cells even under the same environmental conditions.

Dynamic advances in NF-kappaB signaling analysis.

Signaling through the transcription factor nuclear factor kappaB (NF-kappaB) regulates various physiological processes, including immune responses and inflammation. An analysis of NF-kappaB dynamics suggests that the oscillation frequency of NF-kappaB activation determines which downstream genes are expressed, enabling a single pathway to respond to diverse stimuli in distinct ways.

Requirement of multiple lysine residues for the transcriptional activity and the instability of Hes7.

The basic helix-loop-helix (bHLH) gene Hes7 is expressed in an oscillatory manner and regulates the periodic somite formation. Oscillatory expression of Hes7 depends on negative feedback and rapid degradation of the gene products, but the precise mechanisms of how the transcriptional activity and the degradation of Hes7 protein are regulated remain to be analyzed. Here, we found that lysine residues (K22, K52, and K55) in the bHLH domain are essential not only for the instability of Hes7 protein but also for the transcriptional repressor activity. Introduction of lysine-to-arginine mutations into the bHLH domain led to stabilization of Hes7 protein and to abnormalities in either the N box-binding activity or partner preference in heterodimer formation. These results indicate that common amino acid residues are involved in both the transcriptional repressor activity and the instability of Hes7 protein, suggesting of a critical link between the transcription and degradation control.